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Metodos Analisis - Vol.2 OIV
Metodos Analisis - Vol.2 OIV
INTERNATIONAL METHODS
OF WINE AND MUST ANALYSIS
INTERNATIONAL ORGANISATION
OF VINE AND WINE
COMPENDIUM
OF INTERNATIONAL
METHODS OF WINE
AND MUST ANALYSIS
EDITION 2012
VOLUME 2
INCLUDED :
Resolutions adopted in Porto (Portugal)
9th A.G. 24 June 2011
OIV - 18, RUE DAGUESSEAU - 75008 PARIS
Table of contents
Foreword
ANNEX A METHODS OF ANALYSIS OF WINES AND MUSTS
SECTION 1 DEFINITIONS AND GENERAL PRINCIPLES
SECTION 2 PHYSICAL ANALYSIS
SECTION 3 CHIMICAL ANALYSIS
SECTION 3.1 ORGANIC COMPOUNDS
SECTION 3.1.1 SUGARS
SECTION 3.1.2 ALCOHOLS
SECTION 3.1.3 ACIDS
SECTION 3.1.4 GAS
SECTION 3.1.5 OTHER ORGANIC COMPOUNDS
SECTION 3.2 NON ORGANIC COMPOUNDS
SECTION 3.2.1 ANIONS
SECTION 3.2.2 CATIONS
SECTION 3.2.3 OTHER NON ORGANIC COMPOUNDS
SECTION 4 MICROBIOLOGICAL ANALYSIS
SECTION 5 OTHER ANALYSIS
ANNEX D ADVICES
OIV-MA-INT-00-2012
Reference
- Table of contents
Type
method
OIV-MA-INT-00
VOLUME 1
- Foreword
- Layout and wording of OIV method of analysis
OIV-MA-INT-01
OIV-MA-INT-04
OIV-MA-INT-00-2012
OIV-MA-AS2-01A
OIV-MA-AS2-01B
IV
OIV-MA-AS2-02
OIV-MA-AS2-03A
OIV-MA-AS2-03B
IV
OIV-MA-AS2-04
OIV-MA-AS2-05
OIV-MA-AS2-06
OIV-MA-AS2-07A
OIV-MA-AS2-07B
OIV-MA-AS2-08
OIV-MA-AS2-09
I
IV
IV
Withdrawn
IV
IV
Withdrawn
OIV-MA-AS2-10
IV
OIV-MA-AS2-11
OIV-MA-AS2-12
II
OIV-MA-AS2-13
OIV-MA-INT-00-2012
OIV-MA-AS311-01A
OIV-MA-AS311-01B
OIV-MA-AS311-01C
IV
Withdrawn
Withdrawn
OIV-MA-AS311-02
II
OIV-MA-AS311-03
OIV-MA-AS311-04
II
OIV-MA-AS311-05
II
OIV-MA-AS311-06
IV
OIV-MA-AS311-07
III
OIV-MA-AS311-08
IV
OIV-MA-AS312-01A
OIV-MA-AS312-01B
IV
OIV-MA-AS312-02
OIV-MA-AS312-03A
OIV-MA-AS312-03B
OIV-MA-AS312-04
IV
IV
IV
OIV-MA-AS312-05
IV
OIV-MA-AS312-06
II
OIV-MA-AS312-07
IV
OIV-MA-AS313-01
OIV-MA-AS313-02
OIV-MA-AS313-03
I
I
I
OIV-MA-AS313-04
IV
OIV-MA-AS313-05A
OIV-MA-AS313-05B
IV
Withdrawn
OIV-MA-AS313-06
Withdrawn
OIV-MA-AS313-07
II
OIV-MA-AS313-08
IV
OIV-MA-AS313-09
II
OIV-MA-AS313-10
IV
OIV-MA-AS313-11
II
OIV-MA-AS313-12A
II
OIV-MA-AS313-12B
IV
OIV-MA-AS313-13A
IV
OIV-MA-AS313-13B
Withdrawn
OIV-MA-AS313-14A
IV
OIV-MA-AS313-14B
OIV-MA-AS313-14C
OIV-MA-AS313-15
OIV-MA-AS313-16
OIV-MA-AS313-17
OIV-MA-AS313-18
OIV-MA-AS313-20
OIV-MA-AS313-21
IV
IV
I
IV
II
IV
II
III
IV
IV
OIV-MA-AS313-22
II
OIV-MA-AS313-23
IV
OIV-MA-AS314-01
II
OIV-MA-AS314-02
OIV-MA-AS314-03
II
OIV-MA-AS314-04
II
OIV-MA-AS315-01
IV
OIV-MA-AS315-02A
IV
OIV-MA-AS313-19
VOLUME 2
SECTION 3.1.5 OTHER ORGANIC COMPOUNDS
- Acetaldehyde (ethanal) (A 37 revised by 377/2009)
- Ethyl Acetate (GC) (Recueil OIV ed. 1990 revised by
377/2009)
OIV-MA-INT-00-2012
OIV-MA-INT-00-2012
OIV-MA-AS315-02B
OIV-MA-AS315-03
OIV-MA-AS315-04
IV
IV
II
OIV-MA-AS315-05A
IV
OIV-MA-AS315-05B
IV
OIV-MA-AS315-06
II
OIV-MA-AS315-07A
IV
OIV-MA-AS315-07B
IV
OIV-MA-AS315-08
IV
OIV-MA-AS315-09
IV
OIV-MA-AS315-10
II
OIV-MA-AS315-11
II
OIV-MA-AS315-12
OIV-MA-AS315-13
OIV-MA-AS315-14
Withdrawn
OIV-MA-AS315-15
II
OIV-MA-AS315-16
IV
OIV-MA-AS315-17
IV
OIV-MA-AS315-18
II
OIV-MA-AS315-19
IV
OIV-MA-AS315-20
IV
OIV-MA-AS315-21
IV
OIV-MA-AS315-22
IV
OIV-MA-AS315-23
criteria
OIV-MA-AS321-01
OIV-MA-SA321-02
OIV-MA-AS321-03
OIV-MA-AS321-04
OIV-MA-AS321-05A
IV
IV
IV
II
II
IV
II
OIV-MA-INT-00-2012
OIV-MA-AS321-05B
Withdrawn
OIV-MA-AS322-01
OIV-MA-AS322-02A
OIV-MA-AS322-02B
OIV-MA-AS322-02C
OIV-MA-AS322-03A
OIV-MA-AS322-03B
OIV-MA-AS322-04
OIV-MA-AS322-05A
OIV-MA-AS322-05B
OIV-MA-AS322-06
OIV-MA-AS322-07
OIV-MA-AS322-08
OIV-MA-AS322-09
OIV-MA-AS322-10
OIV-MA-AS322-11
OIV-MA-AS322-12
IV
II
III
OIV-MA-AS323-01A
OIV-MA-AS323-01B
OIV-MA-AS323-01C
OIV-MA-AS323-02A
Withdrawn
II
III
II
IV
IV
IV
II
IV
IV
IV
Withdrawn
II
IV
IV
Withdrawn
IV
OIV-MA-AS323-02B
II
OIV-MA-AS323-03
OIV-MA-AS323-04A
OIV-MA-AS323-04B
IV
II
IV
OIV-MA-AS323-04C
IV
OIV-MA-AS323-05
IV
OIV-MA-AS323-06
IV
OIV-MA-AS323-07
II
OIV-MA-AS4-01
IV
OIV-MA-AS4-02A
IV
OIV-MA-AS4-02B
IV
OIV-MA-AS4-02C
IV
OIV-MA-AS4-02D
IV
OIV-MA-AS4-02E
IV
OIV-MA-AS4-02F
IV
OIV-MA-AS4-03
IV
OIV-MA-B1-01
OIV-MA-B1-02
OIV-MA-INT-00-2012
OIV-MA-D1-01
OIV-MA-D1-02
OIV-MA-D1-03
OIV-MA-AS1-05
OIV-MA-AS1-07
OIV-MA-AS1-08
OIV-MA-AS1-09
OIV-MA-AS1-10
OIV-MA-AS1-11
OIV-MA-AS1-12
OIV-MA-AS1-13
OIV-MA-AS1-14
OIV-MA-AS1-15
OIV-MA-INT-00-2012
IV
IV
OIV-MA-F1-01
OIV-MA-F1-02
IV
OIV-MA-F1-03
OIV-MA-F1-04
IV
OIV-MA-F1-05
OIV-MA-F1-06
OIV-MA-F1-07
OIV-MA-F1-08
IV
IV
IV
IV
Foreword
The Compendium of International Methods of Wine Analysis was first published in
1962 and re-published in 1965, 1972, 1978, 1990 and 2000; each time it included
additional material as approved by the General Assembly and produced each year
by the Sub-Commission.
This edition of Compendium of International Methods of Wine and Must Analysis
includes all material as approved by the General Assembly of representatives of the
member governments of the OIV, revised and amended since 2000.
OIV-MA-INT-01
Method OIV-MA-AS315-01
Type IV method
Acetaldehyde
(Resolution Oeno 377/2009)
1. Principle
Acetaldehyde (ethanal) in carbon decolorized wine, reacts with sodium
nitroferricyanide and piperidine and causes a green to violet color change whose
intensity is measured at 570 nm.
2. Apparatus
Spectrophotometer permitting measurement of absorbance at a wavelength of
570 nm with a 1 cm optical cell path.
3. Reagents
3.1 Piperidine solution, (C5H11N) 10% (v/v).
Prepare just before use by mixing 2 mL of piperidine with 18 mL of distilled
water.
3.2 Sodium nitroferricyanide solution, 0.4% (m/v).
In a 250 mL glass volumetric flask, dissolve 1 g of pulverized sodium
nitroferricyanide, Na2 [Fe(CN)5 NO].2H2O in distilled water and make up to
volume.
3.3 Activated carbon
3.4 Dilute hydrochloric acid, 25% (v/v)
3.5 Alkaline solution
Dissolve 8.75 g of boric acid in 400 mL sodium hydroxide solution, 1 M.
Make up to 1 L with distilled water.
4. Procedure
4.1 Sample
Place approx. 25 mL of wine in a 100 mL Erlenmeyer flask, add 2 g of
activated charcoal. Shake vigorously for a few seconds, allow to stand for 2
minutes and filter through a fluted slow filter to obtain a clear filtrate.
OIV-MA-AS315-01 : R2009
Place 2 mL of the clear filtrate into a 100 mL Erlenmeyer flask, add, while
shaking, 5 mL of the sodium nitroferricyanide solution (3.2) and 5 mL of the
piperidine solution (3.1). Mix and place the mixture immediately into a 1 cm
optical cell. The coloration produced, which varies from green to violet, is
measured with reference to air at a wavelength of 570 nm. This color change
increases then decreases rapidly; measure immediately and record the
maximum value of the absorbance that is obtained after about 50 seconds. The
concentration of acetaldehyde in the liquid analyzed is obtained using a
calibration curve.
Note: If the liquid analyzed contains excess free acetaldehyde, it will be necessary,
before beginning the total acetaldehyde determination, to first combine it with sulfur
dioxide. To achieve this, add a small amount of excess free SO2 to a portion of the
liquid to be analyzed and wait for an hour before proceeding.
BIBLIOGRAPHY
REBELEIN H., Dtsch. Lebensmit. Rdsch., 1970, 66, 5-6.
OIV-MA-AS315-01 : R2009
Method OIV-MA-AS315-02A
Type IV method
Ethyl Acetate
2. Method
2.1 Apparatus (see chapter Volatile Acidity).
2.2 Procedure
Prepare an internal standard solution of 4-methyl-2-pentanol, 1 g/L, in ethanol
solution, 10% (v/v).
Prepare the sample solution to be determined by adding 5 mL of this internal
standard solution to 50 mL of wine distillate obtained as indicated in the
chapter on Alcoholic Strength.
Prepare a reference solution of ethyl acetate, 50 mg/L, in ethanol, 10% (v/v).
Add 5 mL of the internal standard to 50 mL of this solution.
Analyze 2 L of the sample solution and the reference solution using gas
chromatography.
Oven temperature is 90C and the carrier gas flow rate is 25 mL per minute.
2.3 Calculation
S = the peak area of ethyl acetate in the reference solution.
Sx = the peak area of the ethyl acetate in the sample solution.
I = the peak area of the internal standard in the sample solution.
I = the peak area of the internal standard in the reference solution.
The concentration of ethyl acetate, expressed in milligrams per liter, is given
by:
S
50 x I x
i S
OIV-MA-AS315-02A : R2009
Method OIV-MA-AS315-02B
Type IV method
Ethyl Acetate
OIV-MA-AS315-02B : R2009
BIBLIOGRAPHY
Usual method:
PEYNAUD E., Analyse et contrle des vins, Librairie Polytechnique Ch.Branger, 1958.
OIV-MA-AS315-02B : R2009
Method OIV-MA-AS315-03
Type IV method
Malvidin diglucoside
1. Principle
Malvidin diglucoside, oxidized by nitric acid, is converted to a substance that, in
an ammonium medium, emits a vivid green fluorescence in ultraviolet light.
The intensity of the fluorescence of the compound formed is measured by
comparison with the fluorescence of a solution titrated with quinine sulfate whose
intensity of fluorescence is standardized with the malvidin diglucoside reference.
Free sulfur dioxide, which attenuates the fluorescence, must previously be
combined with excess acetaldehyde.
2. Qualitative Examination
2.1 Apparatus
2.1.1 Ultraviolet lamp permitting measurement at 365 nm.
2.2 Reagents
2.2.1 Acetaldehyde solution
Crystallizable paraldehyde ..................................... 10 g
Ethanol 96% (v/v) ............................................ 100 mL
2.2.2 Hydrochloric acid, 1.0 M.
2.2.3 Sodium nitrate solution, 10 g/L.
2.2.4 Ethanol, 96% (v/v), containing 5% concentrated ammonia solution
(20 = 0.92 g/mL).
2.2.5 Control wine containing 15 mg of malvidin diglucoside per liter.
2.2.6 Wine containing no malvidin diglucoside.
2.3 Method
Into a test tube add:
- 10 mL of wine
- 1.5 mL of acetaldehyde solution
wait 20 minutes.
Into a 20 mL centrifuge tube place:
- 1 mL of wine reacted with acetaldehyde
- 1 drop of hydrochloric acid
- 1 mL sodium nitrate solution
Stir; wait 2 minutes (5 minutes maximum); add:
OIV-MA-AS315-03 : R2009
- 10 mL ammoniacal ethanol
Treat similarly 10 mL of wine containing 15 mg/L malvidin diglucoside (The
control wine). Stir. Wait 10 minutes and centrifuge.
Decant the clear liquids from the top into calibrated test tubes. Observe the
difference in green fluorescence between the test wine and the control wine
under ultraviolet light at 365 nm.
For rose wines, it is possible to increase the sensitivity using:
- 5 mL of wine treated with acetaldehyde (2.3)
- 0.2 mL hydrochloric acid, 1 M (2.2.2)
- 1 mL sodium nitrate solution, 10 g/L (2.2.3)
- 5.8 mL ammoniacal ethanol (2.2.4)
Treat the control wine in a similar manner.
2.4 Interpretation
Wines that do not fluoresce, or have a distinctly lower fluorescence, than the
control, may be considered to have no malvidin diglucoside. Those whose
fluorescence is slightly less than, equal to, or greater than the control should
have a quantitative determination.
3. Quantitative Determination
3.1. Apparatus
3.1.1. Equipment for measuring fluorescence:
- excitation wavelength 365 nm;
- wavelength of fluorescent radiation 490 nm.
3.1.2. Optical quartz cell (1 cm path length)
3.2 Reagents
3.2.1. See qualitative examination
3.2.2. 2 mg/L quinine sulfate solution
Prepare a solution containing 10 mg very pure quinine sulfate in 100 mL
sulfuric acid, 0.1 M. Dilute 20 mL of this solution to 1 liter with sulfuric acid
solution, 0.1 M.
3.3 Procedure
Treat the wine by the method described in Qualitative examination (2), except
that the aliquot of acetaldehyde treated wine is each case (red wines and roses)
1 mL.
Place the 2 mg/L solution of quinine sulfate in the cell, adjust the fluorometer
to the full range (transmission T, equal to 100%) by adjusting the slit width or
the sensitivity.
OIV-MA-AS315-03 : R2009
Replace this tube with the one containing the test wine: this is the T 1 value.
If the percentage of transmission, T1 is greater than 35, dilute the wine with
wine without malvidin diglucoside whose fluorescence must be less than 6%
(this should be ascertained by previous testing.)
Remarks:
1. Salicylic acid (sodium salicylate) added to the wine for stabilization before
analysis, causes a spurious fluorescence which can be eliminated by an
extraction with ether.
2. Spurious fluorescence is caused by the addition of caramel.
3.4 Calculation
A fluorescence intensity of 1, for wine without SO2, for the operating
conditions above with the exception of the acetaldehyde treatment,
corresponds to 0.426 mg malvidin diglucoside per liter of wine.
On the other hand, red and rose wines, containing no malvidin diglucoside,
give fluorescence corresponding to a T value of the order of 6%.
The amount of malvidin diglucoside in wine in milligrams per liter is
therefore:
(T1 6) 0,426 x
11,5
(T1 6) x 0,49
10
BIBLIOGRAPHY
DORIER P., VERELLE L., Ann. Fals. Exp. Chim., 1966, 59, 1.
GAROGLIO P.G., Rivista Vitic. Enol., 1968, 21, 11.
BIEBER H., Deutsche Lebensm. Rdsch., 1967, 44-46.
CLERMONT Mlle S., SUDRAUD P., F.V., O.I.V., 1976 n 586.
OIV-MA-AS315-03 : R2009
Method OIV-MA-AS315-04
Type II method
Ethyl Carbamate
(Resolution Oeno 8/98)
* For certain wines which are particularly rich, it may be desirable to use a 50m long
capillary column.
** For certain wines which are particularly rich, it may be desirable to carry out a
temperature program of 2C per minute.
OIV-MA-AS315-04 : R2009
sensitivity, SIM acquisition mode, solvent delay and time for the start of
acquisition 22 min., dwell time/ion 100 ms.
2.4 Rotary evaporator under vacuum or concentration system similar to Kuderna
Danish. (Note: the recovery of the ethyl carbamate test sample, (3.7) must be
between 90-110% during the process).
2.5 Flask - pear-shaped, 300 mL, single neck, 24/40 standard taper joint.
2.6 Concentrator tube - 4 mL, graduated, with a standard taper 19/22 Teflon
coated joint and stopper.
3. Reagents
3.1 Acetone - HPLC quality. Note: Check each batch by GC/MS before use with
regard to the absence of response for m/z 62, 74 and 89 ions.
3.2 Dichloromethane - Note: Analyze each batch before use by GC/MS after 200
fold concentration to check the absence of response for m/z 62, 74 and 89
ions.
3.3 Ethanol - anhydrous
3.4 Ethyl carbamate (EC) standard solutions
- (1) Stock solution - 1.00 mg/mL. Weigh 100 mg EC ( 99% purity) in a
volumetric flask of 100 mL and dilute to mark with acetone.
- (2) Standard working solution- 10.0 g/mL. Transfer 1 mL of the EC stock
solution to a 100 mL volumetric flask and dilute with acetone to the mark.
3.5 n-Propyl carbamate (PC), standard solutions.
- (1) Stock solution - 1.00 mg/mL. Weigh 100 mg PC (reagent quality) in a
100 mL volumetric flask and dilute with acetone to the mark.
- (2) Standard working solution- 10.0 g/mL. Transfer 1 mL of the PC stock
solution to a volumetric flask of 100 mL and dilute with acetone to the
mark.
- (3) Internal standard solution PC - 400 ng/mL. Transfer 4 mL of the
standard PC working solution to a volumetric flask of 100 mL and dilute
with water to the mark.
3.6 EC - nPC standard calibration solutions - Dilute the standard working
solutions of EC, 3.4 (2), and PC 3.5 (2), with dichloromethane in order to
obtain:
(1)
100 ng EC and 400 ng nPC/mL,
(2)
200 ng EC and 400 ng nPC/mL,
(3)
400 ng EC and 400 ng nPC/mL,
(4)
800 ng EC and 400 ng nPC/mL,
(5) 1600 ng EC and 400 ng nPC/mL.
OIV-MA-AS315-04 : R2009
OIV-MA-AS315-04 : R2009
Rinse the flask with 1 mL of dichloromethane and transfer the rinsing liquid to the
tube.
Concentrate the sample to 1 mL under a slight nitrogen stream.
If an autosampler is used, transfer the concentrate to a vial for GC/MS analysis.
6. GC/MS Analysis
6.1 Calibration curve - Inject 1l of each calibration standard solution 3.6, into
GC/MS. Plot the graph of the EC-nPC area ratio for the response to m/z 62
ion on the y-axis and the quantity of EC in ng/mL on the x-axis (i.e., 100, 200,
400, 800, 1600 ng/mL).
6.2 EC quantification - Inject 1l of concentrated extract from 5 in the GC/MS
system and calculate the EC-nPC area ratio for m/z 62 ion. Determine the
concentration of EC (ng/mL) in the extract by using the internal standard
standardization curve. Calculate the EC concentration in the test sample
(ng/mL) by dividing the quantity of EC (ng/mL) in the extract by the test
sample volume 3.7.
6.3 Confirmation of EC identity. Determine if the response for m/z 62, 74 and 89
ions appear at the EC retention time. These responses characteristic
respectively of the main fragments (M - C2H3)+ and (M - CH3)+ and
molecular ion (M). The presence of EC is confirmed if the relative ratio of
these ions does not exceed 20% of the ratios of the EC standard. The extract
may need to be further concentrated in order to obtain a sufficient response for
the m/z 89 ion.
7. Method performance.
Sample
Wine over 14
% alcohol
(v/v)
Wine under
14% alcohol
(v/v)
Mean EC Recovery
found, ng/g of added
EC, %
40
80
162
11
25
48
OIV-MA-AS315-04 : R2009
89
90
93
93
sr
SR
1.59
3.32
8.20
0.43
1.67
1.97
4.77
7.00
11.11
2.03
2.67
4.25
RSDr % RSDR%
4.01
4.14
5.05
3.94
6.73
4.10
12.02
8.74
6.84
18.47
10.73
8.86
Method OIV-MA-AS315-05A
Type IV method
Hydroxymethylfurfural (HMF)
1. Principle of the methods
Aldehydes derived from furan, the main one being hydroxymethylfurfural,
react with barbituric acid and para-toluidine to give a red compound which is
determined by colorimetry at 550 nm.
Free sulfurous acid interferes with the determination. When its amount
exceeds 10 mg/L, it must be previously eliminated by combining it with
acetaldehyde whose excess does not interfere with the determination.
2. Colorimetric method
2.1 Apparatus
2.1.1 Spectrophotometer for making measurements between 300 and 700 nm.
2.1.2 Glass cells with optical paths of 1 cm.
2.2 Reagents
2.2.1 Barbituric acid solution, 0.5% (m/v)
Dissolve 500 mg of barbituric acid in distilled water by heating slightly over a
water bath at 100C. Make up to 100 mL with distilled water. This solution
keeps for about a week.
2.2.2 Para-toluidine solution, 10% (m/v).
Place 10 g of para-toluidine in a 100 mL volumetric flask; add 50 mL of isopropanol, CH3CH(OH)CH3, and 10 mL of glacial acetic acid, CH3COOH
(20 = 1.05 g/mL). Make up to 100 mL with iso-propanol. This solution
should be renewed daily.
2.2.3 Acetaldehyde (ethanal) solution, 1% (m/v).
Prepare just before use.
2.2.4 Hydroxymethylfurfural solution, 1 g/L.
Prepare dilutions of the above solution to containing 5, 10, 20, 30 and 40 mg
hydroxymethylfurfural/L. The 1 g/L solution and its dilutions must be freshly
prepared.
OIV-MA-AS315-05A : R2009
2.3 Procedure
2.3.1 Preparation of sample
- Free sulfur dioxide less than 10 mg/L:
Perform the analysis on 2 mL of wine or must. If necessary filter the wine or
must before analysis.
- Free sulfur dioxide greater than 10 mg/L:
15 mL of the test samples are placed in a 25 mL spherical flask with 2 mL
acetaldehyde solution (2.2.3). Stir. Wait 15 minutes. Bring to volume with
distilled water. Filter if necessary. Perform the analysis on 2 mL of this
solution.
2.3.2 Colorimetric determination
Into each of two 25 mL flasks, a and b, fitted with ground glass stoppers, place
2 mL of the sample prepared as in 2.3.1. Place in each flask 5 mL of paratoluidine solution (2.2.2); mix. Add 1 mL of distilled water to flask b (control)
and 1 mL barbituric acid (2.2.1) solution to flask a, shake to mix. Transfer the
contents of the flasks into spectrophotometer cells with optical paths of 1 cm.
Zero the absorbance scale at a wavelength of 550 nm using the contents of
flask b. Follow the variation in the absorbance of the contents of flask a;
record the maximum value A, which is reached after 2 to 5 minutes.
Samples with hydroxymethylfurfural concentrations above 30 mg/L must be
diluted before the analysis.
2.3.3 Preparation of the calibration curve
Place 2 mL of each of the hydroxymethylfurfural solutions of 5, 10, 20, 30 and
40 mg/L into two sets of 25 mL flasks, a and b, and treat them as described in
2.3.2.
The graph representing the variation of absorbance with the
hydroxymethylfurfural concentration in mg/L should be a straight line passing
through the origin.
2.4 Expression of results
The hydroxymethylfurfural concentration is obtained by plotting on the
calibration curve the absorbance determined on the sample analyzed, taking
into account any dilution carried out.
The result is expressed in milligrams per liter (mg/L) to one decimal point.
OIV-MA-AS315-05A : R2009
Method OIV-MA-AS315-05B
Type IV method
Hydroxymethylfurfural (HMF)
1. Principle of the methods
Separation through a column by reversed-phase chromatography and
determination at 280 nm.
Procedures described below are given as examples.
OIV-MA-AS315-05B : R2009
2.3 Procedure
Inject 5 (or 10) L of the sample prepared as described above and 5 (or 10) L
of hydroxymethylfurfural reference solution into the chromatograph. Record
the chromatogram.
The retention time of hydroxymethylfurfural is about six to seven minutes.
2.4 Expression of the Results
The hydroxymethylfurfural concentration is expressed in milligrams per liter
(mg/L) to one decimal point.
OIV-MA-AS315-05B : R2009
Method OIV-MA-AS315-06
Type II method
Cyanide Derivatives
(Resolution Oeno 4/94)
1. Principle
Free and total hydrocyanic acid is liberated by acid hydrolysis and separated by
distillation. After reaction with chloramine T and pyridine, the glutaconic
dialdehyde formed is determined by colorimetry, due to the blue coloration it gives
with 1.3-dimethyl barbituric acid.
2. Equipment
2.1. Distillation apparatus: Use the distillation apparatus described for the
determination of alcohol in wine.
2.2. Round-bottomed 500 mL flask with standard taper joint.
2.3. Water bath, thermostated at 20 C.
2.4. Spectrophotometer permitting the measurement of absorbance at a
wavelength of 590 nm.
2.5. Glass cuvette or disposable cuvettes for one use only, with 20 mm optical
path.
3. Reagents
3.1. Phosphoric acid (H3PO4) at 25 p. 100 (w/v)
3.2. Solution of chloramine T (C7H7 ClNNaO2S.3H2O) 3% (w/v)
3.3. Solution of 1,3-dimethylbarbituric acid: dissolve 3.658 g of 1,3dimethylbarbituric acid (C6H8N2O3) in 15 mL of pyridine and 3 mL of
hydrochloric acid (20 = 1.19 g/mL) and bring to 50 mL with distilled water.
3.4. Potassium cyanide (KCN)
3.5. Solution of potassium iodide (KI) 10% (w/v)
3.6. Solution of silver nitrate (AgNO3), 0.1 M
4. Procedure
4.1 Distillation:
In the 500 mL round-bottomed flask (2.2), place 25 mL of wine, 50 mL of
OIV-MA-AS315-06 : R2009
OIV-MA-AS315-06 : R2009
i.e. approximately 6% . Xi
i.e. approximately 25% . Xi
r = 6.4 g/L
R = 23 g/L
i.e. approximately 8% . Xi
i.e. approximately 29% . Xi
Xi = average concentration of HCN in the wine.
BIBLIOGRAPHY
1) JUNGE C., Feuillet vert N 877 (1990)
2) ASMUS E. GARSCHLAGEN H., Z Anal. Chem. 138, 413-422 (1953)
3) WRDIG G., MLLER TH., Die Weinwissenschaft 43, 29-37 (1988)
OIV-MA-AS315-06 : R2009
Method OIV-MA-AS315-07A
Type IV method
OIV-MA-AS315-07A : R2009
- Benzene
- Ethyl acetate
2.2.2 Chromatography solvents:
Mixture No.1:
Acetone .......................................................
Ethyl acetate ....................................................
Ammonium hydroxide (20= 0.92 g/mL) ..............
Mixture No 2.:
Toluene ..........................................................
Methanol ........................................................
Glacial acetic acid (20= 1.05 g/mL) .....................
60 parts
30 parts
10 parts
90 parts
10 parts
10 parts
2.3 Procedure
2.3.1 Extraction
100 mL of wine, placed in a beaker, are rapidly evaporated by boiling until the
volume is reduced to 30 mL, while directing a current of cold air to the surface
of the flask. Allow to cool. Acidify with 3 mL 50% hydrochloric acid (v/v).
Transfer to a 500 mL conical flask with a ground stopper, add 40 mL of
benzene and stir with a mechanical stirrer for 30 min. Transfer to a separating
funnel to separate the organic phase. If an emulsion is formed, it must be
separated by centrifugation. Place the organic phase in a conical flask with a
ground glass stopper.
Decant the wine previously extracted with benzene, which corresponds to the
lower layer in the separating funnel, into a 500 mL conical flask with a ground
stopper containing 40 mL of ethyl acetate. Agitate for 30 minutes and separate
the organic phase as before taking care to recover only the organic fraction and
not the wine.
On a 100C water bath, evaporate each extraction solvent in 50-60 mm
diameter evaporation dishes, in small amounts while directing a stream of cold
air on the surface of the dishes. Continue the evaporation until the residue has
a syrupy consistency, stopping before the evaporation is complete.
Re-dissolve the benzene extract residue in the evaporation dish with 0.5 mL
ethanol-water (1:1) solution (it is advisable to re-dissolve the residue once with
0.25 mL ethanol-water solution and then to rinse the dish with another portion
of 0.25 mL of the same solution). Place the ethanol-water extract into a small
tube with a ground stopper (extract B).
The residue of the dish in which the ethyl acetate (containing the cyclamate)
has been evaporated, is dissolved with 0.5 mL of water and is poured into a
small separator tube. Wash the dish with 10 mL ether and add the ether to the
contents of the separator tube. Mix vigorously for 2 minutes and separate the
lower layer into a small test tube that contains 0.5 mL ethanol. This comprises
a total of 1 mL of ethanol-water solution that contains the possible cyclamate
(extract A).
2.3.2 Chromatography
2.3.2.1 Saccharine and cyclamate
For examination of the saccharine and cyclamate, use a cellulose plate, with
half of the plate for the identification of cyclamate and the other half for
saccharine.
To do this, spot 5 to 10 L of extract A and 5L of the standard cyclamate
solution. On the second part of the plate spot 5 to 10 L of extract B and 5 L
of the standard saccharine solution. Place the prepared plate in the
chromatography bath containing solvent No.1 (acetone; ethyl acetate;
OIV-MA-AS315-07A : R2009
BIBLIOGRAPHY
TERCERO C., F.V., O.I.V., 1968, n 277 and F.V., O.I.V., 1970, n 352.
Wine Analysis Commission of the Federal Health Department of Germany,
1969, F.V., O.I.V., n 316.
International Federation of Fruit Juice Manufacturers, 1972, F.V., O.I.V., n
40.
SALO T., ALRO E. and SALMINEN K., Z. Lebensmittel Unters. u.
Forschung, 1964, 125, 20.
OIV-MA-AS315-07A : R2009
Method OIV-MA-AS315-07B
Type IV method
2. Method
Examination of saccharine, cyclamate and Dulcin.
2.1 Apparatus
2.1.1 Apparatus for expression by thin layer
2.1.2 Glass plate 20 x 20 cm
Preparation of the plates: mix thoroughly 9 g of dry cellulose powder and 6 g
of polyamide powder. Add, while stirring, 60 mL methanol. Spread on the
plates to a thickness of 0.25 mm. Dry for 10 minutes at 70C. The quantities
prepared are sufficient for the preparation of 5 plates.
2.1.3 Water bath with a temperature regulator or a rotary evaporator,
2.1.4 UV lamp for examination of the chromatography plates.
2.2 Reagents
2.2.1 Petroleum ether (40-60)
2.2.2 Ion exchange resin, for example: Amberlite LA-2
2.2.3 Acetic acid diluted to 20% (v/v)
OIV-MA-AS315-07B : R2009
OIV-MA-AS315-07B : R2009
evaporated dry at 70C on a water bath. It is necessary to avoid heating for too
long and above 70C. The residue is recovered with 1 mL of methanol.
5 to 10 L of this solution and 2 L of the standard solutions are spotted on
the plate. Let the solvent migrate (xylene: n-propanol: acetic acid: formic acid)
(2.2.10) to a height of about 15 cm, which takes about 1 hour.
After air-drying, the dichlorofluorescein solution is thoroughly sprayed on the
plate. The saccharine and the cyclamate appear immediately as light spots on a
salmon colored background. Under examination in ultraviolet light (254 or
360 nm), the three sweeteners appear as a fluorescent blue on a yellow
background.
The sweeteners separate, from the bottom to the top of the plate, in the
following order: cyclamate, saccharine, Dulcin.
The vanillin and the esters of p-hydroxybenzoic acid migrate with the same Rf
as the Dulcin. To identify Dulcin in the presence of these substances, the plate
then must be sprayed with a solution of dimethylaminobenzaldehyde. The
Dulcin appears as an orange spot, whereas the other substances do not react.
Sensitivity - The quantity limitation shown on the chromatography plate is 5 g
for the three substances.
This method permits detection of:
Saccharin ...................................................... 10 mg/L
Cyclamate ......................................................... 50 mg/L
Dulcin ............................................................. 10 mg/L
BIBLIOGRAPHY
TERCERO C., F.V., O.I.V., 1968, n 277 and F.V., O.I.V., 1970, n 352.
Wine Analysis Commission of the Federal Health Department of Germany,
1969, F.V., O.I.V., n 316.
International Federation of Fruit Juice Manufacturers, 1972, F.V., O.I.V., n
40.
SALO T., ALRO E. and SALMINEN K., Z. Lebensmittel Unters. u.
Forschung, 1964, 125, 20.
OIV-MA-AS315-07B : R2009
Method OIV-MA-AS315-08
Type IV method
OIV-MA-AS315-08 : R2009
BIBLIOGRAPHY
TERCERO C., F.V., O.I.V., 1970, n 356.
ARATA P., SAENZ-LASCANO-RUIZ, Mme I., F.V., O.I.V., 1967, n 229.
OIV-MA-AS315-08 : R2009
Method OIV-MA-AS315-09
Type IV method
Diethylene glycol
(2hydoxy-ethoxyethanol)
1. Objective
The detection of diethylene glycol, HOCH2CH2OCH2CH2OH, in wine where its
concentration is equal to or greater than 10 mg/L.
2. Principle
Separation of diethylene glycol from other constituents in wine by gas
chromatography using a capillary column, after extraction with ether.
Note: The operating conditions described below are provided as an example.
3. Apparatus
3.1 Gas chromatograph equipped with:
- split-splitless injector,
- flame ionization detector,
- capillary column coated with a film of polyethyleneglycol (Carbowax
20 M), 50 m x 0.32 mm I.D.
Operating conditions:
Injector temperature: 280C.
Detector temperature: 270C.
Carrier gas: hydrogen.
Flow rate of carrier gas: 2 mL/min.
Flow rate: 30 mL/min.
Injection: splitless.
Injection volume: 2 L.
Injection 35C - flow closed after 40 seconds.
Temperature program: 120C to 170C at 3C/min.
3.2 Centrifuge
4. Reagents
4.1 1,3-propanediol, 1 g/L, in alcohol, 20% (v/v), (internal standard).
4.2 Aqueous solution of diethyleneglycol 20 mg/L.
5. Procedure
Into a 50 mL flask, place:
OIV-MA-AS315-09 : R2009
- 10 mL of wine
- 1 mL of 1,3-propanediol solution
- 25 mL diethyl ether.
Shake and add sufficient quantity of neutral potassium carbonate to saturate the
mixture. Shake. Separate the two phases by centrifugation.
Carry out a second extraction. Eliminate the diethyl ether by evaporation and
recover the residue with 5 mL ethanol.
The yield of the extraction must be at least 90%.
Carry out the chromatography according to the conditions given in 3.1.
6. Results
The diethylene glycol is identified by comparing its retention time to the time of
the reference solution, analyzed under the same conditions as the wine.
The amount is determined by comparison to the reference solution using the
internal standard method.
It is recommended, if the concentration is equal to or less than 20 mg/l, to confirm
the presence by mass spectrometry.
BIBLIOGRAPHY
BANDION F., VALENTA M. & KOHLMANN H., Mitt. Klosterneuburg, Rebe
und Wein, 1985, 35, 89.
BERTRAND A., Conn. vigne vins, 1985, 19, 191.
Laboratoire de la rpression des fraudes et du contrle de la qualit de
Montpellier, F.V., O.I.V., 1986, n 807.
OIV-MA-AS315-09 : R2009
Method OIV-MA-AS315-10
Type II method
1. FIELD OF APPLICATION
This document describes the method used for determining ochratoxine A (OTA) in
red, ros, and white wines, including special wines, in concentrations ranging up 10
g/l using an immunoaffinity column and high performance liquid chromatography
(HPLC) [1].
This method was validated following an international joint study in which OTAs
were measured in white and red wines during the analysis of naturally
contaminated wines and wines with toxins ranging from 0.01 g/l to 3.00 g/l.
This method can apply to semi-sparkling wines and sparkling wines as long as the
samples have been degassed beforehand, through sonication, for example.
2. PRINCIPLE
Wine samples are diluted with a solution containing polyethylene glycol and
sodium hydrogen carbonate. This solution is filtered and purified on the
immunoaffinity column.
OTA is eluted with methanol and quantified by HPLC in inverse state with
fluorimetric detection.
3. REAGENTS
3.1.4 Purified water for laboratories, for example EN ISO 3696 quality
(water for analytical laboratory use Specification and test method [ISO
3696:1987]).
3.1.5 Phosphate buffer (dilution solution)
Dissolve 60g of Na2HPO42H2O (3.1.1) and 8.8g of NaH2PO4H2O (3.1.2)
in 950ml of water and add more water to make up to 1 litre.
3.1.6 Phosphate buffer saline (washing solution)
Dissolve 2.85g of Na2HPO42H2O (3.1.1), 0.55g of NaH2PO4H2O (3.1.2)
and 8.7g of NaCl in 950ml of water and add more water to make up to 1
litre.
3.1.7 Methanol (CH3OH) CAS [67-56-1]
3.2 Reagents for HPLC
3.2.1 Acetonitrile for HPLC (CH3CN) CAS [75-05-8]
3.2.2 Glacial acetic acid (CH3COOH) CAS [64-19-7]
3.2.3 Mobile phase: water: acetonitrile: glacial acetic acid, 99:99:2, v/v/v
Mix 990 ml of water with 990 ml of acetonitrile (3.2.2) and 20 ml of glacial
acetic acid (3.2.3). In the presence of undissolved components, filter through
a 0.45m filter. Degas (with helium, for example) unless the HPLC
equipment used includes a degassing step.
3.3 Reagents for the preparation of the OTA stock solution
3.3.1 Toluene (C6H5CH3) CAS [108-88-3]
3.3.2 Mixture of solvents (toluene: glacial acetic acid, 99:1, v/v).
Mix 99 parts in volume of toluene (3.3.1) with one part volume of glacial
acetic acid (3.2.2).
3.4 OTA stock solution
Dissolve 1 mg of OTA or the same content in a bulb, if the OTA was obtained in
the form of film after evaporation, in the solvent mixture (3.12) to obtain a solution
containing approximately 20 to 30 g/ml of OTA.
To determine the exact concentration, record the absorption spectrum between 300
and 370 nm in a quartz space with 1 cm of optical path while using the solvent
mixture (3.12) as a blank. Identify maximum absorption and calculate the
concentration of OTA (c) in g/ml by using the following equation:
c = Amax M 100 /
OIV-MA-AS315-10: R2011
In which:
Amax = Absorption determined by the longest maximum wave (about 333 nm)
M = OTA molecular mass = 403,8 g/mole
= coefficient d'extinction molaire de l'OTA dans le mlange de solvant (3.12) ( =
544/mole)
= optical pathway (cm)
This solution is stable at -18C for at least 4 years.
3.5 Standard OTA solution (2 g/ml in toluene: acetic acid, 99:1, v/v)
Dilute the stock solution (3.13) with the solvent mixture (3.12) to obtain a standard
solution of OTA with a concentration of 2 g/ml.
This solution can be stored at + 4 C in a refrigerator. The stability should be tested
regularly.
4. EQUIPMENT
Usual laboratory equipment and in particular, the following equipment:
4.1 Glass tubes (4 ml)
4.2 Vacuum pump to prepare the immunoaffinity columns.
4.3 Reservoir and flow tube adapted to immunoaffinity columns.
4.4 Fibre glass filters (for example Whatman GF/A).
4.5 Immunoaffinity columns specifically for OTA.
The column should have the total link capacity of at least 100 ng OTA. This will
allow for a purification yield of at least 85% when a diluted solution of wine
containing 100 ng OTA is passed through.
4.6 Rotating evaporator
4.7 Liquid chromatography, a pump capable of attaining a constant flow of 1
ml/mn isocratic, as with the mobile phase.
4.8 Injection system must be equipped with 100 l loop.
4.9 Column of analytical HPLC in steel 150 4.6 mm (i.d.) filled with a stationary
phase C18 (5 m) preceded with a pre-column or a pre-filter (0,5 m) containing an
appropriate phase. Different size columns can be used provided that they guarantee
a good base line and background noise enabling the detection of of OTA peaks,
among others.
4.10 Fluorescence detector is connected to the column and the excitation
wavelength is set at 333 nm and the emitting wavelength at 460 nm.
OIV-MA-AS315-10: R2011
Std 2
Std 3
Std 4
Std 5
4970
4900
4700
4000
2000
30
0.6
0.06
100
2.0
0.20
300
6.0
0.60
1000
20
2.00
3000
60
6.00
NOTE:
1. If the quantity of OTA in the samples is outside the calibration range, an
appropriate dilution should occur or smaller volumes should be injected. In these
cases, the final (7) should be reviewed on a case by case basis.
2. Due to the great variations in concentrations, it is recommended to pass the
linear calibration by zero in order to obtain an exact quantification for low
concentrations of OTA. (less than 0.1 g/l)
7. CALCULATIONS
Calculate the quantity of OTA in the aliquot of the solution testes and injected in
the HPLC column.
Calculate the concentration of OTA (COTA) in ng/ml (equivalent to g/l) by using
the following formula:
COTA = MA F/V1 V3/V2
Where:
MA is the volume of ochratoxin A (in ng) in the aliquot part of the template
injected on the column and evaluated from the calibration curve.
F is the dilution factor
OIV-MA-AS315-10: R2011
Addition
(g/l)
0.04
0.1
0.2
0.5
1.0
2.0
5.0
10,0
Average of
averages
Red wine
Yield SD* RSD#
(%)
(%)
96.7 2.2
2.3
90.8 2.6
2.9
91.3 0.6
0.7
92.3 0.4
0.5
97.8 2.6
2.6
96.5 1.6
1.7
88.1 1.3
1.5
88,9 0,6
0,7
92.8 3.5
3.8
Ros wine
White wine
#
Yield SD* RSD Yield SD* RSD#
(%)
(%)
(%)
(%)
94.1 6.1
6.5
91.6 8.9
9.7
89.9 1.0
1.1
88.4 0.2
0.2
88.9 2.1
2.4
95.1 2.4
2.5
91.6 0.4
0.4
93.0 0.2
0.2
100.6 .,5
2.5
100.7 1.0
1.0
98.6 1.8
1.8
98.0 1.5
1.5
94.5 5.2
5.5
94.5 4.1
4.3
ITALY
ITALY
ITALY
ITALY
ITALY
ITALY
ITALY
FRANCE
FRANCE
SWEDEN
SWEDEN
GERMANY
CYPRUS
FINLAND
UNITED
KINGDOM
UNITED STATES
11. REFERENCES
[1]
A. Visconti, M. Pascale, G. Centonze. Determination of ochratoxin A in
wine by means of immunoaffinity column clean-up and high-performance liquid
chromatography. Journal of Chromatography A, 864 (1999) 89-101.
[2]
AOAC International 1995, AOAC Official Methods Program, p. 23-51.
OIV-MA-AS315-10: R2011
APPENDIX I
The following data was obtained in inter-laboratory tests, according to harmonised
protocol recommendations for joint studies in view of validating an analysis
method.
WHITE WINE
Sample
White
Inter-laboratory test year
1999
Number of laboratories
16
Number
of
laboratories
retained
after
eliminating
14*
absurd findings
Number
of
eliminated
laboratories
Number of accepted results
28
Average value (g/l)
<0,01
Spread-type/Repeatabilityr
(g/l)
Relative spread-type (Variation
percentage)
/Repeatability
RSDr (%)
Repeatability limit r (g/l)
Spread-type/capacity of being
reproduced sR (g/l)
Relative spread-type (variation
percentage) /capacity of being
reproduced RSDR (%)
Capacity of being reproduced
limit R (g/l)
Extraction yield %
* 2 laboratories were excluded from the
detection limit (= 0,2 g/l).
n.c. = sample naturally contaminated
OIV-MA-AS315-10: R2011
n.c.
1999
16
13*
14
14
15
26
0,102
28
1,000
28
1,768
30
0,283
0.01
0.07
0.15
0.03
10.0
6.6
8.5
10.6
0.028
0.196
0.420
0.084
0.01
0.14
0.23
0.04
14.0
13.6
13.3
14.9
0.028
0.392
0.644
0.112
101.7
90.9
88.4
statistical 'evaluation due to high
APPENDIX II
The following data was obtained in inter-laboratory tests, according to harmonised
protocol recommendations for joint studies in view of validating an analysis
method.
RED WINE
Added OTA (g/l)
samples
White
0.200
0.900
3.000
Inter-laboratory test year
1999
1999
1999
1999
Number of laboratories
15
15
15
15
Number of laboratories retained
14*
12*
14
15
after eliminating absurd findings
Number
of
eliminated
2
1
laboratories
Number of accepted results
28
24
28
30
Average value (g/l)
<0.01
0.187
0.814
2.537
Spread-type/Repeatabilityr (g/l)
0.01
0.08
0.23
Relative spread-type (Variation
percentage) /Repeatability RSDr
5.5
9.9
8.9
(%)
Repeatability limit r (g/l
0.028
0.224
0.644
Spread-type/capacity of being
0.02
0.10
0.34
reproduced sR (g/l )
Relative spread-type (variation
percentage) /capacity of being
9.9
12.5
13.4
reproduced RSDR (%)
Capacity of being reproduced
0.056
0.280
0.952
limit R (g/l)
Extraction yield %
93.4
90.4
84.6
* 1 laboratory was excluded from the statistical evaluation because
detection limits (= 0,2 g/l).
n.c. = naturally contaminated sample
OIV-MA-AS315-10: R2011
n.c.
1999
15
14
1
28
1.693
0.19
10.9
0.532
0.23
13.4
0.644
of high
APPENDIX III
1. Field of application
For information purposes only the method can be applied to grape musts,
partially fermented grape musts, and new wines still under fermentation.
The validation parameters concern wines only.
2. Principle
The method is broken down into two steps. The first step involves
purification and concentration of the OTA in the wine or the must by
capture on an immunoaffinity column followed by elution. The second step
involves quantification of the eluate by HPLC using fluorescence detection.
3. Reagents
3.4 OTA stock solution
The use of OTA in solid form in not recommended; it is recommended to
use a standard solution of OTA (point 3.5)
3.5 Standard OTA solution
Use of a commercial solution of standard concentration (around 50 g/ml)
with an analysis certificate stating the reference value and uncertainty of the
concentration.
In theory the volume of these solutions is not certified, and they must be
sampled with certified pipettes to constitute stock solutions from 0.25 to 1
mg/l in pure ethanol or in the mobile phase of the HPLC method (see 3.2.3).
This solution is stable at -18C for at least 4 years.
4. Equipment
OIV-MA-AS315-10: R2011
10
THE
The standard deviation of the recovery rate calculated in this way represents
not only the variability of the recovery rate of the columns, but also the
standard uncertainty of the measurement system used after use of the
columns (HPLC). It is nevertheless possible to establish a reasonable
estimate of the standard deviation of the recovery rate of the columns by
OIV-MA-AS315-10: R2011
11
deducting the standard uncertainty of the HPLC system from the calculated
recovery error:
- Estimate the standard uncertainty Sv (expressed as the standard deviation)
of the measurement system in the strict sense of the word (without
considering the the immunoaffinity column step).
For this it is possible to use a fidelity study on the OTA solutions.
The standard deviation of the recovery rate Sp is estimated as follows:
OIV-MA-AS315-10: R2011
12
Method OIV-MA-AS315-11
Type II method
(v/v/v)
(v/v/v)
1
Membrane filter for HPLC solvent degassing and for sample preparation to be
analysed.
Reference products for peak identification.
The HPLC analysis of anthocyanins in wine is difficult to perform due to the
absence of commercially available pure products. Furthermore, anthocyanins
are extremely unstable in solution.
The following anthocyanin pigments are commercially available:
Cyanidol-3-glucoside (also couromanin chloride); M = 484.84 g/mol
Peonidol-3-glucoside; M = 498.84 g/mol
Malvidol-3-glucoside (also Oeninchloride); M = 528.84 g/mol
Malvidol-3,5-diglucoside (also Malvinchloride); M = 691.04 g/mol
4. APPARATUS
HPLC system with:
binary gradient pump, injection system for sample volumes ranging from 10 to
200 l,
diode array detector or a UV detector with a visible range,
integrator or a computer with data acquisition software,
furnace for column heating at 40C,
solvent degassing system,
analytical column, for example:
LiChrospher 100 RP 18 (5 m) in LiChroCart 250-4 guard column: for
example RP 18 (30-40 mm) in a cartridge 2 mm in diameter x 20 mm long
5. PROCEDURE
5.1 Preparation of samples
Clear wines are poured directly without any preparation into the sample vials of
the automatic sample changer. Cloudy samples are filtered using a 0.45 m
membrane filter for HPLC sample preparation. The first part of the filtrate
should be rejected.
Since the range of the linearity of absorption depending on the concentration of
anthocyanins is large, it is possible to modulate the injection volumes between
10 and 200 l depending on the intensity of the wine colour. No significant
difference between the results obtained for different injection volumes was
observed.
OIV-MA-AS315-11 : R2007
5.2 Analysis
HPLC conditions
The HPLC analysis is carried out in the following conditions:
Injection Volume:
Flow:
Temperature:
Run time:
Post time:
Detection:
Gradient elution:
Time
(min)
Solvent A
% (v/v)
Solvent B
% (v/v)
0
15
30
35
41
94
70
50
40
94
6
30
50
60
6
To check the column efficiency, the number of theoretical plates (N) calculated
according to malvidol-3-glucoside should not be below 20,000, and the
resolution (R) between peonidol-3-coumaryl glucoside and malvidolin-3coumaryl glucoside should not be lower than 1.5. Below these values, the use
of a new column is recommended.
OIV-MA-AS315-11 : R2007
Peak-N
1
2
3
4
5
Group 2: Acetylated
anthocyanidin-3glucosides:
peonidol-3-acetylglucoside
malvidol-3-acetylglucoside
6
7
Group 3:
Coumarylated
anthocyanidin-3glucosides:
peonidol-3-coumarylglucoside
malvidol-3-coumarylglucoside
8
9
6. EXPRESSION OF RESULTS
Note that the values are expressed as relative amounts of the sum of the nine
anthocyanins defined in this method.
OIV-MA-AS315-11 : R2007
The limit of detection (LD) and the limit of quantification (LQ) depend on the
individual measurement conditions of the chemical analysis and are to be
determined by the user of the method. The Annex gives an example of its
determination with the following results:
hmax = 0,208 [mAU]; LD = 3 x 0,208 [mAU] = 0,62 [mAU].
LQ = 10 x 0, 208 [mAu] = 2,08 [mAU].
Recommendation:
With combined data out of the whole Anthocyanin composition such as the sum of
Acylated Anthocyanins or the ratio of Acetylated to Coumarylated Anthocyanins
the calculation should not be carried out in cases where one of the components is
below the limit of quantification (LQ).
On the other hand measurements below the limit of quantification (LQ) are not
devoid of information content and may well be fit for purpose [1].
Bibliography:
[1] Thompson, M.; Ellison, S.L.R. ; Wood, R., Harmonized Guidelines for SingleLaboratory Validation of Methods of Analysis, Pure Appl. Chem. (2002) 74: 835855
8. FIDELITY PARAMETERS
The repeatability (r) and the reproducibility (R) values for the nine
anthocyanins are given in Table 2 and depend on the amount of the peak area.
The uncertainty measurement of a particular peak area is determined by the
value of r and R which corresponds to the nearest value given in Table 2.
The values made up of validation data can be calculated by following the
appropriate statistical rules. To calculate the total error (sr) for example of the
sum of acetylated anthocyanins, the variances (sr2) of specific the total error of
ratios, for example, that of acetylated to coumarylated anthocyanins the square
of relative errors (=sr/ai) are to be added. By using these rules, all the fidelity
values can be calculated by using the data in Table 2.
OIV-MA-AS315-11 : R2007
OIV-MA-AS315-11 : R2007
Annex A
Bibliography
[1]
[2]
[3]
[4]
[5]
[6]
Mattivi F.; Scienza, A.; Failla, O.; Vika, P.; Anzani, R.; Redesco, G.;
Gianazza, E.; Righetti; P. Vitis vinifera - a chemotaxonomic approach:
Anthocyanins in the skin. Vitis (special issue) 1990, 119-133
[7]
[8]
[9]
[10]
OIV-MA-AS315-11 : R2007
several red grape cultivars and wines made from them. Eur. Food Res.
Technol. 2002, 215, 32-37
[11]
Arozarena, I.; Ayestarn, B.; Cantalejo, M.J.; Navarro, M.; Vera, M.;
Abril, K.; Casp, A. Eur. Food Res. Technol. 2002, 214, 313-309
[12]
Revilla, E.; Garcia-Beneytez, E.; Cabello, F.; Martin-Ortega, G.; Ryan, JM. Value of high-performance liquid chromatographic analysis of
anthocyanins in the differentiation of red grape cultivars and red wines
made from them. J. Chromatogr A 2001, 915, 53-60
[13]
Heier, A.; Blaas, W.; Dro, A.; Wittkowski, R.; Anthocyanin Analysis by
HPLC/ESI-MS, Am.J.Enol.Vitic, 2002, 53, 78-86
[14]
[15]
[16]
Burns, I.; Mullen, W.; Landrault, N.; Teissedre, P.-L.; Lean, M.E.I.;
Crozier, A. Variations in the Profile and Content of Anthocyanins in
Wines made from Cabernet Sauvignon and hybrid grapes. J. Agric. Food
Chem. 2002, 50, 4096-4102
[17]
[18]
OIV-MA-AS315-11 : R2007
Annex B
Statistical results
Method performance study and evaluation
17 laboratories from 5 European Nations participated in the validation study of
the method under the coordination of the German Official State Laboratory for
Food Chemistry in Trier. The participants are listed in Table 3. An example of a
chromatogram is presented in Figure 1 and the detailed results are given in Table
2.
The statistical evaluation followed the Resolution 6/99 and the Standard ISO
5725-1944 [4.5].
The chromatograms sent back with the results sheets fulfilled all requirements
concerning the performance of the analytical column. No laboratory had to be
completely eliminated, for example, because of a wrong peak identification.
The outlier values were searched using Dixon and Grubbs outlier testing
according to the procedure for Harmonised Protocol IUPAC 1994 and the
OIV Resolution OENO 19/2002. The values of sr, sR, r and R were calculated
for 9 major anthocyanins at 5 content levels. For analytical results, the values of
the closest levels should be used.
In order to have a global vision of the method performance, all the values RSDret RSDR- gathered are grouped by range of areas in the following table:
Table 1: Summary of the results of the method performance study
Range of relative peak
areas*[%]
Range of RSDr
[%]
Range of RSDR
[%]
>0.4 1.0
6.8 - 22.4
20.6 - 50.9
>1.1 1.5
4.2 - 18.1
11.8 - 28.1
>1.5 3.5
2.1 7.7
10.6 - 15.6
>3.5 5.5
2.7 5.7
18.7 7.5
>5.5 7.5
2.4 3.9
6.5 - 10.0
>10 14
1.1 2.9
3.7 - 9.2
>14 17
1.0 - 3.9
3.2 - 5.4
>50 76
0.3 - 1.0
2.1 - 3.1
* independent of anthocyanin
OIV-MA-AS315-11 : R2007
4-98
4039
UV_VIS
_1
WVL:518
nm
Mv-3gl
55
0
50
0
45
0
40
0
35
0
30
0
5
0
Po-3cuglMv-3cugl
Cy-3gl
10
0
acgl
15
0
Po-3acgl Mv-3-
Po-3gl
20
0
Pt-3gl
De-3gl
25
0
0
mi
n
50 0,
0
2,
5
5,
0
7,
5
10,
0
12,
5
15,
0
17,
5
OIV-MA-AS315-11 : R2007
20,
0
22,
5
25,
0
27,
5
30,
0
32,
5
35,
0
37,
5
40,
0
42,
5
45,
0
10
OIV-MA-AS315-11 : R2007
sample 4
sample 5
15
16.68
0.142
0.8
0.40
0.704
4.2
1.97
16
3.54
0.108
3.1
0.30
0.490
13.8
1.37
15
1.46
0.110
7.5
0.31
0.180
12.3
0.50
14
0.34
0.031
9.2
0.09
0.158
46.7
0.44
14
12.21
0.097
0.8
0.27
0.469
3.8
1.31
15
6.19
0.196
3.2
0.55
0.404
6.5
1.13
17
7.44
0.232
3.1
0.65
0.602
8.1
1.69
16
4.12
0.174
4.2
0.49
0.532
12.9
1.49
16
52.60
0.298
0.6
0.83
1.606
3.1
4.50
16
61.04
0.377
0.6
1.06
1.986
3.3
5.56
11
sample 1
sample 2
14
1.16
0.064
5.5
0.18
0.511
43.9
1.43
16
1.44
0.062
4.3
0.17
0.392
27.2
1.10
sample 3
sR
RSDR(%)
R
Malvidol-3-acetylglucoside
n
16
17
mean
5.51
4.84
sr
0.176
0.167
RSDr(%)
3.2
3.4
r
0.49
0.47
sR
0.395
0.366
RSDR(%)
7.2
7.6
R
1.11
1.02
Peonidol-3-coumarylglucoside
n
16
14
mean
1.26
0.90
sr
0.130
0.046
RSDr(%)
10.3
5.1
r
0.36
0.13
sR
0.309
0.109
RSDR(%)
24.5
12.2
R
0.86
0.31
Malvidol-3-coumarylglucoside
n
17
17
mean
4.62
2.66
sr
0.159
0.055
RSDr(%)
3.4
2.1
r
0.45
0.15
sR
0.865
0.392
RSDR(%)
18.7
14.7
R
2.42
1.10
n
= N of laboratories retained after eliminating outliers
sr
= standard deviation of repeatability
RSDr(%)
= relative standard deviation of repeatability
r
= repeatability
sR
= standard deviation of reproducibility
RSDR(%)
= relative standard deviation of reproducibility
R
= reproducibility
OIV-MA-AS315-11 : R2007
sample 4
sample 5
14
0.59
0.059
10.1
0.17
0.272
46.4
0.76
16
3.74
0.215
5.8
0.60
0.374
10.0
1.05
17
3.11
0.088
2.8
0.25
0.496
16.0
1.39
16
15.07
0.213
1.4
0.60
0.617
4.1
1.73
17
0.89
0.060
6.8
0.17
0.204
23.0
0.57
16
1.32
0.058
4.4
0.16
0.156
11.8
0.44
17
4.54
0.124
2.7
0.35
0.574
12.6
1.61
16
4.45
0.048
1.1
0.13
0.364
8.2
1.02
12
D
D
UK
A
OIV-MA-AS315-11 : R2007
13
Method OIV-MA-AS315-12
Type IV method
2 PROTOCOL
2.1 Concentration of proteins by precipitation with trichloroacetic acid (TCA)
2.1.1 Reagents
2.1.1.1 Pure trichloroacetic acid (TCA)
2.1.1.2 TCA at 0.1% prepared using 2.1.1.1: 0.1 g in
100 ml of water.
2.1.1.3 TCA at 100% prepared using 2.1.1.1: 100 g in
100 ml of water.
2.1.1.4 Sodium hydroxide 0.5 M
2.1.1.5 Buffer Tris/HCl 0.25 M pH=6.8
30.27 g of Tris-(hydroxymethyl)aminomethane (Tris) are dissolved in
300 ml of distilled water. The pH is adjusted to 6.8 with concentrated
OIV-MA-AS315-12 : R2004
OIV-MA-AS315-12 : R2004
OIV-MA-AS315-12 : R2004
2.2.2 Procedure
The separation gel solution (2.2.1.7) is poured between two glass
plates of 7x10cm. The upper surface of the gel is levelled by the addition of 2
drops of distilled water.
After polymerisation of the separation gel and the elimination of water, 1
ml of concentration gel (2.2.1.10) is deposited on the separation gel using a 1 ml
pipette. Then the comb is set up whose imprints will create deposit wells.
The samples necessary for the calibration range are prepared in a mixture
(1:1), v/v, 0.5% M sodium hydroxide (2.1.1.4) and the buffer solution (2.1.1.9) in
order for the calibration range be between 5 g/ml and 50 g/ml.
20 to 30 l of wine and calibration solution are deposited in the wells.
After migration (at a constant voltage of 90 V) at room temperature for
about 3-4 hours, the gels are removed from the mould. They are immediately
plunged into 50 ml of an aqueous solution of TCA 20% for 30 minutes then in 50
ml of the colouring solution (2.2.1.12).
The proteins appear in the form of blue coloured bands. The gel is then
discoloured with 50 ml of discolouring solution (2.2.1.13). When the bottom of the
gel is transparent, it is placed in distilled water for storage.
OIV-MA-AS315-12 : R2004
3. QUANTITATIVE ANALYSIS
The intensity of each spot is evaluated by using a scanner for gel with an
image analyser software. The quantity of protein on the gel is determined by the
calculation of the average density of the pixels of the band and by integration of
the band width. The protein content of each sample is obtained using a calibration
curve. The points of this curve are obtained by tracing the known concentration
values of plant proteins deposited on the gel depending on the corresponding
integration area.
The detection and quantification limit is about 0.030 ppm for peas and at 0.36 ppm
for gluten, in an environment concentrated 100 times. The coefficient of variation
is always below 5%.
4 SEARCH BY IMMUNOBLOTTING OF THE ANTIGENIC
POTENTIAL OF WINES AND MUSTS TREATED
The antigenic capacity of proteins that could remain in the beverages
treated after racking is then evaluated.
4.1 PRINCIPLE
After electrophoresis, the gels are submitted to the immunoblotting technique. The
proteins are transferred to a membrane where they are adsorbed. An antigen
antibody complex is formed by the addition of a plant anti-protein antibody (for
example antigliadin antibodies if the plant protein is gluten). The method is
revealed by the addition of an antibody directed against the plant anti-protein
antibodies coupled with phosphatase. In the presence of the chromogenic substrate
of the enzyme, a colouration whose intensity will be proportional to the quantity of
immunocomplexes will develop. This immunoreactivity will be quantified using a
calibration curve made with known concentration plant proteins solutions.
4.2 PROTOCOL
4.2.1 : Reagents
4.2.1.1 Transfer buffer
3.03 g of Tris, 14.4 g of glycine (R), 200 ml of methanol (R) are mixed
and completed to 1 l with distilled water.
4.2.1.2 Gelatine 1%
8.77 g of sodium chloride (R), 18.6 g of ethylenediaminetetraacetic acid
(EDTA) for analysis, 6.06 g of Tris and 0.5 ml of Triton X are dissolved in 800 ml
of distilled water. The pH is adjusted to 7.5 with concentrated hydrochloric acid
for analysis. 10 g of gelatine are added and the volume is completed to 1 l.
4.2.1.3 Gelatine 0.25%
8.77 g of sodium chloride (R), 18.6 g of ethylenediaminetetraacetic acid
(EDTA) for analysis, 6.06 g of Tris and 0.5 ml of Triton X are dissolved in 800 ml
OIV-MA-AS315-12 : R2004
OIV-MA-AS315-12 : R2004
ANNEX
Production of polyclonal anti-peas
Anti-peas polyclonal antibodies necessary for the determination of
antigenic capacity of pea proteins in wine and musts treated are being carried out
on animals.
1 Principle
Serums containing polyclonal antibodies are obtained from New Zealand
rabbits after an intradermal injection of antigen.
2 Protocol
2.1 Reagents
2.1.1 PBS pH=7.4 phosphate buffer: 8 g of NaCl, 200 mg of KCl, 1.73 of
Na2HPO4 H2O and 200 mg of KH2PO4 are dissolved in 300 ml of distilled water.
pH is adjusted to 7.4 with sodium hydrate 1 M. The volume is brought to 1 l with
distilled water.
2.1.2 Antigens:
10 mg of pea protein is dissolved in 5 ml of PBS phosphate buffer
(2.1.1). The solution is then filtered under sterile conditions
through 0.2 m and stored at 20C until the day of immunization.
2.2 Procedure
1 ml of 2.1.2. solution is mixed with 1 ml of Freund complete adjuvant. 1
ml of this mixture is injected intradermically to a New Zealand rabbit weighing
approximately 3 kg. This injection is repeated on day 15, day 30 and day 45.
60 days after the first injection, 100l of blood were withdrawn from the
auricular vein which was then tested for its capacity to react to antigens.
Immunoblotting was used for this evaluation as described in Chapter 4.2 of the
analysis method using a gel with a pea protein which migrated on the gel.
After checking the formation of an antigen-antibody complex, 15 ml of blood were
withdrawn from the auricular vein. The blood is placed at 37C for 30 minutes.
The serum containing the anti-pea polyclonal antibodies is withdrawn after
centrifuging the blood at 3000 rpm for 5 minutes.
OIV-MA-AS315-12 : R2004
Method OIV-MA-AS315-13
Type IV method
WITHDRAWN
(Replaced by OIV-MA-AS315-16)
OIV-MA-AS315-13 : R2009
Method OIV-MA-AS315-14
Type IV method
1. Introduction
It is preferable to have an analysis method available for lysozyme which is not
based on enzyme activity.
2. Scope
The method allows the quantification of lysozyme (mg of protein per l) present
in red and white wines independently of the enzyme activity (which could be
inhibited by partial denaturation or by complex formation or coprecipitation
phenomena) found in the test solution.
3. Definition
HPLC provides an analytical approach based on steric, polar or adsorptive
interactions betwen the stationary phase and the analyte, and is therefore not
linked to the actual enzyme activity exhibited by the protein.
4. Principle
The analysis is carried out using HPLC with a spectrophotometric detector
combined with a spectrofluorimetric detector. The unknown quantity in the
wine sample is calculated on the chromatographic peak areas, using the
external calibration method.
5. Reagents
5.1. Solvents and working solutions
HPLC analysis on Acetonitrile (CH3CN)
Pure trifluoroacetic acid (TFA)
deionised water for HPLC analysis
Standard solution: Tartaric acid 1g/L, Ethyl alcohol 10% v/v, adjusted to
pH 3.2 with neutral potassium tartrate.
OIV-MA-AS315-14 : R2007
5.2. Eluents
A: CH3CN 1%, TFA 0.2 %, H2O= 98.8%
B: CH3CN 70%, TFA 0.2 %, H2O= 29.8%
5.3. Reference solutions
Quantities from 1 to 250 mg/L standard lysozyme, dissolved in standard
solution by stirring continuously for at least 12 hours.
6. Equipment
6.1.
HPLC apparatus equipped with a pumping system suitable for
gradient elution
6.2.
Thermostated column compartment (oven)
6.3.
Spectrophotometer combined with spectrofluorimeter
6.4.
20 L loop injection
6.5.
Column: polymer in reverse phase with phenyl functional groups
(diameter of pores = 1000 , exclusion limit = 1000000 Da) Toso
Bioscience TSK-gel Phenyl 5PW RP, 7.5 cm x 4.6 mm ID as an
example
6.6.
Pre-column in the same material as the column: Toso Bioscience
TSK-gel Phenyl 5PW RP Guardgel, 1.5 cm 3.2mm ID as an example
8. Operating conditions
8.1.
Eluent flow-rate: 1mL/min
8.2.
Temperature of column: 30C
8.3.
Spectrophotometric detection: 280 nm
8.4.
Spectrofluorimetric detection:
ex = 276 nm;
em = 345 nm;
Gain = 10
8.5.
Gradient elution sequence
OIV-MA-AS315-14 : R2007
Time (min)
A%
B%
100
100
10
65
35
gradient
isocratic
linear
isocratic
15
65
35
27
40.5
59.5
29
100
linear
linear
isocratic
34
100
36
100
40
100
linear
isocratic
8.6
9. Calculation
The reference solutions containing the following concentrations of lysozyme:
1; 5; 10; 50; 100; 200; 250 mg/L are analysed in triplicate. For each
chromatogram, the peak areas corresponding to the lysozyme are plotted
according to the respective concentrations, in order to obtain the linear
regresssion lines expressed by the formula Y= ax+b. The correlation
coefficient r2 must be > 0.999
10. Characteristics of the method
A validation study was carried out for the purpose of assessing the suitability
of the method for the purpose in question, taking into account linearity, limits
of detection and quantification and the accuracy of the method. The latter
parameter was determined by defining the levels of precision and trueness of
the method.
OIV-MA-AS315-14 : R2007
Linearity
range
(mg/L)
Line
gradient
Correlation
coefficient
(r2)
LD
(mg/L)
LQ
(mg/L)
Repeatability
(n=5)
RSD%
Std1 V.R.2 V.B.3
Reproducibility
(n=5)
RSD%
Std1
UV
5-250
3 786
0,9993
1,86
6,20
4,67
5,54
0,62
1,93
FLD
1-250
52 037
0,9990
0,18
0,59
2,61
2,37
0,68
2,30
standard solution ;
red
OIV-MA-AS315-14 : R2007
Nominal
initial [C]
(mg/L)
Quantity
added
(mg/L)
Theoretical
[C] (mg/L)
[C]
found
Std.Dev.
%age
recovery
UV
280 nm
50
13.1
63.1
62.3
3.86
99
FD
50
13.1
63.1
64.5
5.36
102
UV
280 nm
14.4
19.4
17.9
1.49
92.1
FD
14.4
19.4
19.0
1.61
97.7
OIV-MA-AS315-14 : R2007
11. Bibliography
Claudio Riponi; Nadia Natali; Fabio Chinnici. Quantitation of hens egg white
lysozyme in wines by an improved HPLC-FLD analytical method. Am. J. Enol.
Vit., in press.
OIV-MA-AS315-14 : R2007
Method OIV-MA-AS315-15
Type II method
3-methoxypropane-1,2-diol
Analysis of Variance
Concentration
Cyclic diglycerols
Gas chromatography mass spectrometry
Hydrogen
Internal standard
mass/charge ratio
Matrix calibration level
Standard dilution 1000 ng/L
OIV-MA-AS315-15 : R2007
S1
Standard dilution 100 ng/L
S2
Standard dilution 10 ng/L
4. Principle
The analytes and the internal standard are salted-out by addition of K2CO3, and
extracted using diethyl ether. Extracts are analyzed directly by GC-MS on a polar
column. Detection is then carried out in selected ion monitoring mode.
5. Reagents and Materials
5.1. Chemicals
5.1.1 K2CO3 p.A .
5.1.2 Diethyl ether Uvasol for spectroscopy
5.1.3 Molecular sieve (2 mm diameter, pore size 0.5 nm)
5.1.4 Ethanol (Absolute)
5.2. Standards
5.2.1 Cyclic diglycerol mixture (6 components)
Solvay Alkali GmbH 1,
89.3 %
cis-, trans-2,6-bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,5bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,-hydroxymethyl-6hydroxy-1,4-dioxepane
5.2.2
5.2.3
Solvay Alkali GmbH no longer provides the standard mixture; solutions of the mixture
may be obtained from the BfR. Federal Institute for Risk Assessment, Thielallee 88-92, D14195 Berlin. www.bfr.bund.de; [email protected]
OIV-MA-AS315-15 : R2007
Concentration
1000
100
ng/L
ng/L
3-Methoxypropane-1,2-diol (3-MPD)
Solution
S0
S1
S2
Concentration
1000
100
10
ng/L
ng/L
ng/L
Concentration
1000
100
ng/L
ng/L
OIV-MA-AS315-15 : R2007
ML0
ML1
ML2
ML3
ML4
ML5
IS
3-MPD
CycDs
IS
3-MPD
CycDs
IS
3-MPD
CycDs
IS
3-MPD
CycDs
IS
3-MPD
CycDs
IS
3-MPD
CycDs
IS
3-MPD
CycDs
Spike l
100
100
100
50
100
25
100
100
50
20
100
100
30
100
200
40
ml
10
g/L
0
mg/L
0
S1 10
1000
1.00
S1
S2
S1
S1
S1
S1
S1
S1
S0
S1
S1
S0
S1
S1
S0
1000
100
500
1000
250
1000
1000
500
2000
1000
1000
3000
1000
2000
4000
1.00
0.10
0.50
1.00
0.25
1.00
1.00
0.50
2.00
1.00
1.00
3.00
1.00
2.00
4.00
10
10
10
10
10
6. Apparatus
6.1
Analytical balance.0.0001 g readability.
6.2
Lab centrifuge (at least 4000 rpm/min)
6.3
Gas chromatograph.-With mass spectrometric detector, split-splitless
injector,
6.4
Diverse precision pipettes and volumetric flasks
6.5
Pasteur pipettes
6.6
40 mL centrifugation vials
6.7
GC-vials (1.5 2.0 mL)
6.8
Thermostat
6.9
Shaking machine
OIV-MA-AS315-15 : R2007
7. Sampling
Wine samples for the analysis should be taken in a sufficient size. Volume needed
for one test sample is 10 mL. The wine used for the preparation of the matrixcalibration (5.4) shall be free of analyte.
8. Procedure
8.1. Extraction
Add 100 L internal standard solution S1 (6.3.2) to 10 mL wine to a suitable
centrifugation vial e.g. 40 mL. (This corresponds to a concentration of 1 mg/L
butane-1,4-(2H)8). Carefully add 10 g of K2CO3 and mix. Take care during this
addition as heat is produced due to the release of CO2. After cooling the solution
to approximately 20 C in a water bath, add 1 mL diethyl ether. Homogenise the
mixture for 5 minutes using a vertical-shaking machine. Centrifuge the vials at
4000 rpm for 5 min. For better removal of the organic phase, the extract can be
partially transferred into a vial with a smaller diameter. Using a Pasteur pipette,
transfer the upper organic phase, composed of diethyl ether and ethanol, into a GC
vial. Add approximately 120 mg of molecular sieve into the vial. Close the vial,
leave for at least 2 h and shake well from time to time. The clear supernatant is
transferred to a second GC vial for the GC-MS analysis.
8.2. GC-MS Analysis
Specific parameters for the GC-MS analysis are provided below. Alternative
systems may be used, if they provide a similar chromatographic performance and
adequate sensitivity. The chromatographic system must be able to separate the
internal standard from phenylethanol, a potential interference.
Typical GC conditions
Gas chromatograph: HP 5890 or equivalent
DB-Wax (J&W) column 60 m, 0.32 mm internal diameter, 0.25 m film thickness,
2 m capillary containment same dimensions or equivalent
Carrier gas: H2
Flow: Pressure 60 k Pa column head
Temperature program:
90 C, 2 min., ramp at 10C/min. up until 165 C, held for 6 min., ramp at 4
C/min to 250C, held for 5 min.
Injection temperature: 250 C; Injected volume; 2 L, 90 sec splitless for 90 s.
OIV-MA-AS315-15 : R2007
Specificl MS conditions
Mass spectrometer: Finnigan SSQ 710 or equivalent
Transfer line: 280 C
Source: 150 C
MS detection:
window1.:
0-25 min.:
14.3 min.
3-MPD:
m/z 75, m/z 61
16.7 min
IS:
m/z 78, m/z 61
Acquisition time for each mass is 250 s (dwell time).
Monitor for m/z 91 the separation of the internal standard (IS) peak from
phenylethanol, which also produces a fragment m/z 78.
window 2.
25-40 min.:
32-34.5 min. CycDs:
m/z 57, m/z 117
Acquisition time for each mass is 250 s (dwell time).
It has been observed that the analysis may degrade chromatographiccolumn. In
particular, the injection of the high boiling CycDs mixture is suspected to cause
irreversible damage. Injections of reference standard solutions should be avoided;
analysis should be restricted to salted-out solutions with low analyte
concentrations. In addition it is recommended to use a 1-2 m pre column in order
to protect the analytical column. Nevertheless, the analytical column has to be
considered as a consumable and must be replaced quite regularly.
9. Evaluation
9.1. Identification
Record the relative retention time of each analyte to the IS. Calculate the mean
relative retention time of the analytes in the calibration standards. The relative
retention time of the analyte should be the same as that of the standard within a
margin of 0.5 %. As a confirmation criterion, an ion ratio can be calculated for
each analyte from the selected ion monitoring. This ratio is 117/57 for CycDs,
75/61 for 3-MPD and 78/61 for the IS. The ratio should be within 20 % of that
which is found in the spiked sample. Confirmation of the identity of substances by
full scan using ionsn can also be used.
9.2. Quantification
The quantification is done by a matrix calibration curve prepared according to
appropriate section. The analyte/IS area ratios of the indicated mass ratios are
correlated by linear regression against the concentration of the analyte.
Quantification of the CycDs is achieved by summing the peak area of all six peaks
OIV-MA-AS315-15 : R2007
and calculating the total content, to allow for other distributions of the six
characteristic CycDs than in the standard. The following m/z values are used for
quantification:
3-MPD:
IS:
CycDs:
m/z 75
m/z 78
m/z 117
OIV-MA-AS315-15 : R2007
3-MPD
Sr = 0,060 x
SR = 0,257 x
r = 0,169 x
R = 0,720 x
CycDs
Sr = 0,082 x
SR = 0,092 x + 0,070
r = 0,230 x
R = 0,257 x + 0,197
OIV-MA-AS315-15 : R2007
Participants
11 international laboratories participated in the collaborative study (5). The
participating laboratories have proven experience in the analysis of the byproducts. All of them participated in the pre-trial:
CSL, York, UK
Unione Italiana Vini, Verona, Italy
BfR, Berlin, Germany
BLGL, Wrzburg, Germany
Istituto Sperimentale per l'enologia, Asti, Italy
LUA, Speyer, Germany
Labor Dr. Haase-Aschoff, Bad Kreuznach, Germany
CLUA, Mnster, Germany
Kantonales Laboratorium, Fllinsdorf, Switzerland
LUA, Koblenz, Germany
ISMAA, S. Michele all Adige, Italy
Samples
In November 2002, participating laboratories were sent 11 wine samples consisting
of five sets of blind duplicates and one further single test material. Dry white
wines, dry red wines and a sweet red wine were used for test materials. The
samples were subjected to homogeneity testing previously (ii).
Data analysis
Statistical analysis was carried out according to the Protocol for the Design,
Conduct and Interpretation of Method Performance Studies (iii) using a blind
duplicate model.
1. Determination of outliers was assessed by Cochran, Grubbs and paired Grubbs
tests.
2. Statistical analysis was performed to obtain repeatability and reproducibility
data.
3. Horrat values were calculated.
OIV-MA-AS315-15 : R2007
Mean mg/L
Spiked mg/L
Recovery %
n
nc
outliers
n1
r
sr
RSDr %
Hor
R
sR
RSDr %
HoR
Sample A
White
wine
0.30
0.30
100
10
1
2
7
0.03
0.01
3.20
0.30
0.13
0.05
15.50
0.80
Sample B
Red
wine a
0.145
0.12
121
10 a
1a
0
9a
0.13
0.05
32.67
1.53
Sample C
White
wine
0.25
10
1
0
9
0.05
0.02
7.20
0.60
0.15
0.05
21.20
1.10
Sample F
Sweet red
wine
0.48
10
1
1
8
0.08
0.03
5.80
0.50
0.31
0.11
22.70
1.30
Sample G
White
wine
0.73
0.80
91
10
1
1
8
0.13
0.05
6.57
0.59
0.59
0.21
28.91
1.72
10
0,7
0,6
R = 0,720 x
r and R [ mg/L]
0,5
0,4
R
0,3
0,2
0,1
r = 0,169 x
0
0
0,2
0,4
0,6
0,8
OIV-MA-AS315-15 : R2007
11
Mean mg/L
Spiked mg/L
Recovery %
n
nc
outliers
n1
r
sr
RSDr %
Hor
R
sR
RSDR %
HoR
a
Sample A
White
wine
1.55
1.50
103
11
0
2
9
0.37
0.13
8.50
0.90
0.61
0.22
14.00
0.90
Sample B
Red
winea
0.593
0.53
113
11a
0
0
11a
0.379
0.135
22.827
1.319
Sample D
Red
wine
0.80
Sample F
Sweet red
wine
0.96
11
0
1
10
0.19
0.07
8.60
0.80
0.39
0.13
17.30
1.00
11
0
2
9
0.18
0.07
6.70
0.60
0.41
0.15
15.20
0.90
Sample G
White
wine
0.56
0.50
112
11
0
1
10
0.15
0.05
9.30
0.80
0.34
0.12
21.50
1.20
OIV-MA-AS315-15 : R2007
12
0,7
R = 0,257 x + 0,197
0,6
r and R in [mg/L]
0,5
0,4
R
0,3
0,2
r = 0,230 x
0,1
0
0
0,5
1,5
OIV-MA-AS315-15 : R2007
13
Method OIV-MA-AS315-16
Type IV method
1 SCOPE:
The method of determination of releasable 2,4,6-trichloroanisole (TCA) by cork
stoppers measures the quantity of TCA released by a sample of cork stoppers
macerated in a aqueous-alcoholic solution. The aim of this method is to evaluate
the risk of releasing by the lot of analyzed cork stoppers and to provide a method
for controlling the quality of cork stoppers.
2 PRINCIPLE
The method aims to simulate 2,4,6-trichloroanisole migration phenomena
susceptible of being produced between the cork stopper and wine in bottles. Cork
stoppers are macerated in a wine or a aqueous-alcoholic solution, until a balance is
obtained. The TCA of the head space is sampled from an appropriate part of the
macerate by the solid-phase micro-extraction technique (SPME), then analyzed by
gas chromatography, with detection by mass spectrometer (or by electron-capture
detector).
3 REAGENTS AND PRODUCTS
3.1 White wine with an alcoholic strength ranging between 10 and 12 %
vol. (It can be replaced by an aqueous-alcoholic solution with an alcoholic strength
of 12 % vol). The wine and/or the aqueous-alcoholic solution must be free of
TCA.
3.2 Sodium chloride 99.5 %
3.3 2,4,6-trichloroanisole (TCA)-d5 purity 98% for GC/MS; 2,6dibromoanisole or 2,3,6-trichloroanisole purity 99 % for GC/ECD
3.4 2,4,6-trichloroanisole (TCA) purity 99.0%
3.5 Absolute ethanol
3.6 Pure de-ionised water void of TCA (Standard EN ISO 3696)
3.7 Aqueous-alcoholic solution at 12 % vol.
Prepared using absolute ethanol (3.5) and de-ionised water void of TCA
(3.6).
3.8 Internal standard stock solution (500 mg/L)
OIV-MA-AS315-16 : R2009
Add either 0.050 g of 2,4,6-trichloroanisole-d5 (or 2,6-dibromoanisole or 2,3,6trichloroanisole (3.3) to approximately 60 ml of absolute ethanol (3.5). After
dissolution, adjust the volume to 100 mL with absolute ethanol (3.5). It can
be kept in a glass bottle with a metallic or glasscover.
3.9 Intermediate solution of internal standard (5.0 mg/L)
Add 1 mL of a solution of either 2,4,6-trichloroanisole-d5 (or 2,6dibromoanisole or 2,3,6-trichloroanisole) at 500 mg/L (3.8) to
approximately 60 mL of absolute ethanol (3.5). Adjust the volume to 100
mL with absolute ethanol (3.5). It can be kept in a glass bottle with a
metallic or glass cover.
3.10 Internal standard solution (2.0 g/L)
Add 40 L of a solution of either 2,4,6-trichloroanisole-d5 (or 2,6dibromoanisole or 2,3,6 trichloroanisole) at 5.0 mg/L (3.9) to approximately
60 mL of absolute ethanol (3.5). Adjust the volume to 100 ml with absolute
ethanol (3.5). It can be kept at an ambient temperature in a glass bottle with
a metallic or glass cover.
3.11 Stock solution of TCA standard (40 mg/L)
Add 0.020g of 2,4,6-trichloroanisole to approximately 400 ml of absolute
ethanol (3.5). Following dissolution, adjust volume to 500 mL with absolute
ethanol (3.5).
3.12 Intermediate solution A of TCA standard (80 g/L)
Add 1 mL of 2,4,6-trichloroanisole solution at 40 mg/L (3.11) to
approximately 400 mL of absolute ethanol (3.5). Following dissolution, adjust
volume to 500 mL with absolute ethanol (3.5).
3.13 Intermediate solution B of TCA standard (160 ng/L)
Add 1 mL of solution 2,4,6-trichloroanisole at 80 g/L (3.12) to
approximately 400 mL of pure de-ionised water (3.6). Following dissolution,
adjust the volume to 500 mL with pure de-ionised water (3.6)
3.14 Use the standard-addition technique to make up a range of standard
solutions of TCA. Standard solutions in the range from 0.5 ng/L to 50 ng/L can be
used, by making additions with a solution of 2,4,6-trichloroanisole at 160 ng/L
(3.13) to 6 ml of absolute ethanol (3.5). Following dissolution, adjust volume to 50
mL with pure de-ionised water (3.6)
The calibration curve obtained should be evaluated regularly and in any case
whenever there is a major change in the GC/MS or GC/ECD systems.
3.15 Carrier gas: Helium, chromatographic purity ( 99.9990 %)
OIV-MA-AS315-16 : R2009
4. APPARATUS
4.1 Laboratory glassware
4.1.1 Graduated 100-mL flask
4.1.2 100-L microsyringe
4.1.3 Wide-neck glass jar of a capacity adapted to the sample size, closed
with a glass or metallic stopper or a material which does not bind TCA.
4.1.4 20-mL glass sample bottle closed with a perforated capsule and a
liner with one side Teflon-coated.
4.2 Solid-phase microextraction system (SPME) with a fiber coated with a
polydimethylsiloxane film 100 m thick
4.3 Heating system for sample bottle (4.1.4)
4.4 Stirring system for sample bottle (4.1.4)
4.5 Gas chromatograph equipped with a "split-splitless" injector and a
mass spectrometer detector (MS) or an electron-capture detector (ECD)
4.6 Data-acquisition system
4.7 If required, an automatic sampling and injection system operating with
an SPME system
4.8 Capillary column coated with an apolar stationary phase, of the
phenylmethylpolysiloxane type (e.g.: 5 % phenyl methylpolysiloxane, 30 m x 0,25
mm x 0,25 m film thickness or equivalent.)
5. SAMPLE PREPARATION
The corks are placed whole in a glass closed container. The container capacity
(4.1.3), the same as the quantity of wine or aqueous-alcoholic solution (3.1 or 3.7),
must be chosen in accordance to the sample size while ensuring that the corks are
completely covered and immersed in the maceration container.
Example 1: 20 corks (45x24) mm, in a 1 L container;
Example 2: 50 corks (45x24) mm, in a 2 L container.
Most of the TCA released during maceration of the groups of stoppers is
generally derived from a very low percentage of these stoppers. In order to obtain
the best representation of a batch of stoppers, a number of appropriate analyses
according to sampling rules and risk with regard to wine contamination should be
carried out.
6. OPERATING METHOD
6.1 Extraction
OIV-MA-AS315-16 : R2009
aqueous-alcoholic solutions (3.7) containing known concentrations of 2,4,6trichloroanisole, as a function of the concentrations of these solutions (3.14).
The results are given in ng/L of TCA present in the maceration, rounded off
to the nearest 0.1 ng/L.
8. CHARACTERISTICS OF THE METHOD
As an indication, the detection limit of the analysis of the macerations must be
lower than 0.5 ng/L, and the quantification limit close to 1 ng/L. The coefficient of
variation is lower than 5% for 5 ng/L, when the selected internal standard is the
deuterated analogue TCA-d5.
An interlaboratory trial was carried out in order to validate the method. This
interlaboratory trial was not carried out according to the OIV protocol and the
validation parameters mentioned in the FV 1224.
9. BIBLIOGRAPHY
HERV E., PRICE S., BURNS G., Chemical analysis of TCA as a quality control
tool for natural corks. ASEV Annual Meeting. 1999.
ISO standard 20752:2007 Cork stoppers Determination of releasable 2, 4, 6trichloroanisol (TCA).
FV 1224 - Rsultats de lanalyse collaborative Ring test 3-TCA SPME.
OIV-MA-AS315-16 : R2009
Method OIV-MA-AS315-17
Type IV method
1. SCOPE
All wines, cork stoppers, bentonites (absorption traps) and wood.
2. PRINCIPLE
Determination
of
2,4,6-trichloroanisol,
2,4,6-trichlorophenol,
2,3,4,6tetrachloroanisol,
2,3,4,6-tetrachlorophenol,
pentachloroanisol
and
pentachlorophenol by gas chromatography, by injecting a hexane extract of the
wine and an ether/hexane extract of the solid samples to be analyzed and internal
calibration.
3. REAGENTS
Preliminary remark: all the reagents and solvents must be free of the compounds
to be determined listed in 2 at the detection limit.
3.1 Purity of hexane > 99 %
3.2 Purity of ethylic ether > 99 %
3.3 Ether/hexane mixture (50/50; v/v)
3.4 or 2,5-dibromophenol purity 99 %
3.5 Pure ethanol
3.6 Pure deionized water, TCA free, type II in accordance with ISO standard EN
3696
3.7 50 % vol. aqueous-alcoholic solution. Place 100 ml of absolute ethanol (3.<5)
in a graduated 200-ml flask (4.9.9), add 200 ml of deionized water (3.6), and
homogenize.
3.8 Internal standard:
3.8.1 200 mg/l stock solution. Place 20 mg of internal standard (3.4) in a
graduated 100-ml flask (4.9.8), add the 50 % volume aqueous-alcoholic solution
(3.7) and homogenize.
OIV-MA-AS315-17 : R2009
OIV-MA-AS315-17 : R2009
4. APPARATUS
4.1 Gas phase chromatograph with Split-splitless injector coupled to an electron
capture detector. (It is likewise possible to use a mass spectrometer)
4.2 Capillary tube of non-polar steady-state phnylmethylpolysiloxane type: (0.32
mm x 50 m, thickness of film 0.12 m or the equivalent
4.3 Chromatographic conditions, as an example:
4.3.1 Injection in "split-splitless" mode (valve closing time 30 seconds)
4.3.2 Carrier gas flow rate: 30 ml/min including 1 ml in the column
Hydrogen U 2 (It is likewise possible to use helium)
4.3.3 Auxiliary gas flow rate: 60 ml/min Nitrogen with
chromatographic purity ( 99,9990 %). It is also possible to use argon
methane.
4.3.4 Furnace gradient temperature for information purposes:
- from 40 C to 160 C at a rate of 2 C/min
- from 160 C to 200 C at a rate of 5 C/min
- step at 220 C for 10 min
4.3.5 Injector temperature: 250 C
43.6 Detector temperature: 250 C
4.4 Acquisition and integration: acquisition is by computer. The peaks of the
various compounds identified by comparison with the reference are then
integrated.
4.5 Magnetic agitator.
4.6 Vortex with adaptation for 30-ml flask (4.9.3)
4.7 Precision balance to within 0.1 mg
4.8 Manual or electric household grate
4.9 Laboratory equipment:
4.9.1 100-l micro-syringe
4.9.2 10-l micro-syringe
4.9.3 30-ml flask closing with a screwed plug and cover with one side
Teflon-coated
4.9.4 10-ml stick pipette graduated 1/10 ml
4.9.5 5-ml stick pipette graduated 1/10 ml
4.9.6 1-ml precision pipette
4.9.7 Graduated 50-ml flask
OIV-MA-AS315-17 : R2009
6.2.5 Carry out the extraction using the magnetic stirrer (4.5) for 5 min.
6.2.6 Elutriate into the funnel (4.9.10)
6.2.7 Recover the organic phase with the emulsion in a 30-ml flask
(4.9.3) and aqueous phase in the 100-ml graduated flask (4.9.8)
6.2.8 Repeat the extraction of the wine or calibration solution using 2 ml
of hexane (3.1)
6.2.9 Carry out the extraction using the magnetic stirrer (4.5) for 5 min.
6.2.10 Elutriate into the funnel (4.9.10)
6.2.11 Recover the organic phase with the emulsion in the same 30-ml
flask mentioned
in 6.2.7 (containing the organic phase obtained
upon the first extraction)
6.2.12 Break the emulsion of the organic phase by centrifugation (4.9.13)
by eliminating the lower aqueous phase using a Pasteur pipette (4.9.11)
fitted with a propipette pear.
6.2.13 Final wine extract and calibration solutions: the residual organic
extract
6.3 Analyze:
6.3.1 Add final extract (6.1.11 or 6.2.13) 100 l (4.9.1) of the pyridine acetic
anydride reagent mixture (3.10) for the derivatisation.
6.3.2 Mix using a magnetic stirrer (4.5) for 10 min.
6.3.3 Inject 2 l of derivatised final extract (6.3.2) into the chromatograph
7. CALCULATION:
Product peak area
Concentration of product = ------ * Response factor
Peak area of internal standard
OIV-MA-AS315-17 : R2009
Mean
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
1.2
26
1.77
2.59
23.3
7.39
Standard
deviation
0.1
3.3
0.44
0.33
2.9
1.91
Repeatability
0.28
9.24
1.23
0.92
8.12
5.35
Standard deviation
1.9
1.9
2.6
3.3
2.7
3.6
Repeatability
5.3
5.3
7.4
9.3
7.5
10.1
Standard deviation
Repeatability
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
0,4
2,1
0,6
4
1,2
6,5
OIV-MA-AS315-17 : R2009
1,1
5,9
1,7
11,2
3,4
18,2
In bentonite with15ng/g
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
Standard deviation
Repeatability
0,9
4
1,2
5,2
4,3
12,1
2,5
11,2
3,4
14,6
12,0
33,9
9.3 Detection limits (DL) and quantification limits (QL) calculated according to
the OIV method:
9.3.1 Wood
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
DL in ng/g
0.72
0.62
0.59
1.12
0.41
0.91
QL in ng/g
2.4
2.0
2.0
3.74
1.4
3.1
DL in ng/g
0.5
1
0.5
1
0.5
Not det.
QL in ng/g
1
3
1
3
1
Not det.
9.3.2 Bentonite
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
9.3.3 Stopper
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
OIV-MA-AS315-17 : R2009
DL in ng/g
0.5
1
0.5
1
0.5
1
QL in ng/g
1.5
2
1.5
2
1.5
2
9.3.4 Wine
2,4,6-trichloroanisol
2,4,6-trichlorophenol
2,3,4,6-tetrachloroanisol
2,3,4,6-tetrachlorophenol
pentachloroanisol
pentachlorophenol
DL in ng/l
0.3
1
0.3
0.3
0.5
1
QL in ng/l
1
3
1
1
3
3
2 Air Liquide
OIV-MA-AS315-17 : R2009
Method OIV-MA-AS315-18
Type II method
1.
SCOPE
This method can be applied for analysing biogenic amines in musts and wines:
Ethanolamine: up to 20 mg/l
Histamine: up to 15 mg/l
Methylamine: up to 10 mg/l
Serotonin: up to 20 mg/l
Ethylamine: up to 20 mg/l
Tyramine: up to 20 mg/l
Isopropylamine: up to 20 mg/l
Propylamine: normally absent
Isobutylamine: up to 15 mg/l
Butylamine: up to 10 mg/l
Tryptamine: up to 20 mg/l
Phenylethylamine: up to 20 mg/l
Putrescine or 1,4-diaminobutane: up to 40 mg/l
2-Methylbutylamine: up to 20 mg/l
3-Methylbutylamine: up to 20 mg/l
Cadaverine or 1,5-diaminopentane: up to 20 mg/l
Hexylamine: up to 10 mg/l
2.
DEFINITION
PRINCIPLE
The biogenic amines are directly determined by HPLC using a C18 column after Ophthalaldehyde (OPA) derivatization and fluorimetric detection.
4.
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20
4.21
4.22
4.23
4.24
OIV-MA-AS315-18 : R2009
4.25
4.26
mg/l)
OIV-MA-AS315-18 : R2009
Histamine
Methylamine
Serotonin
20
Ethylamine
Tyramine
Isopropylamine
Propylamine
2.5
Isobutylamine
Butylamine
Tryptamine
10
Phenylethylamine
Putrescine
12
2- Methylbutylamine
3- Methylbutylamine
Cadaverine
13
1.6 Diaminohexane
Hexylamine
The true concentration of the calibration solution is recorded with the batch
number of the products used.
Certain biogenic amines being in salt form, the weight of the salt needs to be taken
into account when determining the true weight of the biogenic amine.
The stock solution is made in a 100-ml volumetric flask (5.8).
The surrogate solution is made in a 250-ml volumetric flask (5.10).
4.32
1,6 Diaminohexane internal standard
Weigh exactly 119 mg in a 25-ml Erlenmeyer flask (5.1) on a balance (5.26).
Transfer to a 100-ml volumetric flask (5.8) and top up to the filling mark with 0.1
N hydrochloric acid (4.30).
OIV-MA-AS315-18 : R2009
4.33
2-Mercaptoethanol - Purity 99 %.
5.
APPARATUS
5.1
25-ml Erlenmeyer flasks
5.2
250-ml Erlenmeyer flasks
5.3
100-ml beakers
5.4
150-ml beakers
5.5
50-ml beaker
5.6
25-ml beaker
5.7
50-ml volumetric flasks
5.8
100-ml volumetric flasks
5.9
2,000-ml volumetric flasks
5.10
250-ml volumetric flask
5.11
1-litre bottles
5.12
2-litre bottle
5.13
2-ml screw cap containers suitable for the sample changer
5.14
50-ml syringe
5.15
Needle
5.16
Filter holder
5.17
0.45 m cellulose membrane
5.18
0.8 m cellulose membrane
5.19
1.2 m cellulose membrane
5.20
5 m cellulose membrane
5.21
Cellulose pre-filter
5.22
1-ml automatic pipette
5.23
5-ml automatic pipette
5.24
10-ml automatic pipette
5.25
Cones for 10-ml, 5-ml and 1-ml automatic pipettes
5.26
Filtering system
5.27
Balances for weighing 0 to 205 g at 0.01 mg
5.28
pH meter
5.29
Electrode
5.30
Magnetic stirrer
5.31
HPLC pump
5.32
Changer-preparer equipped with an oven
Note: An oven is indispensable, if a changer-preparer is used for injecting several
samples one after another. This operation may likewise be done manually) the
results may be less precise;
OIV-MA-AS315-18 : R2009
5.33
Injection loop
5.34
5 m C18 column, 250 mm 4 (which must lead to a similar
chromatogram as presented in annex B);
5.35
Fluorimetric detector
5.36
Integrator
5.37
Borosilicic glass tube with a stopper and closure cap covered with PTFE
(ex Sovirel 15).
6.
PREPARATION OF SAMPLES
6.4
Routine cleaning
Syringe (5.13) and needle (5.14) rinsed with demineralised water (4.1) after each
sample;
filter holder (5.16) rinsed with hot water, then MeOH (4.6). Leave to drain and
dry.
7.
PROCEDURE
%A
%B
80
70
60
50
35
35
80
80
20
30
40
50
65
65
20
20
Note: The gradient can be adjusted to obtain a chromatogram close to the one
presented in annex B
Flow rate: 1 ml/min;
Column temperature: 35 C (5.32);
Detector (5.35): Exc = 356 nm, Em = 445 nm (5.30);
Internal calibration
The calibration solution is injected for each series;
Calibration by internal standard;
Calculation of response factors:
RF = Ccis area i / area is Cci
OIV-MA-AS315-18 : R2009
8.
EXPRESSION OF RESULTS
Results are expressed in mg/l with one significant digit after the decimal point.
9.
RELIABILITY
r (mg/l)
R (mg/l)
Histamine
0.07x + 0.23
0.50x + 0.36
Methylamine
0.11x + 0.09
0.40x + 0.25
Ethylamine
0.34x - 0.08
0.33x + 0.18
Tyramine
0.06x + 0.15
0.54x + 0.13
Phenylethylamine
0.06x + 0.09
0.34x + 0.03
Diaminobutane
0.03x + 0.71
0.31x + 0.23
2-methylbutylamine et 3methylbutylamine
0.38x + 0.03
0.38x + 0.03
Diaminopentane
0.14x + 0.09
0.36x + 0.12
The details of the interlaboratory trial with regard to reliability of the method are
summarised in appendix A.
OIV-MA-AS315-18 : R2009
10.
The influence of certain wine components: amino acids are released at the
beginning of the analysis and do not impede in detection of biogenic amines.
The limit of detection (LOD) and limit of quantification (LOQ) according to an
intralaboratory study
11.
Histamine
0,01
0,03
Methylamine
0,01
0,02
Ethylamine
0,01
0,03
Tyramine
0,01
0,04
Phenylethylamine
0,02
0,06
Diaminobutane
0,02
0,06
2-methylbutylamine
0,01
0,03
3-methylbutylamine
0,03
0,10
Diaminopentane
0,01
0,03
QUALITY CONTROL
Quality controls may be carried out with certified reference materials, with wines
the characteristics of which result from a consensus or spiked wines regularly
inserted into analytical series and by following the corresponding control charts.
OIV-MA-AS315-18 : R2009
Annex A
Statistical data obtained from the results of interlaboratory trials
The following parameters were defined during an interlaboratory trial. This
trial was carried out by the Oenology Institute of Bordeaux (France) under
the supervision of the National Interprofessional Office of Wine (ONIVINS
France).
Year of interlaboratory trial: 1994
Number of laboratories: 7
Number of samples: 9 double blind samples
(Bulletin de lO.I.V. November-December 1994, 765-766, p.916 to 962) numbers
recalculated in compliance with ISO 5725-2:1994.
Types of samples: white wine (BT), white wine (BT) fortified = B1, white wine
(BT) fortified = B2, red wine n1 (RT), red wine fortified = R1, red wine (RT)
fortified = R2, red wine n2 (CT), red wine (CT) fortified = C1 and red wine (CT)
fortified = C2. fortified in mg/l.
HistN
MetN
EthN
TyrN
PhEtN
DiNbut
IsoamN
DiNpen
wine B1
wine BT
+ 0,5
vine BT
+ 0,12
wineBT
+ 0,13
wine BT
+ 0,36
vine BT
+ 0,15
wine BT
+ 0,5
wine BT
+ 0,28
wineBT
+ 0,25
wine B2
wine BT
+2
wine BT
+ 0,40
wine BT
+ 0,50
wine BT
+ 1,44
wine BT
+ 0,60
wine BT
+2
Wine BT
+ 0,1,74
wine BT
+ 1,04
wine C1
wine CT
+2
wine CT
+ 0,1
wine CT
+ 0,18
wine CT
+ 0,72
wine CT
+ 0,15
wine CT
+2
wine CT
+ 0,29
wine CT
+ 0,26
wineC2
wine CT
+4
wine CT
+ 0,41
wine CT
+ 0,50
wine CT
+ 2,90
wine CT
+ 0,58
wine CT
+8
wine CT
+ 1,14
wine CT
+ 1,04
wine R1
wine RT
+2
wine RT
+ 0,14
wine RT
+ 0,13
wine RT
+ 1,45
wine RT
+ 0,19
wine RT
+3
wine RT
+ 0,0,57
wine RT
+ 0,51
wine R2
wine RT
+5
wine RT
+ 0,41
wine RT
+ 0,50
wine RT
+ 2,88
wine RT
+ 0,59
wine RT
+ 10
wine RT
+ 2,28
wine RT
+ 2,08
10
OIV-MA-AS315-18 : R2009
11
BIBLIOGRAPHY
TRICARD C., CAZABEIL J.-M., SALAGOTI M.H. (1991): Dosage des amines
biognes dans les vins par HPLC, Analusis, 19, M53-M55.
PEREIRA MONTEIRO M.-J. et BERTRAND A. (1994): validation d'une mthode
de dosage Application l'analyse des amines biognes du vin. Bull. O.I.V., (765766), 916-962.
OIV-MA-AS315-18 : R2009
12
Method OIV-MA-AS315-19
Type IV method
4. Apparatus
4.1 Capillary electrophoresis
Capillary electrophoresis equipped with a hydrostatic-type injector is coupled with
a laser-induced fluorescence detector with an excitation wavelength similar to the
absorption wavelength of the MBB-GSH adduct: e.g.= 390 nm (e.g. Zetalif
detector).
4.2 The capillary tube
The total length of the non-grafted silica capillary tube is 120 cm. Its effective
length is 105 cm, and its internal diameter is 30 m.
5. Preparation of samples
The method of determination used consists of the derivatization of the SH
functions by the monobromobimane (MBB) (Radkowsky & Kosower, 1986).
Samples of musts or non-bottled wines are clarified by centrifugation prior to
analysis. Bottled wines are analysed without prior clarification.
Preparation of samples:
In a 1.5-ml test tube (Eppendorf type), put successively:
- 200 l of the sample,
- 10 l of the DTT solution (3.2.4) - final concentration of 0.25 mM,
- 145 l of CHES (3.2.3) - final concentration of 179 mM,
- 50 l of MBB (3.2.2) - final concentration of 6.2 mM.
After stirring the reagent mixture, the derivatization of thiol functions by the MBB
requires a 20-minute incubation period in the dark at ambient temperature. In these
analytical conditions, the MBB-SR derivatives thus formed are relatively unstable;
CE-LIF determination should be carried out immediately after incubation.
6. Procedure
6.1 Capillary tube preparation
Before being used for the first time and as soon as migration times increase, the
capillary tube (4.2) should be treated in the following way:
6.1.1. Rinse with 0.1 M sodium hydroxide (3.2.5) for 3 minutes,
6.1.2. Rinse with ultra-pure water (3.1.12) for 3 minutes,
6.1.3. Rinse with the electrophoretic phosphate buffer (3.2.1) for 3 minutes.
OIV-MA-AS315-19 : R2009
6.2.2 Analysis.
A voltage of +30 kV, applied throughout separation, generates a current of 47 A.
These conditions are reached in 20 s. Separation is carried out at a constant
temperature of 21 C.
6.2.3 Rinsing the capillary tube
The capillary tube should be rinsed after each analysis, successively with:
- 0.1M sodium hydroxide (3.2.5) for 3 minutes,
- ultra-pure water (3.1.12) for 3 minutes,
- electrophoretic phosphate buffer (3.2.1) for 3 minutes.
7. Results
At the concentration ultimately used in the sample, the presence of DTT during
derivatization makes it possible to stabilise the unstable functions of thiols that
have an alkaline pH and are very easily oxidized by quinines produced by phenolic
compound auto-oxidation, but does not break the disulphide bonds. Thus, under
these analytical conditions, the reduced glutathione content (GSH) found in a wine
with or without the addition of 10 mg/l of oxidized glutathione (GSSG) is strictly
comparable (Figure 1). This method therefore makes it possible to determine
glutathione content in its reduced form alone.
control
control GSSG
OIV-MA-AS315-19 : R2009
Wine is used as a matrix to produce calibration curves and repeatability tests for
each compound. Each concentration is calculated based on the average of three
determinations obtained by using the right of the calibration curb regression.
Results are expressed in mg/L.
Linear regressions and correlation coefficients are calculated according to the least
squares method. The stock solutions of the various thiols are produced from an
HCl/EDTA solution, allowing them to be stored at +6 C for several days with no
loss. Successive dilutions of these solutions allow the threshold limits for detection
in wine to be estimated, for a signal-to-noise ratio of three of more.
Homocysteine
Cysteine
Glutathione
N-acetyl-cysteine
Linearity spectrum
Linear regression
0 - 15 mg/l
0 - 15 mg/l
0 - 40 mg/l
0 - 10 mg/l
Y= 0.459X 0.231
Y = 0.374X 0.131
Y = 0.583X 0.948
Y = 0.256X 0.085
Correlation
coefficient
0.9987
0.9979
0.9966
0.9982
OIV-MA-AS315-19 : R2009
9. Bibliography
Noctor, G. and C. Foyer, 1998. Simultaneous measurement of foliar glutathione,
gamma-glutamylcysteine, and amino acids by high-performance liquid
chromatography: comparison with two other assay methods for glutathione,
Analytical Biochemistry, 264, 98-110.
Kosower, N.S., Kosower E. M., Newton G. L.,and Ranney H. M., 1979. Bimane
fluorescent labels: Labeling of normal human red cells under physiological
conditions. Proc. Natl. Acad. Sci., 76 (7), 3382-3386.
Newton, G.L., R. Dorian, and R.C. Fahey, Analysis of biological thiols:
derivatisation with monobromobimane and separation by reverse-phase highperformance liquid chromatography. Anal. Biochem., 1981. 114: p. 383-387.
Cheynier, V., J.M. Souquet, and M. Moutounet, 1989. Glutathione content and
glutathione to
hydroxycinnamique acid ration in Vitis vinifera grapes and musts. Am.
J.Enol.Vitic,. 40 (4), 320-324.
Mills, B.J., Stinson C. T., Liu M. C. and Lang C. A., 1997. Glutathione and
cyst(e)ine profiles of vegetables using high performance liquid chromatography
with dual electrochemical detection. Journal of food composition and analysis, 10,
90-101.
Ivanov, A.R., I.V. Nazimov, and L. Baratova, 2000. Determination of biologically
active low molecular mass thiols in human blood. Journal of Chromatogr. A,. 895,
167-171.
OIV-MA-AS315-19 : R2009
Method OIV-MA-AS315-20
Type IV method
1. Introduction
The principal -dicarbonyl compounds found in wine (Fig 1) are: glyoxal,
methylglyoxal, diacetyl and pentane-2,3-dione, but only -diketones are
relatively abundant in wine. Carbonyl compounds exist in all types of
wines, particularly after malolactic fermentation and in red wines. In
addition, sweet white wines produced with botrytized grapes can contain
high levels of glyoxal and methylglyoxal.
OIV-MA-AS315-20: R2010
2. Applicability
This method applies to all types of wines (white, red, sweetened or
fortified), for dicarbonyl compounds with a content that ranges from 0.05
mg/l to 20 mg/l .
3. Principle
The method is based on the formation of derivatives of the quinoxaline type
based on the -dicarbonyl compounds of the wine with 1,2-diaminobenzene
(Figure 2).
R
NH2
O C
+
NH2
1,2-diaminobenzene
O C
dicarbonyl
quinoxaline
OIV-MA-AS315-20: R2010
OIV-MA-AS315-20: R2010
25
40
60
92
95
96
93
97
98
94
98
100
Diacetyl
25
40
60
23
64
85
77
89
100
87
94
100
2,3-Hexanedione
25
40
60
17
55
69
67
79
93
79
88
100
solvent A
80
50
25
0
0
100
80
80
OIV-MA-AS315-20: R2010
solvent B
20
50
75
100
100
0
20
20
2.211
1.034
1.854
0.698
0.227
0.102
0.046
0.091
10.30
9.91
2.49
13.09
OIV-MA-AS315-20: R2010
- Linearity. The linearity of the method was tested using standard solutions
(using an aqueous-alcoholic solution at 12% vol. as a matrix) (Table 4). The
quantitative analysis of the additions of dicarbonyl compounds showed that
the method is linear for the four compounds and that its precision is
satisfactory.
Table 4. Study of the linearity and recovery tests with standard solutions
(water-ethanol at 12% v/v) Value of the correlation coefficient
Glyoxal
Methylglyoxal
Diacetyl
Pentane-2,3-dione
R = 0.997
R = 0.999
R = 0.999
R = 0.992
- The recovery of additions carried out in red and white wines demonstrated
the satisfactory performance of the method . Contained in the 92% - 116%
range for extreme values
- The quantification limit of the dicarbonyl compounds is very low, the best
results being obtained with diacetyl, whose detection limit is 10 times lower
than that of the other compounds (Table 5).
Table 5. Performance of the method by HPLC for the quantification of
dicarbonyl compounds
Limits
Glyoxal
Methylglyoxal
Diacetyl
2.3-Pentanedione
detectiona
0.015
0.015
0.002
0.003
determinationa
quantificationa
0.020
0.020
0.002
0.004
0.028
0.027
0.003
0.006
OIV-MA-AS315-20: R2010
OIV-MA-AS315-20: R2010
Bibliography
Bartowski E.J. and Henschke P.A., The buttery attribute of wine diacetyl
desirability spoilage and beyond. Int. J. Food Microbiol. 96: 235-252
(2004).
Bednarski W., Jedrychowski L., Hammond E., and Nikolov L., A method
for determination of -dicarbonyl compounds. J. Dairy Sci. 72:2474-2477
(1989).
Leppannen O., Ronkainen P., Koivisto T. and Denslow J., A
semiautomatic method for the gas chromatographic determination of vicinal
diketones in alcoholic beverages. J. Inst. Brew. 85:278- 281 (1979).
Martineau B., Acree T. and Henick-Kling T., Effect of wine type on the
detection threshold for diacetyl. Food Res. Int. 28:139-143 (1995).
Moree-Testa P. and Saint-Jalm Y., Determination of -dicarbonyl
compounds in cigarette smoke. J. Chromatogr. 217:197-208 (1981).
De Revel G., Pripis-Nicolau L., Barbe J.-C. and Bertrand A., The detection
of -dicarbonyl compounds in wine by the formation of quinoxaline
derivatives. J. Sci. Food Agric. 80:102-108 (2000).
De Revel G. and Bertrand A., Dicarbonyl compounds and their reduction
products in wine. Identification of wine aldehydes. Proc. 7th Weurman
Flavour Research Symp, Zeist, June, pp 353-361 (1994).
De Revel G. and Bertrand A., A method for the detection of carbonyl
compounds in wine: glyoxal and methylglyoxal. J. Sci. Food Agric. 61:267272 (1993).
Voulgaropoulos A., Soilis T. and Andricopoulos N., Fluorimetric
determination of diacetyl in wines after condensation with 3,4diaminoanisole. Am. J. Enol. Vitic. 42:73-75 (1991).
Gilles de Revel et Alain Bertrand Analyse des composs -dicarbonyles
du vin aprs drivation par le 2,3-diaminobenzne OIV FV 1275
OIV-MA-AS315-20: R2010
Type IV method
1. Introduction
The principal -dicarbonyl compounds found in wine (Fig 1) are: glyoxal,
methylglyoxal, diacetyl and 2,3-pentanedione, but only -diketones are
relatively abundant in wine. Carbonyl compounds exist in all types of
wines, particularly after malolactic fermentation and in red wines. In
addition, sweet white wines produced with botrytized grapes can contain
high levels of glyoxal and methylglyoxal.
OIV-MA-AS315-21: R2010
O C
+
NH2
1,2-diaminobenzene
O C
di-carbonyl
quinoxaline
4.3 Water for HPLC (for example microfiltered and with a resistivity of
18.2
M)
4.4 Pure ethanol for HPLC (CAS n 64-17-5)
4.5 Sodium hydroxide M. (CAS n 1310-73-2)
4.6 Sulphuric acid 2M (CAS n 7664-93-9)
4.7 Dichchloromethane (CAS n 75-09-2)
4.8 Anhydrous sodium sulphate (CAS n 7757-82-6)
4.9 Aqueous-alcoholic solution at 50% vol .
Mix 50 ml of pure ethanol for HPLC (4.4) with 50 ml of water (4.3)
4.10 Solution of internal standard 2,3-hexanedione at 2.0 g/L
Place 40 mg of 2,3-hexanedione (4.2) in a 30-ml flask, dilute in 20
ml of aqueous-alcoholic solution to 50% vol (4.9) and stir until it has
completely dissolved.
4.11 Anhydrous sodium sulphate (CAS n 7757-82-6)
5. Equipment
5.1 Gas chromatograph with detection by mass spectrometry (GC-MS) or a
nitrogen-specific detector.
5.1.1 Relatively polar, polyethylene glycol capillary column (CW 20M,
BP21 etc.) with the following characteristics (as an example): 50 m
x 0.32 mm x 0.25 m.
5.1.2 Data acquisition system.
5.2 pH measuring apparatus
5.3 Magnetic stirrer
5.4 Balance with a precision of 0.1 mg.
5. 5 Oven which can be set to 60C
5.6 Standard laboratory glassware including pipettes, screw-cap flasks, and
microsyringes.
6. Preparation of the sample
No specific preparation is necessary
7. Procedure
Place 50 ml of wine in a flask (5.6)
OIV-MA-AS315-21: R2010
OIV-MA-AS315-21: R2010
OIV-MA-AS315-21: R2010
Derivative
Mass spectrum
(principal ions and
abundance)
Glyoxal
Quinoxaline
Methylglyoxal
2-Methylquinoxaline
Diacetyl
2,3-Dimethylquinoxaline
2,3-Pentanedione
2,3-Hexanedione
2,3-Diethylquinoxaline
compound
OIV-MA-AS315-21: R2010
OIV-MA-AS315-21: R2010
Bibliography
Bartowski E.J. and Henschke P.A. The buttery attribute of wine diacetyl
desirability spoilage and beyond. Int. J. Food Microbiol. 96: 235-252
(2004).
Bednarski W., Jedrychowski L., Hammond E., and Nikolov L., A method
for determination of -dicarbonyl compounds. J. Dairy Sci. 72:2474-2477
(1989).
Leppannen O., Ronkainen P., Koivisto T. and Denslow J. A semiautomatic
method for the gas chromatographic determination of vicinal diketones in
alcoholic beverages. J. Inst. Brew. 85:278- 281 (1979).
Martineau B., Acree T. and Henick-Kling T., Effect of wine type on the
detection threshold for diacetyl. Food Res. Int. 28:139-143 (1995).
Moree-Testa P. and Saint-Jalm Y., Determination of -dicarbonyl
compounds in cigarette smoke. J. Chromatogr. 217:197-208 (1981).
De Revel G., Pripis-Nicolau L., Barbe J.-C. and Bertrand A., The detection
of -dicarbonyl compounds in wine by the formation of quinoxaline
derivatives. J. Sci. Food Agric. 80:102-108 (2000).
De Revel G. and Bertrand A. Dicarbonyl compounds and their reduction
products in wine. Identification of wine aldehydes. Proc 7th Weurman
Flavour Research Symp., Zeist, June, pp 353-361 (1994).
De Revel G. and Bertrand A., A method for the detection of carbonyl
compounds in wine: glyoxal and methylglyoxal. J. Sci. Food Agric. 61:267272 (1993).
Voulgaropoulos A., Soilis T. and Andricopoulos N., Fluorimetric
determination of diacetyl in wines after condensation with 3,4diaminoanisole. Am. J. Enol. Vitic. 42:73-75 (1991).
Gilles de Revel et Alain Bertrand, Analyse des composs -dicarbonyles
du vin aprs drivation par le 1-2-diaminobenzne OIV FV 1275
OIV-MA-AS315-21: R2010
Type of method: IV
1. Introduction
Carboxymethyl cellulose (CMC) is a polymer derived from natural cellulose that
has been routinely used for many years now as a food additive (INS 466) in
products such as ice creams and pre-cooked meals [1], to give them smoothness.
The use of CMC in white wines and sparkling wines to contribute to their tartaric
stabilisation [2] was recently accepted by the OIV in resolution Oeno 2/2008
provided that the dose added to the wine is less than 100 mg/l. A specific method
for determination of CMC in white wine has therefore been developed based on the
method of H.D Graham published in 1971 [3].
2. Field of application
The method applies to white wines (still and sparkling).
3. Principle
Once the CMC has been isolated from the wine by dialysis, it is hydrolysed
in an acid medium to form glycolic acid which is then degraded to form
formaldehyde. 2,7-Dihydroxynaphthalene (DHN) is added to form 2,2,7,7tetrahydroxydinaphthylmethane in the presence of formaldehyde. The
complex formed develops a purple-blue colour under the action of
concentrated sulphuric acid, at 100 C, allowing colorimetric measurement
at 540nm (Figure 1).
OIV-MA-AS315-22: R2010
4. Reagents
Sodium carboxymethylcellulose [N CAS 9004-32-4] (21902 - average
viscosity 400-1000 mPas, substitution degree 0.60-0.95)
2,7-Dihydroxynaphthalene [N CAS 582-17-2] (purity > 98,0 % - HPLC)
95 % concentrated sulphuric acid
Purified water for laboratory use (example of quality: EN ISO 3696)
OIV-MA-AS315-22: R2010
5. Equipment
Laboratory glassware
Dialysis membrane (6000 to 8000 Da)
Temperature-controlled bath
Double-beam UV-visible spectrophotometer
6. Operating procedure
6.1 Preparation of the reagent
Place 50 mg of DHN weighed to within 1 mg in a calibrated 100 mL phial.
Add concentrated sulphuric acid up to the gauge line.
Place the calibrated phial in a temperature-controlled bath at 28 C for 4h
(without stirring).
After heating, decant the reagent into a brown flask and store it in a refrigerator
at 4 C.
6.2 Preparation of wine test specimens
Insert 20 mL of wine, after degassing, into the dialysis membrane.
Place the dialysis membrane containing the wine in a 6-litre flask filled with
distilled water.
Leave to dialyse for 24h, changing the dialysis water twice.
6.3 Colour reaction
Place 1 mL of dialysed wine into a test tube.
Add 9 mL of reagent.
Place the test tube in a temperature-controlled bath at 100 C for 2h.
Analyse the coloured solution by UV-visible spectrophotometer at 540nm and
read the absorbance value.
6.4 Calculation of the wines CMC content
Recording the absorbance value read in point 6.3 on the calibration curve
obtained for a wine (see figure 2)
OIV-MA-AS315-22: R2010
7.2 Repeatability
The repeatability of the determination of CMC in white wines was defined on the
basis of the results achieved on 22 samples of wine that underwent 2 successive
analyses, so as to be analysed in identical conditions. The results are given in table
1.
OIV-MA-AS315-22: R2010
calculated values
Repeatability:
standard deviation
CV in %
r-limit
r-limit in %
0,075
7,2 %
0,21
20 %
0,082
9,6 %
0,23
27 %
7.4 Specificity
OIV-MA-AS315-22: R2010
Sample
Wine 1
Wine 1
Wine 1
Wine 2
Wine 2
Wine 2
Wine 3
Wine 3
Wine 3
Wine 4
Wine 4
Wine 4
Added
concentration
(mg/l)
50
50
50
75
75
75
100
100
100
150
150
150
Resulting
concentration
(mg/l)
33
51
24
78
90
69
109
97
103
163
149
159
Recovery
rate
66 %
102 %
77 %
104 %
121 %
92 %
109 %
97 %
103 %
109 %
100 %
106 %
The detection limits (LD) and quantification limits (LQ) were calculated for
an untreated wine that underwent 10 analyses. The detection limit thus
determined is of 14 mg/l and the quantification limit is of 61 mg/l.
The method therefore enables to detect the adding of CMC into white wine
in quantities exceeding 20 mg/l and to quantify the addition when it exceeds
60 mg/l; this is not highly satisfactory but remains compatible with the
maximum authorised dose of 100 mg/l.
7.5 Uncertainty
The uncertainty was calculated at 3 different concentration levels (25, 75 and 150
mg/l) based on the analysis results for wines that have undergone CMC treatment,
OIV-MA-AS315-22: R2010
8. Bibliography
[1] Regulation (CE) N 1333/2008 of the 16th of December, 2008
concerning food additives
[2] Stabilisation tartrique des vins par la carboxymthylcellulose - Bulletin de
lOIV 2001, vol 74, n841-842, p151-159.
[3] Determination of carboxymethycellulose in food products - H.D Graham,
Journal of food science 1971, p 1052-1055
OIV-MA-AS315-22: R2010
OIV-MA-AS315-23
Trueness
r=
Sr =
RSDr=
R=
OIV-MA-AS315-23: R2010
RSDR =
HoR =
B0 =
Mean blank
LOD =
LOQ =
2. General Aspects
Requirement
The method of analysis must be associated with specific oenological
practices
Additives or processing aids containing allergenic proteins
Each product must be characterized from the chemical point of view and
quality control is strictly necessary
Class of analytical methods
Generally speaking, immunoenzymatic approaches are considered the most
suitable and easy methods for routine control of allergens.
OIV-MA-AS315-23: R2010
3. Type of methods
Specific methods for the determination of fining agent proteins in wine are
not prescribed yet. Several ELISA methods are already available and can be
applied.
Laboratories shall use a method validated to OIV requirements that fulfils
the performance criteria indicated in Table 1. Wherever possible, the
validation shall include a certified reference material in the collaborative
OIV-MA-AS315-23: R2010
Before the assay, the antibody preparations must be purified and conjugated.
Indirect
1.
2.
3.
4.
OIV-MA-AS315-23: R2010
Direct
1.
2.
3.
4.
5.
Before the assay, both antibody preparations must be purified and one must
be conjugated.
Indirect
1. Prepare a surface to which capture antibody is bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen-containing sample or standard to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specifically to the antigen.
6. Wash the plate, so that primary antibody in excess (unbound) is
removed.
7. Apply enzyme-linked antibodies (secondary antibodies) that bind
specifically to the primary antibody.
8. Wash the plate, so that the enzyme-linked antibodies in excess
(unbound) are removed.
9. Apply a chemical which is converted by the enzyme into a color or
fluorescent or electrochemical signal.
10. Measure the absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and
quantity of antigen.
8
OIV-MA-AS315-23: R2010
Before the assay, all the antibody preparations must be purified and one of
them must be conjugated.
OIV-MA-AS315-23: R2010
Detection limit
(expressed in mg/L)
Casein: at least 0,5
Isinglass : at least 0,5
Lysozyme : at least 0,5
Ovalbumin : at least 0,5
Limit of quantification
(expressed in mg/L)
Casein: at least 1
Isinglass : at least 1
Lysozyme : at least 1
Ovalbumin : at least 1
Precision
Recovery
Specificity
Trueness
x m < 1,96 *
sR ( lab ) sr ( lab ) * (1 1 n )
10
OIV-MA-AS315-23: R2010
11
Method OIV-MA-AS321-01
Type IV method
Total Bromide
1. Principle
The wine is ashed at 525 oC in presence of an excess of soda lime. A solution of the
residue (at pH 4.65) is treated with chloramine T to liberate bromide. The bromide
is reacted with phenolsulfonephthalein to form phenoltetra-bromophthalein-3-3disulfonic acid, which is determined by spectrophotometer at 590 nm.
2. Apparatus
2.1 Boiling water-bath 100C
2.2 Temperature-controlled electric furnace
2.3 Spectrophotometer capable of measuring absorbance at wavelengths between
300 and 700 nm
3. Reagents
3.1 Sodium hydroxide solution, 50% (m/m)
3.2 Calcium hydroxide suspension containing 120 g of CaO per liter
3.3 Phenolsulfonephthalein solution:
0.24 g of phenolsulfonephthalein (phenol red) are dissolved in 24 mL sodium
hydroxide solution, 0.1 M, and made up to the liter with distilled water.
3.4 pH 4.65 buffer solution:
Acetic acid, 2 M ...................................
500 mL
Sodium hydroxide, 2 M ...............................
250 mL
Distilled water to ......................................
1L
3.5 Oxidizing solution:
Chloramine T ......................................
2g
Distilled water to ................................
1L
Prepare this solution 48 hours before use
Storage: two weeks at 4 oC
3.6 Reducing solution:
Sodium thiosulfate . 25 g/L.
Distilled water to ....................................
1L
3.7 Sulfuric acid, 10%(v/v): sulfuric acid (20 = 1.84 g/mL) diluted 1/10.
3.8 Sulfuric acid, 1%(v/v): sulfuric acid (20 = 1.84 g/mL) diluted 1/100.
3.9 Potassium bromide solution corresponding to 1 g of bromide per liter. 1.489g
of potassium bromide, KBr, is dissolved in distilled water and made up to one
liter.
OIV-MA-AS321-01 : R2009
4. Procedure
4.1 How to obtain ash and ash solution
Place 50 mL of wine in a silica dish of 7 cm diameter, add 0.5 mL 50% sodium
hydroxide solution, (3.1), and 1 mL calcium hydroxide suspension (3.2).
Check that the pH is at least pH 10. Leave the dish covered with a watch glass
for 24 hours. Evaporate the liquid until dry on a boiling water bath. To
accelerate the evaporation, a hot air current can be used in the final stages.
Ash as follows: place the dish 30 minutes in a furnace (2.2) at 525C . After
cooling, mix the residue with a little distilled water. Evaporate on the boiling
water-bath. Ash again at 525C. Repeat the operation until the ash is
gray/white.
Mix the residue with 5 mL boiling distilled water. Add using a burette: first
10% sulfuric acid (3.7), then sufficient 1% sulfuric acid (3.8) to bring the pH
to between 4 and 5 as measured by indicator paper. Let X mL = the volume
added of sulfuric acid (3.7 & 3.8). Add 10.2-(X+5) mL of distilled water.
Crush the precipitated calcium sulfate with a glass rod. Transfer the content of
the dish to a centrifugation tube. Centrifuge for 10 min. Place 8 to 9 mL of
the clear supernatant into a test tube.
4.2 Qualitative test
This test is performed to determine if the bromide content of the wine is
between 0 and 1 mg/L, which would enable the determination to be performed
on the undiluted ash solution.
Place in a small test tube:
- 1 mL of ash solution
- 1 drop of pH 4.65 buffer solution
- 1 drop of phenolsulfonephthalein solution
- 1 drop of chloramine T solution
After exactly 1 minute, stop the reaction by adding 1 drop of sodium
thiosulfate solution.
If the coloration obtained is yellow, brownish yellow or greenish yellow, the
ash solution can be used undiluted.
If the obtained coloration is blue, purple or violet, the wine contains more than
1 mg of bromide per liter and the ash solution must be diluted 1/12 or 1/5 until
the coloration obtained corresponds to the conditions above.
4.3 Quantitative method
Place in a test tube:
- 5 mL of ash solution, diluted or undiluted, add:
- 0.25 mL of pH 4.65 buffer solution
- 0.25 mL of phenolsulfonephthalein solution
- 0.25 mL T chloramine solution
OIV-MA-AS321-01 : R2009
BIBLIOGRAPHY
DAMIENS A., Bull. Sci. Pharmacologiques, 1920, 27, 609; Ibid, 1921, 28, 37, 85
et 105.
BALANTRE P., J. Pharm. Chem., 1936, 24, 409.
PERRONET M., ROCQUES Mme S., Ann. Fals. Fraudes, 1952, 45, 347.
CABANIS J.-C., Le brome dans les vins, Thse doct. Pharm., Montpellier, 1962.
JAULMES P., BRUN Mme S., Cabanis J.-C., Chim anal., 1962, 327.
STELLA C., Riv. Viticolt. Enol., Conegliano, 1967, 5.
OIV-MA-AS321-01 : R2009
Method OIV-MA-AS321-02
Type II method
Chloride
1. Principle
Chloride is determined directly in the wine by potentiometry using an Ag/AgCl
electrode.
2. Apparatus
2.1 pH/mV meter graduated at intervals of at least 2 mV.
2.2 Magnetic stirrer.
2.3 Ag/AgCl electrode with a saturated solution of potassium nitrate as
electrolyte.
2.4 Microburette graduated in 0.01 mL.
2.5 Chronometer.
3. Reagents
3.1 Standard chloride solution: 2.1027 g of potassium chloride, KCl (max.
0.005% Br), dried before use, by leaving in a desiccator for several days, is
dissolved in distilled water and made up to one liter. 1 mL of this solution
contains 1 mg Cl-.
3.2 Silver nitrate solution: 4.7912 g of analytical grade silver nitrate, AgNO 3, is
dissolved in ethanol solution, 10% (v/v) and made up to one liter. 1 mL of this
solution corresponds to 1 mg Cl-.
3.3 Nitric acid, not less than 65% (20 = 1.40 g/mL).
4. Procedure
4.1 Place 5.0 mL of standard chloride solution (3.1) into a 150 mL cylindrical
vessel placed on a magnetic stirrer (2.2), dilute with distilled water to
approximately 100 mL and acidify with 1.0 mL of nitric acid (3.3). After
immersing the electrode, add silver nitrate solution (3.2) with the
microburette, with moderate stirring using the following procedure: begin by
adding the first 4 mL in 1 mL fractions and read the corresponding millivolt
values. Add the next 2 mL in fractions of 0.20 mL. Finally, continue the
addition in fractions of 1 mL until a total of 10 mL has been added. After
each addition, wait for approximately 30 sec before reading the corresponding
millivolt value. Plot the values obtained on a graph against the corresponding
milliliters of titrant and determine the potential corresponding to the
equivalence point.
OIV-MA-AS321-02 : R2009
r = 1.2 mg Cl/L
r = 0.03 mEq/L
r = 2.0 mg NaCl/L
R= 4.1 mg/L
R= 0.12 mEq/L
R= 6.8 mg NaCl/L
OIV-MA-AS321-02 : R2009
V = V + Vi
E1
E1 E 2
Where:
V = volume of titrant at the equivalence point;
V = volume of titrant before the largest potential change;
Vi = constant volume of the increments of titrant, i.e. 0.2 mL;
E1 = second difference in potential before the largest potential change;
E2 = second difference in potential after the largest potential change.
Example:
Volume of AgNO3
titrating solution
E potential in
mV
Difference
E
Second difference
E
204
208
212
218
224
230
238
250
272
316
350
376
396
4
4
6
6
6
8
12
22
44
34
26
20
0
2
0
0
2
4
10
22
10
8
6
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
In this example, the end point of the titration is between 1.6 and 1.8 mL: the
largest potential change ( E = 44 mV) occurs in this interval. The volume of
silver nitrate titrant consumed to measure the chlorides in the test sample is:
V 1. 6 0. 2
22 1. 74mL
22 10
BIBLIOGRAPHY
MIRANDA PATO C. de, F.V., O.I.V., 1959, no12.
HUBACH C.E., J. Ass. Off. Agric. Chem., 1966, 49, 498.
Fdration internationale des Producteurs de jus de fruits, F. O.I.V.,1968, no37.
JUNGE Ch., F.V., O.I.V., 1973, no440.
OIV-MA-AS321-02 : R2009
Method OIV-MA-AS321-03
Type II method
SCOPE
This method is applicable to the analysis of fluoride in all wines. With
proper dilution, the range of detection is 0.1 mg/l to 10.0 mg/l.
2.
PRINCIPLE
The concentration of fluoride in the sample is measured after addition of a
buffer, using a fluoride ion selective electrode. The buffer provides a high,
constant background ionic strength; complexes iron and aluminium (which
would otherwise complex with fluoride); and adjusts the pH to a level that
minimises the formation of a HFHF complex. The matrix effects are then
minimised using standard addition.
3.
REAGENTS
3.1 Deionized or distilled water
3.2 Sodium chloride 99.0% purity
3.3 Trisodic citrate 99.0% purity
3.4 CDTA (1,2-diaminocyclohexane-N,N,N,N- tetracetic hydrate acid)
98.0% purity3.5 Sodium hydroxide to 98.0% purity
3.6 Sodium hydroxide solution 32% (w/v) made from 3.5
3.7 Glacial acetic acid 99.0% purity
3.8 Sodium fluoride 99.0% purity
OIV-MA-AS321-03 : R2004
3.9 Commercial Total Ionic Strength Adjustment Buffer (TISAB) (i.e. IIIOrion Research Inc. Cat. # 940911) or equivalent (See 4.2).
OIV-MA-AS321-03 : R2004
3.12 Wine blank : a wine known to be fluoride free is used as a matrix blank
3.13 1 mg/l spiked wine standard - Place 10 ml (4.11) of 100 mg/l fluoride stock
standard solution(3.11.1)into a 1 l volumetric flask (4.10) and
bring to volume with fluoride free wine (3.12).
OIV-MA-AS321-03 : R2004
4.
APPARATUS
4.1
4.2
4.3
4.4
4.5
Magnetic stirrer
4.6
4.7
4.8
4.9
Ultrasonic bath
4.10
4.11
OIV-MA-AS321-03 : R2004
Calibration standards
Measure the potential of each of the calibration solutions, using the meter
(4.1), fluoride selective electrode (4.2), and reference electrode (4.2). The
final reading must be taken when the readings have stabilised (stability is
obtained when the potential varies by not more than 0.2 to 0.3 mV/ 3
minutes). Record the readings for each of the calibration standards.
The log10 of each of the standard concentrations versus the millivolt
reading measured for each standard concentration is plotted on graph paper
in order to determine the slope of the electrode.
7.2
Wine samples
Measure and record the potential expressed in mV (E1) of the sample (6.1
or 6.2) after the readings have stabilised. Add 500 l (4.8) of 100 mg/l
fluoride standard (3.11.1) to the sample (6.1 or 6.2). After the readings
have stabilised, read and record the potential expressed in mV (E2) of the
wine solution.
OIV-MA-AS321-03 : R2004
CALCULATION
The fluoride content of the sample solution expressed in mg/l is obtained
by using the following formula:
Cf
Va Ca
1
Vo
((anti log E / S ) 1)
Cf DF Ca
1
((anti log E / S ) 1)
OIV-MA-AS321-03 : R2004
Ca =
Vstd C std
Vsamp
where
Ca = concentration (in mg/l) of fluoride added to the sample volume (Vo ) obtained
by multiplying the standard volume (3.11.1) added to the solution (Vstd) by the
concentration (Cstd) of standard (3.11.1) and divided by the sample volume (25 ml)
using (6.1) or (6.2)
Vstd = volume added standard (3.11.1) (0.5 ml)
Vsamp = sample volume used in (6.1) or (6.2), Vsamp = 25 ml
Cstd = standard concentration (3.11.1)
Calculation example:
(1) for a sample prepared as in (6.2) and measured as in (7.2)
DF = 1
Ca =
Vstd C std
0.5 ml 100 mg / l
=
= 2 mg /l
Vsamp
25 ml
E = 19.6 mV
S = -58.342
1
((anti log E / S ) 1)
1
C f 1 2 mg / l
((anti log 19.6 / 58.342) 1)
C f 1 2 mg / l 0.856 = 1.71 mg / l of fluoride
Cf DF Ca
OIV-MA-AS321-03 : R2004
Ca =
Vstd C std
0.5 ml 100 mg / l
=
= 2 mg /l
Vsamp
25 ml
E = 20.4 mV
S = -55.937
1
((anti log E / S ) 1)
1
C f 2 2 mg / L
((anti log 20.4 / 55.937) 1)
Cf DF Ca
9. PRECISION
The details of inter laboratory study are given in Annex B. the Horrat (HoR) ranges
from 0.30 to 0.97 and indicates a very good reproducibility among participants.
The results of the statistical calculations are given in Annex B table 2.
The standard deviation of repeatability (RDSr) ranges from 1.94% to 4.88%. The
standard deviation of reproducibility (RDSR) ranges from 4.15% to 18.40%.
Average % recovery ranged between 99.8% and 100.3% of the mean target.
10. QUALITY ASURANCE AND MANAGEMENT
10.1 Analyse a standard solution from 1.0 mg/l (3.11.2) at the beginning and end
of each series of measurement. The results must be 1.0 0.1 mg/l.
10.2 Before each measurement series analyse a blank sample (3.12) and for the
internal quality control (CQI) a overloaded wine (3.13). The blank sample must
not be over 0.0 mg/l 0.1 mg/l. and the CQI must not be over 1.0 mg/l 0.2 mg/l.
OIV-MA-AS321-03 : R2004
Annex A
References
1. AOAC International, AOAC Official Methods Program, Associate Referees
Manual On Development, Study, Review, and Approval Process,1997
2. Postel, W.; Prasch, E., Wein-Wissenschoft, (1975) 30 (6), 320-326
3. Office International de la Vigne et du Vin, Compendium of International
Methods of Wine Analysis, 255257
4. Gil Armentia, J. M.; Arranz, J. F.; Barrio, R. J.; Arranz, A., Anales de
Bromatologia, (1988) 40 (1) 71-77
5. Gran, G; Analyst (1952) 77, 661
6. Corning fluoride ion selective electrode Instruction Manual, 1994
7. Corning Instruction Manual pH/ion analyzer 455, 109121-1 Rev. A, 11/96
8. Horwitz,W.; Albert, R.; Journal of the Association of Official Analytical
Chemists, (1991) 74 (5) 718
OIV-MA-AS321-03 : R2004
Annex B
Inter laboratory Study
VALIDATION OF A FLUORIDE ION SELECTIVE ELECTRODE, STANDARD
ADDITION METHOD FOR THE MEASUREMENT OF FLUORIDE IN WINE
B.1 Introduction
The validation by collaborative trial of a fluoride selective ion electrode, standard
addition method for the determination of fluoride in wine is described. The
collaborative trial involved a total of twelve participants, six European and six
Americans, who took part in the study. The collaborative study was performed
using the AOAC, Youden protocol(1).
B2 Participants
The twelve participants of this validation consisted of laboratories from Austria,
France, Germany, Spain, and the United States and comprised of the following:
BATF Alcohol and Tobacco LaboratoryAlcohol Section, SF, Walnut Creek,
CA., United States; BATF, National Laboratory Ctr., Rockville, MD, United
States; Bundesinstitut fr Gesundheitlichen Verbraucherschutz, Berlin, Germany;
Canandaigua Winery, Madera, CA, United States; CIVC, Epernay, France; E. & J.
Gallo Winery-Analytical Services Laboratory, Modesto, CA, United States; E. &
J. Gallo Winery-Technical Analytical Services Laboratory, Modesto, CA, United
States; ETS Labs, St. Helena, CA, United States; Hhere Bundeslehranstalt &
Bundesamt fr Wein und Obstbau, Klosterneuburg, Austria; Institut Catala de la
Vinya i el Vi, Vilafranca del Penedes (Barcelona),Spain; Laboratorio Arbitral
Agroalimentario, Madrid, Spain; and Sutter Home Winery, St. Helena, CA.,
United States.
B3 Samples used in the trial
The samples used in the trial are given in Appendix I. They were distributed as
twelve wine samples (six Youden pairs of samples comprised of three red wines
and three white wines).
OIV-MA-AS321-03 : R2004
10
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Sample description
White wine with no fortification (total of 0.6 mg/l F-)
White wine fortified with 0.3 mg /l (total of 0.9 mg/l F-)
White wine fortified with 0.9 mg /l (total de 1,5 mg/l F-)
White wine fortified with 1.2 mg /l (total de 1,8 mg/l F-)
White wine fortified with 1.4 mg /l (total de 2,0 mg/l F-)
White wine fortified with 1.7 mg /l (total de 2,3 mg/l F-)
Red wine with no fortification (total de 0,2 mg/l F-)
Red wine fortified with 0.3 mg /l (total de 0,5 mg/l F-)
Red wine fortified with 0.8 mg /l (total de 1,0 mg/l F-)
Red wine fortified with 1.1 mg /l (total de 1,3 mg/l F-)
Red wine fortified with 2.5 mg /l (total de 2,7 mg/l F-)
Red wine fortified with 2.8 mg /l (total de 3,0 mg/l F-)
8.4 Results
A summary of the results obtained by the twelve participants is given in Table I.
None of the laboratories reported any difficulties with the analysis. One Youden
pair from one laboratory was determined to be an outlier, using the Cochrans test.
These results are noted(c) in Table I, and were not used in the statistical analysis.
OIV-MA-AS321-03 : R2004
11
Table 1
Collaborative data for the determination of fluoride in wine by fluoride selective
electrode, standard additiona
Lab
Number
1
2
3
4
5
6
7
8
9
10
11
12
N of cases
Minimum
Maximum
Range
Mean
Median
Std Dev
Pair 1b
1
2
0.55 0.80
0.52 0.81
0.52 0.81
0.62 0.98
0.48 0.78
0.53 0.84
0.53 0.76
0.57 0.88
0.51 0.81
0.54 0.84
0.60 0.93
0.65 0.94
White Wine
Pair 2b
3
4
1.33 1.56
1.39 1.64
1.40 1.70
1.48 1.64
1.34 1.64
1.45 1.74
1.27 1.64
1.51 1.85
1.40 1.71
1.43 1.71
1.48 1.75
1.54 1.79
Pair 3b
5
6
1.86 2.24
1.86 2.31
1.92 2.25
1.85 2.14
1.84 2.11
1.97 2.30
1.89 2.06
2.11 2.33
1.90 2.20
1.93 2.22
1.98 2.32
2.05 2.32
Pair 4b
7
8
0.19 0.45
0.19 0.46
0.14 0.42
0.28 0.56
0.12 0.39
0.13 0.43
0.14 0.40
0.48c 0.48c
0.13 0.42
0.18 0.44
0.25 0.57
0.21 0.52
Red Wine
Pair 5b
9
10
0.89 1.17
0.92 1.20
0.96 1.22
1.00 1.32
0.88 1.16
0.92 1.21
0.88 1.12
1.01 1.32
0.90 1.19
0.96 1.23
1.06 1.31
1.03 1.24
Pair 6b
11
12
2.54 2.77
2.58 2.77
2.64 2.95
2.64 2.72
2.56 2.82
2.66 2.93
2.44 2.83
2.64 3.08
2.60 2.86
2.66 2.87
2.68 2.82
2.81 3.07
12
0.48
0.65
0.17
0.55
0.54
0.050
12
1.27
1.54
0.27
1.42
1.42
0.079
12
1.84
2.11
0.27
1.93
1.91
0.084
11
0.12
0.28
0.16
0.18
0.18
0.052
12
0.88
1.06
0.18
0.95
0.94
0.061
12
2.44
2.81
0.37
2.62
2.64
0.090
12
0.76
0.98
0.22
0.85
0.83
0.069
12
1.56
1.85
0.29
1.70
1.71
0.079
12
2.06
2.33
0.27
2.23
2.25
0.091
11
0.39
0.57
0.18
0.46
0.44
0.063
12
1.12
1.32
0.20
1.22
1.22
0.065
12
2.72
3.08
0.36
2.87
2.85
0.114
OIV-MA-AS321-03 : R2004
12
Table 2
Statistical data from the collaborative study on the analysis of fluoride in wine by
fluoride selective ion electrode, standard addition method
White Wine
STATISTIC
Pair 1
Pair 2
Red
Wine
Pair 3
Pair 4
Pair 6
Total # of Labs
12
12
12
11d
Pair 5
12
Number of "replicates
per lab
Mean (split levels)
0.55
0.85
0.0006
0.0235
1.42
1.70
0.0015
0.0382
1.93
2.23
0.0026
0.5106
0.18
0.46
0.0002
0.0156
0.95
1.22
0.0005
0.0211
2.62
2.87
0.0049
0.0703
3.35 %
2.45 %
2.45 %
4.88 %
1.94 %
2.55 %
0.0039
0.0070
0.0089
0.0034
0.0042
0.0130
0.0625
0.0835
0.0945
0.0587
0.0647
0.1141
8.92 %
5.36 %
4.54 %
18.39 %
5.95 %
4.15 %
16.88
14.97
14.33
19.00
15.80
13.74
0.53
0.36
0.32
0.97
0.38
0.30
93.1
94.6
96.7
91.0
94.4
96.4
Repeatability variance
Repeatability Standard
Deviation
Relative standard
deviation RSDr,
repeatability
Reproducibility
variance
Reproducibility
standard deviation
Relative standard
deviation RSDR,
reproducibility
Horwitz Equation
Applied (as RSDR)
HORRAT Value HoR
(RSDR
(measured)/RSDR
(Horwitz))
Average % recovery
12
One lab pair was deleted from data set by Cochrans Test
OIV-MA-AS321-03 : R2004
13
Method OIV-MA-AS321-04
Type IV method
Total Phosphorus
1. Principle
After nitric oxidation and ashing, and dissolution in hydrochloric acid, phosphoric
acid is determined colorimetrically as the yellow phospho-vanadomolybdate
complex.
2. Apparatus
2.1 Boiling water-bath 100C
2.2 Hot plate
2.3 Temperature-controlled electric furnace.
2.4 Spectrophotometer measuring absorbance at wavelengths between 300 and
700 nm
3. Reagents
3.1 Nitric acid, (20 = 1.39 g/mL).
3.2 Hydrochloric acid, approx. 3 M; hydrochloric acid (20 = 1.15 - 1.18 g/mL)
diluted 1/4 with water.
3.3 Vanadomolybdate reagent:
Solution A: dissolve 40 g of ammonium molybdate, (NH4)6Mo7O24.4H2O, in
400 mL water.
Solution B: dissolve 1 g of ammonium vanadate, NH4VO3, in 300 mL water
and 200 mL nitric acid (20 = 1.39g/L) (3.1). Leave to cool.
Vanadomolybdate reagent: place first solution B then solution A into a 1 liter
flask, and make up to the mark with water. Reagent to be used within 8 days
of preparation.
3.4 P2O5 solution, 0.1 g/L.
Prepare a P2O5 solution 1 g/L by dissolving 2.454 g of di-potassium hydrogen
phosphate, K2HPO4, in a liter of water. Dilute 10% (v/v).
4. Procedure
4.1 Ashing
OIV-MA-AS321-04 : R2009
BIBLIOGRAPHY
A.F.N.O.R., Norme U, 42-246, Tour Europe, Paris.
SUDRAUD P., Bull. O.I.V., 1969, 462-463, 933.
A 5 mL sample volume is suitable for P2O5 content, of between 100 and 500 mg/L.
Outside these concentration limits, increase or decrease the sample volume.
OIV-MA-AS321-04 : R2009
Method OIV-MA-AS321-05A
Type II method
Sulfates
1. Principle
Gravimetric determination following precipitation of barium sulfate. The
barium phosphate precipitated at the same time is eliminated by washing the
precipitate in hydrochloric acid.
In the case of musts or wine rich in sulfur dioxide, prior de-sulfiting by boiling
in an airtight vessel is recommended.
2. Method
2.1 Reagents
2.1.1 Hydrochloric acid, 2 M.
2.1.2 Barium chloride solution, BaCl2.2H2O, 200 g/L.
2.2 Procedure
2.2.1 General procedure:
Introduce 40 mL of the sample to be analyzed into a 50 mL centrifuge tube;
add 2 mL hydrochloric acid, 2 M (2.1.1), and 2 mL of barium chloride
solution, 200 g/L (2.1.2). Stir with a glass stirrer; rinse the stirrer with a little
distilled water and leave to stand for five min. Centrifuge for five min, then
carefully decant the supernatant liquid.
Wash the barium sulfate precipitate as follows: add 10 mL hydrochloric acid,
2 M (2.1.1), place the precipitate in suspension and centrifuge for five min,
then carefully decant the supernatant liquid. Repeat the washing procedure
twice as before using 15 mL distilled water each time.
Quantitatively transfer the precipitate, with distilled water, into a tared
platinum capsule and place over a water bath at 100C until fully evaporated.
The dried precipitate is calcined several times briefly over a flame until a white
residue is obtained. Leave to cool in a desiccator and weigh.
Let m = mass in milligrams of barium sulfate obtained.
2.2.2 Special procedure: sulfited must and wine with a high sulfur dioxide
content.
Elimination of sulfur dioxide.
Measure 25 mL of water and 1 mL of concentrated hydrochloric acid (20=
1.15 to 1.18 g/mL) into a 500 mL conical flask equipped with a dropping
funnel and an outlet tube. Boil the solution to remove the air and introduce
OIV-MA-AS321-05A : R2009
100 mL of wine through the dropping funnel. Continue boiling until the
volume of liquid in the flask has been reduced to about 75 mL and
quantitatively transfer, after cooling, to a 100 mL volumetric flask. Make up
to mark with water. Determine the sulfate in the 40 mL sample as indicated in
2.2.1.
2.3. Expression of results
2.3.1 Calculations:
The sulfate content, expressed in milligrams per liter of potassium sulfate, K 2SO4
is given by:
18.67 x m
The sulfate content in musts or wine is expressed in milligrams per liter of
potassium sulfate, to the nearest whole number.
2.3.2 Repeatability (r):
up to 1000 mg/L:
approx. 1500 mg/L:
r = 27 mg/L
r = 41 mg/L
R = 51 mg/L
R = 81 mg/L
BIBLIOGRAPHY
DEIBNER L , BNARD P., Ind. alim. agric., 1954, 71, no1, 23; no5, 427; 1955, 72,
no9-10, 565 et no11, 673.
DEIBNER L., Rv. ferm. ind. alim., 1959, 14 no5, 179 et no6, 227.
BLAREZ Ch., Vins et spiritueux, 1908, 149, Maloine d., Paris.
DER HEIDE X. von, SCHITTHENNER F., Der Wein, 1922, 320, Vieweg & Sohn
Verlag, Braunschweig.
JAULMES P., Analyse des vins, 1924, 73, Dubois et Poulain, d., Montpellier; 2e
dition, 1951, 112.
SIMONEAU G., tude sur les mots concentrs de raisins, 1946, Thse pharm.,
Montpellier, 49.
RIBREAU-GAYON J., PEYNAUD E., Analyse et contrle des vins, 1947, 244, Ch.
Branger d., Paris-Lige.
FROLOV-BAGREEV A., AGABALIANTZ G., Chimie du vin, 1951, 369, Moscou,
Laboratoire de chimie de l'tat de Wrzburg (Allemagne), F.V., O.I.V., 1969, no321.
OIV-MA-AS321-05A : R2009
Method OIV-MA-AS321-05B
Sulfates
Quick test method
Wines are classified into several categories using the so-called limits
method, based on the precipitation of barium sulfate using a barium ion titrant.
WITHDRAWN
OIV-MA-AS321-05B : R2009
Method OIV-MA-AS322-01
Type IV method
Ammonium
1. Principle
Retention of the ammonium cation on a weak cation exchange resin, elution using
an acidic solution, distillation of the eluent and determination of the ammonia in
the distillate by titration with a standardized solution of hydrochloric acid.
2. Apparatus
2.1 Cation exchange resin column
A 50 mL burette with a glass stopcock fitted with a glass wool plug containing
25 g of weak cation exchange resin (e.g. Amberlite IR-50, 80-100 mesh).
Wash alternately with 1 M sodium hydroxide solution and 1 M hydrochloric
acid solution. Wash the resin with distilled water until a negative reaction of
chloride ion with silver nitrate is obtained. Pass 50 mL of neutral buffer
slowly through the glass column, rinse with distilled water until phosphates
begin to elute as detected using a saturated solution of lead acetate.
2.2 Distillation apparatus
Use the apparatus described in the chapter on Alcoholic Strength 3.1
The condensate is transferred to the conical flask through a drawn-out tube
touching the bottom of the vessel.
Alternatively, it is possible to use the steam distillation apparatus used in the
chapter on Volatile Acidity 4.1 or other apparatus that can be used for the
following experiments which check the purity of the reagents.
a) Place 40-45 mL of 30 % sodium hydroxide solution (v/v), 50 mL of water
and 50 mL hydrochloric acid, 1 M, in the distillation flask. Distil half the
volume and collect the distillate in 30 mL of boric acid solution, 40 g/L to
which 5 drops of methyl red have been added. Adjust the color to pink by
the addition of 0.1 mL of 0.1 M hydrochloric acid.
b) A test (similar to that described in a) is conducted using, 10 mL 0.05 M
ammonium sulfate solution, containing 3.55 g/L of anhydrous ammonium
sulfate, (NH4)2SO4. In this case, between 10 and 10.1 mL 0.1 M
hydrochloric acid must be used to obtain the change of color of the
indicator.
3. Reagents
3.1 Hydrochloric acid solution, 1 M.
OIV-MA-AS322-01 : R2009
OIV-MA-AS322-01 : R2009
Elute the cations retained on the resin with 50 mL of 1 M hydrochloric acid, (3.1)
and rinse with 50 mL distilled water.* The eluate and the water washings are
combined in a 1 liter round bottom distillation flask.
Add one drop of phenolphthalein, 1% (m/v), and sufficient quantity of 30%
sodium hydroxide solution (m/v)(3.4), to obtain a true alkaline reaction, constantly
cooling the flask during this addition.
Distil about half the volume of the liquid from the distillation flask, into 30 mL of
4% boric acid (m/v)(3.9).
The distillate is titrated with 0.1 M hydrochloric acid (3.5), in the presence of
bromocresol green or methyl red. Record the volume of hydrochloric acid used (n).
5. Expression of results
The content of ammonium (NH4) ions is expressed in milligrams per liter to the
nearest whole number.
5.1 Calculation
The content of ammonium ions, expressed in milligrams per liter is:
36 x n
When wines with low ammonium content are analyzed, the determination is
conducted using 100 mL of wine. In this case the quantity of ammonium is given
by:
18 x n
BIBLIOGRAPHY
Usual Method:
JAULMES P., Analyse des vins, 1951, 220, Montpellier
KOURAKOU Mme S., Ann. Fals. Exp. Chim., 1960, 53, 337.
* The column should be washed with 50 mL of neutral buffer solution and rinsed with water
before using the column for another determination.
OIV-MA-AS322-01 : R2009
Method OIV-MA-AS322-02A
Type II method
Potassium
1. Principle
Potassium is determined directly in diluted wine by atomic absorption
spectrophotometry after the addition of cesium chloride to suppress ionization
of potassium.
2 Method
2.1 Apparatus
Atomic absorption spectrophotometer, equipped with an air-acetylene burner
Potassium hollow cathode lamp
2.2 Reagents
2.2.1 Solution containing 1 g of potassium per liter.
Use a standard commercial solution containing 1 g of potassium per liter. This
solution may be prepared by dissolving 4.813 g of potassium hydrogen tartrate
(C4H5KO6) in distilled water and making up the volume to 1 liter with water.
2.2.2 Matrix (model) solution:
citric acid monohydrate .......................................
3.5 g
sucrose ...........................................................
1.5 g
glycerol ..........................................................
5.0 g
anhydrous calcium chloride, (CaCl2) ..............
50 mg
anhydrous magnesium chloride (MgCl2) ............ 50 mg
absolute alcohol .................................................
50 mL
water to ............................................................
500 mL
2.2.3 Cesium chloride solution containing 5% cesium:
Dissolve 6.33 g of cesium chloride, CsCl, in 100 mL of distilled water.
2.3 Procedure
2.3.1 Preparation of sample
Pipette 2.5 mL of wine (previously diluted 1/10) into a 50 mL volumetric flask,
add 1 mL of the cesium chloride solution and make up to the mark with
distilled water.
OIV-MA-AS322-02A : R2009
2.3.2 Calibration
Introduce 5.0 mL of the matrix solution into each one of five of 100mL
volumetric flasks and add 0, 2.0, 4.0, 6.0 and 8.0 mL respectively of the 1 g/L
potassium solution (previously diluted 1/10). Add 2 mL of the cesium chloride
solution to each flask and make up to 100 mL with distilled water.
The standard solutions contain 0, 2, 4, 6 and 8 mg of potassium per liter
respectively and each contains 1 g of cesium per liter. Keep these solutions in
polyethylene bottles.
2.3.3 Determination
Set the wavelength to 769.9 nm. Zero the absorbance scale using the zero
standard solution (2.3.2). Aspirate the diluted wine (2.3.1) directly into the
spectrophotometer, followed in succession by the standard solutions (2.3.2).
Record the absorbance for each solution and repeat.
2.4 Expression of results
2.4.1 Method of calculation
Plot a graph showing the variation in absorbance as a function of potassium
concentration in the standard solutions.
Record the mean absorbance obtained with diluted wine on this graph and
determine its potassium concentration C in milligrams per liter.
The potassium concentration, expressed in milligrams per liter of the wine to
the nearest whole number, is F x C, where F is the dilution factor (here 200).
2.4.2 Repeatability (r):
r = 35 mg/L.
OIV-MA-AS322-02A : R2009
Method OIV-MA-AS322-02B
Potassium
1. Principle
Potassium is determined directly in diluted wine by flame photometry.
Note: The gravimetric determination of potassium tetraphenylborate
precipitated from the solution of the ash of wine is a precise method for the
determination of potassium and is described in the annex.
2. Method
2.1 Apparatus
2.1.1 Flame photometer supplied with an air-butane mixture.
2.2 Reagents
2.2.1 Reference solution containing 100 mg potassium per liter
Absolute alcohol .............................................
10 mL
Citric acid C6H8O7, H2O ...........................
700 mg
Sucrose ..........................................................
300 mg
Glycerol .........................................................
1000 mg
Sodium chloride, NaCl . ....................................
50.8 mg
Anhydrous calcium chloride, CaCl2 ......................
10 mg
Anhydrous potassium hydrogen tartrate ....... 481.3 mg
water to .........................................................
1000 mL
Dissolve the potassium hydrogen tartrate in 500 mL of very hot distilled water,
mix this solution with 400 mL of distilled water in which the other chemicals
have already been dissolved, and make up to one liter.
2.2.2 Dilution solution
Absolute alcohol ..............................................
10 mL
Citric acid anhydrous ........................................ 700 mg
Sucrose ........................................................
300 mg
Glycerol ........................................................
1000 mg
Sodium chloride, NaCl .......................................
50.8 mg
Anhydrous calcium chloride, CaCl2 ..................
10 mg
Anhydrous magnesium chloride, MgCl2 ..........
10 mg
Tartaric acid ...................................................
383 mg
Water to ..........................................................
1000 mL
Preserve the solutions in polyethylene bottles by adding two drops of allyl
isothiocyanate (3-isothiocyanato-1-propene; CH2=CHCH2NCS).
OIV-MA-AS322-02B : R2009
2.3 Procedure
2.3.1 Calibration
Place 25, 50, 75 and 100 mL of the reference solution into a set of four 100 mL
volumetric flasks and make up to 100 mL with the dilution solution to give
solutions containing 25, 50, 75 and 100 mg of potassium per liter respectively.
2.3.2 Determination
Make measurements at 766 nm. and adjust the 100% transmission using
distilled water. Successively aspirate the standard solutions directly into the
burner of the photometer, followed by wine diluted 1/10 with distilled water
and note the readings. If necessary, the wine already diluted 1/10 may be
further diluted with the dilution solution (2.2.2).
2.4 Expression of results
2.4.1 Method of calculation
Plot a graph of the variation in percentage transmission as a function of the
potassium concentration in the standard solutions. Record the transmission
obtained for the sample of diluted wine on this graph and determine the
corresponding potassium concentration C.
The potassium concentration in mg potassium per liter to the nearest whole
number will be:
FxC
where F is the dilution factor.
2.4.2 Repeatability (r):
r = 17 mg/L.
OIV-MA-AS322-02B : R2009
Method OIV-MA-AS322-02C
Potassium
(Resolution Oeno 377/2009)
WITHDRAWN
OIV-MA-AS322-02C : R2009
Method OIV-MA-AS322-03A
Type II method
Sodium
1. Principle
Sodium is determined directly in the wine by atomic absorption
spectrophotometry after the addition of cesium chloride to suppress ionization
of sodium.
2. Method
2.1 Apparatus
Atomic absorption spectrophotometer equipped with an air-acetylene
burner.
Sodium hollow cathode lamp.
2.2 Reagents
2.2.1 Solution containing 1 g of sodium per liter:
The use of a commercial standard solution containing 1 g of sodium per liter is
preferred.
Alternatively, this solution may be prepared by dissolving 2.542 g of
anhydrous sodium chloride (NaCl) in distilled water and making up to a
volume of 1 liter.
Keep this solution in a polyethylene bottle.
2.2.2 Matrix (model) solution:
Citric acid monohydrate, (C6H8O7.H2O) ..................... 3.5 g
Sucrose ...........................................................
1.5 g
Glycerol .........................................................
5.0 g
Anhydrous calcium chloride (CaCl2) ....................... 50 mg
Anhydrous magnesium chloride, (MgCl2) ................. 50 mg
Absolute alcohol ................................................
50 mL
De-ionized water to .............................................
500 mL
2.2.3 Cesium chloride solution containing 5% cesium
Dissolve 6.330 g of cesium chloride, CsCl, in 100 mL of distilled water.
2.3 Procedure
2.3.1 Preparation of the sample
Pipette 2.5 mL of wine into a 50 mL volumetric flask, add 1 mL of the cesium
chloride solution (2.2.3) and make up to the mark with distilled water.
OIV-MA-AS322-03A : R2009
2.3.2 Calibration
Place 5.0 mL of the matrix solution in each one of five 100 mL volumetric
flasks and add 0, 2.5, 5.0, 7.5 and 10 mL respectively of a 1:100 dilution of the
1 g/L sodium solution. Add 2 mL of the cesium chloride solution (2.2.3) to
each flask and make up to 100 mL with distilled water.
The standard solutions prepared in this way contain 0.25, 0.50, 0.75 and 1.00
mg of sodium per liter respectively and each contains 1 g of cesium per liter.
Keep these solutions in polyethylene bottles.
2.3.3 Determination
Set the absorbance wavelength to 589.0 nm. Zero the absorbance scale using
the zero standard solution. Aspirate the diluted wine (2.3.1) directly into the
spectrophotometer, followed in succession by the standard solutions (2.3.2).
Record each absorbance and repeat each measurement.
2.4 Expression of results
2.4.1 Method of calculation
Plot a graph of measured absorbance versus the sodium concentration in the
standard solutions.
Record the absorbance obtained with the diluted wine on this graph and
determine its sodium concentration C in milligrams per liter.
The sodium concentration in milligrams per liter of the wine will then be F x
C, expressed to the nearest whole number, where F is the dilution factor.
2.4.2. Repeatability (r):
r = 1 + 0.024 xi mg/L.
xi = concentration of sodium in the sample in mg/L.
2.4.3. Reproducibility (R):
R = 2.5 + 0.05 xi mg/L.
xi = concentration of sodium in the sample in mg/L.
OIV-MA-AS322-03A : R2009
Method OIV-MA-AS322-03B
Sodium
1. Principle
Sodium is determined directly in diluted wine (at least 1 mL:10 mL) by flame
photometry.
2. Method
2.1 Apparatus
2.1.1. Flame photometer supplied with an air-butane mixture.
2.2 Reagents
2.2.1 Reference solution containing 20 mg sodium per liter
Absolute alcohol ................................................................
10 mL
Citric acid monohydrate (C6H8O7 H2O) ........................ 700 mg
Sucrose ..........................................................................
300 mg
Glycerol ......................................................................
1000 mg
Potassium hydrogen tartrate .................................
481.3 mg
Anhydrous calcium chloride, CaCl2 ..........................
10 mg
Anhydrous magnesium chloride, MgCl2 ......................
10 mg
Dry sodium chloride, NaCl ...................................
50.84 mg
Water to ............................................................................
1000 mL
2.2.2 Dilution solution
Absolute alcohol ..................................................
10 mL
Citric acid monohydrate (C6H8O7.H2O) ................... 700 mg
Sucrose ..........................................................
300 mg
Glycerol .......................................................................
1000 mg
Potassium hydrogen tartrate ....................................
481.3 mg
Anhydrous calcium chloride, CaCl2 .........................
10 mg
Anhydrous magnesium chloride, MgCl2 .....................
10 mg
Water to .......................................................
1000 mL
To prepare 2.2.1 and 2.2.2, dissolve the potassium hydrogen tartrate in
approximately 500 mL of very hot distilled water, mix with 400 mL of distilled
water into which the other chemicals have already been dissolved, and make
up to one liter.
Preserve the solutions in polyethylene bottles by adding two drops of allyl
isothiocyanate to each.
OIV-MA-AS322-03B : R2009
2.3 Procedure
2.3.1 Calibration
Place 5, 10, 15, 20 and 25 mL of the reference solution in each of five 100 mL
volumetric flasks and make up to 100 mL with the dilution solution to give
solutions containing 1, 2, 3, 4 and 5 mg of sodium per liter respectively.
2.3.2 Determination
Carry out measurements at 589.0 nm and adjust the 100% transmission using
distilled water. Successively aspirate the standard solutions directly into the
photometer, followed by the wine diluted 1:10 with distilled water and note the
percentage transmission of each. If necessary, the wine already diluted 1:10
may be further diluted with dilution solution.
2.4 Expression of results
2.4.1 Calculation method
Plot a graph of the percentage transmittance versus sodium concentration of
the standard solutions. Record the transmission obtained for the diluted wine
sample on this graph and note the concentration, C, of sodium in the wine.
The sodium concentration in mg of sodium per liter will be:
FxC
where F is the dilution factor.
2.4.2 Repeatability (r)
r = 1.4 mg/L (except for liqueur wine)
r = 2.0 mg/L for liqueur wine.
2.4.3. Reproducibility (R)
R = 4.7 + 0.08 xi mg/L.
xi = sodium concentration in the sample in mg/L.
OIV-MA-AS322-03B : R2009
Method OIV-MA-AS322-04
Type II method
Calcium
1. Principle
Calcium is determined directly on diluted wine by atomic absorption
spectrophotometry after the addition of an ionization suppression agent.
2. Apparatus
2.1 Atomic absorption spectrophotometer fitted with an air-acetylene burner.
2.2 Calcium hollow cathode lamp.
3. Reagents
3.1 Calcium standard solution 1 g/L. Use of a standard commercial calcium
solution, 1 g/L, is preferred.
Alternatively this solution may be prepared by dissolving 2.5 g of calcium
carbonate, CaC03, in sufficient hydrochloric acid (concentrated hydrochloric
acid diluted 1:10) to dissolve it completely and making up to one liter with
distilled water.
3.2 Dilute calcium standard solution, 50 mg/L
Note : Store the calcium solutions in polyethylene containers.
3.3 Dilute lanthanum standard solution, 50 g/L
Dissolve 13.369 g of lanthanum chloride, LaCl3.7H2O in distilled water; add 1
mL, of dilute hydrochloric acid (concentrated hydrochloric acid diluted 1/10)
and make up to 100 mL with distilled water.
4. Procedure
4.1 Preparation of sample
Place 1 mL of wine and 2 mL of the lanthanum chloride solution (3.3) in a 20
mL volumetric flask and make up to the mark with distilled water. The diluted
wine contains 5 g lanthanum per liter.
Note: For sweet wines, 5 g lanthanum per liter is sufficient provided that the
dilution reduces the sugar content to less than 2.5 g/L. For wines with higher
concentrations of sugar, the lanthanum concentration should be increased to 10
g/L.
4.2 Calibration
Place 0, 5, 10, 15 and 20 mL, of dilute standard calcium solution (3.2)
respectively into each of five 100 mL volumetric flasks, followed by 10 mL of
OIV-MA-AS322-04 : R2009
the lanthanum chloride solution (3.3) and make up to 100 mL with distilled
water. The solutions prepared in this way contain 0, 2.5, 5.0, 7.5 and 10 mg of
calcium per liter respectively, and each contains 5 g of lanthanum per liter.
These solutions should be stored in polyethylene bottles.
4.3 Determination
Set the absorbance wavelength to 422.7 nm. Zero the absorbance scale using
the zero standard (4.2). Aspirate the diluted wine directly into the
spectrophotometer, followed in succession by the five standard solutions (4.2)
and record the absorbance. Repeat each measurement.
5. Expression of results
5.1 Method of calculation
Plot a graph showing the variation in absorbance as a function of the calcium
concentration in the standard solutions.
Record the mean value of the absorbance obtained with the sample of diluted
wine on this graph and read its calcium concentration C. The calcium
concentration in milligrams per liter of the wine to the nearest whole number is
given by:
20 x C.
5.2 Repeatability (r)
Concentration<60mg/L:
Concentration > 60 mg/L:
r=2.7mg/L.
r = 4 mg/L.
OIV-MA-AS322-04 : R2009
Method OIV-MA-AS322-05A
Type IV method
Iron
1. Principle
After suitable dilution of the wine and removal of alcohol, iron is determined
directly by atomic absorption spectrophotometry.
2. Method
2.1 Apparatus
2.1.1 Rotary evaporator with thermostatically controlled water bath.
2.1.2 Atomic absorption spectrophotometer equipped with an air-acetylene
burner.
2.1.3 Iron hollow cathode lamp.
2.2 Reagents
2.2.1 Concentrated standard iron solution containing 1 g Fe (III) per liter.
Use a standard commercial solution, 1 g/L. This solution may be prepared by
dissolving 8.6341 g of ferric ammonium sulfate, FeNH4 (SO4)2.12H20, in
distilled water slightly acidified with hydrochloric acid, 1 M, and making up to
one liter.
2.2.2 Dilute standard iron solution containing 100 mg iron per liter.
2.3 Procedure
2.3.1 Preparation of sample
Remove the alcohol from the wine by reducing the volume of the sample to
half its original size using a rotary evaporator (50 to 60 C). Make up to the
original volume with distilled water.
If necessary, dilute prior to analysis with distilled water.
2.3.2 Calibration
Place 1, 2, 3, 4 and 5 mL of the solution containing 100 mg iron per liter
(2.2.2) respectively into each of five 100 mL volumetric flasks and make up to
100 mL with distilled water. The solutions prepared in this way contain 1, 2, 3,
4 and 5 mg of iron per liter respectively. These solutions should be stored in
polyethylene bottles.
2.3.3 Determination
Set the absorption wavelength to 248.3 nm. Zero the absorbance scale using
distilled water. Aspirate the diluted sample directly into the spectrophotometer,
OIV-MA-AS322-05A : R2009
OIV-MA-AS322-05A : R2009
Method OIV-MA-AS322-05B
Type IV method
Iron
1. Principle
After digestion in hydrogen peroxide, 30%, the total iron, present as Fe (III) state,
is reduced to the Fe (II) and quantified by the formation of a colored
orthophenanthroline complex.
2. Method
2.1 Apparatus
2.1.1 Kjeldahl flask, 100 mL.
2.1.2 Spectrophotometer enabling measurements to be made at a wavelength of
508 nm.
2.2 Reagents
2.2.1 Hydrogen peroxide, H202, 30% (m/v), solution, iron free.
2.2.2 Hydrochloric acid, 1 M, iron free.
2.2.3 Ammonium hydroxide (20 = 0.92 g/mL).
2.2.4 Pumice stone grains, pretreated with boiling hydrochloric acid diluted 1/2
and washed with distilled water.
2.2.5 Hydroquinone solution, C6H602, 2.5%, acidified with 1 mL concentrated
sulfuric acid (20 = 1.84 g/mL) per 100 mL of solution. This solution must be
kept in an amber bottle in the refrigerator and discarded at the slightest sign of
darkening.
2.2.6 Sodium sulfite solution, Na2S03, 20%, prepared from neutral anhydrous
sodium sulfite.
2.2.7 ortho-phenanthroline solution, C12H8N2, 0.5%, in alcohol, 96% vol.
2.2.8 Ammonium acetate solution, CH3COONH4, 20% (m/v).
2.2.9 Fe (III) solution containing 1 g of iron per liter. Use of a commercial
solution is preferred. Alternatively, a 1000 mg/L Fe (III) solution can be
prepared by dissolving 8.6341 g of ferric ammonium sulfate, FeNH4
(SO4)2.12H20, in 100 mL of hydrochloric acid, 1 M, and making up the volume
to one liter with the hydrochloric acid, 1 M.
2.2.10 Dilute standard iron solution containing 100 milligrams of iron per liter.
OIV-MA-AS322-05B : R2009
2.3 Procedure
2.3.1 Digestion
2.3.1.1 For wines with sugar content below 50 g/L
Combine 25 mL of the wine, 10 mL of the hydrogen peroxide solution and a
few grains of pumice into the 100 mL Kjeldah1 flask. Concentrate the mixture
to a volume of 2 to 3 mL by heating. Allow to cool and add sufficient
ammonium hydroxide to make the residue alkaline thus precipitating
hydroxides while taking care not to wet the walls of the flask.
After cooling, carefully add hydrochloric acid, to the alkaline liquid to dissolve
the precipitated hydroxides and transfer the resulting solution to a 100 mL
volumetric flask. Rinse the Kjeldahl flask with hydrochloric acid, and
combined the solutions in the volumetric flask and make up to 100 mL.
2.3.1.2 For musts and wines with sugar content above 50 g/L
If the sugar content is between 50 and 200 g/L, the 25 mL wine sample is
treated with 20 mL of hydrogen peroxide solution. Continue as in 2.3.1.1.
If the sugar content is greater than 200 mg/L, the samples of wine or must
should be diluted 1/2 or possibly 1/4 before being treated with 20 mL of
hydrogen peroxide solution. Continue as in 2.3.1.1.
2.3.2 Blank experiment
Carry out a blank trial with distilled water using the same volume of hydrogen
peroxide solution as the amount used for the mineralization, following the
experimental protocol described in 2.3.1.1.
2.3.3 Determination
Introduce 20 mL of the hydrochloric acid wine digest solution and 20 mL, of
the hydrochloric acid solution obtained from the 'blank experiment' into two
separate 50 mL volumetric flasks. Add 2 mL of hydroquinone solution, 2 mL
of sulfite solution and 1 mL of ortho-phenanthroline. Allow to stand for 15
minutes, during which time Fe (III) is reduced to Fe (II). Then add 10 mL of
ammonium acetate solution, make each up to 50 mL with distilled water and
shake the two volumetric flasks. Use the solution originating from the blank
experiment to zero the absorbance scale at 508 nm and measure the absorbance
of the wine solution at the same wavelength.
2.3.4 Calibration
Place 0.5, 1, 1.5 and 2 mL of the 100 mg of iron per liter solution into each of
four 50 mL volumetric flasks, and add 20 mL of distilled water to each. Carry
out the procedure described in 2.3.3 to measure the absorbance of each of these
OIV-MA-AS322-05B : R2009
standard solutions, which contain 50, 100, 150 and 200 micrograms of iron
respectively.
2.4 Expression of results
2.4.1 Method of calculation
Plot a graph giving the variation in absorbance as a function of the iron
concentration in the standard solutions. Record the absorbance of the test
solution and read off the iron concentration C in the hydrochloric acid
digestion solution, i.e. in 5 mL of the wine being analyzed.
The iron concentration in milligrams per liter of the wine to one decimal place
is given by:
200 C
If the wine (or must) has been diluted, the iron concentration in milligrams per
liter of the wine to one decimal place is given by:
200 F C
where F is the dilution factor.
OIV-MA-AS322-05B : R2009
Method OIV-MA-AS322-06
Type IV method
Copper
(Resolution Oeno 377/2009)
1. Principle
The method is based on the use of atomic absorption spectrophotometry.
2. Apparatus
2.1 Platinum dish.
2.2 Atomic absorption spectrophotometer.
2.3 Copper hollow cathode lamp.
2.4 Gas supplies: air-acetylene or nitrous oxide/acetylene.
3. Reagents
3.1 Metallic copper.
3.2 Nitric acid (20 = 1.38 g/mL), 65%.
3.3 Nitric acid (3.2), diluted 1/2 (v/v) with water.
3.4 Solution containing 1g of copper per L.
Use of a standard commercial copper solution is preferred. Alternatively this
solution may be prepared by weighing 1.000 g of metallic copper and
transferring it without loss to a 1000 mL volumetric flask. Add just enough
dilute nitric acid to dissolve the metal, add 10 mL of concentrated nitric acid
and make up to the mark with double distilled water.
3.5 Solution containing copper at 100 mg/L
Transfer 10 mL, of the 1 g/L solution 3.4. into a 100 mL volumetric flask, and
make up to the mark with double-distilled water.
3.6 Double-distilled water
4. Procedure
4.1 Preparation of sample and determination of copper
Place 20 mL sample in a 100 mL volumetric flask and make up to 100 mL with
double-distilled water. Modify the dilution if necessary to obtain a response
within the dynamic range of the detector.
OIV-MA-AS322-06 : R2009
Measure the absorbance at 324.8 nm. Set the zero with double distilled water.
4.2 Constructing a standard curve
Pipette 0.5, 1 and 2 mL of copper solution into each of three 100 mL
volumetric flasks and make to the volume with double distilled water; the
solutions contain 0.5, 1 and 2 mg of copper per liter respectively. Measure the
absorbance of standard solutions and the sample prepared in and repeat each
measurement. Plot a graph showing the variation in absorbance as a function of
the copper concentration in the standard solutions.
5 Expression of results
5.1 Method of calculation
Using the measured absorbance of the samples read off the concentration C in
mg/L from the calibration curve.
If F is the dilution factor, the concentration of the copper present is given in
milligrams per liter by:
F x C.
It is quoted to two decimal places.
Notes:
a) Select a sample dilution appropriate to the sensitivity of the apparatus to be
used and the concentration of the copper present in the sample.
b) Proceed as follows when very low copper concentrations are expected in the
sample to be analyzed: Place 100 mL of the sample in a platinum dish and
evaporate on a water bath at 100 C until it becomes syrupy. Add 2.5 mL of
concentrated nitric acid drop wise, covering the bottom of the dish completely.
Carefully ash the residue on an electric hotplate or over a low flame; then place
the dish in a muffle furnace set at 500 25C and leave for about one hour.
After coo1ing, moisten the ash with 1 mL of concentrated nitric acid while
crushing it with a glass rod; allow the mixture to evaporate and ash again as
before. Place the dish in the muffle furnace again for 15 min; repeat the
treatment with nitric acid at least three times. Dissolve the ash by adding 1 mL
of concentrated nitric acid and 2 mL of double distilled water to the dish and
transfer to a 10 mL flask. Wash the dish three times using 2 mL of double
distilled water each time. Finally, make to volume with double distilled water.
Proceed to analyze the sample as in 4.1 but use 10 mL of solution. Take into
account the change in dilution factor when calculating the results.
OIV-MA-AS322-06 : R2009
Method OIV-MA-AS322-07
Type II method
Magnesium
1. Principle
Magnesium is determined directly on diluted wine by atomic absorption
spectrophotometry.
2. Apparatus
2.1 Atomic absorption spectrophotometer fitted with an air-acetylene burner.
2.2 Magnesium hollow cathode lamp.
3. Reagents
3.1 Concentrated magnesium standard solution containing 1 g/L
Use of a standard commercial magnesium solution (1 g/L) is preferred.
Alternatively, this solution may be prepared by dissolving 8.3646 g of
magnesium chloride, MgC12.6H2O, in distilled water and making up to 1 liter.
3.2 Dilute magnesium standard solution, 5 mg/L.
Note: Keep the standard magnesium solutions in polyethylene bottles.
4. Procedure
4.1 Preparation of sample
The wine is diluted 1/100 with distilled water.
4.2 Calibration
Place 5, 10, 15 and 20 mL of the dilute standard magnesium solution into each
one of a set of four 100 ml. volumetric flasks and make up to 100 mL with
distilled water. The standard solutions prepared in this way contain 0.25, 0.50,
0.75 and 1.0 mg of magnesium per liter respectively. These solutions should be
kept in polyethylene bottles.
4.3 Determination
Set the absorption wavelength to 285 nm. Zero the absorbance scale using
distilled water. Aspirate the diluted wine directly into the spectrophotometer,
followed in succession by the standard solutions (4.2).
Record the absorbance of each solution and repeat each measurement.
OIV-MA-AS322-07 : R2009
5. Expression of results
5.1 Method of calculation
Plot a graph showing the variation in absorbance as a function of the
magnesium concentration in the standard solutions.
Record the mean value of absorbance with the diluted sample of wine on this
graph and read off the magnesium concentration C in milligrams per liter. The
magnesium concentration in milligrams per liter of the wine to the nearest
whole number is given by:
100 x C
5.2 Repeatability (r):
r = 3 mg/L.
R = 8 mg/L.
OIV-MA-AS322-07 : R2009
Method OIV-MA-AS322-08
Type IV method
Zinc
1. Principle
After removal of alcohol, zinc is determined directly in the wine by atomic
absorption spectrophotometry.
2. Apparatus
2.1 Rotary evaporator and thermostatically controlled water bath.
2.2 Atomic absorption spectrophotometer equipped with an air-acetylene burner.
2.3 Zinc hollow cathode lamp.
3. Reagents
The water used must be double distilled in borosilicate glass apparatus or of an
equivalent degree of purity.
3.1 Standard solution containing zinc, 1 g/L
Use of a commercial standard zinc solution is preferred. Alternatively this solution may be prepared by dissolving 4.3975 g of zinc sulfate, ZnS04.7H20, in
water and making up the volume to one liter.
3.2 Dilute standard solution containing 100 mg of zinc per liter.
4. Procedure
4.1 Preparation of sample
Remove the alcohol from 100 mL of wine by reducing the volume of the
sample to half its original value using a rotary evaporator (50 to 60 C). Make
up to the original volume of 100 ml, with double distilled water.
4.2 Calibration
Place 0.5, 1, 1.5 and 2 ml, of the solution containing 100 mg zinc per liter into
each one of four 100 mL volumetric flasks and make up to the mark with
double distilled water. The solutions prepared in this way contain 0.5, 1, 1.5
and 2 mg of zinc per liter respectively.
4.3 Determination
Set the absorbance wavelength to 213.9 nm. Zero the absorbance scale using
double distilled water. Aspirate the wine directly into the burner of the
OIV-MA-AS322-08 : R2009
OIV-MA-AS322-08 : R2009
Method OIV-MA-AS322-09
Type IV method
Silver
1. Principle
The method is based on the use of atomic absorption spectrophotometry after
ashing the sample.
2. Apparatus
2.1 Platinum dish.
2.2 Water bath, thermostatically controlled to 100 C
2.3 Furnace set at 500 to 525 C.
2.4 Atomic absorption spectrophotometer.
2.5 Silver hollow cathode lamp.
2.6 Gas supplies: air, acetylene.
3. Reagents
3.1 Silver nitrate, AgN03.
3.2 Nitric acid, (20 = 1.38 g/mL), 65%.
3.3 Nitric acid, diluted 1/10 (v/v) with distilled water.
3.4 Solution containing 1 g of silver per L.
Use of a standard commercial silver solution is preferred. Alternatively this
solution may be prepared by dissolving 1.575 g of silver nitrate in dilute nitric
acid and making up to a volume of 1,000 mL with dilute nitric acid (3.3).
3.5 Solution containing 10 mg of silver per L.
Take 10 mL of the 1 mg/L solution and make up to 1 L with dilute nitric acid.
4. Procedure
4.1 Preparation of sample
Place 20 mL of the sample in a platinum dish and evaporate to dryness over a
boiling water bath. Ash in the furnace at a temperature of 500 to 525 C.
Moisten the white ash with 1 mL of concentrated nitric acid (3.2). Evaporate
over a boiling water bath, repeat the addition of 1 mL nitric acid (3.2) and
evaporate a second time. Add 5 mL of dilute nitric acid (3.3) and heat gently
until dissolved.
OIV-MA-AS322-09 : R2009
4.2 Calibration
Pipette 2, 4, 6, 8, 10 and 20 mL of solution (3.5) respectively into each of size
100 mL volumetric flasks and make up to the mark with dilute nitric acid (3.3):
the solutions contain 0.20, 0.40, 0.60, 0.80, 1.0 and 2.0 mg of silver per liter
respectively.
4.3 Set the absorbance wavelength to 328.1 nm. Adjust zero using double distilled
water. Measure the absorbance directly of successive standard solutions (4.2)
and carry out in duplicate.
5. Expression of results
Plot a graph showing the variation in absorbance as a function of the silver
concentration in the standard solutions.
Using the measured absorbance of the sample read the concentration C in mg/L
from the calibration curve.
The concentration of silver in the wine is given in milligrams per liter by
0.25 x C.
It is quoted to two decimal places.
Note: Select the concentration of the solutions for the preparation of the calibration
curve. The volume of sample taken and the final volume of the liquid should be
appropriate for the sensitivity of the apparatus to be used.
OIV-MA-AS322-09 : R2009
Method OIV-MA-AS322-10
Type IV method
Cadmium
1. Principle
Cadmium is determined directly in the wine by graphite furnace atomic absorption
spectrophotometry.
2. Apparatus
All the glassware must be washed in concentrated nitric acid prior to use, heated to
70 to 80 C and rinsed in double distilled water.
2.1 Atomic absorption spectrophotometer equipped with a graphite furnace,
background correction and a recorder.
2.2 Cadmium hollow cathode lamp
2.3 5 l micropipettes with special tips for atomic absorption measurement.
3. Reagents
The water used must be double distilled prepared using borosilicate glass
apparatus, or water of a similar purity. All reagents must be of recognized
analytical reagent grade and, in particular, free of cadmium.
3.1 Phosphoric acid (20 = 1.71 g/mL), 85%.
3.2 Phosphoric acid solution obtained by diluting 8 mL of phosphoric acid with
water to 100 mL.
3.3 0.02 M Ethylenediaminetetraacetic acid disodium (EDTA) solution.
3.4 pH 9 buffer solution: dissolve 5.4 g of ammonium chloride in a few milliliters
of water in a 100 mL volumetric flask, add 35 mL of 25% (v/v) ammonium
hydroxide solution. Ammonium hydroxide solution, 20 = 0.92 g/mL, diluted
to 25% (v/v) and made up to 100 mL with water.
3.5 Eriochrome black T, 1% (m/m) solution in sodium chloride.
3.6 Cadmium sulfate, 3CdSO4.8H20.
The concentration of the cadmium sulfate must be verified using the
following method:
Weigh exactly 102.6 mg of the cadmium sulfate sample into a beaker with
some water and shake until dissolved; add 5 mL of the pH 9 buffer solution
OIV-MA-AS322-10 : R2009
BIBLIOGRAPHY
OIV-MA-AS322-10 : R2009
Method OIV-MA-AS322-11
Lead
(Resolution Oeno 3/94)
1. Principle of the method
Lead is determined directly in wine by flameless atomic absorption
spectrophotometry.
WITHDRAWN
OIV-MA-AS322-11 : R2009
Method OIV-MA-AS322-12
Type II method
1.1
Trueness the closeness of agreement between the average value obtained from a
large series of test results and an accepted reference value
r=
Repeatability limit, the value below which the absolute difference
between 2 single test results obtained under repeatability conditions (i.e., same
sample, same operator, same apparatus, same laboratory, and short interval of
time) may be expected to lie within a specific probability (typically 95%) and
hence r = 2.8 x sr.
Sr =
Standard deviation, calculated from results generated under
repeatability conditions.
RSDr= Relative standard deviation, calculated from results generated under
repeatability conditions [(Sr/ x ) x 100], where x is the average of results over
all laboratories and samples.
R=
Reproducibility limit, the value below which the absolute difference
between single test results obtained under reproducibility conditions (i.e., on
identical material obtained by operators in different laboratories, using the
standardised test method), may be expected to lie within a certain probability
(typically 95%); R = 2.8 x sR.
SR =
Standard deviation, calculated from results under reproducibility
conditions.
RSDR = Relative standard deviation calculated from results generated under
reproducibility conditions [(SR/ x x 100]
HoR = HORRAT value: the observed RSDR value divided by the RSDR value
calculated from the Horwitz equation [1].
OIV-MA-AS322-12 : R2006
2
Method of analysis to be used by the laboratory and laboratory
control requirements
2.1
Requirements
Specific methods for the determination of lead in wine are not prescribed.
Laboratories shall use a method (Type II) validated to OIV requirements [2]
that fulfils the performance criteria indicated in Table 1 e.g. GFAA or ICP-MS
methods are applicable provided they meet the performance criteria outlined
below. Wherever possible, the validation shall include a certified reference
material in the collaborative trial test materials. If not an alternative estimation
of trueness should be used. Examples of suitably validated methods for the
determination of lead in wine are provided in Appendices 1 & 2.
2.2
General considerations
All apparatus which comes into contact with the sample shall be made of an
inert material (e.g. polypropylene, polytetrafluoroethylene [PTFE], etc.). The
use of ceramic materials is not advisable because of the possibility that lead
might be present. If it is not certain that the materials available are free from the
analytes in question, their use shall be assessed by means of ad hoc studies,
which should be considered as an integral part of the validation of the method
of analysis. All plastic ware including sample containers shall be acid cleaned.
If possible, equipment used for preparing samples should be reserved for lead
analyses only.
OIV-MA-AS322-12 : R2006
Value/Comment
Suitable for determining lead in wine for official
purposes.
Detection limit
Limit of
quantification
Precision
Recovery
Specificity
Trueness
xm
< 1,96 *
sR ( lab ) sr ( lab ) * 1 1 n
2.3
OIV-MA-AS322-12 : R2006
References
[1] W Horwitz, Evaluation of Analytical Methods for Regulation of Foods
and Drugs, Anal. Chem., 1982, 54, 67A - 76A
[2] Protocol for the design, conduct and interpretation of methodperformance studies, FV 1061, OIV, 1998
[3] ISO 5725-6:1994, 4.2.3. International Organisation for Standardisation,
case Postal 56, CH-1211, Genve 20, Switzerland.
[4] ISO/AOAC/IUPAC Harmonised Guidelines for the Use of Recovery
Information in Analytical Measurement. Edited Michael Thompson, Steven L R
Ellison, Ales Fajgelj, Paul Willetts and Roger Wood, Pure Appl. Chem., 1999,
71, 337 348
OIV-MA-AS322-12 : R2006
EXAMPLE 1
DETERMINATION OF LEAD IN WINE BY ATOMIC
ABSORPTION SPECTROMETRY
1
The method can be used for red, white, still, sparkling and fortified wines.
DEFINITION
The lead content of wine: the content of lead determined by this procedure
expressed as mg/L.
3
PRINCIPLE
REAGENTS
Diluent solution
NOTE 1: The exact composition of the diluent used may need adjustment to suit
the specific model of instrument and graphite furnace employed. If problems
are experienced with the suggested modifier composition adjust the phosphate
and nitrate concentrations to give:
i) a stable element signal at the optimum ashing temperature and
ii) atomisation with a single reproducible analyte peak which is time separated
from the background signal.
Equipment with VDU facilities will allow analysts to confirm time separation of
the sample and background peaks (See Annex). The following is an example of
a technique for determining the absorbance versus time profile:
Measure the full peak width at half maximum height (FWHM) of a sample peak
and compare it to the FWHM of a calibration standard with a similar maximum
absorbance. If the peak shapes are visibly different then the composition of the
matrix modification modifier needs to be adjusted.
The following are examples of diluents utilised for:
(a) a Perkin-Elmer 3030 equipped with deuterium arc background corrector
with an HGA 500 furnace; and (b) a Thermo-Electron Video 12E equipped
with Smith-Hieftje background corrector, a CTF 188 furnace and a
FASTAC
sample deposition system.
4.1.1 Perkin-Elmer 3030 diluent:
To 187 g of water in a 250 ml plastic bottle (5.1) add 11 g ethanol (4.1.3.), 1.1 g
of glucose (4.1.4.), 1.1 g of fructose (4.1.5.) and 0.28 g of sodium chloride
(4.1.6.). Shake to dissolve the solids. Then add 22 ml nitric acid (4.1.7.) and 4.4
g ammonium dihydrogen orthophosphate (4.1.8.). Shake until all the phosphate
has dissolved. Finally add 0.88 g magnesium nitrate (4.1.9.) and shake again
until no undissolved solid remains.
OIV-MA-AS322-12 : R2006
5.
APPARATUS
All glass and plastic ware used must be acid cleaned (soaked in 20 % nitric acid
for at least 24 hours), rinsed thoroughly with distilled water prior to use and
kept covered (with cling-film if appropriate) to prevent aerial contamination.
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
OIV-MA-AS322-12 : R2006
OIV-MA-AS322-12 : R2006
Step
Temperature
(C)
Ramp (s)
Hold (s)
Gas
Gas flow
(mL/min)
Read (2.5s
integration)
1
200
2
1100
3
1100
4
1800
5
2400
6
20
5
60
Ar
50
20
20
Ar
50
1
2
Ar
0
0
3
Ar
0
1
6
Ar
300
1
25
Ar
300
(b)
(c)
Step
Temperature (C)
Ramp (s)
Hold (s)
Gas
Gas flow (mL/min)
Read (2.5s integration)
1
150
0
2
Ar
50
2
350
30
0
Ar
50
3
650
15
5
Ar
0
4
100
0
5
240
0
1
4
Ar
0
X
10
Ar
300
OIV-MA-AS322-12 : R2006
10
6.
PROCEDURE
6.1.
Preparation of wine
Shake the wine container to thoroughly mix the contents before sub-sampling.
Sparkling wines should be transferred to a clean beaker and placed in an
ultrasonic bath until gas is no longer evolved prior to use.
6.2
Measurement solutions
Measurement
Determinations are carried out in batches. Each batch is to contain at least four
replicates of the reagent blank and three spiked replicates of samples for
recovery estimate purposes. The lead calibration solutions are distributed
evenly amongst the unknowns on the auto-sampler tray. Transfer the samples
and standards to the auto-sampler sample containers using a Pasteur pipette
(5.3). Discard the first filling of the container and measure the second filling (if
there is not enough sample solution, care will have to be taken to ensure that
the sample containers are scrupulously clean). Wash the Pasteur pipette four or
five times with 1% nitric acid (4.4.) between each standard and sample transfer.
OIV-MA-AS322-12 : R2006
11
6.4.
Quantification of lead
EXPRESSION OF RESULTS
Calculation
Obtain from the calibration graph, the lead content of all the measurement
solutions. Calculate the lead content of the wine samples and spiked wine
samples using the following calculation:
Pb concentration (mg/l) =
(Cm - Cb) x Vt
--------------------------V
m
where:
C is the mean lead concentration of the measurement solution (mg/l).
m
C is the mean measured lead concentration of the reagent blank solutions
b
(mg/l).
V is the final total volume of the measurement solution (ml).
t.
V is the volume of the wine sample taken (ml).
m
7.2
Recovery (%) =
OIV-MA-AS322-12 : R2006
12
where:
C is the calculated mean lead concentration of the spiked wine sample (mg/l).
s
C is the calculated mean lead concentration of the unspiked wine (mg/l).
a
V is the volume of wine to which the spike is added (ml).
s
S is the amount of spike added (g).
7.3
Cw x 100
----------------R
a
where:
C is the calculated lead concentration of the wine sample (mg/l).
w
R is the average in-batch recovery (%).
a
OIV-MA-AS322-12 : R2006
13
SAMPLE SCHEME
Sample Code
Sample Description
5&9
3 & 11
7&8
6 & 10
2 & 12
OIV-MA-AS322-12 : R2006
14
F1
Code
5, 9
3, 11
7, 8
6, 10
2, 12
16
16
16
16
15*
n (-outl)
16
15
14
16
15
16
Targ.
56
24
279
67
192
143
153
Mean
50.8
27.2
298
70.6
189
143
149
23
15
51
38
13.6
r
Sr
RSDr
8.1
16
Hor
R
5.3
19
1.0
42
SR
RSDR
15.1
HoR
1.2
30
1.1
25
8.8
28
1.2
16
24
32
8.7
11.8
18.2
17
10
0.2
1.1
0.7
0.7
83
57
29.8
20.3
55.2
28.2
10
29
29
19
0.5
1.2
154
1.4
79
0.9
OIV-MA-AS322-12 : R2006
15
r
Repeatability limit, the value below which the absolute difference
between 2 single test results obtained under repeatability conditions (i.e., same
sample, same operator, same apparatus, same laboratory, and short interval of
time) may be expected to lie within a specific probability (typically 95%) and
hence r = 2.8 x sr.
Sr
The standard deviation of the repeatability.
RSDr
The relative standard deviation of the repeatability
100/MEAN).
(S
Hor
The observed RSD divided by the RSD value estimated from the
r
r
Horwitz equation using the assumption r=0.66R.
R
Reproducibility limit, the value below which the absolute difference
between single test results obtained under reproducibility conditions (i.e., on
identical material obtained by operators in different laboratories, using the
standardised test method), may be expected to lie within a certain probability
(typically 95%); R = 2.8 x sR.
SR
The standard deviation of the reproducibility (between laboratory
variation).
RSDR
The relative standard deviation of the reproducibility (S x
R
100/MEAN).
HoR
The observed RSD value divided by the RSD value calculated
R
R
from the Horwitz equation.
RSDR = 2(1-0.5log10C)
decimal)
HORRAT(4) values are:
For repeatability, the observed RSDr divided by the RSDr value estimated from
the Horwitz equation using the assumption r = 0.66R.
For reproducibility, the observed RSDR divided by the RSDR value estimated
from the Horwitz equation.
8. REFERENCES
8.1 Paul A. Brereton, Paul Robb, Christine M Sargent, Helen M. Crews and
Roger Wood. Determination of Lead in Wine by Graphite Furnace Atomic
Absorption Spectrometry: Interlaboratory Study. JAOAC Int., 1997, 80, No 6,
OIV-MA-AS322-12 : R2006
16
1287-1297.
8.2 "Protocol for the Design, Conduct and Interpretation of Collaborative
Studies." Editor W Horwitz, Pure & Appl. Chem., Vol. 67, No., 2, pp.331-343,
1995
8.3 Horwitz W, Evaluation of Methods Used for Regulation of Foods and
Drugs, Analytical Chemistry, 1982, 57, 67A-76A.
8.4Peeler J T, Horwitz W and Albert R, Precision Parameters of Standard
Methods
of Analysis for Dairy Products, JAOAC, 1989, 72, No 5, 784-806.
OIV-MA-AS322-12 : R2006
17
Annex
Absorbance vs. Time profiles for the measurement of lead in wine
using a Perkin-Elmer 3030 atomic absorbtion spectrometer
with deuterium arc background correction.
0,5
Absorbance, AU
0,4
0,3
0,2
0,1
0
0
0,5
1,5
2,5
Time, seconds
(i)
OIV-MA-AS322-12 : R2006
18
0,5
Absorbanc, AU
0,4
0,3
0,2
0,1
0
0
0,5
1,5
2,5
Time, seconds
(ii)
wine sample
Key:
OIV-MA-AS322-12 : R2006
19
EXAMPLE 2
DETERMINATION OF LEAD IN WINE BY ATOMIC
ABSORPTION SPECTROMETRY
1.
FIELD OF APPLICATION :
This analysis method can be applied to all types of wine, given the maximum
limit set by the O.I.V.
2. REFERENCES:
2.1. Journal Officiel des Communauts Europennes (3 octobre 1990). Mthode
de dosage du plomb dans le vin (p. 152 et 153).
2.2. Teissdre P.L., Brun S., Mdina B. (1992). Dosage du plomb dans les vins
/ Proposition de modifications la mthode du Recueil. Feuillet Vert de
lO.I.V., n928, 1997/151292.
2.3. Moreira Balio da Silva M., Gaye J., Mdina B. (1996). Comparaison de six
mthodes de dosage du plomb dans les vins par absorption atomique en four
graphite. Feuillet Vert de lO.I.V. n 1013, 2310/190196.
2.4. Brereton P., Robb P., Sargent C., Crews H., Wood R. (1996). Validation of
a graphite furnace atomic absorption spectrometry method for the detection of
lead in wine. Feuillet Vert de lO.I.V. n 1016, 2913/230196.
2.5. Bourguignon J.B., Douet Ch., Gaye J., Mdina B. (1997). Dosage du
plomb dans le vin / Interprtation des rsultats de lessai interlaboratoire.
Feuillet Vert 1055 de lO.I.V. n 2456/190397.
3. PRINCIPLE:
The wine will undergo no preparations, except dilution in the case of white
sweet wines.
Adding ammonium dihydrogeno-phosphate enables the lead contained in wine
to be stable at high temperatures, which leads to eliminating interferences and
to acting in an identical manner to the standard solution.
The atomizer is a pyrolytic graphite equipped with a platform heated by the
Joule effect.
The wavelength of the ray used is 283.3 nanometres.
The non specific absorption correction can be done by the Zeeman effect or by
using a deuterium discharge lamp.
OIV-MA-AS322-12 : R2006
20
The type of lead determination in wine is a direct dosage method with external
calibration.
4. REAGENTS
4.1. Demineralised water: ultra pure; with resistivity above 18 M/cm.
4.2. Nitric acid: 65 % ; suprapur quality acid.
4.3. Ammonium dihydrogeno-phosphate NH4H2PO4 for analysis.
4.4. Lead standard solution: at 1000 g/ml (or 1 g/l) in 2% nitric acid
(commercial solution, ready to use).
5. APPARTUS
5.1
5.2
Glass ware:
21
dissolved. Dissolve acid. Fill the solution up to 50 ml. This solution is stored in
a plastic flask.
5.3.1.2 Tantalisation of a platform: the platform is placed inside the graphite
tube. These items are placed together on a spectrophotometer atomization unit.
10 l tantalum solution is injected on a platform using an auto-sampler. The
temperature cycle is set according to the following program: drying at 150C
for 40 s; mineralization at 900C for 60 s; atomization at 2600C for 2.5 s.
Argon is used as an inert gas.
6. PROCEDURE
6.1 Test portion: The neck of the wine bottle with a tinned lead capsule must
be carefully cleaned before uncorking.
6.2 Sample preparation: In general, no preparation of wine is necessary;
samples are placed directly in the automatic sampler buckets. Cloudy wine
needs to be filtered. To prolong the utilisation period of the platforms, sweet
white wines are diluted for sugar contents between 10 to 50 g/L, dilute by 1/2;
for contents above 50 g/l, dilute by 1/4.
6.3
Preparation of solutions:
OIV-MA-AS322-12 : R2006
22
OIV-MA-AS322-12 : R2006
lecture du
signal
oui
oui
23
volumes injected in l
sample
Pb solution
Calibration blank
Standard 1
Standard 2
Standard 3
Sample
0
0
0
0
2
100 g/l
0
1
2
3
0
"white"
dilution
5
4
3
2
3
Matrix
modifier
1
1
1
1
1
6.4.2 Tracing of calibration curve: the automatic distributor cycle enables the
preparation of standards from 100 g/l (Table II) lead solutions. The calibration
graph is drawn up: absorbency according to lead concentration in micrograms
per litre.
7. EXPRESSION OF RESULTS
7.1 Concentration of lead in injected solution:
calibration curve (6.4.2.).
24
8.1
Typ
T.A.V.
(% Vol.)
R1
R2
Ro1
Ro2
Bs1
Bs2
D1
Red
Red
Ros
Ros
Dry white
Dry white
Sweet
white
Sweet
white
D2
11,86
12,54
12,23
11,43
11,65
12,32
12,94
Total
acidity
(g/l H2SO4)
4,43
3,77
5,30
4,88
4,62
4,57
3,72
Volatile
acidity
(g/l H2SO4)
1,57
0,34
0,44
0,45
0,37
0,31
0,67
Reducing
sugar
(g/l)
1,2
1,5
1,2
1,1
2,2
0,9
76,4
12,66
4,70
0,45
62,8
OIV-MA-AS322-12 : R2006
25
8.2
Statistics of results:
R1
R2
Ro1
Ro2
Bs1
Bs2
D1
D2
11
11
11
11
11
11
11
11
10
11
11
10
10
11
10
44
162
28
145
52
138
60
145
18
12
17
13
28
6,4
4,3
2,5
6,1
2,1
4,6
10
2,5
14,5
2,8
9,2
4,2
4,2
3,4
16,5
1,8
0,6
0,1
0,3
0,2
0,2
0,2
0,7
0,1
34
105
23
86
30
101
86
144
12,3
37,5
8,2
30,8
10,7
35,9
30,6
51,6
28
23,1
29,3
21,2
20,6
26
51
35,6
1,1
1,1
1,1
0,8
1,2
2,1
1,7
Out of the 11 laboratories which participated in the trial, 7 declared that they
had followed the proposed method and 4 modified some of the parameters.
OIV-MA-AS322-12 : R2006
26
Lead
concentration
(g/l)
9.4
Certified value
(B.C.R. 1992)
Average value
(series: 10 results)
Red wine
BCR E
Dry white
wine
BCR C
Sweet white
wine
BCR D
36,1 4,9
65,1 9,1
132,4 32
41,0 3,8
66,0 4,4
128,3 14,1
Control card
A control card can be drawn up for each reference material used. Control limits
are equal to: +/- 2 SRintra (SR intra: reproductibility standard deviation).
OIV-MA-AS322-12 : R2006
27
Control card
CarteLead
de
of BCRcontrle
E
Plomb du BCR E
80
Lead in g/l
plomb en g/l
70
limites
: + =2SR
= 63,9 g/l
Limits : + 2SR
63.9 g/l
2SR
=
28,7 g/l
- 2SR = 28.7 g/l
60
50
40
30
20
15/12/95
26/06/96
29/04/97
08/12/97
09/10/98
date
moyenne
: 46,3 g/l
Mean : 46.3 g/l
10.
BIBLIOGRAPHY
10.1. Zatka V. (1978). Treated graphite atomizer tubes for atomic absorption
spectrometry. Analytical Chemistry, vol. 50, n3.
10.2. US Bureau of Alcohol, Tobacco and Firearms (1991). Analysis of lead in
wines and related products by graphite furnace atomic absorption
spectrometry. Note dinformation de lO.I.V. du 21 aot 1991 : Plomb dans
les vins aux U.S.A. .
10.3. Mindak W.R. (1994). Determination of lead in table wines by graphite
furnace atomic absorption spectrometry. Journal of A.O.A.C. International,
vol. 77, n4, p. 1023-1030.
10.4. Mdina B. (1994). Apport de nouvelles techniques au dosage des mtaux
dans les vins. Congrs des nologues de France Bordeaux.
10.5. Norme franaise NF ISO 5725-2 (1994). Application de la statistique :
Exactitude (justesse et fidlit) des rsultats et mthodes de mesure.
10.6. Jorhem L., Sundstrm B. (1995). Direct determination of lead in wine
using graphite furnace AAS. Atomic Spectroscopy, September/October 1995.
OIV-MA-AS322-12 : R2006
28
Method OIV-MA-AS323-01A
Type IV method
1. PRINCIPLE
After evaporating ethyl alcohol and reducing the arsenic V in arsenic III, wine
arsenic is measured by hydride generation and by atomic absorption
spectrometry.
2. EQUIPMENT
2.1. Glass ware:
2.1.1. Graduated flask 50, 100 ml (class A)
2.1.2. Graduated pipettes 1, 5, 10, 25 ml (class A)
2.2. Water bath at 100C
2.3. Filters without ashes
2.4. Spectrophotometer :
2.4.1. Atomic absorption spectrophotometer
2.4.2. Instrumental parameters
2.4.2.1. Air-acetylene oxidising flame
2.4.2.2 Hollow cathode lamp (arsenic)
2.4.2.3. Wave length: 193.7 nm
2.4.2.4. Split width: 1.0 nm
2.4.2.5. Intensity of hollow cathode lamp: 7 mA
2.4.2.6. Correction of non-specified absorption with a deuterium
lamp
2.5. Accessories:
2.5.1. Hydride absorption cell, placed on an air-acetylene burner.
2.5.2. Vapour generator (liquid gas separator)
2.5.3. Neutral gas (argon)
OIV-MA-AS323-01A : R2009
Peristaltic
pump
Reaction
loop
Reference or sample
Hydrochloric acid Sodium 10%
borohydride
Liquid gas
separator
Towards sink
Flow controller
3. REAGENTS
3.1. Ultra-pure demineralised water
3.2. Ultra-pure 65% nitric acid
3.3. Potassium iodide (KI)
3.4. 10% . Potassium iodide (m/v)
3.5. Concentrated hydrochloric acid (R)
3.6. 10% Hydrochloric acid (R)
3.7. Sodium borohydride (NaBH4)
3.8. Sodium hydroxide (NaOH)
3.9. 0.6% Sodium borohydride (containing sodium hydroxide: 0.5% (m/v))
3.10. Calcium Chloride CaCl2 (used as a drying agent)
3.11. 1 g/l Arsenic stock solution prepared in the following manner :
dissolve 1.5339 g of AS2O5 in demineralised water, adjust to 1 l.
OIV-MA-AS323-01A : R2009
4. SAMPLE PREPARATION
25 ml of water is evaporated over a 100 C water bath. This is then brought to 50
ml in the presence of 5 ml of 10% potassium iodide and 5 ml of concentrated
hydrochloric acid; leave for 1 hour; filter on an ashless filter.
Make a blank reference sample.
5. DETERMINATION
The peristaltic pump sucks in the borohydride solution, the 10% hydrochloric acid
solution and the sample solution.
Present the calibration standards in succession (3.14.); take an absorbency reading
for 10 seconds; take two readings; the operating software establishes a calibration
curve (absorbency according to concentration of arsenic in g/l).
Then present the samples (4) ; the software establishes the samples arsenic
concentration in g/l; deduct the arsenic concentration in the wine in g/l taking
into account that the solution be diluted by 1 / 2 .
6. QUALITY CONTROL
Quality control is assured by placing a control sample of internal quality (*) in a
regular manner in 5 samples, or after the set of calibration solutions, or in the
middle of a series or at the end the measurement.
Two deviation types are accepted compared to known value.
(*) Samples from the Bureau Communautaire de Rfrence (Community Bureau of
reference): red wine, dry white wine and sweet white wine.
OIV-MA-AS323-01A : R2009
7. BIBLIOGRAPHY
Varian Techtron, 1972. Analytical methods for flame spectroscopy.
Hobbins B., 1982. Arsenic Determination by Hydride Generation. Varian
Instruments at Work.
Le Houillier R., 1986. Use of Drierite Trap to Extend the Lifetime of Vapor
Generation Absorption Cell. Varian Instruments at Work.
Varian, 1994. Vapor Generation Accessory VGA-77.
OIV-MA-AS323-01A : R2009
Method OIV-MA-AS323-01B
Type IV method
Arsenic
(Resolution Oeno 377/2009)
1. Principle
After mineralization, using sulfuric and nitric acids, arsenic V is reduced to
arsenic III by means of potassium iodide in hydrochloric acid and the arsenic
is transformed into arsenic III hydride (H3As) using sodium borohydride. The
arsenic III hydride formed is carried by nitrogen gas and determined by
flameless atomic absorption spectrophotometry at high temperature.
2. Method
2.1 Apparatus
2.1.1 Kjeldahl flask (borosilicate glass)
2.1.2 Atomic absorption spectrophotometer equipped with arsenic hollow
cathode lamp, hydride generator, background corrector and a chart recorder.
The hydride generator includes a reaction flask (which can eventually be put
onto a magnetic stirrer) connected by a tube to a nitrogen gas supply (flow
rate: 11 L/min) and by a second tube, to a quartz cell which can be brought to
a temperature of 900 oC. The reaction flask also has an opening for the
introduction of the reagent (borohydride).
2.2 Reagents
All reagents must be of recognized analytically pure quality, and in particular
free of arsenic. Double distilled water prepared using a borosilicate glass
flask or water of similar purity should be used.
2.2.1 Sulfuric acid (20= 1.84 g/mL) arsenic free
2.2.2 Nitric acid (20= 1.38 g/mL) arsenic free
2.2.3 Hydrochloric acid (20= 1.19 g/mL), arsenic free
2.2.4 10% (m/v) Potassium iodide solution
2.2.5 2.5% (m/v) Sodium borohydride solution obtained by dissolving 2.5 g of
sodium borohydride in 100 mL of 4 % (m/v) of sodium hydroxide solution.
This solution must be prepared at the time of use.
2.2.6 Arsenic reference solution 1 g/L. Use of a commercial standard arsenic
solution is preferred.
OIV-MA-AS323-01B : R2009
2.4.1 Calculation
Plot the curve showing the variation in absorbance as a function of the arsenic
concentration in the standard solutions. The relationship is linear. Note the
average absorbance of the sample solutions on the graph and read the arsenic
concentration C.
The arsenic concentration in wine, expressed in micrograms per liter is given
by: 2 C.
BIBLIOGRAPHY
JAULMES P. et HAMELLE G., Trav. Soc. Pharm. Montpellier, 1967, 27, no 3,
213-225.
JAULMES P., F.V., O.I.V., 1967, no 238
MEDINA B et SUDRAUD P., F.V., O.I.V., 1983, no 770.
OIV-MA-AS323-01B : R2009
B
A
Glass wool
mm
80 mm
OIV-MA-AS323-01B : R2009
Method OIV-MA-AS323-01C
Arsenic
(Resolution Oeno 377/2009)
1. Principle
After mineralization using sulfuric and nitric acids, arsenic V is reduced to
arsenic III using tin II chloride. The arsenic is then converted into arsenic III
hydride by the action of the hydrogen produced. Arsenic III hydride is
detected by reaction with mercury II bromide (limit sample).
WITHDRAWN
OIV-MA-AS323-01C : R2009
Method OIV-MA-AS323-02A
Type II method
1 - FIELD OF APPLICATION
This method can be applied to the analysis of total nitrogen in musts and
wine within the range of 0 to 1000 mg/l.
OIV-MA-AS323-02A : R2009
11
1
1
1
1
1
6
6
He
11
10
1
0
22
77
33
55
88
99
44
4 - Apparatus
4.1 - Centrifuge with 25 ml pots;
4.2 Nitrogen analyser;
4.3 Metallic crucible;
4.4 - Quartz reaction tube (2) ;
4.5 Precision balance between 0.5 mg and 30 g at 0.3 mg ;
4.6 Boat carrier;
4.7.- Furnace;
4.8 Apparatus for folding boats;
4.9 Sample changer;
4.10 Computer and printer.
5 - SAMPLING
Degas by nitrogen bubbling (3.1) for 5 to 10 mn, sparkling wine. The musts
are centrifuged (4.1) for 10 mn at 10C, at 4200 g.
6 OPERATING INSTRUCTIONS
- Open the apparatus programme (4.2 and 4.10) ;
- Put the heating on the apparatus (4.2).
6.1 Principle analytical parameters
Nitrogen analyser (4.2) under the following conditions:
gas carrier: helium (3.2) ;
metallic crucible (4.3) to be emptied every 80 analyses ;
OIV-MA-AS323-02A : R2009
18359
. 0.6
14.007 = 7.864 g/l
OIV-MA-AS323-02A : R2009
7 - EXPRESSION OF RESULTS
Results are expressed in g/l to the fourth decimal.
8 CHECKING RESULTS
Splicing by mass, temperature, and volume.
Average contents
Repeatability
Reproductibility
591 mg/l
43 mg/l
43 mg/l
10 - BIBLIOGRAPHY
Dumas A. (1826) : Annales de chimie, 33,342.
Buckee G.K. (1994) : Determination of total nitrogen in Barley, Malt and Beer
by Kjeldahl procedures and the Dumas combustion method. Collaborative
trial. J. Inst. Brew., 100, 57-64.
OIV-MA-AS323-02A : R2009
Method OIV-MA-AS323-02B
Type IV method
Total Nitrogen
(Resolution Oeno 377/2009)
1. Principle
The sample is wet ashed using sulfuric acid in the presence of a catalyst. The
ammonia liberated by sodium hydroxide is determined titrimetrically.
2. Apparatus
2.1 Digestion apparatus
300 mL Kjeldahl flask. Place on a metal heating mantle. Appropriate stand to
hold this apparatus, the neck bent at 45 degrees.
2.2 Distillation apparatus
1 liter round bottomed flask, fitted with a small rectifying column 30 cm long
by 2.5 cm diameter or any other equivalent apparatus. The vapor emitted from
the end of this apparatus enters into the top part of the cylindrical condenser,
held vertically, of 30 cm length and 1 cm internal diameter. The condensed
liquid is brought to the receiving conical flask by a drawn-out tube placed at
the bottom alternatively one can use a steam distillation apparatus such as
described in Volatile Acidity, or any other apparatus relating to the test
described in paragraph "Blank tests or sample tests".
3. Reagents
3.1 Sulfuric acid free of ammonia (20 = 1.83 - 1.84g/mL)
3.2 Benzoic acid
3.3 Catalyst:
Copper sulfate, CuSO4, .................................................
10 g
Potassium sulfate, K2SO4, .............................................
100 g
3.4 30%Sodium hydroxide solution. Sodium hydroxide (20 =1.33 g/mL) diluted
30% (m/m).
3.5 0.1 M Hydrochloric acid solution
3.6 Indicator:
Methyl red ..............................................
100 mg
Methylene blue .........................
50 mg
Ethanol (50%) .............................................
100 mL
3.7 Boric acid solution:
OIV-MA-AS323-02B : R2009
OIV-MA-AS323-02B : R2009
Method OIV-MA-AS323-03
Type IV method
Boron
Rapid Colorimetric Method
1. Principle
The alcohol content of the wine is removed by reducing the volume by half by
rotary evaporation. The wine is then passed through a column of
polyvinylpolypyrrolidone, which retains the coloring agents. The eluate is
collected quantitatively and the boron concentration determined by complexation
with azomethine H at pH 5.2 followed by spectroscopic analysis at 420 nm.
2. Apparatus
2.1. Rotary evaporator
2.2. Spectrophotometer capable of measuring absorbance wavelengths between
300 and 700 nm
2.3. Cells of 1 cm optical path
2.4.
Glass column of 1 cm internal diameter and 15 cm in length containing an
8 cm layer of polyvinylpolypyrrolidone.
3. Reagents
3.1. Azomethin H (4-hydroxy-5-(2-hydroxybenzylideneamino)2,7-napthalenedisulfonic acid)
3.2. Azomethin H solution
Place 1 g of azomethin H and 2 g of ascorbic acid in a 100 mL volumetric
flask and add 50 mL double distilled water. Warm slightly to dissolve and
make up to the mark with double distilled water. The reagent is stable for
2 days if kept cold.
3.3. Buffer solution pH 5.2
Dissolve 3g of EDTA (disodium salt of ethylenediaminetetraacetic acid) in
150 mL of double distilled water. Add 125 mL acetic acid (20 = 1.05 g/mL)
and 250 g of ammonium acetate, NH4CH3COO, and dissolve. Check the pH
with a pH meter and adjust if necessary to pH 5.2.
OIV-MA-AS323-03 : R2009
As = absorbance of sample
Ab = absorbance of blank
OIV-MA-AS323-03 : R2009
5. Calculations
The g of boron contained in 5 mL of eluate, (corresponding to 0.5 mL of wine)
obtained from interpolating the net absorbance values of (As - Ab) on the
calibration graph is E. The content, B, in milligrams of boron per liter is given by:
B mg/L = E
0 .5
BIBLIOGRAPHY
WOLF B., Soil Science and Plant Analysis, 1971, 2(5), 363-374 et 1974, 5(1), 39-44.
CHARLOT C. and BRUN S., F.V., O.I.V., 1983, no771.
OIV-MA-AS323-03 : R2009
Method OIV-MA-AS323-04A
Type II method
Sulfur dioxide
(Resolution Oeno 377/2009)
1. Definitions
Free sulfur dioxide is defined as the sulfur dioxide present in the must or wine in
the following forms: H2SO3, HSO3, whose equilibrium as a function of pH and
temperature is:
H2SO3
H+ + HSO3
H2SO3 represents molecular sulfur dioxide.
Total sulfur dioxide is defined as the total of all the various forms of sulfur dioxide
present in the wine, either in the free state or combined with their constituents.
2. Free and Total Sulfur Dioxide
2.1 Principle
Free sulfur dioxide is carried over by a stream of air or nitrogen and is fixed
and oxidized by bubbling through a dilute and neutral solution of hydrogen
peroxide. The sulfuric acid formed is determined by titration with a standard
solution of sodium hydroxide. Free sulfur dioxide is purged from the wine by
entrainment at low temperature (10 C).
Total sulfur dioxide is purged from the wine by entrainment at high
temperature (approximately 100 C).
2.2 Method
2.2.1 Apparatus
The apparatus to be used should conform to the diagram overleaf, especially
with regard to the condenser (see Fig 1).
The gas supply tube to the bubbler B ends in a small sphere of 1 cm diameter
with 20 holes 0.2 mm in diameter around its largest horizontal circumference.
Alternatively, this tube may end in a sintered glass plate that produces a large
number of very small bubbles and thus ensures good contact between the
liquid and gaseous phases.
The gas flow through the apparatus should be approximately 40 L/h. The bottle
situated on the right of the apparatus is intended to restrict the pressure
reduction produced by the water pump to 20 30 cm water. In order to
regulate the flow rate, a flow meter with a semi-capillary tube should be
installed between the bubbler and the bottle.
OIV-MA-AS323-04A : R2009
Pump
FIGURE 1: The dimensions are given in millimeters. The internal diameters of the
4 concentric tubes making up the condenser are: 45, 34, 27 and 10 mm.
2.2.2 Reagents
2.2.2.1 Phosphoric acid: phosphoric acid 85% (20 =1.71 g/mL), diluted to 25%
2.2.2.2 Hydrogen peroxide solution, 9.1 g H2O2/L (3 volumes)
2.2.2.3 Indicator reagent:
Methyl Red ......................................... 100 mg
Methylene Blue .................................... 50 mg
Ethanol 50% (v/v) ................................ 100 mL
2.2.2.4 0.01 M Sodium hydroxide solution
OIV-MA-AS323-04A : R2009
OIV-MA-AS323-04A : R2009
diameter in it. This is to avoid overheating substances extracted from the wine
that are deposited on the walls of the flask.
Maintain boiling while passing a current of air (or nitrogen). Within 15
minutes the total sulfur dioxide is carried over and oxidized. Determine the
sulfuric acid formed by titration with 0.01 M sodium hydroxide solution.
Let n be the volume used.
2.2.4.2 Expression of results.
2.2.4.2.1 Calculation
Total sulfur dioxide in milligrams per liter:
- Samples low in sulfur dioxide (50 mL test sample): 6.4 n
- Other samples (20 mL test sample): 16 n
2.2.4.3 Repeatability (r):
(< 50 mg/L) 50 mL test sample,
(> 50 mg/L) 20 mL test sample,
2.2.4.4 Reproducibility (R):
(< 50 mg/L) 50 mL test sample,
(> 50 mg/L) 20 mL test sample,
r = 1 mg/L
r = 6 mg/L
R = 9 mg/L
R = 15 mg/L
BIBLIOGRAPHY
Reference method
PAUL F., Mitt. Klosterneuburg, Rebe u. Wein, 1958, ser. A, 821.
OIV-MA-AS323-04A : R2009
Method OIV-MA-AS323-04B
Type IV method
Sulfur dioxide
(Resolution Oeno 377/2009)
1. Definitions
Free sulfur dioxide is defined as the sulfur dioxide present in the must or wine in
the following forms: H2SO3, HSO3, whose equilibrium as a function of pH and
temperature is:
H2SO3
H+ + HSO3
H2SO3 represents molecular sulfur dioxide.
Total sulfur dioxide is defined as the total of all the various forms of sulfur dioxide
present in the wine, either in the free state or combined with their constituents.
2. Free and Total Sulfur Dioxide
2.1 Principle
Free sulfur dioxide is determined by direct titration with iodine. The combined
sulfur dioxide is subsequently determined by iodometric titration after alkaline
hydrolysis. When added to the free sulfur dioxide, it gives the total sulfur
dioxide.
2.2 Rapid Method
2.2.1 Reagents
2.2.1.1 EDTA: ethylenediaminetetraacetic acid, di-sodium salt
2.2.1.2 4 M Sodium hydroxide solution (160 g/L).
2.2.1.3 Dilute sulfuric acid: 10% sulfuric acid (20 = 1.84 g/mL) diluted 10%
(v/v).
2.2.1.4 Starch solution, 5 g/L.
Mix 5 g starch with approx. 500 mL water. Bring to a boil stirring
continuously and keep boiling for 10 minutes. Add 200 g of sodium chloride.
Cool and make to 1 liter.
2.2.1.5 0.025 M Iodine solution
2.2.2 Free sulfur dioxide
Place in a 500 mL conical flask place:
- 50 mL of wine
- 5 mL starch solution
- 30 mg EDTA
- 3 mL H2SO4
OIV-MA-AS323-04B : R2009
Immediately titrate with 0.025 M iodine, until the blue color persists clearly
for 10 to 15 seconds. Let n mL be the volume of iodine used.
2.2.3 Combined sulfur dioxide
Add 8 mL of 4 M sodium hydroxide solution, shake the mixture once and
allow to stand for 5 minutes. Add, with vigorous stirring and in one operation,
the contents of a small beaker in which 10 mL of sulfuric acid have been
placed. Titrate immediately with the 0.025 M iodine solution; let n' be the
volume used.
Add 20 mL of sodium hydroxide solution, shake once and allow to stand for
5 minutes. Dilute with 200 mL of ice-cold water.
Add, while stirring vigorously and in one operation, the contents of a test tube
in which 30 mL sulfuric acid has previously been placed. Titrate the free
sulfur dioxide immediately with the 0.025 M iodine, and let n" be the volume
of iodine used.
2.2.4 Expression of the results
2.2.4.1 Calculation
Free sulfur dioxide in milligrams per liter is given by:
32 n
Total sulfur dioxide in milligrams per liter is given by:
32 (n + n' + n")
Remarks:
1. For red wines with low SO2 concentrations, the 0.025 M iodine may be
diluted (for example: 0.01 M). In this case, replace the coefficient 32 by
12.8 in the above formula.
2. For red wines, it is useful to illuminate the wine from below with a beam of
yellow light from an ordinary electric light bulb shining through a solution
of potassium chromate or from a sodium vapor lamp. The determination
should be carried out in a dark room and the transparency of the wine
observed: it becomes opaque when the starch endpoint is reached.
3. If the quantity of sulfur dioxide found is close to or exceeds the legal limit,
the total sulfur dioxide should be determined with the reference method.
4. If the determination of free sulfur dioxide is specifically required, carry out
a determination on a sample kept under anaerobic conditions for two days at
20 C before analysis. Carry out the determination at 20 C.
5. Because certain substances are oxidized by iodine in an acid medium, the
quantity of iodine used in this way must be assessed for more accurate
determinations. To achieve this, combine the free sulfur dioxide in an
OIV-MA-AS323-04B : R2009
BIBLIOGRAPHY
Rapid method:
RIPPER M., J. Prakt. Chem., 1892, 46, 428.
JAULMES, P., DIEUZEIDE J.-C., Ann. Fals. Fraudes, 1954, 46, 9; Bull.
O.I.V., 1953, 26, n 274, 52.
KIELHOFER E., AUMANN H., Mitt. Klosterneuburg, Rebe u. Wein, 1957, 7, 289.
JAULMES P., HAMELLE Mme G., Ann. Fals. Exp. Chim., 1961, 54, 338
OIV-MA-AS323-04B : R2009
Method OIV-MA-AS323-04C
Type IV method
Sulfur dioxide
(Resolution Oeno 377/2009)
1. Definitions
Free sulfur dioxide is defined as the sulfur dioxide present in the must or wine in
the following forms: H2SO3, HSO3, whose equilibrium as a function of pH and
temperature is:
H2SO3
H+ + HSO3
H2SO3 represents molecular sulfur dioxide.
Total sulfur dioxide is defined as the total of all the various forms of sulfur dioxide
present in the wine, either in the free state or combined with their constituents.
2 Molecular Sulfur Dioxide
2.1 Principle of the Method
The percentage of molecular sulfur dioxide, H2SO3, in free sulfur dioxide, is
calculated as a function of pH, alcoholic strength and temperature.
For a given temperature and the alcoholic strength:
H2SO3
H+ + HSO3
[H2SO3] =
L
10 (pH pkM ) 1
(1)
where
L = [H2SO3] + [HSO3]
pkM = pkT
A I
I B I
I
= ionic strength
A & B = Coefficients which vary according to temperature and alcoholic strength.
kT
= Thermodynamic dissociation constant; the value of pkT is given in Table 1
for various alcoholic strengths and temperatures.
kM = Mixed dissociation constant
Taking a mean value 0.038 for the ionic strength I, Table 2 gives the values of
pkM for various temperatures and alcoholic strengths.
OIV-MA-AS323-04C : R2009
The molecular sulfur dioxide content calculated by the relationship given in (1)
is presented in Table 3 for various values of pH, temperature and alcoholic
strength.
2.2 Calculations
Knowing the pH of wine and its alcoholic strength, the percentage of
molecular sulfur dioxide is given in Table 3 for a temperature t C. Let this be
X %.
The amount of molecular sulfur dioxide in mg/L is given by: X C
C = the free sulfur dioxide in mg/L
Table I
Values of the thermodynamic constant pkT
Temperature oC
Alcohol
% by volume
20
25
30
35
40
0
5
10
15
20
1.798
1.897
1.997
2.099
2.203
2.000
2.098
2.198
2.301
2.406
2.219
2.299
2.394
2.503
2.628
2.334
2.397
2.488
2.607
2.754
2.493
2.527
2.606
2.728
2.895
Table II
Values of the Mixed Dissociation Constant pkM (I= 0.038)
Temperature C
Alcohol
% by volume
20
25
30
35
40
0
5
10
15
20
1.723
1.819
1.916
2.014
2.114
1.925
2.020
2.116
2.216
2.317
2.143
2.220
2.311
2.417
2.538
2.257
2.317
2.405
2.520
2.663
2.416
2.446
2.522
2.640
2.803
OIV-MA-AS323-04C : R2009
Table III
Molecular Sulfur Dioxide as a Percentage of Free Sulfur Dioxide (I=0.038)
T = 20 oC
Alcohol % by volume
pH
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
7.73
6.24
5.02
4.03
3.22
2.58
2.06
1.64
1.31
1.04
0.83
10
15
20
9.46
7.66
6.18
4.98
3.99
3.20
2.56
2.04
1.63
1.30
1.03
11.55
9.40
7.61
6.14
4.94
3.98
3.18
2.54
2.03
1.62
1.29
14.07
11.51
9.36
7.58
6.12
4.92
3.95
3.16
2.53
2.02
1.61
17.09
14.07
11.51
9.36
7.58
6.12
4.92
3.95
3.16
2.53
2.02
17.15
14.12
11.55
9.40
7.61
6.14
4.94
3.97
3.18
2.54
2.03
20.67
17.15
14.12
11.55
9.40
7.61
6.14
4.94
3.97
3.18
2.54
24.75
22.71
17.18
14.15
11.58
9.42
7.63
6.16
4.55
3.98
3.18
24.49
20.48
16.98
13.98
11.44
9.30
7.53
6.08
4.89
3.92
3.14
29.28
24.75
20.71
17.18
14.15
11.58
9.42
7.63
6.16
4.95
3.98
35.36
30.29
25.66
21.52
17.88
14.75
12.08
9.84
7.98
6.44
5.19
T = 25 oC
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
11.47
9.58
7.76
6.27
5.04
4.05
3.24
2.60
2.07
1.65
1.32
14.23
11.65
9.48
7.68
6.20
4.99
4.00
3.20
2.56
2.05
1.63
T = 30 oC
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
18.05
14.89
12.20
9.94
8.06
6.51
5.24
4.21
3.37
2.69
2.16
20.83
17.28
14.23
11.65
9.48
7.68
6.20
4.99
4.00
3.21
2.56
OIV-MA-AS323-04C : R2009
pH
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
10
15
20
22.27
18.53
15.31
12.55
10.24
8.31
6.71
5.44
4.34
3.48
2.78
24.75
20.71
17.18
14.15
11.58
9.42
7.63
6.16
4.95
3.98
3.18
28.71
24.24
20.26
16.79
13. 82
11.30
9.19
7.44
6.00
4.88
3.87
34.42
29.42
24.88
20.83
17.28
14.23
11.65
9.48
7.68
6.20
4.99
42.18
36.69
31.52
26.77
22.51
18.74
15.49
12.71
10.36
8.41
6.80
34.52
29.52
24.96
20.90
17.35
14.29
11.70
9.52
7.71
6.22
5.01
40.89
35.47
30.39
25.75
21.60
17.96
14.81
12.13
9.88
8.01
6.47
50.14
44.74
38.85
33.54
28.62
24.15
20.19
16.73
13.77
11.25
9.15
T = 40 oC
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
29.23
24.70
20.67
17.15
14.12
11.55
9.40
7.61
6.14
4.94
3.97
30.68
26.01
21.83
18.16
14.98
12.28
10.00
8.11
6.56
5.28
4.24
BIBLIOGRAPHY
OIV-MA-AS323-04C : R2009
Method OIV-MA-AS323-05
Type IV method
Sulphur dioxide
Reference method: Procedure for grape juice
(Resolution Oeno 377/2009)
1. Apparatus:
See 2.2.1., from OIV-MA-AS323-04A
2. Reagents:
Phosphoric acid (20 =1.71 g/ml) diluted at 25% (m/v).
For other reagents, see 2.2.2., from OIV-MA-AS323-04A
3. Procedure:
Introduce 50 ml of grape juice and 5 ml of phosphoric acid diluted 25% (m/v)
in a 250 ml balloon A control trainer. Set up the balloon.
Continue as indicated as in 2.2.4.1., from the OIV-MA-AS323-04A form.
4. Calculation:
Given n as the number of milliliters of 0.01 M sodium hydroxide solution used,
the total sulphur dioxide content of grape juice in milliliters per liter:
6.4 x n
OIV-MA-AS323-05 : R2009
Method OIV-MA-AS323-06
Type IV method
1. FIELD OF APPLICATION
This method applies to the analysis of mercury in wines with a concentration
range between 0 to 10 ug/l.
2. DESCRIPTION OF TECHNIQUE
2.1. Principle of the method
2.1.1 Mineralisation of wine takes place in an acid environment: heating
under reflux;
mineralisation is achieved with a potassium permanganate.
2.1.2. Reduction of non-consumed permanganate by hydroxylamine
hydrochlorate
2.1.3. Reduction in mercury II (metal mercury by stannous chloride (II).
2.1.4. Mercury pick up by an argon current at ambient temperature
2.1.5. Dosage of mercury in monoatomic vapour state by atomic
flourescence spectometre with wavelength of 254 nm. Mercury atoms
are excited by a mercury vapour lamp; the atoms thus excited emit a
radiation called flourescent which allows the quantification of
mercury present using a photonics detector to obtain good linearity
while eliminating memory effects.
OIV-MA-AS323-06 : R2009
peristaltic
Peristaltic
pump
perist
Air
peristaltic
Stannous
chloride
c
pump
Blank
Reference or
Sample
Hygroscopic
membrane
Stannous
Valve
open
chloride
Upon sample
position
Argon
flowmeter
Towards
sink
Gas-liquid
separator
Florescence
detector
OIV-MA-AS323-06 : R2009
4. APPARATUS
4.1 Glass ware
4.1.1 Volumetric flasks 100, 200, and 1000 ml (class A)
4.1.2 Volumetric pipette 0.5,1.0, 2.0, 5, 10 and 20 ml (class A)
4.1.3 Precautionary action: Before using, the glass ware must be
washed with 10% nitric acid, leave in contact 24 hours, then
rinse with demineralised water.
4.2 Mineralisation apparatus (figure 2)
4.3 Temperature controlled heating mantle
4.4 Squeeze pump
4.5 Cold vapour generator
4.5.1 Liquid gas separator
4.6 Desiccant (Hygroscopic membrane) covered by an air current
(supplied from a compressor) and placed before the detector
4.7 Spectrofluorimeter
OIV-MA-AS323-06 : R2009
OIV-MA-AS323-06 : R2009
Refrigerant
with balls
Circulation
cold water
Vapour
Recuperation
chamber
Introduction
of wine
and reagents
Reaction Balloon
(wine + sulphuric acid
+ nitric acid)
Heating mantle
Mineralisation apparatus
6. OPERATING PROCEDURE
6.1 Analytical measurement
Turn on the fluorimeter; the apparatus is stable after 15 minutes. The
squeeze pump absorbs the white (3.3), the stannous lead II (3.13) and the
sample calibrations (5.1) or (5.2.) Verify that bubbling occurs in the liquid
gas separator. Present the calibration samples successively (5.1); set off
the vapour generator program. The computer software establishes a
calibration curve (percentage of fluorescence according to concentration
of mercury ug/l). Then present the samples (5.2).
OIV-MA-AS323-06 : R2009
7. EXPRESSION OF RESULTS
Results are provided by the computer software and expressed in ug/l. Deduct
the mercury concentration in wine in ug/l keeping into account 1/5 dilution.
8. CHECKING RESULTS
Quality control is carried out by placing reference material in which the
mercury content is known, following the set of calibrations and every 5
samples. Following the analytical series, the reference material is red wine, dry
white wine or sweet white wine.
The check card is set for each reference material used. The check limits are set
at: +/- 2SR intra (2SR intra : reproducibility spread-type)
The uncertainly calculation, carried out on check cards, resulted in a red wine
reference of: 3.4 +/- 0.8 ug/l and for reference dry white wine : 2.8+/-0.9 ug/l.
9. BIBLIOGRAPHY
CAYROL M., BRUN S., 1975. Dosage du mercure dans les vins. Feuillet Vert de
lO.I.V. n371.
REVUELTA D., GOMEZ R., BARDON A., 1976. Dosage du mercure dans le vin
par la mthode des vapeurs froides et spectromtrie dabsorption atomique. Feuillet
Vert de lO.I.V. n494.
CACHO J., CASTELLS J.E., 1989. Determination of mercury in wine by flameless
atomic absorption spectrophotometry. Atomic Spectroscopy, vol. 10, n3.
STOCKWELL P.B., CORNS W.T., 1993. The role of atomic fluorescence
spectrometry in the automatic environmental monitoring of trace element analysis.
Journal of Automatic Chemistry, vol. 15, n3, p 79-84.
SANJUAN J., COSSA D., 1993. Dosage automatique du mercure total dans les
organismes marins par fluorescence atomique. IFREMER, Rapport dactivit.
AFNOR, 1997. Dosage du mercure total dans les eaux par spectromtrie de
fluorescence atomique. XPT 90-113-2.
GAYE J., MEDINA B., 1998. Dosage du mercure dans le vin par analyse en flux
continu et spectrofluorimtrie. Feuillet Vert de lO.I.V. n1070.
OIV-MA-AS323-06 : R2009
OIV-MA-AS323-07
Type of Method II
1.
344-2010)
SCOPE OF APPLICATION
This method can be applied to the analysis of the elements present in wines within
the range indicated and featured in the following list:
-
The sample sometimes requires mineralization. This is the case, for example, of
wines with more than 100 g/L of sugar where it can be necessary to realise
mineralization of the sample before. In this case, it is recommended to perform a
digestion with nitric acid in a microwave.
The technique can also be applied to musts, after mineralization.
2.
BASIS
3.
4.
OIV-MA-AS323-07: R2010
Note: material that will come into contact with the sample, such as, for example,
tubes and tips, must remain for at least 24 hours in a nitric acid solution (3.4) at a
concentration of 10% and must subsequently be rinsed several times in water
(3.1).
5.
SAMPLE PREPARATION
Samples of sparkling wine must be degasified. This can be done through nitrogen
bubbling (3.6) for 10 minutes or by using an ultrasound bath.
Remove the bung carefully to ensure that the wine is not contaminated. Wash the
bottle neck in an acid solution (2% HNO3). Wine samples are taken directly from
the bottle.
Use a micropipette (4.4) to insert 0.5 ml of wine, 0.1 to 0.5 ml of nitric acid (3.4)
and 100 l of internal standard solution (3.8) into a 10 ml tube (3.5).Level off with
water (3.1) and homogenize.
For certain elements a higher dilution may be necessary owing to their high natural
content in the sample.
Br has high ionization potential and its ionization in plasma may be incomplete
because of the presence of high concentrations of other elements in wines with low
ionization potential. This may result in the incorrect quantification of Br and
therefore a 1/50 dilution is recommended to avoid this effect (in the event of
another dilution being used, confirm the results by checking recovery after an
addition).
When the standards are prepared gravimetrically, the final dilution of the sample
must also be obtained gravimetrically.
6.
PROCEDURE
OIV-MA-AS323-07: R2010
and finally the blank to ensure that there is no memory effect. Read the samples in
duplicate. For the internal control, use a wine of known concentrations (3.10) to
confirm that the results are coherent.
Element
m/z*
Aluminium
27
Boron
11
Bromine
79
Cadmium
114
Cobalt
59
Copper
63
Strontium
88
Iron
56/57
Lithium
Magnesium
24
Manganese
55
Nickel
60
Lead
Rubidium
85
Sodium
23
Vanadium
51
Zinc
64
* The above table is given by way of example. Other isotopes may be required,
depending on the equipment.
In the event of using equipment with no collision/reaction cell, correction
equations may be necessary for some elements.
7.
RESULTS
OIV-MA-AS323-07: R2010
C=
Cm Vt
Vm
Where:
C=
Concentration of the element in the sample
Cm = Concentration of the elements in the diluted sample
Vt = Final volume of the measurement solution, in ml
Vm = Aliquot volume of wine, in ml.
8.
QUALITY CONTROL
9.
PRECISION
The results of the statistical parameters of the collaborative trial are shown in
Appendix A.
9.1 Repeatability (r)
The difference between two independent results, obtained using the same method,
in the same sample, in the same laboratory, by the same operator, using the same
OIV-MA-AS323-07: R2010
Reproducibility (R)
The difference between two results, obtained using the same method, in the same
sample, in a different laboratory, by a different operator and with different
equipment. R results are given in Tables 1 to 17 of the Appendix A.
Table 1 represents the % of the relative standard deviation of Repeatability and
Reproducibility (RSDr% et RSDR%) of the method. (*) C = Concentration
Element
Concentration
RSDr %
RSDR %
Aluminium
10
Boron
10 - 40 mg/l
3,8
6,3
4,1
2.1
0,06 C*+0,18
1,5
16,3
8,0
10
10
Cobalt
3,2
13,2
Copper
3,8
2,0
11,4
11,4
Strontium
2,5
7,5
Iron
4,2
4,2
15,7
7,8
Lithium
12
Magnesium
50 - 300 mg/l
Manganese
0,50-1,5 mg/l
Nickel
Lead
8
2
7
7
Rubidium
5 - 10 mg/l
10 - 30 mg/l
0,003 0,010 mg/l
0,010 0,20 mg/l
2
0,3 C*-2,5
8
3
10
10
10
10
Bromine
Cadmium
Sodium
Vanadium
OIV-MA-AS323-07: R2010
Zinc
12
10.
BIBLIOGRAPHY
OIV-MA-AS323-07: R2010
The method has been checked with two collaborative trials, by evaluating
precision in accordance with ISO 5725.The trueness of the method has been
obtained through recovery studies.
1st Collaborative Trial
8 samples (A, B, C, D, E, F, MH1 and MH2) were used from the following origins:
Three samples of red wine, with and without addition.
Three samples of white wine, with and without addition.
Two samples of synthetic hydroalcoholic mixture, prepared with ethanol and
water.
Hydroalcoholic sample MH1 presented problems of instability during the trial and
the results have not been taken into account.
MH2
Metal
(mg/l)
Hydroalcoholic
mixture
RW2
RW3
WW2
WW3
Natural
red wine
Aluminium
0.5
Cadmium
0.001
0.005
0.02
0.05
0.01
Strontium
0.300
No
addition
No
addition
No
addition
No
addition
Lithium
0.020
0.01
0.02
0.04
0.01
Magnesium
50
100
200
50
25
Manganese
0.500
0.5
0.5
Nickel
0.070
0.025
0.2
0.1
0.1
Lead
0.010
0.05
0.1
0.15
0.05
Rubidium
1.0
No
addition
No
addition
No
addition
No
addition
Sodium
20
10
10
20
Vanadium
0.010
0.05
0.2
0.1
0.1
Zinc
0.500
0.1
0.5
0.5
OIV-MA-AS323-07: R2010
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
F
Natural
white
wine
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
No
addition
Code
F-N
White wine
C-I
A-O
B-K
Liqueur wine
E-L
D-M
Red wine
H-J
Sparkling
wine
G-P
Addition
No
addition
Addition
1
Addition
2
No
addition
Addition
3
No
addition
Addition
4
No
addition
OIV-MA-AS323-07: R2010
B
mg/l
Co
g/l
Cu
mg/l
Fe
Mg/l
0.0
0.0
0.0
0.0
5.0
5.0
5.0
1.0
10.0
10.0
1.0
2.0
0.0
0.0
0.0
0.0
15.0
20.0
1.5
3.0
0.0
0.0
0.0
0.0
20.0
50.0
2.0
5.0
0.0
0.0
0.0
0.0
10
LAB.
No.
Accepted
Vrf
Sr
RSD r
(%)
11
11
11
11
11
11
11
10
9
9
10
10
10
8
0,68
2,1
2,1
1,2
0,34
0,27
5,2
0,020
0,043
0,032
0,041
0,014
0,006
0,26
0,06
0,12
0,09
0,12
0,04
0,02
0,73
2,9
2,0
1,5
3,4
4,1
2,2
5,0
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
11
9,4
9,5
10
12
13
8,2
Horratr
SR
RSDR
(%)
0,26
0,22
0,16
0,34
0,34
0,17
0,60
0,077
0,21
0,21
0,10
0,029
0,028
0,56
0,22
0,61
0,59
0,29
0,08
0,08
1,6
11
10
10
8,3
8,5
10
11
Horwitz
R
RSDR
(%)
17
14
14
16
19
20
13
HorratR
0,66
0,71
0,69
0,56
0,46
0,52
0,86
11
LAB.
No.
Accepted
Vrf
Sr
RSD r
(%)
8
8
8
8
8
8
7
8
6
4
4
7
5
5
4
5
18
4,5
13
11
21
8,3
3,1
31
0,77
0,27
0,31
0,26
0,47
0,43
0,094
1,0
2,2
0,76
0,89
0,74
1,3
1,2
0,27
3,0
4,3
6,0
2,4
2,4
2,2
5,2
3,0
3,2
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
6,8
8,4
7,2
7.4
6.7
7.7
8.9
6.3
Horratr
SR
RSDR
(%)
0,62
0,72
0,33
0,31
0,33
0,68
0,34
0,54
0,94
0,40
0,33
1,1
0,85
0,47
0,18
1,6
2,69
1,14
0,94
3,11
2,43
1,34
0,51
4,43
5,2
8,9
2,5
10
4,0
5,7
5,8
5,2
Horwitz
R
RSDR
(%)
10
13
11
11
10
12
14
9,6
HorratR
0,50
0,70
0,24
0,90
0,40
0,48
0,43
0,52
12
LAB.
No.
Accepted
Vref
Sr
RSD r
(%)
6
5
6
6
6
6
6
2
2
3
4
3
3
2
1,21
0,19
0,81
0,38
1,72
0,22
2,30
0,028
0,006
0,017
0,017
0,030
0,014
0,061
0,08
0,02
0,05
0,05
0,09
0,04
0,17
2,3
2,9
2,1
4,5
1,7
6,4
2.7
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
10,3
13,6
10,9
12,2
9,7
13,3
9,3
Horratr
SR
RSDR
(%)
0,22
0,21
0,19
0,37
0,17
0,48
0,28
0,041
0,0043
0,062
0,066
0,22
0,046
0,092
0,12
0,012
0,18
0,19
0,62
0,13
0,26
3,4
2,3
7,7
17,4
12,8
20,9
4
Horwitz
R
RSDR
(%)
15,6
20,5
16,5
18,5
14,8
20,1
14.1
HorratR
0,22
0,11
0,47
0,94
0,86
1
0.28
13
LAB.
No.
Accepted
Vrf
Sr
RSD r
(%)
12
12
12
12
8
8
9
11
11
9
10
7
6
5
6
16
40
10
0,3
0,3
0,9
0,2
0,4
0,4
0,3
0,20
0,04
0,08
0,6
1
1
0,8
0,6
0,1
0,2
3,3
2,5
1,0
3,0
67
13
8,9
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
15
15
15
15
15
15
15
Horratr
SR
0,22
0,17
0,07
0,20
4,47
0,87
0,59
1
2
3
0,9
0,20
0,20
0,10
R
3
6
8
3
0,67
0,45
0,29
RSDR
(%)
17
13
7,5
9,0
67
67
11
Horwitz
R
RSDR
(%)
22
22
22
22
22
22
22
HorratR
0,77
0,59
0,34
0,41
3,05
3,05
0,50
14
LAB.
No.
Accepted
Vrf
Sr
RSD r
(%)
10
10
10
10
10
10
9
10
6
6
8
3
8
7
5
6
22
8
19
3
27
12
2
49
0,5
0,3
0,4
0,07
1
0,5
0,2
0,5
1
0,9
1
0,2
3
2
0,5
1
2,3
3,8
2,1
2,3
3,7
4,2
10
2,3
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
15
15
15
15
15
15
15
15
Horratr
SR
RSDR
(%)
0,15
0,25
0,14
0,15
0,25
0,28
0,67
0,15
2
1
3
0,1
3
1
0,3
6
6
4
7
0,3
9
4
0,8
18
9,1
13
16
3,3
11
8,3
15
12
Horwitz
R
RSDR
(%)
22
22
22
22
22
22
22
22
HorratR
0,41
0,59
0,73
0,15
0,50
0,38
0,68
0,55
15
LAB.
No.
Accepted
10
10
10
10
10
10
9
10
8
8
7
8
9
7
4
7
Vrf
1,1
0,21
0,74
0,14
1,7
0,16
0,042
2,1
OIV-MA-AS323-07: R2010
Sr
RSD r
(%)
0,013
0,006
0,009
0,007
0,061
0,006
0,004
0,018
0,040
0,020
0,030
0,020
0,17
0,020
0,010
0,050
1,2
2,9
1,2
5,0
3,6
3,8
9,5
0,86
Horwitz
RSDr
(%)
10
13
10
14
7,8
14
15
9,5
Horratr
SR
RSDR
(%)
0,12
0,22
0,12
0,36
0,5
0,27
0,63
0,09
0,11
0,021
0,046
0,015
0,16
0,029
0,006
0,24
0,32
0,060
0,13
0,043
0,46
0,083
0,017
0,69
10
10
6,2
11
9,0
18
14
11
Horwitz
R
RSDR
(%)
16
20
17
22
15
21
22
14
HorratR
0,63
0,50
0,36
0,50
0,60
0,86
0,64
0,79
16
SAMPLE
Vrf
RSD r
(%)
33
93
3,0
66
188
10
18
5,8
1,8
20
28
80
3
7
Accepted
12
11
1091
12
1139
12
328
12
10
313
12
10
1176
F
MH2
12
10
293
12
352
OIV-MA-AS323-07: R2010
Horwitz
LAB. N
Sr
HorwitzR
Horrat
Horratr
SR
RSDR
(%)
0,30
78
222
7,2
16
0,45
69
195
19
54
6,1
5,8
16
13
0,58
0,14
19
0,38
0,31
2,2
13
0,17
22
61
7,0
19
0,37
2,4
10
0,24
86
243
7,3
16
0,46
1,0
13
0,08
22
62
7,5
19
0,39
19
2,0
12
0,17
24
69
6,8
19
0,36
RSDr
(%)
10
RSDR (%)
17
LAB.
No.
Accepted
Vrf
Sr
RSD r
(%)
10
10
10
10
10
10
9
10
6
6
5
5
5
6
6
7
3,2
1,5
2,1
3,1
4,3
1,1
0,83
7,8
0,017
0,085
0,036
0,033
0,120
0,051
0,024
0,180
0,05
0,24
0,10
0,094
0,34
0,15
0,07
0,52
0,53
5,7
1,7
1,1
2,8
4,6
2,9
2,3
OIV-MA-AS323-07: R2010
Horwitz
RSDr
(%)
8,9
9,9
9,4
8,9
8,5
10
11
7,8
Horratr
SR
RSDR
(%)
0,06
0,58
0,18
0,12
0,33
0,46
0,26
0,29
0,23
0,11
0,18
0,29
0,29
0,16
0,14
1,2
0,66
0,31
0,51
0,83
0,83
0,46
0,40
3,52
7,2
7,3
8,6
9,4
6,7
15
17
15
Horwitz
R
RSDR
(%)
13
15
14
14
13
16
16
12
HorratR
0,55
0,49
0,61
0,67
0,52
0,94
1,06
1,25
18
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
11
10
34
5,9
(%)
15
0,39
11
11
(%)
22
11
11
42
7,1
15
0,47
12
10
22
0,45
11
11
47
2,1
15
0,14
13
9,8
22
0,45
11
11
18
5,6
15
0,37
14
22
0,64
11
11
25
4,0
15
0,27
12
22
0,55
F
MH2
11
0,3
3,8
15
0,25
0,6
7,2
22
0,33
11
22
4,6
15
0,31
5,3
22
0,24
OIV-MA-AS323-07: R2010
0,50
19
Accepted
10
182
2,9
8,1
10
280
3,9
11
10
104
2,4
10
85
10
F
MH2
10
10
SAMPLE
:
Horwit
z
Horwitz
RSDR
Horrat
Horratr
SR
1,6
(%)
4,3
RSDR
(%)
0,37
9,3
26
5,1
7,3
0,70
1,4
4,5
0,31
6,0
17
2,1
6,9
0,30
6,9
2,3
5,3
0,43
6,8
19,25
6,5
8,0
0,81
1,4
4,0
1,7
5,4
0,31
2,2
6,1
2,6
8,2
0,32
94
2,2
6,2
2,3
5,3
0,43
5,5
16
5,9
8,1
0,73
65
0,95
2,7
1,5
5,6
0,27
3,8
11
5,9
8,5
0,69
51
0,90
2,5
1,8
5,8
0,31
2,4
6,9
4,7
8,9
0,53
Vrf
OIV-MA-AS323-07: R2010
Sr
RSD r
(%)
RSDr
(%)
20
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
11
10
1,3
0,014
0,040
1,1
(%)
10
0,11
0,13
0,37
10
(%)
15
11
1,8
0,14
0,40
7,8
9,7
0,80
0,20
0,56
11
15
0,73
11
1,5
0,028
0,080
1,9
9,9
0,19
0,084
0,24
5,6
15
0,37
11
1,0
0,035
0,10
3,5
11
0,32
0,049
0,14
4,9
16
0,31
11
0,84
0,019
0,050
2,3
11
0,21
0,057
0,16
6,8
16
0,43
F
MH2
11
0,59
0,015
0,040
2,5
11
0,23
0,031
0,090
5,3
17
0,31
11
0,52
0,029
0,080
5,6
12
0,47
0,037
0,10
7,1
18
0,39
OIV-MA-AS323-07: R2010
0,67
21
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
11
10
40
5,0
(%)
15
0,33
13,90
13
(%)
22
12
10
194
20
3,6
14
0,26
17
48,96
8,8
21
0,42
12
148
10
2,7
14
0,19
15,12
3,4
21
0,16
12
157
12
2,6
14
0,19
23,10
5,1
21
0,24
11
15
0,6
4,0
15
0,27
3,33
6,7
22
0,30
F
MH2
12
66
1,5
15
0,10
10,58
6,1
22
0,28
11
71
14
7,0
15
0,47
11,41
5,6
22
0,25
OIV-MA-AS323-07: R2010
0,59
22
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
12
59
1,7
(%)
15
0,11
5,1
(%)
22
12
10
109
1,8
15
0,12
23
7,3
22
0,33
12
136
2,2
14
0,16
13
37
9,6
22
0,44
12
119
1,7
15
0,11
13
4,2
22
0,19
12
10
13
7,7
15
0,51
7,7
22
0,35
F
MH2
12
92
1,1
15
0,07
11
4,4
22
0,20
12
10
13
7,7
15
0,51
7,7
22
0,35
OIV-MA-AS323-07: R2010
0,23
23
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
11
717
14
41
2,0
(%)
11
0,18
13
36
1,8
(%)
17
11
799
25
70
3,1
11
0,28
30
86
3,8
17
0,22
11
677
10
27
1,5
11
0,14
34
96
5,0
17
0,29
11
612
18
51
2,9
11
0,26
18
50
2,9
17
0,17
11
741
19
53
2,6
11
0,24
66
187
8,9
17
0,52
F
MH2
11
617
10
28
1,6
11
0,15
43
123
7,0
17
0,41
11
1128
10
28
0,89
10
0,09
64
181
5,7
16
0,36
OIV-MA-AS323-07: R2010
0,11
24
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
10
19
0,59
1,7
3,1
(%)
6,8
0,46
2,2
5,7
12
(%)
10
10
20
1,3
3,6
6,5
6,7
0,97
2,2
6,3
11
10
1,10
10
28
0,33
0,93
1,2
6,4
0,19
1,9
5,4
6,8
9,7
0,70
10
11
0,24
0,68
2,2
7,4
0,30
1,1
3,0
10
11
0,91
10
9,8
0,19
0,53
1,9
7,5
0,25
0,89
2,5
9,1
11
0,83
F
MH2
10
6,1
0,093
0,26
1,5
8,1
0,19
0,74
2,1
12
12
1,00
10
24
1,8
5,0
7,5
6,6
1,14
2,6
7,2
11
9,9
1,11
OIV-MA-AS323-07: R2010
1,20
25
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
12
11
46
2,2
(%)
15
0,15
13
11
(%)
22
12
11
167
15
3,0
14
0,21
19
54
11
21
0,52
12
11
93
3,2
15
0,21
12
33
13
22
0,59
12
96
3,1
15
0,21
22
8,3
22
0,38
10
0,2
0,7
6,7
15
0,45
0,3
0,9
10
22
0,45
F
MH2
10
0,2
0,6
6,7
15
0,45
0,2
0,7
6,7
22
0,30
12
11
0,3
2,7
15
0,18
0,9
8,2
22
0,37
OIV-MA-AS323-07: R2010
0,50
26
LAB.
Accepted
No.
Vrf
Sr
RSD r
(%)
Horwitz
RSDr
Horratr
SR
RSDR
(%)
HorwitzR
RSDR
Horrat
R
11
405
22
61
5,4
(%)
12
0,45
45
128
11
(%)
18
11
1327
49
138
3,7
10
0,37
152
429
11
15
0,73
11
990
14
41
1,4
11
0,13
86
243
8,7
16
0,54
11
1002
28
79
2,8
11
0,25
110
310
11
16
0,69
11
328
13
37
4,0
13
0,31
79
224
24
19
1,26
F
MH2
11
539
15
42
2,8
12
0,23
61
172
11
18
0,61
11
604
72
204
12
11
1,09
89
251
15
17
0,88
OIV-MA-AS323-07: R2010
0,61
27
Method OIV-MA-AS4-01
Type IV Method
Objective:
Microbiological analysis is aimed at following alcoholic fermentation and/or
malolactic fermentation and detecting microbiological infections, and allowing
the detection of any abnormality, not only in the finished product but also during
the different phases of manufacture.
Comments:
All experiments must be carried out under normal microbiological aseptic
conditions, using sterilized material, close to a Bunsen burner flame or in a
laminar flow room and flaming the openings of pipettes, tubes, flasks, etc.
Before carrying out microbiological analysis, it is necessary to ensure that the
samples to be analyzed are taken correctly.
Field of application:
Microbiological analysis can be applied to wines, musts, mistelles and all similar
products even when they have been changed by bacterial activity. These
methods may also be used in the analysis of industrial preparations of selected
microorganisms, such as dry active yeasts and lactic bacteria.
Microbiological analysis techniques:
1.
2.
3.
4.
4.1
4.2
4.3
4.3.1
4.3.2
5.
Microscopic techniques for the detection, differentiation of microorganisms and direct counting of yeasts
5.1. Microscopic examination of liquids or deposits
5.2. Gram staining for the differentiation of bacteria isolated from colonies (see
paragraph 6)
5.3. Catalase Test for the differentiation of bacteria isolated from colonies (see
paragraph 6)
5.4. Yeast cell count haemocytometry
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
2H2O
+ O2
Reagent:
12 Volume hydrogen peroxide solution (3%)
Preparation: Measure 10 mL of 30% by volume hydrogen peroxide in a 100 mL
calibrated flask and fill with freshly boiled distilled water. Stir and keep in the
refrigerator in a dark flask. The solution must be freshly prepared.
Operating method:
Place a drop of 3% by volume hydrogen peroxide on a slide and add a small
sample of young colony. If gas is released, it can be concluded that catalase
activity is occurring in the culture . It is sometimes difficult to observe gas
clearing immediately, particularly with bacterial colonies. It is therefore
advisable to examine the culture through a microscope (objective x10).
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
OIV-MA-AS4-01 : R2010
T C
x100
T
5.5.7 References
European Brewery Convention. Analytica Microbiologica EBC. Fachverlag Hans
Carl, 2001
OIV-MA-AS4-01 : R2010
10
OIV-MA-AS4-01 : R2010
11
OIV-MA-AS4-01 : R2010
12
Bottled or filtered wines, and concentrated musts after dilution in sterile peptone
water, are analyzed with membrane filter technique.
6.1.7.4 Plating
The necessary serial dilutions are prepared for the number of samples to be plated.
Multiple serial dilutions can be prepared, if many samples have to be plated, but
any dilution must be plated within 20 minutes.
Inoculate each plate with 0.1 or 0.2 ml of the three lowest dilutions prepared, as
follows:
Unfermenting musts
dilutions -2; -3; -4.
Fermenting musts
dilutions -5; -6; -7.
Unfiltered wines during ageing
dilutions 0; -1; -2.
In doubt, inoculate a higher number of dilutions, never a lower.
Under aseptic conditions (preferably under a laminar flow cabinet) spread the
sample on the surface of the culture media before the liquid is absorbed (usually
within 1-2 minutes) with a sterile bent glass rod (Drigalski rods) or a single-use
one. A separate hockey stick must be used for each plate, or the plate must be
spread starting with the most diluted sample and proceeding to the least dilute
ones. Leave the plates some minutes under sterile air flow, until the liquid is
absorbed.
Note 1: Plating 0.,2 ml instead of 0.1 ml, as frequently reported, allows an
easier spreading and a delayed one. Calculations must consider this.
Note 2: For the enumeration of yeast Bacterial growth is avoided by adding 50
mg/l chloramphenicol (or equivalent if validated) to growth media, after
autoclaving it, and the mold by adding biphenyl 150mg/L (or equivalent if
validated).
Note 3: For the enumeration of lactic acid bacteria, yeasts growth is prevented
by the addition of natamycin (pimaricin) (0.1 g/L) (or equivalent if validated)
and acetic bacteria by anaerobic incubation.
Note 4: For the enumeration of acetic bacteria, the growth of yeast is prevented
by the addition of natamycin (pimaricin) (0.1 g/L) (or equivalent if validated)
and that of lactic acid bacteria with the addition of penicillin (12.5 mg/L) (or
equivalent if validated) .
The addition of antibiotics is done after the autoclave sterilization.
If a specific research of non-Saccharomyces yeast is performed, inoculate as
previously described, three Lysine Agar plates and three WL Differential Agar plates
with the appropriate dilutions
- Incorporation method (alternative method).
OIV-MA-AS4-01 : R2010
13
OIV-MA-AS4-01 : R2010
14
V 1,1 d
where:
is the sum of colonies counted on the two dishes retained from two
OIV-MA-AS4-01 : R2010
15
Kp
| C1 C 2 |
C1 C 2
Number of.
colonies
Lower
Lower
OIV-MA-AS4-01 : R2010
Upper
Upper
16
-97
-88
-79
-73
-68
-63
-60
-57
-54
-52
-50
-48
-47
-45
-44
457
261
192
156
133
118
106
97
90
84
79
75
71
68
65
If the colony count is >10, the confidence limit at a p probability level is calculated
with the following equation:
C = Ci Kp Ci,
where Ci is the number of colonies on the plate, and Kp is the coverage factor.
Usually the coverage factor is 2, or 1.96. C value is calculated from each plate
and multiplied by the number of dilutions, together with the result of the count.
6.2. Culture in liquid medium- "Most Probable Number" (MPN)
6.2.1 Objective
The purpose of this technique is to evaluate the number of viable
microorganisms in wines having high contents of solid particles in suspension
and/or high incidence of plugging.
6.2.2 Principle
This technique is based on the estimation of the number of viable
microorganisms in liquid medium, starting from the principle of its normal
distribution in the sample.
6.2.3 Diluents and liquid culture media (see Appendices 4 and 5)
6.2.4 Operating method
Several quantitative and successive solutions are prepared and following this,
after incubation, a certain proportion of tests will not lead to any growth
(negative tests), while others will begin to grow (positive tests). If the sample
and the dilutions are homogeneous, and if the number of dilutions is sufficiently
high, it is possible to treat the results statistically, using suitable tables (tables
based on McCrady's probability calculations), and to extrapolate this result to
the initial sample.
6.2.5 Preparation of dilutions
Starting from a sample of homogenized wine, prepare a series of decimal
1
dilutions ( /10) in the diluent.
OIV-MA-AS4-01 : R2010
17
OIV-MA-AS4-01 : R2010
18
Table:
Number of positive tubes for each dilution
Example
10
10
10
10
10
Typical number
3-1-0
3
3
3
1
0
3-2-0
a
3
3
2
0
0
3-2-1
a
3
2
1
0
0
3-0-1
a
3
0
1
0
0
3-2-3
a
3
2
2
1
0
3-2-3
b
3
2
1
1
0
3-2-2
b
2
2
2
2
0
2-2-2
c
0
1
0
0
0
0-1-0
d
Example a : take the greatest dilution for which all the tubes are
positive and the two following ones.
Example b : if a positive result is achieved for a dilution that is bigger
than the last chosen dilution, it must be added.
Example c : if no dilution achieves three positive tubes, take the
dilutions that correspond to the last three positive tubes.
Example d : instance of a very small number of positive tubes. Choose
the typical number so that the positive dilution is in the tens
row.
OIV-MA-AS4-01 : R2010
19
7. BIBLIOGRAPHY
ISO 4833:2003. Microbiology of food and animal feeding stuffs
Horizontal medium for the enumeration of microorganisms Colony count
technique at 30C.
ISO 7218:2007 - Microbiology of food and animal feeding stuff - General
rules for microbiological examinations.
ISO 7667:1983. Microbiology - Standard layout for methods of
microbiological examination.
Pallman, C., J. B. Brown, T. L. Olineka, L. Cocolin, D. A. Mills and L. F.
Bisson. 2001. Use of WL medium to profile native flora fermentations. American
Journal of Enology and Viticulture 52:198-203;
A. Cavazza, M. S. Grando, C. Zini, 1992. Rilevazione della flora
microbica di mosti e vini. Vignevini, 9-1992 17-20.ANDREWS,
W.
et
MESSER, J. (1990). Microbiological Methods. in : AOAC Official Methods of
Analysis, 15th edition, 1, 425-497, Association of Analytical Chemist,
Washington.
BIDAN, P. (1992). Analyses Microbiologiques du Vin. F.V. O.I.V. n 910,
Paris.
BOURGEOIS, C.M. et LEVEAU, J.Y. (1991). Techniques d'analyse et de
contrle dans les industries agro alimentaires, 2me dition, 3. Le Contrle
Microbiologique Lavoisier, Tec. & Doc., APRIA Ed. Paris.
CARR, J. G. (1959). Acetic acid bacteria in ciders. Ann. Rep. Long Ashton
Res. Sta., 160.
DE MAN, J. C. (1975). The probability of most probable number.
European Journal of Applied Microbiology, 1, 67-78.
LAFON-LAFOURCADE, S. et al. (1980). Quelques observations sur la
formation d'acide actique par les bactries lactiques. Conn. Vigne Vin, 14, 3,
183-194.
MAUGENET, J. (1962). Les Actobacter du cidre. Identification de
quelques souches. An. Technol. Agric., 11, 1, 45-53.
PLARIDIS et LAFON-LAFOURCADE, S. (1983). Contrle microbiologique
des vins. Bull. O.I.V., 618, 433-437, Paris.
RIBREAU-GAYON, J. et PEYNAUD, E. (2004). Trait d'Oenologie, Tome
2, Librairie Polytechnique CH. Branger, Paris et Lige.
Standard Methods for the Examination of Water and Waste Water
(1976). 14th edition, American Public Health Association, Incorporated, New
York.
Standard Methods for the Examination of Water and Waste Water
(1985). 16th edition, American Public Health Association, DC 20005,
Washington.
VAZ OLIVEIRA, M., BARROS, P. et LOUREIRO, V. (1995). Analyse
microbiologique du vin. Technique des tubes multiples pour l'numration de
micro-organismes dans les vins - "Nombre le plus probable" (NPP), F.V. O.I.V.
n 987, Paris.
VAZ OLIVEIRA, M. et LOUREIRO, V. (1993). L'numration de microorganismes dans les vins ayant un indice de colmatage lev, Compte rendu des
OIV-MA-AS4-01 : R2010
20
OIV-MA-AS4-01 : R2010
21
Appendix 2
Preparation of dilutions and inoculations
OIV-MA-AS4-01 : R2010
22
0,1
mL
0
0
1
1
2
0
0
0
1
1
2
2
3
0
0
0,01
mL
0
1
0
1
0
0
1
2
0
1
0
1
0
0
1
Positive tubes
MPN
1 mL
0,0
0,3
0,3
0,6
0,6
0,4
0,7
1,1
0,7
1,1
1,1
1,5
1,6
0,9
1,4
1
mL
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
0,1
mL
0
1
1
1
2
2
2
2
3
3
3
0
0
0
1
0,01
mL
2
0
1
2
0
1
2
3
0
1
2
0
1
2
0
Positive tubes
MPN
1 mL
2,0
1,5
2,0
3,0
2,0
3,0
3,5
4,0
3,0
3,5
4,0
2,5
4,0
6,5
4,5
1
mL
1
3
3
3
3
3
3
3
3
3
3
0.1
mL
1
1
1
2
2
2
2
3
3
3
3
0,01
mL
1
2
3
0
1
2
3
0
1
2
3
MPN
1 mL
7,5
11,5
16,0
9,5
15,0
20,0
30,0
25,0
45,0
110,0
>140,0
Adapted from the Standard Methods for the Examination of Water and Waste Water (1976)
OIV-MA-AS4-01 : R2010
23
OIV-MA-AS4-01 : R2010
24
Glucose
50 g
Peptone
5g
Yeast extract 3 g
Malt extract
3g
Agar-agar
20 g
Water: up to
1000 ml
If necessary add 100 mg chloramphenicol to suppress bacterial growth and 150
mg biphenyl to suppress mould growth.
1.2 YEPD
Glucose
20 g
Peptone
20 g
Yeast extract 10 g
Agar-agar
20 g
Water: up to
1000 ml
If necessary add 100 mg chloramphenicol to suppress bacterial growth and 150
mg biphenyl to suppress mould growth.
1.3 WL Nutrient Agar
Glucose
Peptone
Yeast extract
Potassium phosphate monobasic (KH2PO4)
Potassium chloride (KCl)
Calcium chloride (CaCl2)
Magnesium sulphate (MgSO4)
Ferric chloride (FeCl3)
Manganese sulphite (MnSO4)
Bromcresol green
Agar bacteriological
Water: up to
pH
50 g
5g
4g
0.55 g
0.425 g
0.125 g
0.125 g
0.0025 g
0.0025 g
0.022 g
12 g
1000 ml
5.5
OIV-MA-AS4-01 : R2010
25
10 g
OIV-MA-AS4-01 : R2010
26
250 ml
Solution B:
Cysteine HCl
1g
Water: up to
100 ml
pH
4.8
Sterilize by membrane filtration.
Add 100mg / L natamycin (pimaricin) to inhibit the growth of yeasts, just before
use.
Add 1 ml of solution B to 20 ml of solution A at the moment of use
2.4 Lafon-Lafourcade medium
Glucose
20 g
Yeast extract 5 g
Beef extract
10 g
Peptone
10 g
Sodium acetate 5 g
Tri-ammonium citrate 2 g
0.2 g
Magnesium sulphate 6H2O
0.05 g
Manganese sulphate 4H2O
"Tween 80"
1 ml
Agar-agar
20 g
Water: up to
1000 ml
pH
5.4
Add 100mg / L natamycin (pimaricin) to inhibit the growth of yeasts, after
autoclaving, just before use.
2.5 Dubois medium (Medium 104)
Tomato juice 250 ml
Yeast extract 5 g
Peptone
5g
Malic acid
3g
0.05 g
Magnesium sulphate 6H2O
0.05 g
Manganese sulphate 4H2O
Agar-agar
20 g
Water: up to
1000 ml
pH
4.8
Add 100mg / L natamycin (pimaricin) to inhibit the growth of yeasts, after
autoclaving, just before use.
OIV-MA-AS4-01 : R2010
27
50 g
10 g
30 g
Agar
25 g
Water: up to
1000 ml
Add 100mg / L natamycin (pimaricin) to inhibit the growth of yeasts, and 12.5
mg/L of penicillin to eradicate the growth of lactic acid bacteria, after
autoclaving, just before use.
3.2 Medium G2
Yeast extract
1.2 g
Ammonium phosphate 2 g
Apple juice
500 ml
Agar
20 g
Water: up to
1000 ml
pH
5.0
Add 100mg / L natamycin (pimaricin) to inhibit the growth of yeasts, and 12.5
mg/L of penicillin to eradicate the growth of lactic acid bacteria after
autoclaving, just before use.
3.3 Kneifel medium
Yeast extract 30 g
Ethanol
20 ml*
Agar
20 g
Bromocresol green 2.2%
OIV-MA-AS4-01 : R2010
1mL
28
OIV-MA-AS4-01 : R2010
29
3) The 10 mL volume is used instead of the 5 mL volume as with yeasts, due to the
greater sensitivity of lactic bacteria to oxygen.
OIV-MA-AS4-01 : R2010
30
OIV-MA-AS4-01 : R2010
31
OIV-MA-AS4-01 : R2010
32
Method OIV-MA-AS4-02A
Type IV method
OIV-MA-AS4-02A : R2009
Method OIV-MA-AS4-02B
Type IV method
1.1.3 Reagents
Diethyl ether
Methanol
Ethanol, 96% (v/v).
Sulfuric acid diluted to 20% (v/v)
Anhydrous sodium sulfate
Polyamide powder for chromatography (e.g., Macherey-Nagel or Merck).
Fluorescent indicator (F254 Merck or equivalent).
Solvent:
n-Pentane ..................................................10 vol.
n-Hexane .................................................. ..10 vol.
Glacial acetic acid ......................................... 3 vol.
Standard solutions:
Prepare standard solutions containing 0.1 g/100 mL of 96% ethanol (v/v)
of the following acids: sorbic, p-chlorobenzoic, salicylic, phydroxybenzoic and its esters.
OIV-MA-AS4-02B : R2009
1.1.4 Procedure
Place 50 mL of wine in a separatory funnel; acidify with dilute 20% sulfuric
acid (1.1.3.4), and extract 3 times using 20 mL diethyl ether (1.1.3.1) per
extraction. Combine the washed solutions in a separatory funnel and wash
with a few milliliters of distilled water. Dry the ether with the anhydrous
sodium sulfate (1.1.3.2). Evaporate the ether dry using a 100C water bath, or a
rotary evaporator. If the evaporation is accomplished on a water bath, it is
advisable to hasten the evaporation using a mild current of air until 2 or 3
milliliters remain, then finish the evaporation cold.
Dissolve the residue in 1 mL ethanol, deposit 3 to 5 L of this solution on the
polyamide plate, as well as 3 to 5 L of the various preservative standard
alcoholic solutions (1.1.3.9). Place the plate in a chromatography tank, and
saturate with solvent vapors. Let the solvent migrate to a height of about 15
cm, which takes from 1.5 to 2.5 hours.
Remove the plate from the tank and allow to dry at normal temperature.
Examine in ultraviolet light, at a wavelength of 254 nm. The preservatives
appear from the bottom of the plate upward in the following order: phydroxybenzoic acid, esters of p-hydroxybenzoic, salicylic acid, pchlorobenzoic acid, benzoic acid, sorbic acid.
With the exception of salicylic acid, which has a light blue fluorescence, other
preservatives give dark spots on a fluorescent yellow-green background.
Sensitivity - This technique allows determination of the following minimum
quantities of the miscellaneous preservatives expressed in milligrams per liter:
Salicylic acid .............................................. 3
Sorbic acid ............................................... 5
Esters of p-hydroxybenzoic acid ......................
5
p-Hydroxybenzoic acid ................................ 5-10
p-Chlorobenzoic acid .................................... 5-10
Benzoic acid ............................................. 20
1.2 High performance liquid chromatography
1.2.1 Procedure
The method is performed directly on the wine, without sample preparation. It
is necessary to dilute red wines before injecting them in order to preserve the
column.
OIV-MA-AS4-02B : R2009
BIBLIOGRAPHY
JUNGE Ch., Zeits. Unters. Lebensmit., 1967, 133, 319
OIV-MA-AS4-02B : R2009
Method OIV-MA-AS4-02C
Type IV method
OIV-MA-AS4-02C : R2009
1.4 Procedure
Place 100 mL of test wine in an extraction flask with a ground glass stopper;
add 2 mL hydrochloric acid (1.3.2) and 100 mL diethyl ether (1.3.1). Shake
the contents vigorously for a few seconds by hand, then for 1 h with a
mechanical stirrer (1.2.2). Transfer to a separating funnel, allow to separate
and recover the ether layer.
Shake the ether extract with 8 to 10 g of anhydrous sodium sulfate (1.3.3) for a
few seconds.
Transfer the extract to the separating funnel, add 10 mL sodium hydroxide
solution, 0.5 M (1.3.5); shake for 1 min. Allow to settle.
Remove 0.5 mL of the alkaline extract and check, by titration with sulfuric
acid, 0.05 M, so that the strength falls between 0.4 and 0.6 M. Transfer the
alkaline extract contained in the separating funnel into a test tube containing 1
mL of thiosalicylic acid solution. Adjust, if necessary the strength of the
alkaline extract in order to bring it to the limits indicated, using a stronger
sodium hydroxide solution of known strength. Shake the contents of the test
tube for 30 seconds and transfer to an evaporating dish.
Place the dish on a water bath at 100C blowing its surface with a current of
cold air. Maintain the dish on the water bath at 100C for exactly 1 hour; the
residue may become practically dry in a shorter amount of time. If a crust
forms on the surface of the residue during the evaporation, it is advisable to
break or grind it up with a thin glass rod to facilitate the evaporation.
Place the dish in an oven maintained at 200 2C for exactly 30 minutes.
After cooling, recover the contents of the dish with 4 mL of water; transfer into
a separation funnel, add to the dish 3 mL of potassium ferricyanide solution to
fully dissolve any remaining residue and add to the separating funnel. Shake
for 30 seconds to facilitate oxidation. Add 5 mL chloroform, mix using 3 to 4
inversions. Allow to separate.
A pink or red color (according to the quantity of thioindigo formed) indicates
the presence of monohalogen derivatives of acetic acid.
Sensitivity - The method allows detection of 1.5 to 2 mg monochloroacetic acid
per liter of wine and corresponding quantities of the other derived
monohalogens. Since the yield of miscellaneous extractions is not quantitative,
this method cannot be used for determining the amount of these monohalogen
derivatives in the wines.
OIV-MA-AS4-02C : R2009
BIBLIOGRAPHY
FRIEDLANDER, Ber. Deutsch. Chem. Gesell., 1906, 39, 1062.
RAMSEY L.L., PATTERSON W.I., J. Ass. Off. Agr. Chem., 1951, 34, 827.
PERONNET M., ROCQUES S., Ann. Fals. Fraudes, 1953, 21-23.
Trait de chimie organique, edited by V. Grignard, 1942, 19, 565-566.
Official Methods of Analysis of the Association of Official Analytical Chemists,
11th dition, publie par l'Association of Official Analytical Chemists,
Washington, 1970, 340-341.
TERCERO C., F.V., O.I.V., 1967, n 224.
OIV-MA-AS4-02C : R2009
Method OIV-MA-AS4-02D
Type IV method
and
determination
of
ethyl
pyrocarbonate
(diethyl
1.1 Principle
The diethyl carbonate formed by degradation of ethyl pyrocarbonate (diethyl
ester of pyrocarbonic acid) in the presence of ethanol is extracted from wine
using carbon disulfide and the quantity determined by gas chromatography.
Either of the procedures described below may be used.
1.2 Apparatus
1.2.1 Gas chromatography with flame ionization detector.
1.2.2 Columns:
- Capillary column coated with Carbowax 1540
Column length: 15.24 m
Inside diameter: 0.51 mm
- Polypropyleneglycol on Celite 545 (15:100), 60-100 mesh
Column length: 2 m
Interior diameter: 3 mm
1.3 Reagents
1.3.1 Anhydrous sodium sulfate
1.3.2 Carbon disulfide
The carbon disulfide must contain no impurities in the critical retention zone
(5 to 7 min.) for maximum sensitivity in accordance with the conditions of gas
chromatography as indicated in paragraph 1.4.2.
1.4 Procedure
1.4.1 Use of the capillary column.
Place 100 mL wine in a 250 mL separating funnel with 1 mL of carbon
disulfide (1.3.2). Mix vigorously for 1 min. The carbon disulfide phase
separated is rapidly centrifuged, then dried with anhydrous sodium sulfate
(1.3.1).
Inject 10 l of the clear liquid supernatant into the chromatograph.
Chromatography conditions:
Detector gases:
OIV-MA-AS4-02D : R2009
hydrogen: 37 mL/min.
air: 250 mL/min.
Gas flow:
nitrogen: 40 mL/min.
A 1/10 splitter sends to the detector the gas mixture with a flow rate
of 3 to 5 mL/min.
Temperature:
injector: 150 C; oven: 80 C; detector: 150 C
Detection limits:
0.05 mg/L of wine
1.4.2 Use of the column for polypropyleneglycol.
Add 20 mL of wine and 1 mL of carbon disulfide (1.3.2) into a conical
centrifuge tube with a stopper. Agitate vigorously for 5 minutes, then
centrifuge for 5 minutes applying a centrifugal force of 1000 to 1200 g. The
liquid supernatant produced is aspirated by a thin-tipped pipette; the carbon
disulfide phase is dried with a small quantity of anhydrous sodium sulfate,
added while stirring with a glass rod. Inject 1 L of the clear liquid into the
gas chromatograph.
Chromatography conditions.
Detector gas:
hydrogen: 35 mL/min.
air: 275 mL/min.
Carrier gas flow:
nitrogen: 25 mL/min.
Temperature:
injector: 240 C
oven: 100 C
detector: 240 C
Sensitivity range:
12 x 10-11 A to 3 x 10-11 A
Chart speed:
1 cm/min.
Detection limit:
0.10 - 0.05 mg/L of wine
Under these exact conditions, diethyl carbonate displays a retention time of
about 6 min.
The calibration of the apparatus is carried out using solutions of 0.01 and
0.05% (m/v) diethyl carbonate in carbon disulfide (1.3.2).
OIV-MA-AS4-02D : R2009
1.5 Calculation
Quantitative determination of diethyl carbonate is carried out preferably using the
internal standard method, referring to the peaks of the iso-butyl alcohol or iso-amyl
alcohol which are close to that of diethyl carbonate.
Prepare two samples of test wine: one of wine with 10 mL 10% ethanol (v/v)
added, the other the same wine to which has been added 1 mg diethyl carbonate
per liter using 10 mL of a 100 mg/L solution of diethyl carbonate in 10% ethanol
(v/v).
Treat these two samples according to one or the other of the techniques above
according to the column used.
Let:
S
Sx
i
I
OIV-MA-AS4-02D : R2009
BIBLIOGRAPHY
KIELHOFER E., WURDIG G., Dtsch. Lebensmit. Rdsch., 1963, 59, 197-200 &
224-228.
PRILLINGER F., Weinberg u. Keller, 1967, 14, 5-15.
REINHARD C., Dtsch. Lebensmit. Rdsch., 1967, 5, 151-153.
BANDION F., Mitt. Klosterneuburg, Rebe u. Wein, 1969, 19, 37-39.
OIV-MA-AS4-02D : R2009
Method OIV-MA-AS4-02E
Type IV method
BIBLIOGRAPHY
HALLER H.E., JUNGE Ch., F.V., O.I.V., 1972, n 397, Mitt. Bl. der Gd CH,
Fachgruppe, Lebensmitt. u. gerichtl. Chem., 1971, 25, n 5, 164-166.
OIV-MA-AS4-02E : R2009
Method OIV-MA-AS4-02F
Type IV method
OIV-MA-AS4-02F : R2009
1.1.4 Procedure
1.1.4.1 Preparation of the sample.
Into a spherical flask with a ground glass neck, place 100 mL of wine, distill
by plunging the end of the condenser in 10 mL of 5% sodium hydroxide
solution (1.1.3), to which are added a few drops of reagent indicator. Distill
until 40-50 mL of distillate is recovered.
Transfer the distillate into another spherical flask (1.1.2.2), rinse the flask
twice with 20 mL of water and add water to bring to 100 mL. To eliminate the
ethanol, attach the flask to the distillation apparatus and eliminate about 50 mL
of distillate (reduce the volume by half).
Cool the flask completely. Acidify with 10% sulfuric acid. Distill, recover the
distillate into a 10 mL flask with a ground glass stopper containing 1 mL of
water, and immerse in an iced bath. Stop the distillation when the total volume
reaches 10 mL.
1.1.4.2 Derivitization
Mix 1 mL distillate (1.1.4.1), 0.5 mL of acetonitrile, 0.2 mL buffer solution
and 30 L of derivatizing reagent and stir vigorously; leave for five minutes.
1.1.4.3 Chromatography
Inject 20 L in accordance with the conditions specified, the hydrazoic acid
derivative has a retention time of about 11 minutes. Detection limit: 0.01 mg/L.
Note: Sometimes another substance not derivatized can simulate hydrazoic
acid. It is necessary to verify a positive result as follows: inject 20 L of
distillate directly; a disappearance of the peak indicates the presence of
hydrazoic acid.
1.1.5 Calculation
To determine the concentration of sodium azide, compare the sample response
to that of the standard solution after derivatization. Take into account the
concentration factor 10 of the sample of wine at the time of analysis.
1.2 Colorimetric method
1.2.1 Principle
Hydrazoic acid, which is very volatile, is separated by double distillation,
permitting the elimination of ethanol, acetic acid and sulfur dioxide. Then the
amount is determined colorimetrically after forming a colored complex with
ferric chloride (maximum absorbance at 465 nm).
1.2.2 Apparatus
1.2.2.1 Simple distillation apparatus, consisting of a 500 mL flask with a
ground glass neck and a condenser ending in a pointed tube
1.2.2.2 Spectrophotometer with optical glass cells 1 cm path length
OIV-MA-AS4-02F : R2009
1.2.3 Reagents
1.2.3.1 Sodium hydroxide solution, 1 M
1.2.3.2 Sulfuric acid, 1 M
1.2.3.3 Hydrogen peroxide, 3% (v/v), whose strength must be adjusted just
before use using a solution of potassium permanganate, 0.02 M; where p mL
equals the volume which oxidizes 1 mL of the hydrogen peroxide solution, 3%
1.2.3.4 Ferric chloride solution at 20 g per liter of Fe III: (weigh 96.6 g of
FeCl3.6H2O, or more as this salt is very hygroscopic; control the concentration
of Fe III of the solution and adjust if necessary to 20 0.5 g per liter)
1.2.3.5 Stock solution of sodium azide, NaN3, at 1 g per liter in distilled water
1.2.3.6 200 mg per liter sodium azide solution prepared by dilution of the
solution at 1 g per liter
1.2.4. Procedure
a) Into a 500 mL flask with a ground glass neck, place 200 mL of wine, distill,
recover the distillate in a 50 mL volumetric flask, containing 5 mL water,
which is immersed in an iced bath. Stop the distillation when the total
volume reaches about 50 mL.
b) Transfer quantitatively the distillate into another 500 mL flask with a
stopper and rinse the 50 mL flask twice with 20 mL of water.
Neutralize using 1 M sodium hydroxide solution (1.2.3.1) (using pH
indicator paper).
Acidify using 10 mL 1 M sulfuric acid (1.2.3.2), mix, then oxidize the
sulfur dioxide by adding 3% hydrogen peroxide solution (1.2.3.3.).
If the wine contains S mg per liter of sulfur dioxide, and if p mL is the
volume of 0.02 M potassium permanganate solution necessary to oxidize 1
mL of 3% hydrogen peroxide solution, then for 200 mL of wine use the
following calculation:
S
S mL of H2O2 solution
5 x 3.2p 16p
OIV-MA-AS4-02F : R2009
BIBLIOGRAPHY
HPLC method:
SEARIN S.J. & WALDO R.A., J. Liquid. Chrom., 1982, 5(4), 597-604.
BATTAGLIA R. & MITISKA J., Z. Lebensm. Unters. Forsch., 1986, 182, 501-502.
Colorimetric method:
CLERMONT S. & CHRETIEN D., F.V., O.I.V., 1977, n 627.
OIV-MA-AS4-02F : R2009
Type IV method
Method OIV-MA-AS4-03
OIV-MA-AS4-03: R2011
Until now two regions specific to the species have been used as targets. One
region is the encoding gene for the 26S ribosomal RNA (ribonucleic acid)
and the other the RAD4 gene [2, 3]. As with the FISH method, PCR is
specific to Brettanomyces bruxellensis but has the advantage of being less
expensive.
The distinctive feature of qPCR is that it is possible to read, after each
amplification cycle, the fluorescence which increases exponentially as the
DNA amplification proceeds. Many fluorescence techniques have been
developed for this application. The SYBR Green fluorophore has been
found to be suitable for use with Brettanomyces.
SYBR Green fluorophore
This agent fluoresces strongly when it inserts itself non-specifically between
the nucleotides in the double-stranded DNA. In contrast, it fluoresces only
weakly when unbound. Using this technology, a merged curve can be
generated at the end of the amplification that validates the specificity of the
reaction.
Internal standard
In order to validate the DNA extraction and amplification stages, an internal
standard has been integrated into the method (Lip4 Yarrowia lipolytica).
4. Reagents and products
All plastic consumables must be autoclaved beforehand to destroy any
DNases (deoxyribonucleases), as must the Tris-HCl and TE (Tris EDTA,
ethylene diamine tetra-acetic acid) buffer solutions, the ammonium acetate
and the ultrapure water ( 18 M). All the aqueous solutions are prepared
using ultrapure water (18 M). Some solutions are sterilized in an autoclave
(indicated as "autoclaved"). Sterile ultrapure water (18 M) is used, if
possible, to prepare any solutions which are not autoclaved. It is not then
necessary to work under sterile conditions.
PVPP(eg:ISPPolyclarSuperRorSigmaP6755100G),
Solutions at room temperature: Tris-HCl buffer, 10mM pH8, solution I
(Tris-HCl 10mM pH8, EDTA 1mM, NaCl 100mM, SDS 1% (sodium
OIV-MA-AS4-03: R2011
PCR substances
4.1 ammonium acetate
>98%
CAS Number
631-61-8
Ultra
136112-00-0
39450-01-6
4.3 proteinase K
4.4 SDS
>99% Ultra
151-21-3
>99.8% Ultra
77-86-1
4.6 BSA
9048-46-8
4.2 phenol:chloroform:IAA
(24:25:1)
Specifications
108-95-2
9003-39-8
4.9 RNase A
70 U/mg in solution
9001-99-4
Ultra
Tris : 77-86-1
4.10 TE pH8
EDTA : 6000-4
OIV-MA-AS4-03: R2011
5. Apparatus
Plastic consumables: 2mL screw-capped microtubes, 1.5 and
1.7mL microtubes, white (10 L), yellow (200 L) and blue (1000
L) pipette tips for micropipettes P20, P200, P1000, P5000, 96-well
PCR microplates and optical film, non-powdered gloves
Glass beads ( 500 m)
Bottle (20mL) autoclaved (for ultrapure [18 M] sterilized water,
one per qPCR plate),
15 and 50 mL Centrifuge tubes
Equipment:
automatic pipettes (P20, P200, P1000, P5000)
microtube centrifuge
automatic stirrer to split cells (eg. GenieDisruptor)
Thermocycler coupled to a spectrofluorimeter (optical system to
detect the fluorescence generated during the real-time PCR
reactions)
Magnetic stirrer
Stop watch
dry bath set to 37C
autoclave
100mL volumetric flasks
50mL volumetric flasks
10mL volumetric flasks
100mL beakers
50mL beakers
10mL beakers
Magnetic stirring bars
OIV-MA-AS4-03: R2011
OIV-MA-AS4-03: R2011
OIV-MA-AS4-03: R2011
One tube will be used for extracting the DNA and the other will be
stored at 20C until validated results have been obtained.
7.2. Extracting the DNA
From a fresh or frozen pellet. Do not process more than 24 samples at
the same time.
-
add PVPP (1% of final mass/volume) by weighing add 0.3 g of 200500m glass beads
add 200L of solution I
add 200L of phenol:chloroform:IAA (24:25:1)
disrupt the cells with the automatic stirrer (for example a GenieDisruptor)
4x80 sec. with cold intervals (-20C refrigerated unit) lasting for about
80sec between each disruption phase
add 200L of TE
centrifuge for 5min at 15700g.
carefully collect 400L of the upper aqueous phase in a 1.7mL microtube.
If the two phases mix, repeat the centrifugation step.
add 1mL of absolute ethanol and mix the tube by inversion 4-5 times * (a
few hours, room T)
7.3. qPCR
For each sample of wine, provide 2 wells with Brett rad3/4 primers and 2
internal standard wells with YAL primers. For each plate, provide a
negative control with TE for each pair of primers to be carried out as the
final operation. Also perform a positive control on the Brettanomyces
bruxellensis DNA available at -20C. To prepare the positive control,
add 5L stock solution (4.5 UG / ml) in a final reaction volume of 25 L.
PCR amplification programme:
Cycle number
1
40
Time (seconds)
Temperature (C)
180
95
30
95
10
64.6
The merged curve is established after 90C by reducing the heat by
0.5C every 10 seconds
OIV-MA-AS4-03: R2011
number of
samples
number of
wells
water at18
M (L)
iQ SYBR Green
Supermix (L)
Mixture of 4 M
primers (L)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
26.3
36.8
47.3
57.8
68.3
78.8
89.3
99.8
110.3
120.8
131.3
141.8
152.3
162.8
173.3
183.8
194.3
204.8
215.3
225.8
236.3
246.8
257.3
65.6
91.9
118.1
144.4
170.6
196.9
223.1
249.4
275.6
301.9
328.1
354.4
380.6
406.9
433.1
459.4
485.6
511.9
538.1
564.4
590.6
616.9
643.1
13.1
18.4
23.6
28.9
34.1
39.4
44.6
49.9
55.1
60.4
65.6
70.9
76.1
81.4
86.6
91.9
97.1
102.4
107.6
112.9
118.1
123.4
128.6
OIV-MA-AS4-03: R2011
remove the Brett 4 M and the YAL 4 M primers from the freezer
remove the SYBR Green (4C if tube in current use, otherwise 20C)
prepare a Brett mixture and a YAL mixture using the quantities shown
in the table above as a function of the number of samples.
apply 20L of mixture to the bottom of each well
add 5L of homogenized DNA solution to the automatic stirrer (or
5L of water for the negative controls)
adjust the optical film and load the plate
remove the plate and place it directly in the bag for disposal (do not open
it)
set the baseline to 100.
analyze (in the order indicated below):
o the negative controls, which should not produce a signal. If a Ct of
less than 37 is observed, repeat the process, changing all the
solutions,
o the positive control on Brett: its Ct must be approximately 25, with
a melting point of 82.5C ( 0.5C),
o YAL internal standards: if a Ct is obtained, check the melting point
of the product (84C 0.5C). If the product does not conform, the
absence of a Brett signal cannot be interpreted,
o samples: check the Tm of the Brettanomyces bruxellensis product
(82C 0.5C). If and only if the Tm is acceptable, check the
exponential profile of the amplification. Then record the Ct values
and plot them onto the standard curve.
NB: the Ct represents the time needed for the fluorescence of the target
sequence to reach a threshold value. Consequently, it is the minimum
number of PCR cycles required for the fluorescent signal to emerge from the
background noise.
8. Calculations (Results)
Five Brettanomyces bruxellensis strains were inoculated at different
concentrations, from 3,1 x 105 to 3 UFC/mL, on 14 wines (3 white wines, 2
ros wines, 9 red wines whose phenolic compound content varied widely).
The DNA was then extracted in the presence of 1% PVPP.
OIV-MA-AS4-03: R2011
10
A standard curve was established from the set of results obtained on the
different combinations of wines and strains.
The results are obtained in GU/mL (genetic unit/mL) from the standard
curve
logGU = log(2.03)Ct + 12.34
Standard curve
8,00
7,00
logUG
6,00
5,00
4,00
3,00
2,00
I > 1,2log
1,00
I = 1,2log
(I max =1,6log)
I > 1,2log
0,00
20
22
24
26
28
30
32
34
36
38
Ct
11
Fidelity of the method was compared to that obtained with the classical
culture method. Three operators prepared DNA extracted from a wine
inoculated with the L02I1 strain at two levels: 1.9x104 (high) or 1.9x102
(low) CFU/mL. Four repeats of PCR were performed for each DNA extract.
The standard deviation for repeatability and reproducibility, respectively Sr
and SR, were calculated from log GU values for both levels (table below).
For the qPCR method, both Sr and SR were similar for the low population
level, but SR was greater than Sr at high population levels. Both standard
deviations were twice as high as those obtained with the classical
microbiology method. This effect was attributed to the increased number of
steps during the qPCR method.
Table
Parameter
Regression equation
Range (CFU/mL)
Slope (SD)
Intercept (SD)
Regression model
Model error
Values
0 to 2x105
0.957 (0.044)
-0.049 (0.142)
Fobs>F(1.18) : Linear model accepted
Fobs<F(4.18) : No model error
Fidelity
Sr qPCR (low/high)
Sr microbio (low/high)
SR qPCR (low/high)
SR microbio (low/high)
0.26/0.25
0.17/0.04
0.29/0.41
0.17/0.04
Accuracy
Mean 43 samples (D)
SR D
Equality test W=D/SR D
12
background. We thus used two other approaches to evaluate LoD and LoQ.
The first method uses slope, intercept and standard error on intercept
obtained from linearity validation experiments. With this method, LoD and
LoQ values of 3 and 31 GU/mL respectively were obtained. In the second
approach, the LD was obtained from the population level resulting in one
negative result from 10 independent measurements. Analysis of our data
obtained from 14 wines inoculated with five strains revealed that 96% of
samples (48/50) containing 101 to 250 CFU/mL resulted in positive signals,
while 83% (49/59) were positive if they contained 26 to 100 CFU/mL and
65% (44/68) if 5 to 25 CFU/mL. Thus the limit of detection evaluated from
this method would be in the range of 26-100 CFU/mL. By the systematic
repetition of each PCR assay, an LoD of 5 CFU/mL was certified thanks to
probability calculations (1 p)2. Indeed for 5 CFU/mL, 88% of samples
were positive. This increased to 97% for 25 CFU/mL.
10.
References
1. Phister T.G., Mills D.A., 2003. Real-time PCR assay for detection and
enumeration of Dekkera bruxellensis in wine. Applied and
Environmental Microbiology, 69: 7430-7434.
2. Cocolin L., Rantsiou K., Iacumin L., Zironi R., Comi G., 2003.
Molecular detection and identification of Brettanomyces/Dekkera
bruxellensis Brettanomyces/Dekkera anomalus in spoiled wines.
Applied and Environmental Microbiology, 70: 1347-1355.
3. Ibeas J.I., Lozano I., Perdigones F., Jimenez J., 1996. Detection of
Dekkera-Brettanomyces strains in sherry by a nested PCR method.
Applied and Environmental Microbiology, 62: 998-1003.
4. Tessonnire H., Vidal S., Barnavon L., Alexandre H., Remize F., 2009.
Design and performance testing of a real-time PCR assay for sensitive
and reliable direct quantification of Brettanomyces in wine.
International Journal of Food Microbiology, 129: 237-243.
OIV-MA-AS4-03: R2011
13
Method OIV-MA-AS5-01
Type IV method
0.25 g/L
0.5 g/L
0.5 g/L
1.0 g/L
1.0 g/L
3.3 Procedure
3.3.1 Preparation of the ion exchange column.
See chapter on Tartaric acid, usual method in 3.3.1.
3.3.2 Isolation of the organic acid of citramalic acid
OIV-MA-AS5-01 : R2009
Proceed as indicated in the chapter Tartaric acid, usual method in 3.3.2. for
the fixation of organic acids on the ion exchanger.
Then elute the fixed acids using the 4 M formic acid solution (100 mL),
collecting the eluate in a 100 mL volumetric flask.
Concentrate the eluate dry in a rotary evaporator at 40C recovering the
residue with 1 mL of distilled water.
3.3.3 Chromatography
The cellulose plate must be activated by placing it in the oven at 100C for 2
hours.
Deposit on the starting line of the cellulose plate in a band 2 cm wide, 10 L of
this solution as well as 10 L of the standard solutions of citramalic acid and
the other organic acids.
Place the plate in the chromatography bath, above the solvent, for 45 minutes.
Proceed with the development and let the solvent migrate to a height of 15 cm.
3.3.4 Development of the chromatogram
Maintain the plate at ambient temperature under an air current, until the formic
acid of the solvent is eliminated. Yellow spots appear on a blue background,
indicating the presence of the acids.
Detect the presence or absence of citramalic acid in the product analyzed by
comparing the spots of this chromatogram to the spots of standard solutions of
citramalic acid and the other organic acids.
BIBLIOGRAPHY
Method of Screening:
HARVALIA A., F.V., O.I.V., 1980, n 728 bis.
Chromatography of citramalic acid:
DIMOTAKI-KOURAKOU V., Ann. Fals. Exp. Chim., 1960, 53, 149.
DIMOTAKI-KOURAKOU V., C. R. Ac. Sci., Paris 1962, 254, 4030.
CARLES J., LAMAZOU-BETBEDER M. & PECH M., C. R. Ac. Sci., Paris 1958,
246, 2160.
CASTINO M., Riv. Vit. Enol., 1967, 6, 247.
KOURAKOU V., F.V., O.I.V., 1977, n 642.
JUNGE Ch F.V., O.I.V., 1978, n 679.
ROUEN J., F.V., O.I.V., 1979, n 691.
OIV-MA-AS5-01 : R2009
Annex B
Certificates of analysis
OIV-MA-ANNEX-B
OIV-MA-B1-01
Certificates of Analysis
Certificate No. 1
- Color
- Clarity
- Specific gravity at 20C
- Alcoholic content at 20C
- Total dry extract g/L
- Sugar g/L
- Total sulfur dioxide mg/L
- pH
- Total acidity meq/L
- Volatile acidity meq/L
- Test for malvidin diglucoside
- Over pressure measurement of carbon dioxide in sparkling wines
- Differentiation of very sweet wine and fortified must in the case of
sweet wines
Certificate No. 2
Certificate No. 1 is completed and the following determinations are added:
- Ash and alkaline ash g/L
- Potassium g/L
- Iron mg/L
- Copper mg/L
- Free sulfur dioxide mg/L
- Sorbic acid mg/L
- Verification of malolactic fermentation
- Citric acid mg/L
- Tartaric acid g/L
- Folin-Ciocalteu Index
- Chromatic Indexes
The following determinations are optional:
- Excess sodium mg/L
- Calcium, magnesium mg/L
- Sulfates mg/L
- Test of fermentability
- Test for artificial colorants
OIV-MA-B1-02
Annex C
Maximum acceptable limits
of various substances
OIV-MA-ANNEX-C
Citric acid:
1 g/L
Volatile acidity:
20 milliequivalents/L
The volatile acidity of various specially fortified old
wines (wines subject to special legislation and
controlled by the government) may exceed this
limit.
Arsenic:
0.2 mg/L
Boron:
Bromine:
Cadmium:
0.01 mg/L
Copper:
1 mg/L
(oeno 434-2011)
Diethylene glycol:
Malvidol diglucoside:
Silver
OIV-MA-C1-01: R2011
Total sulfur dioxide at the time of sale to the consumer: (oeno 9/98)
10 mg/L
Methanol:
(oeno 19/2004)
Ochratoxin A :
(CST 1/2002)
Propan-1,2-diol/propylene
glycol
(oeno 20/2003)
Excess sodium:
(oeno 12/2007)
OIV-MA-C1-01: R2011
80 mg/L
Sulfates:
(expressed
sulfate)
1 g/L
as
Zinc
OIV-MA-C1-01: R2011
1.5
g/L
2.0
g/L
voile"
2.5
g/L
5 mg/L
Annex D
Advices
OIV-MA-ANNEX-D
Gluconic acid
Resolution oeno 4/91
OIV-MA-D1-01
NOTICE
OIV-MA-D1-02
The level of Chloride and sodium ions in wines essentially depends on the
geographic, geologic and climatic conditions of vine culture.
As a general rule, the levels of these ions are low.
the content of these elements is increased in wines coming from vineyards which
are near the sea coast, which have brackish subsoil or which have arid ground
irrigated with salt water and the molar ratio cf Cl/Na+ therefore varies significantly
and can even have a value close to one (1) which could imply the addition of salt
(NaCl) to the wine.
When wine contains excess sodium (excess sodium is equal to the content of
sodium ions less the content of chloride ions expressed as sodium), it is generally
less than 60 mg/L, a limit which may be exceeded in exceptional cases.
The laboratories and official control agencies, confronted with elevated levels of Cl
and/or Na+, must take the above conclusions into account and possib1y make
inquiries to the official agencies of the country of origin before expelling these
wines.
OIV-MA-D1-03
Annex E
Laboratory quality
assurance
OIV-MA-ANNEX-E
OIV-MA-AS1-05 : R1998
Collaborative Study
The purpose of the collaborative study is to give a quantified indication of the
precision of method of analysis, expressed as its repeatability r and reproducibility
R.
Repeatability: the value below which the absolute difference between two single
test results obtained using the same method on identical test material, under the
same conditions (same operator, same apparatus, same laboratory and a short
period of time) may be expected to lie within a specified probability.
Reproducibility: the value below which the absolute difference between two single
test results obtained using the same method on identical test material, under
different conditions (different operators, different apparatus and/or different
laboratories and/or different time) may be expected to lie within a specified
probability.
The term "individual result" is the value obtained when the standardized trial
method is applied, once and fully, to a single sample. Unless otherwise stated, the
probability is 95%.
General Principles
The method subjected to trial must be standardized, that is, chosen from the
existing methods as the method best suited for subsequent general use.
The protocol must be clear and precise.
The number of laboratories participating must be at least ten.
The samples used in the trials must be taken from homogeneous batches of
material.
The levels of the analyte to be determined must cover the concentrations
generally encountered.
Those taking part must have a good experience of the technique employed.
For each participant, all analyses must be conducted within the same laboratory
by the same analyst.
The method must be followed as strictly as possible. Any departure from the
method described must be documented.
The experimental values must be determined under strictly identical conditions:
on the same type of apparatus, etc.
They must be determined independently of each other and immediately after
each other.
The results must be expressed by all laboratories in the same units, to the same
number of decimal places.
Five replicate experimental values must be determined, free from outliers. If an
experimental value is an outlier according to the Grubbs test, three additional
measurements must be taken.
OIV-MA-AS1-07 : R2000
Statistical Model
The statistical methods set out in this document are given for one level
(concentration, sample). If there are a number of levels, the statistical evaluation
must be made separately for each. If a linear relationship is found (y = bx or y = a
+ bx) as between the repeatability (r) or reproducibility (R) and the concentration
( x ), a regression of r (or R) may be run as a function of x
The statistical methods given below suppose normally-distributed random values.
The steps to be followed are as follows:
A/ Elimination of outliers within a single laboratory by Grubbs test. Outliers are
values which depart so far from the other experimental values that these
deviations cannot be regarded as random, assuming the causes of such
deviations are not known.
B/ Examine whether all laboratories are working to the same precision, by
comparing variances by the Bartlett test and Cochran test. Eliminate those
laboratories for which statistically deviant values are obtained.
C/ Track down the systematic errors from the remaining laboratories by a
variance analysis and by a Dixon test identify the extreme outlier values.
Eliminate those laboratories for which the outlier values are significant.
D/ From the remaining figures, calculate standard deviation of repeatability); Sr.,
and repeatability r standard deviation of reproducibility SR and reproducibility
R.
Notation:
The following designations have been chosen:
m
Number of laboratories
i(i = 1, 2... m)
Index (No. of the laboratory)
ni
Number of individual values from the ith laboratory
m
N n i
i l
xi
x
si
1
ni
ni
x
i =1
1 m ni
xi
N i =1 i =1
1 ni
x i x i
ni-1 i 1
OIV-MA-AS1-07 : R2000
xi* xi
si.
Calculate PG =
x *i = suspect value
Compare PG with the corresponding value shown in Table 1 for P = 95%.
If PG < value as read, value x * is not an outlier and s can be calculated.
i
i
*
If PG > value as read, value x probably is an outlier therefore make a further
three determinations.
Calculate the Grubbs test for x * with the eight determinations.
i
If PG > corresponding value for P = 99%, regard x * as a deviant value and
calculate s without x * .
i
i
B/ Comparison of variances among laboratories
- Bartlett Test
The Bartlett test allows us to examine both major and minor variances. It serves to
test the null hypothesis of the equality of variances in all laboratories, as against
the alternative hypothesis whereby the variances are not equal in the case of some
laboratories.
At least five individual values are required per laboratory.
Calculate the statistics of the test:
PB
m
1
m
ln
ln 2
S
r f i si
C
i 1
f N m
C i 1
3m1
f s
2
i I
2
r
i 1
N m
OIV-MA-AS1-07 : R2000
fi = ni - 1 degrees of freedom of s .
i
Compare PB with the value x2 indicated in table 2 at m - 1 degrees of freedom.
If PB > the value in the table, there are differences among the variances.
The Cochran test is used to confirm that the variance from one laboratory is
greater than that from other laboratories.
Calculate the test statistics:
PC
si max
m
2
i
i 1
OIV-MA-AS1-07 : R2000
PF
sZ
2
sI
1 m
n i x
m-1 i =1 xi
1 m ni
xi xi
N m i =1 i =1
2
Z
2
I
Z 2Z 1
Z H Z 1
oder
Z H Z H 1
Z H Z 1
Z 2Z 1
Z H 1Z 1
oder
Z H Z H 1
Z H Z 2
Z 3Z 1
Z H 2Z 1
oder
Z H Z H 2
Z H Z 3
3 to 7
Q10
8 to 12
Q11
13 plus
Q22
Compare the greatest value of Q with the critical values shown in table 5.
If the test statistic is > the table value at P = 95%, the mean in question can be
regarded as an outlier.
If there is a significant result in the R A Fischer variance analysis or the Dixon
test, eliminate one of the extreme values and calculate the test statistics again with
the remaining values. As regards repeated application of the tests, see the
explanations in paragraph (B).
OIV-MA-AS1-07 : R2000
sr
1 m
2
f is i
N m i 1
r sr2 2
sR
1 2
sZ a 1s2I
a
Rs R2 2
2
m
1
n i
N
m1
i 1 N
If there is no difference between the means from the laboratories, then there is no
difference between sr and sR or between r and R. But, if we find differences
among the laboratory means, although these may be tolerated for practical
considerations, we have to show sr and sR, and r and R.
OIV-MA-AS1-07 : R2000
BIBLIOGRAPHY
OIV-MA-AS1-07 : R2000
P = 95%
3
4
5
6
7
8
9
10
11
12
1,155
1,481
1,715
1,887
2,020
2,126
2,215
2,290
2,355
2,412
P 99%
1,155
1,496
1,764
1,973
2,139
2,274
2,387
2,482
2,564
2,636
X2
3,84
5,99
7,81
9,49
11,07
12,59
14,07
15,51
16,92
18,31
19,68
21,03
22,36
23,69
25,00
26,30
27,59
28,87
30,14
31,41
OIV-MA-AS1-07 : R2000
f(m - 1)
X2
21
22
23
24
25
26
27
28
29
30
35
40
50
60
70
80
90
100
32,7
33,9
35,2
36,4
37,7
38,9
40,1
41,3
42,6
43,8
49,8
55,8
67,5
79,1
90,5
101,9
113,1
124,3
2
3
4
5
6
7
8
9
10
ni =
99%
0.993
0.968
0.928
0.883
0.838
0.794
0.754
0.718
2
95%
0.967
0.906
0.841
0.781
0.727
0.680
0.638
0.602
ni= 3
99%
95%
0.995 0.975
0.942 0.871
0.864 0.768
0.788 0.684
0.722 0.616
0.664 0.561
0.615 0.516
0.573 0.478
0.536 0.445
ni = 4
99%
95%
0.979 0.939
0.883 0.798
0.781 0.684
0.696 0.598
0.626 0.532
0.568 0.480
0.521 0.438
0.481 0.403
0.447 0.373
ni = 5
99%
95%
0.959 0.906
0.834 0.746
0.721 0.629
0.633 0.544
0.564 0.480
0.508 0.431
0.463 0.391
0.425 0.358
0.393 0.331
ni = 6
99%
95%
0.937 0.877
0.793 0.707
0.676 0.590
0.588 0.506
0.520 0.445
0.466 0.397
0.423 0.360
0.387 0.329
0.357 0.303
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.684
0.653
0.624
0.599
0.575
0.553
0.532
0.514
0.496
0.480
0.465
0.450
0.437
0.425
0.413
0.402
0.391
0.382
0.372
0.363
0.355
0.347
0.339
0.332
0.325
0.318
0.312
0.306
0.300
0.294
0.570
0.541
0.515
0.492
0.471
0.452
0.434
0.418
0.403
0.389
0.377
0.365
0.354
0.343
0.334
0.325
0.316
0.308
0.300
0.293
0.286
0.280
0.273
0.267
0.262
0.256
0.251
0.246
0.242
0.237
0.504
0.475
0.450
0.427
0.407
0.388
0.372
0.356
0.343
0.330
0.318
0.307
0.297
0.287
0.278
0.270
0.262
0.255
0.248
0.241
0.235
0.229
0.224
0.218
0.213
0.208
0.204
0.200
0.196
0.192
0.418
0.392
0.369
0.349
0.332
0.316
0.301
0.288
0.276
0.265
0.255
0.246
0.238
0.230
0.222
0.215
0.209
0.202
0.196
0.191
0.186
0.181
0.177
0.172
0.168
0.165
0.161
0.157
0.154
0.151
0.366
0.343
0.322
0.304
0.288
0.274
0.261
0.249
0.238
0.229
0.220
0.212
0.204
0.197
0.190
0.184
0.179
0.173
0.168
0.164
0.159
0.155
0.151
0.147
0.144
0.140
0.137
0.134
0.131
0.128
0.332
0.310
0.291
0.274
0.259
0.246
0.234
0.223
0.214
0.205
0.197
0.189
0.182
0.176
0.170
0.164
0.159
0.154
0.150
0.145
0.141
0.138
0.134
0.131
0.127
0.124
0.121
0.119
0.116
0.114
0.417
0.392
0.371
0.352
0.335
0.319
0.305
0.293
0.281
0.270
0.261
0.252
0.243
0.235
0.228
0.221
0.215
0.209
0.203
0.198
0.193
0.188
0.184
0.179
0.175
0.172
0.168
0.164
0.161
0.158
OIV-MA-AS1-07 : R2000
0.348
0.326
0.307
0.291
0.276
0.262
0.250
0.240
0.230
0.220
0.212
0.204
0.197
0.191
0.185
0.179
0.173
0.168
0.164
0.159
0.155
0.151
0.147
0.144
0.140
0.137
0.134
0.131
0.129
0.126
0.308
0.288
0.271
0.255
0.242
0.230
0.219
0.209
0.200
0.192
0.185
0.178
0.172
0.166
0.160
0.155
0.150
0.146
0.142
0.138
0.134
0.131
0.127
0.124
0.121
0.119
0.116
0.113
0.111
0.108
0.281
0.262
0.246
0.232
0.220
0.208
0.198
0.189
0.181
0.174
0.167
0.160
0.155
0.149
0.144
0.140
0.135
0.131
0.127
0.124
0.120
0.117
0.114
0.111
0.108
0.106
0.103
0.101
0.099
0.097
10
11
12
13
14
15
4052 4999 5403 5625 5764 5859 5928 5981 6023 6056 6083 6106 6126 6143 6157
98.5 99.0 99.2 99.3 99.3 99.3 99.4 99.4 99.4 99.4 99.4 99.4 99.4 99.4 99.4
34.1 30.8 29.4 28.7 28.2 27.9 27.7 27.5 27.3 27.2 27.1 27.1 27.0 26.9 26.9
21.2 18.0 16.7 16.0 15.5 15.2 15.0 14.8 14.7 14.5 14.5 14.4 14.3 14.2 14.2
16.3 13.3 12.1 11.4 11.0 10.7 10.5 10.3 10.2 10.1 9.96 9.89 9.82 9.77 9.72
13.7 10.9 9.78 9.15 8.75 8.47 8.26 8.10 7.98 7.87 7.79 7.72 7.66 7.60 7.56
12.2 9.55 8.45 7.85 7.46 7.19 6.99 6.84 6.72 6.62 6.54 6.47 6.41 6.36 6.31
11.3 8.65 7.59 7.01 6.63 6.37 6.18 6.03 5.91 5.81 5.73 5.67 5.61 5.56 5.52
10.6 8.02 6.99 6.42 6.06 5.80 5.61 5.47 5.35 5.26 5.18 5.11 5.05 5.01 4.96
10.0 7.56 6.55 5.99 5.64 5.39 5.20 5.06 4.94 4.85 4.77 4.71 4.65 4.60 4.56
9.64 7.20 6.21 5.67 5.31 5.07 4.88 4.74 4.63 4.54 4.46 4.39 4.34 4.29 4.25
9.33 6.93 5.95 5.41 5.06 4.82 4.64 4.50 4.39 4.30 4.22 4.16 4.10 4.05 4.01
9.07 6.70 5.74 5.21 4.86 4.62 4.44 4.30 4.19 4.10 4.02 3.96 3.90 3.86 3.82
8.86 6.51 5.56 5.04 4.69 4.46 4.28 4.14 4.03 3.94 3.86 3.80 3.75 3.70 3.66
8.68 6.36 5.42 4.89 4.56 4.32 4.14 4.00 3.89 3.80 3.73 3.67 3.61 3.56 3.52
8.53 6.23 5.29 4.77 4.44 4.20 4.03 3.89 3.78 3.69 3.62 3.55 3.50 3.45 3.41
8.40 6.11 5.18 4.67 4.34 4.10 3.93 3.79 3.68 3.59 3.52 3.46 3.40 3.35 3.31
8.29 6.01 5.09 4.58 4.25 4.01 3.84 3.71 3.60 3.51 3.43 3.37 3.32 3.27 3.23
8.18 5.93 5.01 4.50 4.17 3.94 3.77 3.63 3.52 3.43 3.36 3.30 3.24 3.19 3.15
8.10 5.85 4.94 4.43 4.10 3.87 3.70 3.56 3.46 3.37 3.29 3.23 3.18 3.13 3.09
8.02 5.78 4.87 4.37 4.04 3.81 3.64 3.51 3.40 3.31 3.24 3.17 3.12 3.07 3.03
7.95 5.72 4.82 4.31 3.99 3.76 3.59 3.45 3.35 3.26 3.18 3.12 3.07 3.02 2.98
7.88 5.66 4.76 4.26 3.94 3.71 3.54 3.41 3.30 3.21 3.14 3.07 3.02 2.97 2.93
7.82 5.61 4.72 4.22 3.90 3.67 3.50 3.36 3.26 3.17 3.09 3.03 2.98 2.93 2.89
7.77 5.57 4.68 4.18 3.85 3.63 3.46 3.32 3.22 3.13 3.06 2.99 2.94 2.89 2.85
7.72 5.53 4.64 4.14 3.82 3.59 3.42 3.29 3.18 3.09 3.02 2.96 2.90 2.86 2.81
7.68 5.49 4.60 4.11 3.78 3.56 3.39 3.26 3.15 3.06 2.99 2.93 2.87 2.82 2.78
7.64 5.45 4.57 4.07 3.75 3.53 3.36 3.23 3.12 3.03 2.96 2.90 2.84 2.79 2.75
7.60 5.42 4.54 4.04 3.73 3.50 3.33 3.20 3.09 3.00 2.93 2.87 2.81 2.77 2.73
7.56 5.39 4.51 4.02 3.70 3.47 3.30 3.17 3.07 2.98 2.91 2.84 2.79 2.74 2.70
7.31 5.18 4.31 3.83 3.51 3.29 3.12 2.99 2.89 2.80 2.73 2.66 2.61 2.56 2.52
7.17 5.06 4.20 3.72 3.41 3.19 3.02 2.89 2.78 2.70 2.62 2.56 2.51 2.46 2.42
7.07 4.98 4.13 3.65 3.34 3.12 2.95 2.82 2.72 2.63 2.56 2.50 2.44 2.39 2.35
7.01 4.92 4.07 3.60 3.29 3.07 2.91 2.78 2.67 2.59 2.51 2.45 2.40 2.35 2.31
6.96 4.88 4.04 3.56 3.25 3.04 2.87 2.74 2.64 2.55 2.48 2.42 2.36 2.31 2.27
6.92 4.85 4.01 3.53 3.23 3.01 2.84 2.72 2.61 2.52 2.45 2.39 2.33 2.29 2.24
6.89 4.82 3.98 3.51 3.21 2.99 2.82 2.69 2.59 2.50 2.43 2.37 2.31 2.27 2.22
6.75 4.71 3.88 3.41 3.11 2.89 2.73 2.60 2.50 2.41 2.34 2.27 2.22 2.17 2.13
6.69 4.65 3.82 3.36 3.05 2.84 2.68 2.55 2.44 2.36 2.29 2.22 2.17 2.12 2.07
6.63 4.61 3.78 3.32 3.02 2.80 2.64 2.51 2.41 2.32 2.25 2.18 2.13 2.08 2.04
OIV-MA-AS1-07 : R2000
10
16
17
18
19
20
30
40
50
60
70
80
6169 6182 6192 6201 6209 6261 6287 6303 6313 6320 6326 6335 6350 6361 6366
99.4 99.4 99.4 99.4 99.5 99.5 99.5 99.5 99.5 99.5 99.5 99.5 99.3 99.5 99.5
26.8 26.8 26.8 26.7 26.7 26.5 26.4 26.4 26.3 26.3 26.3 26.2 26.2 26.1 26.1
14.2 14.1 14.1 14.0 14.0 13.8 13.7 13.7 13.7 13.6 13.6 13.6 13.5 13.5 13.5
9.68 9.64 9.61 9.58 9.55 9.38 9.29 9.24 9.20 9.18 9.16 9.13 9.08 9.04 9.02
7.52 7.48 7.45 7.42 7.40 7.23 7.14 7.09 7.06 7.03 7.01 6.99 6.93 6.90 6.88
6.28 6.24 6.21 6.18 6.16 5.99 5.91 5.86 5.82 5.80 5.78 5.75 5.70 5.67 5.65
5.48 5.44 5.41 5.38 5.36 5.20 5.12 5.07 5.03 5.01 4.99 4.96 4.91 4.88 4.86
4.92 4.89 4.86 4.83 4.81 4.65 4.57 4.52 4.48 4.46 4.44 4.41 4.36 4.33 4.31
4.52 4.49 4.46 4.43 4.41 4.25 4.17 4.12 4.08 4.06 4.04 4.01 3.96 3.93 3.91
4.21 4.18 4.15 4.12 4.10 3.94 3.86 3.81 3.77 3.75 3.73 3.70 3.65 3.62 3.60
3.97 3.94 3.91 3.88 3.86 3.70 3.62 3.57 3.54 3.51 3.49 3.47 3.41 3.38 3.36
3.78 3.74 3.72 3.69 3.66 3.51 3.42 3.37 3.34 3.32 3.30 3.27 3.22 3.19 3.17
3.62 3.59 3.56 3.53 3.51 3.35 3.27 3.22 3.18 3.16 3.14 3.11 3.06 3.03 3.00
3.49 3.45 3.42 3.40 3.37 3.21 3.13 3.08 3.05 3.02 3.00 2.98 2.92 2.89 2.87
3.37 3.34 3.31 3.28 3.26 3.10 3.02 2.97 2.93 2.91 2.89 2.86 2.81 2.78 2.75
3.27 3.24 3.21 3.19 3.16 3.00 2.92 2.87 2.83 2.81 2.79 2.76 2.71 2.68 2.65
3.19 3.16 3.13 3.10 3.08 2.92 2.84 2.78 2.75 2.72 2.70 2.68 2.62 2.59 2.57
3.12 3.08 3.05 3.03 3.00 2.84 2.76 2.71 2.67 2.65 2.63 2.60 2.55 2.51 2.49
3.05 3.02 2.99 2.96 2.94 2.78 2.69 2.64 2.61 2.58 2.56 2.54 2.48 2.44 2.42
2.99 2.96 2.93 2.90 2.88 2.72 2.64 2.58 2.55 2.52 2.50 2.48 2.42 2.38 2.36
2.94 2.91 2.88 2.85 2.83 2.67 2.58 2.53 2.50 2.47 2.45 2.42 2.36 2.33 2.31
2.89 2.86 2.83 2.80 2.78 2.62 2.54 2.48 2.45 2.42 2.40 2.37 2.32 2.28 2.26
2.85 2.82 2.79 2.76 2.74 2.58 2.49 2.44 2.40 2.38 2.36 2.33 2.27 2.24 2.21
2.81 2.78 2.75 2.72 2.70 2.54 2.45 2.40 2.36 2.34 2.32 2.29 2.23 2.19 2.17
2.78 2.75 2.72 2.69 2.66 2.50 2.42 2.36 2.33 2.30 2.28 2.25 2.19 2.16 2.13
2.75 2.71 2.68 2.66 2.63 2.47 2.38 2.33 2.29 2.27 2.25 2.22 2.16 2.12 2.10
2.72 2.68 2.65 2.63 2.60 2.44 2.35 2.30 2.26 2.24 2.22 2.19 2.13 2.09 2.06
2.69 2.66 2.63 2.60 2.57 2.41 2.33 2.27 2.23 2.21 2.19 2.16 2.10 2.06 2.03
2.66 2.63 2.60 2.57 2.55 2.39 2.30 2.25 2.21 2.18 2.16 2.13 2.07 2.03 2.01
2.48 2.45 2.42 2.39 2.37 2.20 2.11 2.06 2.02 1.99 1.97 1.94 1.87 1.85 1.80
2.38 2.35 2.32 2.29 2.27 2.10 2.01 1.95 1.91 1.88 1.86 1.82 1.76 1.71 1.68
2.31 2.28 2.25 2.22 2.20 2.03 1.94 1.88 1.84 1.81 1.78 1.75 1.68 1.63 1.60
2.27 2.23 2.20 2.18 2.15 1.98 1.89 1.83 1.78 1.75 1.73 1.70 1.62 1.57 1.54
2.23 2.20 2.17 2.14 2.12 1.94 1.85 1.79 1.75 1.71 1.69 1.65 1.58 1.53 1.49
2.21 2.17 2.14 2.11 2.09 1.92 1.82 1.76 1.72 1.68 1.66 1.62 1.55 1.50 1.46
2.19 2.15 2.12 2.09 2.07 1.89 1.80 1.74 1.69 1.66 1.63 1.60 1.52 1.47 1.43
2.09 2.06 2.03 2.00 1.97 1.79 1.69 1.63 1.58 1.55 1.52 1.48 1.39 1.33 1.28
2.04 2.00 1.97 1.94 1.92 1.74 1.63 1.56 1.52 1.48 1.45 1.41 1.31 1.23 1.16
2.00 1.97 1.93 1.90 1.88 1.70 1.59 1.52 1.47 1.43 1.40 1.36 1.25 1.15 1.00
OIV-MA-AS1-07 : R2000
11
Critical values
m
95%
99%
3
4
5
6
7
0,970
0,829
0,710
0,628
0,569
0,994
0,926
0,821
0,740
0,680
8
9
10
11
12
0,608
0,564
0,530
0,502
0,479
0,717
0,672
0,635
0,605
0,579
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0,611
0,586
0,565
0,546
0,529
0,514
0,501
0,489
0,478
0,468
0,459
0,451
0,443
0,436
0,429
0,423
0,417
0,412
0,407
0,402
0,397
0,393
0,388
0,384
0,381
0,377
0,374
0,371
0,697
0,670
0,647
0,627
0,610
0,594
0,580
0,567
0,555
0,544
0,535
0,526
0,517
0,510
0,502
0,495
0,489
0,483
0,477
0,472
0,467
0,462
0,458
0,454
0,450
0,446
0,442
0,438
Q10 =
OIV-MA-AS1-07 : R2000
12
Analysis
Lab
n
Sample
Individual values x1
1
n1
x1
s1
s21
551 6,47
41,8
302 3,83
563 3,51
12,3
555 4,16
17,3
568 5,89
34,7
- =
6 550 546 549 557 588 570 576 568
7
555 5,63
31,7
550 6,28
39,5
556 5,63
31,7
10
553
30,2
5,5
Statistical Figures:
Bartlett Test:
Analysis of variance:
sr = 5.37
r = 15 sR = 7.78
OIV-MA-AS1-07 : R2000
R = 22
13
OF
RESULTS
OIV-MA-AS1-08 : R1999
sample which does not contain (or not yet contain) the substance by means of a
recovery test (dosed addition) on one of the samples to analyze.
Quality Control is assured by adding reference material to each series of samples,
and analyzing these pairs (test samples and reference material). This verifies
correct implementation of the method and should be independent of the analytical
calibration and protocol as its goal is to verify the aforementioned.
Series means a number of samples analyzed under repeatable conditions. Internal
controls serve to ensure the appropriate level of uncertainty is not exceeded.
If the analytical results are considered to be part of a normal population whose
mean is m and standard deviation is s, only around 0.3% of the results will be
outside the limits m 3s. When aberrant results are obtained (outside these limits),
the system is considered to be outside statistical control (unreliable data).
The control is graphically represented using Shewhart Control Graphs. To produce
these graphical results, the measured values obtained from the reference material
are placed on the vertical axis while the series numbers are placed on the horizontal
axis. The graph also includes horizontal lines representing the mean, m, m 2
(warning limits) and m 3 (action limits) (Figure 1).
To estimate the standard deviation, a control should be analyzed, in pairs, in at
least 12 trials. Each analytical pair shall be analyzed under repeatable conditions
and randomly inserted in a sample series. Analyses will be duplicated on different
days to reflect reasonable changes from one series to another. Variations can have
several causes: modification of the reactants composition, instrument re-calibration
and even different operators. After eliminating aberrant data using the Grubbs test,
calculate the standard deviation to construct the Shewhart graphs. This standard
deviation is compared to that of the reference method. If a published precision
level is not obtained for the reference method, caused should be investigated.
The precision limits of the laboratory should be periodically revised by repeating
the indicated procedure.
Once the Quality Control graph is constructed, graph the results obtained from
each series for the control material.
A series is considered outside statistical control if:
I)
II)
OIV-MA-AS1-08 : R1999
A Shewhart Control Graph can also be produced for the differences between
analytical pairs in the same sample, especially when reference material does not
exist. In this case, the absolute difference between two analyses of the same
sample is graphed. The graph's lower line is 0 and the attention limit is 1.128Sw
while the action limit is 3.686Sw where Sw = the standard deviation of a series.
This type of graph only accounts for repeatability. It should be no greater than the
published repeatability limit for the method.
In the absence of control material, it sometimes becomes necessary to verify that
the reproducibility limit of the reference method is not exceeded by comparing the
results obtained to those of obtained by an experimental laboratory using the same
sample.
Each laboratory performs two tests and the following formula is used:
Cr D95 ( y1 - y2 )=
R -
r2
2
CrD95 =
Critical difference (P=0,95)
y1
=
Means of 2 results obtained by lab 1
y2
=
Means of 2 results obtained by lab 2
R
=
Reproducibility of reference method
r
=
Repeatability of reference method
If the critical difference has been exceeded, the underlying reason is to be found
and the test is to be repeated within one month.
2) EVALUATION OF ANALYTIC RESULTS INDICATING THAT A
LEGAL LIMIT HAS BEEN EXCEEDED.
When analytical results indicated that a legal limit has been exceeded, the
following procedure should be followed:
1) In the case of an individual result, conduct a second test under repeatable
conditions. If it is not possible to conduct a second test under repeatable
conditions, conduct a double analysis under repeatable conditions and use these
data to evaluate the critical difference.
2) Determine the absolute value of the difference between the mean of the results
obtained under repeatable conditions and the legal limit. An absolute value of
the difference which is greater than the critical distance indicates that the
sample does not fit the specifications.
Critical difference is calculated by the formula:
C r D95 ( y - m0 ) =
OIV-MA-AS1-08 : R1999
1 2 2 2 n -1
R -r
n
2
y
=
Mean of results obtained
m0
=
Limit
n
=
Number of analyses
R
=
reproducibility
r
=
repeatability
In other words, this is a maximal limit where the average of the results obtained
should not be greater than:
m0 + CrD95(y - m0)
If the limit is a minimum, the average of the results obtained should not be less
than:
m0 - CrD95(y - m0)
3) COMPARING RESULTS OBTAINED USING TWO OR MORE
LABORATORIES AND COMPARING THESE RESULTS TO A
REFERENCE VALUE
To determine whether or not data originating in two laboratories are in agreement,
calculate the absolute difference between the two results and compare to the critical
difference:
1
1
)
2 n1 2 n2
Cr D95 ( y1 - y2 )= 2 R2 - r 2 (1y1
y2
n1
n2
R
r
=
=
=
=
=
=
If the result is the average of two tests, the equation can be simplified to:
Cr D95 ( y1 - y2 )= 2 R2 -
r2
2
y=
OIV-MA-AS1-08 : R1999
1
yi
p
4
The mean of all laboratories is compared with the reference value. If the absolute
difference exceeds the critical difference, as calculated using the following
formula, we conclude the results are not in agreement with the reference value:
Cr D95 ( y - m0 ) =
1
2p
R2 - r 2 (1 -
1 1
)
p n1
CrD95 =
Critical difference, calculated as indicated in point 2, for the reference
method.
For example, the reference value can be the value assigned to a reference material
or the value obtained by the same laboratory or by a different laboratory with a
different method.
4) EVALUATING ANALYTICAL RESULTS OBTAINED USING NONVALIDATED METHODS
A provisional reproducibility value can be assigned to a non-validated method by
comparing it to that of a second laboratory:
R prov =
y1 0
y2
r
=
=
=
( y1 - y 2 ) +
r2
2
RSDR % = 21-0,5log10 C
RSDR % = Standard deviation for reproducibility
(expressed as a percentage of the mean)
C
OIV-MA-AS1-08 : R1999
This equation was empirically obtained from more than 3000 collaborative studies
including a diverse group of analyzed substances, matrices and measurement
techniques. In the absence of other information, RSDR values that are lower or
equal to the RSDR values calculated using the Horwitz equation can be considered
acceptable.
OIV-MA-AS1-08 : R1999
RSDR %
10-9
45
10-8
32
10-7
23
10-6
16
10-5
11
10-4
10-3
5,6
10-2
10-1
2,8
If the result obtained using a non-validated method is close to the limit specified by
legislation, the decision on the limit shall be decided as follows (for upper limits):
S = m0 + {(Rrout/Rref)-1} CrD95
and, for lower limits,
S = m0 - {(Rrout/Rref)-1} CrD95
S
m0
Rrout
Rref
CrD95
=
=
=
=
=
decision limit
legal limit
provisional reproducibility for non-validated method
reproducibility for reference method
critical difference, calculated as indicated in point 2, for the reference
method
The result which exceeds the decision limit should be replaced with a final result
obtained using the reference method.
Critical differences for probability levels other than 95%
OIV-MA-AS1-08 : R1999
Multiplicative coefficient
0,82
1,00
1,16
1,29
1,40
OIV-MA-AS1-08 : R1999
OIV-MA-AS1-08 : R1999
BIBLIOGRAPHY
- "Harmonized Guidelines for Internal Quality Control in Analytical Chemistry
Laboratories". IUPAC. Pure and App. Chem. Vol 67, n 4, 649-666, 1995
- "Shewhart Control Charts" ISO 8258. 1991.
- "Precision of test methods - Determination of repeatability and reproducibility for
a standard test method by inter-laboratory tests". ISO 5725, 1994.
- "Draft Commission Regulation of establishing rules for the application of
reference and routine methods for the analysis and quality evaluation of milk and
milk products". Commission of the European Communities, 1995.
- "Harmonized protocols for the adoption of standardized analytical methods and for
the presentation
of their performance characteristics". IUPAC. Pure an App. Chem., Vol. 62, n 1,
149-162. 1990.
OIV-MA-AS1-08 : R1999
10
INTRODUCTION
After a number of meetings and workshops, a group of representatives from
27 organizations adopted by consensus a "Protocol for the design, conducts and
interpretation of collaborative studies" which was published in Pure & Appl.
Chem. 60, 855-864, 1995. A number of organizations have accepted and used this
protocol. As a result of their experience and the recommendations of the Codex
Committee on Methods of Analysis and Sampling (Joint FAO/WHO Food
Standards Programme, Report of the Eighteenth Session, 9-13 November, 1992;
FAO, Rome Italy, ALINORM 93/23, Sections 34-39), three minor revisions were
recommended for incorporation into the original protocol. These are: (1) Delete the
double split level design because the interaction term it generates depends upon the
choice of levels and if it is statistically significant, the interaction cannot be
physically interpreted. (2) Amplify the definition of "material". (3) Change the
outlier removal criterion from 1% to 2.5%.
The revised protocol incorporating the changes is reproduced below. Some minor
editorial revisions to improve readability have also been made. The vocabulary and
definitions of the document 'Nomenclature of Interlaboratory Studies
(Recommendations 1994)' [published in Pure Appl Chem., 66, 1903-1911 (1994)]
has been incorporated into this revision, as well as utilizing, as far as possible, the
appropriate terms of the International Organization for Standardization (ISO),
modified to be applicable to analytical chemistry.
PROTOCOL
1
Preliminary work
Method-performance (collaborative) studies require considerable effort and should
be conducted only on methods that have received adequate prior testing. Such
within-laboratory testing should include, as applicable, information on the
following:
1.1
Preliminary estimates of precision
Estimates of the total within-laboratory standard deviation of the analytical results
over the concentration range of interest as a minimum at the upper and lower limits
OIV-MA-AS1-09 : R2000
OIV-MA-AS1-09 : R2000
1.6
Method comparison
The results of comparison of the application of the method with existing tested
methods intended for similar purposes.
1.7
Calibration Procedures
The procedures specified for calibration and for blank correction must not
introduce important bias into the results.
1.8
Method description
The method must be clearly and unambiguously written.
1.9
Significant figures
The initiating laboratory should indicate the number of significant figures to be
reported, based on the output of the measuring instrument.
NOTE: In making statistical calculations from the reported data, the full power of
the calculator or computer is to be used with no rounding or truncating until the
final reported mean and standard deviations are achieved. At this point the standard
deviations are rounded to 2 significant figures and the means and related standard
deviations are rounded to accommodate the significant figures of the standard
deviation. For example, if SR = 0.012, c is reported as 0.147, not as 0. 1473 or 0.
15, and RSDR is reported as 8.2%. (Symbols are defined in Appendix L) If
standard deviation calculations must be conducted manually in steps, with the
transfer of intermediate results, the number of significant figures to be retained for
squared numbers should be at least 2 times the number of figures in the data plus 1.
2.
2.1
Number of materials
For a single type of substance, at least 5 materials (test samples) must be used; only
when a single level specification is involved for a single matrix may this minimum
required number of materials to be reduced to 3. For this design parameter, the two
portions of a split level and the two individual portions of blind replicates per
laboratory are considered as a single material.
NOTE 1: A material is an 'analyte/matrix/concentration' combination to which the
method-performance parameters apply. This parameter determines the applicability
of a method. For application to a number of different substances, a sufficient
OIV-MA-AS1-09 : R2000
sr = ((di ) / 2(n - 1)
2
where di, the difference between the 2 individual values from the split level for
each laboratory and n is the number of laboratories. In this special case, S R, the
among laboratories standard deviation, is merely the average of the two SR values
calculated from the individual components of the split level, and it is used only as a
check of the calculations.
NOTE 4: The blank or negative control may be a material or not depending on the
usual purpose of the analysis. For example, in trace analysis, where very low levels
(near the limit of quantitation) are often sought, the blanks are considered as
materials and are necessary to determine certain 'limits of measurement.' However,
if the blank is merely a procedural control in macro analysis (e.g., fat in cheese), it
would not be considered a material.
2.2
Number of laboratories
At least 8 laboratories must report results for each material; only when it is
impossible to obtain this number (e.g., very expensive instrumentation or
specialized laboratories required) may the study be conducted with less, but with
an absolute minimum of 5 laboratories. If the study is intended for international
use, laboratories from different countries should participate. In the case of methods
requiring the use of specialized instruments, the study might include the entire
population of available laboratories. In such cases, "n" is used in the denominator
for calculating the standard deviation instead of "(n - 1)". Subsequent entrants to
the field should demonstrate the ability to perform as well as the original
participant.
2.3
Number of Replicates
Split Level
OIV-MA-AS1-09 : R2000
For each level that is split and which constitutes only a single material for purposes
of design and statistical analysis, use 2 nearly identical test samples that differ only
slightly in analyte concentration (e.g., <1-5%). Each laboratory must analyse each
test sample once and only once.
NOTE: The statistical criterion that must be met for a pair of test samples to
constitute a split level is that the reproducibility standard deviation of the two parts
of the single split level must be equal.
2.3.2 Combination blind replicates and split level
Use split levels for some materials and blind replicates for other materials in the
same study (single values from each submitted test sample).
2.3.3 Blind replicates
For each material, use blind identical replicates, when data censoring is impossible
(e.g., automatic input, calculation, and printout) non-blind identical replicates may
be used.
2.3.4 Known replicates
For each material, use known replicates (2 or more analyses of test portions from
the same test sample), but only when it is not practical to use one of the preceding
designs.
2.3.5 Independent analyses
Use only a single test portion from each material (i.e., do not perform multiple
analyses) in the study, but rectify the inability to calculate repeatability parameters
by quality control parameters or other within-laboratory data obtained
independently of the method-performance study.
3.
Statistical analysis (See Flowchart, A.4. 1)
For the statistical analysis of the data, the required statistical procedures listed
below must be performed and the results reported. Supplemental, additional
procedures are not precluded.
3.1
Valid data
Only valid data should be reported and subjected to statistical treatment. Valid data
are those data that would be reported as resulting from the normal performance of
laboratory analyses; they are not marred by method deviations, instrument
malfunctions, unexpected occurrences during performance, or by clerical,
typographical and arithmetical errors.
3.2
One-way analysis of variance
One-way analysis of variance and outlier treatments must be applied separately to
each material (test sample) to estimate the components of variance and
repeatability and reproducibility parameters.
OIV-MA-AS1-09 : R2000
3.3
Initial estimation
Calculate the mean, c (= the average of laboratory averages), repeatability relative
standard deviation, RSDr, and reproducibility relative standard deviation, RSDR
with no outliers removed, but using only valid data.
3.4
Outlier treatment
The estimated precision parameters that must also be reported are based on the
initial valid data purged of all outliers flagged by the harmonized 1994 outlier
removal procedure. This procedure essentially consists of sequential application of
the Cochran and Grubbs tests (at 2.5% probability (P) level, 1-tail for Cochran and
2-tail for Grubbs) until no further outliers are flagged or until a drop of 22.2% (=
219) in the original number of laboratories providing valid data would occur.
NOTE: Prompt consultation with a laboratory reporting suspect values may result
in correction of mistakes or discovering conditions that lead to invalid data, 3.1.
Recognizing mistakes and invalid data per se is much preferred to relying upon
statistical tests to remove deviate values.
3.4.1 Cochran test
First apply Cochran outlier test (1-tail test a P = 2.5%) and remove any laboratory
whose critical value exceeds the tabular value given in the tale, Appendix A.3. 1,
for the number of laboratories and replicates involved.
3.4.2 Grubbs tests
Apply the single value Grubbs test (2 tail) and remove any outlying laboratory. If
no laboratory is flagged, then apply the pair value tests (2 tail) - - 2 at the same end
and 1 value at each end, P = 2.5% overall. Remove any laboratory(ies) flagged by
these tests whose critical value exceeds the tabular value given in the appropriate
column of the table Appendix A.3.3. Stop removal when the next application of the
test will flag as table, A outliers more that 22.2% (2 of 9) of the laboratories.
NOTE: The Grubbs tests are to be applied one material at a time to the set of
replicate means from all laboratories, and not to the individual values from
replicated designs because the distribution of all the values taken together is
multimodal, not Caussian, i.e., their differences from the overall mean for that
material are not independent.
3.4.3 Final estimation
Recalculate the parameters as in 3.3 after the laboratories flagged by the
preceding procedure have been removed. If no outliers were removed by the
Cochran-Grubbs sequence, terminate testing. Otherwise, reapply the CochranGrubbs sequence to the data purged of the flagged outliers until no further outliers
are flagged or until more than a total of 22.2% (2 of 9 laboratories) would be
removed in the next cycle. See flowchart A.3.4.
OIV-MA-AS1-09 : R2000
4.
Final report
The final report should be published and should include all valid data. Other
information and parameters should be reported in a format similar (with respect to
the reported items) to the following, as applicable:
[x] Method-performance tests carried out at the international level in [year(s)] by
[organisation] in which [y and z] laboratories participated, each performing [k]
replicates, gave the following statistical results:
TABLE OF METHOD-PERFORMANCE PARAMETERS
Analyte; Results expressed in [units]
Material [Description and listed in columns across top of table in increasing order
of magnitude of means]
Number of laboratories retained after eliminating outliers
Number of outlying laboratories
Code (or designation) of outlying laboratories
Number of accepted results
Mean
True or accepted value, if known
Repeatability standard deviation (Sr)
Repeatability relative standard deviation (RSDR)
Repeatability limit, r (2.8 x Sr)
Reproducibility standard deviation (SR)
Reproducibility relative standard deviation (RSDR)
Reproducibility limit, R (2.8 X SR)
4.1
Symbols
A set of symbols for use in reports and publications is attached as Appendix 1
(A.1.).
4.2
Definitions
A set of definitions for use in study reports and publications is attached as
Appendix 2 (A.2.).
4.3
Miscellaneous
4.3.1 Recovery
Recovery of added analyte as a control on method or laboratory bias should be
calculated as follows:
[Marginal] Recovery, %=
(Total analyte found - analyte originally present) x 100/(analyte added)
OIV-MA-AS1-09 : R2000
Although the analyte may be expressed as either concentration or amount, the units
must be the same throughout. When the quantity of analyte is determined by
analysis, it must be determined in the same way throughout.
Analytical results should be reported uncorrected for recovery. Report recoveries
separately.
4.3.2
5.
REFERENCES
Horwitz, W. (1988) Protocol for the design, conduct, and interpretation of method
performance studies. Pure & Appl. Chem. 60, 855-864.
Pocklington, W.D. (1990) Harmonized protocol for the adoption of standardized
analytical methods and for the presentation of their performance characteristics.
Pure and Appl. Chem. 62, 149-162.
International Organization for Standardization. International Standard 5725-1986.
Under revision in 6 parts; individual parts may be available from National
Standards member bodies.
OIV-MA-AS1-09 : R2000
A. APPENDICES
APPENDIX 1. - SYMBOLS
Use the following set of symbols and terms for designating parameters developed
by a method-performance study.
Mean (of laboratory averages)
Standard deviations:
Repeatability
'Pure' between-laboratory
Reproducibility
x
s (estimates)
Sr
SL
SR
Variances:
2
2
2
SR = SL + Sr
k (general)
k (for a balanced design)
r = (2.8 x Sr)
R = (2.8 X SR)
Number of laboratories
L
Number of materials (test samples)
m
Total number of values in a given assay n (= kL for a balanced design)
Total number of values in a given study N (= kLm for an overall balanced design)
____________________
If other symbols are used, their relationship to the recommended symbols should
be explained fully.
OIV-MA-AS1-09 : R2000
APPENDIX 2. - DEFINITIONS
Use the following definitions. The first three definitions utilize the 1UPAC
document "Nomenclature of Interlaboratory Studies" (approved for publication
1994). The next two definitions are assembled from components given in ISO
3534-1:1993. All test results are assumed to be independent, i.e., 'obtained in a
manner not influenced by any previous result on the same or similar test object.
Quantitative measures of precision depend critically on the stipulated conditions.
Repeatability and reproducibility conditions are particular sets of extreme
stipulated conditions.'
A.2.1 Method-performance studies
An interlaboratory study in which all laboratories follow the same written protocol
and use the same test method to measure a quantity in sets of identical test items
[test samples, materials]. The reported results are used to estimate the performance
characteristics of the method. Usually these characteristics are within-laboratory
and among-laboratories precision, and when necessary and possible, other pertinent
characteristics such as systematic error, recovery, internal quality control
parameters, sensitivity, limit of determination, and applicability.
A.2.2. Laboratory-performance study
An interlaboratory study that consists of one or more analyses or measurements by
a group of laboratories on one or more homogeneous, stable test items, by the
method selected or used by each laboratory. The reported results are compared
with those of other laboratories or with the known or assigned reference value,
usually with the objective of evaluating or improving laboratory performance.
A.2.3 Material certification stud
An interlaboratory study that assigns a reference value ('true value') to a quantity
(concentration or property) in the test item, usually with a stated uncertainty.
A.2.4 Repeatability limit (r)
When the mean of the values obtained from two single determinations with the
same method on identical test items in the same laboratory by the same operator
using the same equipment within short intervals of time, lies within the range of the
mean values cited in the Final Report, 4.0, the absolute difference between the two
test results obtained should be less than or equal to the repeatability limit (r) [= 2.8
x s,) that can generally be inferred by linear interpolation of sr from the Report.
NOTE: This definition, and the corresponding definition for reproducibility limit,
has been assembled from five cascading terms and expanded to permit application
by interpolation to a test item whose mean is not the same as that used to establish
the original parameters, which is the usual case in applying these definitions. The
term 'repeatability [and reproducibility] limit' is applied specifically to a probability
OIV-MA-AS1-09 : R2000
10
of 95% and is taken as 2.8 x s, [or SRI. The general term for this statistical concept
applied to any measure of location (e.g., median) and with other probabilities (e.g.,
99%) is "repeatability [and reproducibility] critical difference".
A.2.5 Reproducibility limit (R)
When the mean of the values obtained from two single determinations with the
same method on identical test items in different laboratories with different
operators using different equipment, lies within the range of the mean values cited
in the Final Report, 4.0, the absolute difference between the two test results
obtained should be less than or equal to the reproducibility limit (R) [= 2.8 x sR]
that can generally be inferred by linear interpolation of SR from the Report.
NOTE 1: When the results of the interlaboratory test make it possible, the value of
r and R can be indicated as a relative value (e.g., as a percentage of the determined
mean value) as an alternative to the absolute value.
NOTE 2: When the final reported result in the study is an average derived from
more than a single value, i.e., k is greater than 1, the value for R must be adjusted
according to the following formula before using R to compare the results of a
single routine analyses between two laboratories.
R' = (R2 + r2 (1 - [l /k])1/2
Similar adjustments must be made for replicate results constituting the final values
for SR and RSDR, if these will be the reported parameters used for quality control
purposes.
NOTE 3: The repeatability limit, r, may be interpreted as the amount within which
two determinations should agree with each other within a laboratory 95% of the
time. The reproducibility limit, R, may be interpreted as the amount within which
two separate determinations conducted in different laboratories should agree with
each other 95% of the time.
NOTE 4: Estimates Of SR can be obtained only from a planned, organized method
performance study; estimates of Sr can be obtained from routine work within a
laboratory by use of control charts. For occasional analyses, in the absence of
control charts, within-laboratory precision may be approximated as one half SR
(Pure and Appl. Chem., 62, 149-162 (1990) , Sec. L3, Note.).
A.2.6 One-way analysis of variance
One-way analysis of variance is the statistical procedure for obtaining the estimates
of within laboratory and between-laboratory variability on a material-by-material
basis. Examples of the calculations for the single level and single-split-level
designs can be found in ISO 5725-1986.
OIV-MA-AS1-09 : R2000
11
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12
r=2
r=3
r=4
r=5
r=6
94.3
88.6
83.2
78.2
73.6
69.3
65.5
62.2
59.2
56.4
53.8
51.5
49.5
47.8
46.0
44.3
42.8
41.5
40.3
39.1
37.9
36.7
35.5
34.5
33.7
33.1
32.5
29.3
26.0
21.6
81.0
72.6
65.8
60.2
55.6
51.8
48.6
45.8
43.1
40.5
38.3
36.4
34.7
33.2
31.8
30.5
29.3
28.2
27.2
26.3
25.5
24.8
24.1
23.4
22.7
22.1
21.6
19.5
17.0
14.3
72.5
64.6
58.3
52.2
47.4
43.3
39.9
37.2
35.0
33.2
31.5
29.9
28.4
27.1
25.9
24.8
23.8
22.9
22.0
21.2
20.5
19.9
19.3
18.7
18.1
17.5
16.9
15.3
13.5
11.4
65.4
58.1
52.2
47.3
43.0
39.3
36.2
33.6
31.3
29.2
27.3
25.7
24.4
23.3
22.4
21.5
20.7
19.9
19.2
18.5
17.8
17.2
16.6
16.1
15.7
15.3
14.9
12.9
11.6
9.7
62.5
53.9
47.3
42.3
38.5
35.3
32.6
30.3
28.3
26.5
25.0
23.7
22.0
21.2
20.4
19.5
18.7
18.0
17.3
16.6
16.0
15.5
15.0
14.5
14.1
13.7
13.3
11.6
10.2
8.6
Tables A.3.1 and A.3.3 were calculated by R. Albert (October, 1993) by computer
simulation involving several runs of approximately 7000 cycles each for each
value, and then smoothed. Although Table A.3.1 is strictly applicable only to a
balanced design (same number of replicates from all laboratories), it can be applied
to an unbalanced design without too much error, if there are only a few deviations.
A.3.2 Calculation of Cochran maximum variance outlier ratio
OIV-MA-AS1-09 : R2000
13
Compute the within-laboratory variance for each laboratory and divide the largest
of these variances by the sum of the all of the variances and multiply by 100. The
resulting quotient is the Cochran statistic which indicates the presence of a
removable outlier if this quotient exceed the critical value listed above in the
Cochran table for the number of replicates and laboratories specified.
A.3.3 Critical values for the Grubbs extreme deviation outlier tests at the 2.5% (2tail), 1.25% (1tail) rejection level, expressed as the percent reduction in standard
deviations caused by the removal of the suspect value(s).
No. of labs
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
40
50
One highest
or lowest
86.1
73.5
64.0
57.0
51.4
46.8
42.8
39.3
36.3
33.8
31.7
29.9
28.3
26.9
25.7
24.6
23.6
22.7
21.9
21.2
20.5
19.8
19.1
18.4
17.8
17.4
17.1
13.3
11.1
OIV-MA-AS1-09 : R2000
Two highest
or two lowest
98.9
90.9
81.3
73.1
66.5
61.0
56.4
52.5
49.1
46.1
43.5
41.2
39.2
37.4
35.9
34.5
33.2
31.9
30.7
29.7
28.8
28.0
27.1
26.2
25.4
24.7
24.1
19.1
16.2
14
OIV-MA-AS1-09 : R2000
15
APPENDIX 4
A.4.1. Flowchart for outlier removal
OIV-MA-AS1-09 : R2000
16
No
Possible
blank
No
Yes
Recording
Stray
peak
Yes
No
Yes
No
RT of
Stable stray
peak
Yes
Straight
regression
line
DL = 3b
LQ= 10b
Results approach
OIV-MA-AS1-10 : R2000
Hmax
method
Haverage
method
Hmax or
haverage
method
Graph approach
4 - Methodology
4.1 "Results" approach
When the analytical method produces no recorded graph, but only numerical values
(i.e., colorimetry), the detection limit (LD) and the quantification limit (LQ) are
estimated using one of the two following methods.
4.1.1 - Method 1:
Directly read n measurements (analyte quantity or response) of separate analytic
blank samples that contain all of the constituents, with the exception of the
substance to be tested for.
LD = mblank + 3Sblank and
LQ = mblank + 10Sblank
where mblank and Sblank are the mean and standard deviation for n measurements.
Note: A multiplication factor of 3 corresponds to a 0.13% chance of concluding
that the substance sought is present, when, in fact, it is lacking. 10 corresponds to
a 0.5% chance.
4.1.2 - Method 2:
Using the straight calibration line: Y = a + bX
The detection limit is the smallest concentration of a substance that can be
distinguished from the blank, with a 0.13% risk of retaining samples containing
nothing ; in other words, the value beginning at which a statistical test comparing
the response to 0 becomes significant with an error level of 0.13%. Hence:
YDL = a +3Sa
XDL = (a +3Sa)/b
Where Sa is the standard deviation on the ordinate at the origin of the straight
regression line. The logic is the same for LQ,, where the multiplication factor is 10
(risk of 0.5%).
4.2 - "Graph" Approach
For analytical methods which generate graphs (i.e., chromatography), the detection
limit is estimated based on the ground noise of the analytic blank recording for a
given sample.
LD = 3 x h x R (associated risk is below 0.13%) and
LQ = 10 x h x R (associated risk is below 0.5%), where
h is the average or maximum amplitude of the signal window corresponding
to 10 width s of the mid-height peak on either side of the retention time, as a
function of stability.
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OIV-MA-AS1-10 : R2000
CONTENTS
1. INTRODUCTION
1.1 Basic concepts
1.2. Scope of this document
1.3. Internal quality control and uncertainties
2. DEFINITIONS
2.1. International definitions
2.2. Definition of terms specific to this document
7. RECOMMENDATIONS
8. CONCLUSIONS
9. REFERENCES
OIV-MA-AS1-11 : R2002
1. INTRODUCTION
1.1 Basic concept
This document sets out guidelines for the implementation of internal
quality control (IQC) in analytical laboratories. IQC is one of a number of
concerted measures that analytical chemists can take to ensure that the data
produced in the laboratory are fit for their intended purpose. In practice,
fitness for purpose is determined by a comparison of the accuracy achieved
in a laboratory at a given time with a required level of accuracy. Internal
quality control therefore comprises the routine practical procedures that
enable the analytical chemist to accept a result or group of results as fit for
purpose, or reject the results and repeat the analysis. As such, IQC is an
important determinant of the quality of analytical data, and is recognised as
such by accreditation agencies.
Internal quality control is undertaken by the inclusion of particular
reference materials, here called "control materials", into the analytical
sequence and by duplicate analysis. The control materials should,
wherever possible, be representative of the test materials under
consideration in respect of matrix composition, the state of physical
preparation and the concentration range of the analyte. As the control
materials are treated in exactly the same way as the test materials, they are
regarded as surrogates that can be used to characterise the performance of
the analytical system, both at a specific time and over longer intervals.
Internal quality control is a final check of the correct execution of all of the
procedures (including calibration) that are prescribed in the analytical
protocol and all of the other quality assurance measures that underlie good
analytical practice. IQC is therefore necessarily retrospective. It is also
required to be as far as possible independent of the analytical protocol,
especially the calibration, that it is designed to test.
Ideally both the control materials and those used to create the calibration
should be traceable to appropriate certified reference materials or a
recognised empirical reference method. When this is not possible, control
materials should be traceable at least to a material of guaranteed purity or
other well characterised material. However, the two paths of traceability
must not become coincident at too late a stage in the analytical process.
For instance, if control materials and calibration standards were prepared
from a single stock solution of analyte, IQC would not detect any
inaccuracy stemming from the incorrect preparation of the stock solution.
OIV-MA-AS1-11 : R2002
OIV-MA-AS1-11 : R2002
OIV-MA-AS1-11 : R2002
2. DEFINITIONS
2.1 International definitions
Quality assurance. All those planned and systematic actions necessary to
provide adequate confidence that a product or service will satisfy given
requirements for quality(10).
Trueness: closeness of the agreement between the average value obtained
from a large series of test results and an accepted reference value(11).
Precision: closeness of agreement between independent test results
obtained under prescribed conditions(12).
Bias: difference between the expectation of the test results and an accepted
reference value(11).
OIV-MA-AS1-11 : R2002
OIV-MA-AS1-11 : R2002
10
OIV-MA-AS1-11 : R2002
11
2
x
2x = 20 + 12
where
20 = variance of the random error (within run) and
12 = variance of the run effect.
The model could be extended if necessary to include other features of the analytical system
OIV-MA-AS1-11 : R2002
12
The variances of the true value and the persistent bias are both zero. An
analytical system in control is fully described by 20 , 12 and the value of
the persistent bias. Gross errors are implied when the analytical system
does not comply with such a description.
There is no intention here of estimating the standard deviation of repeatability r from the IQC data or of
comparing estimates: there would usually be too few results for a satisfactory outcome. Where such an estimate is
needed the formula sr d 2 / 2n can be used.
OIV-MA-AS1-11 : R2002
13
14
15
Where a CRM is not available traceability only to a reference method or to a batch of a reagent supplied by a
manufacturer may be necessary.
OIV-MA-AS1-11 : R2002
16
17
hoc analysis is executed. A test portion of the test material spiked with a
known amount of the analyte and analysed alongside the original test
material. The recovery of the added analyte (known as the "marginal
recovery") is the difference between the two measurements divided by the
amount that is added. The obvious advantages of recovery checks are that
the matrix is representative and the approach is widely applicable - most
test materials can be spiked by some means. However, the recovery check
suffers from the disadvantage previously noted regarding the speciation,
binding and physical distribution of the analyte. Furthermore, the
assumption of an equivalent recovery of the analyte added as a spike and of
the native analyte may not be valid. However, it can normally be assumed
that a poor performance in a recovery check is strongly indicative of a
similar or worse performance for the native analyte in the test materials.
Spiking and recovery testing as an IQC method must be distinguished from
the method of standard additions, which is a measurement procedure: a
single spiking addition cannot be used to fulfil the roles of both
measurement and IQC.
6.4
Blank determinations
Blank determinations are nearly always an essential part of the analytical
process and can conveniently be effected alongside the IQC protocol. The
simplest form of blank is the "reagent blank", where the analytical
procedure is executed in all respects apart from the addition of the test
portion. This kind of blank, in fact, tests more than the purity of the
reagents. For example it is capable of detecting contamination of the
analytical system originating from any source, e.g., glassware and the
atmosphere, and is therefore better described as a "procedural blank". In
some instances, better execution of blank determinations is achieved if a
simulated test material is employed. The simulant could be an actual test
material known to be virtually analyte-free or a surrogate (e.g., ashless
filter paper used instead of plant material). Where it can be contrived, the
best type of blank is the "field blank", which is a typical matrix with zero
concentration of analyte.
An inconsistent set of blanks in a run suggests sporadic contamination and
may add weight to IQC evidence suggesting the rejection of the results.
When an analytical protocol prescribes the subtraction of a blank value, the
blank value must be subtracted also from the results of the control
materials before they are used in IQC.
OIV-MA-AS1-11 : R2002
18
7. RECOMMENDATIONS
The following recommendations represent integrated approaches to IQC that are
suitable for many types of analysis and applications areas. Managers of laboratory
quality systems will have to adapt the recommendations to the demands of their
own particular requirements. Such adaption could be implemented, for example, by
adjusting the number of duplicates and control material inserted into a run, or by
the inclusion of any additional measures favoured in the particular application area.
The procedure finally chosen and its accompanying decision rules must be codified
in an IQC protocol that is separate from the analytical system protocol.
The practical approach to quality control is determined by the frequency with
which the measurement is carried out and the size and nature of each run. The
following recommendations are therefore made. The use of control charts and
decision rules are covered in Appendix 1.
In each of the following the order in the run in which the various materials are
analysed should be randomised if possible. A failure to randomise may result in an
underestimation of various components of error.
(i) Short (e.g., n<20) frequent runs of similar materials. Here the concentration
range of the
analyte in the run is relatively small, so a common value of standard deviation can
be assumed.
Insert a control material at least once per run. Plot either the individual values
obtained, or
The mean value, on an appropriate control chart. Analyse in duplicate at least half
of the
Test materials, selected at random. Insert at least one blank determination.
OIV-MA-AS1-11 : R2002
19
(ii) Longer (e.g., n>20) frequent runs of similar materials. Again a common level
of standard
deviation is assumed.
Insert the control material at an approximate frequency of one per ten test
materials. If the run size is likely to vary from run to run it is easier to standardise
on a fixed number of insertions per run and plot the mean value on a control chart
of means. Otherwise plot individual values.
Analyse in duplicate a minimum of five test materials selected at random. Insert
one blank
determination per ten test materials.
(iii) Frequent runs containing similar materials but with a wide range of analyte
concentration.
Here we cannot assume that a single value of standard deviation is applicable.
Insert control materials in total numbers approximately as recommended above.
However, there should be at least two levels of analyte represented, one close to the
median level of typical test materials, and the other approximately at the upper or
lower decile as appropriate. Enter values for the two control materials on separate
control charts. Duplicate a minimum of five test materials, and insert one
procedural blank per ten test materials.
(iv) Ad hoc analysis. Here the concept of statistical control is not applicable. It is
assumed, however, that the materials in the run are of a single type, i.e., sufficiently
similar for general conclusions on errors to be made.
Carry out duplicate analysis on all of the test materials. Carry out spiking or
recovery tests or use a formulated control material, with an appropriate number of
insertions (see above), and with different concentrations of analyte if appropriate.
Carry out blank determinations. As no control limits are available, compare the
bias and precision with fitness for purpose limits or other established criteria..
8. CONCLUSIONS
Internal quality control is an essential aspect of ensuring that data released from a
laboratory are fit for purpose. If properly executed, quality control methods can
monitor the various aspects of data quality on a run-by-run basis. In runs where
performance falls outside acceptable limits, the data produced can be rejected and,
after remedial action on the analytical system, the analysis can be repeated.
OIV-MA-AS1-11 : R2002
20
It must be stressed, however, that internal quality control is not foolproof even
when properly executed. Obviously it is subject to "errors of both kinds", i.e., runs
that are in control will occasionally be rejected and runs that are out of control
occasionally accepted. Of more importance, IQC cannot usually identify sporadic
gross errors or short-term disturbances in the analytical system that affect the
results for individual test materials. Moreover, inferences based on IQC results are
applicable only to test materials that fall within the scope of the analytical method
validation. Despite these limitations, which professional experience and diligence
can alleviate to a degree, internal quality control is the principal recourse available
for ensuring that only data of appropriate quality are released from a laboratory.
When properly executed it is very successful.
Finally, it must be appreciated that a perfunctory execution of any quality system
will not guarantee the production of data of adequate quality. The correct
procedures for feedback, remedial action and staff motivation must also be
documented and acted upon. In other words, there must be a genuine commitment
to quality within a laboratory for an internal quality control programme to succeed,
i.e., the IQC must be part of a total quality management system.
9. REFERENCES
1
"Protocol for the Design, Conduct and Interpretation of Method
Performance Studies", Edited W Horwitz, Pure Appl. Chem., 1988, 60,
855- 864. (Revision in press)
2
"The International Harmonised Protocol for the Proficiency Testing of
(Chemical) Analytical Laboratories", Edited M Thompson and R Wood,
Pure Appl. Chem., 1993, 65, 2123-2144. (Also published in J. AOAC
International, 1993, 76, 926-940.
3
"IFCC approved recommendations on quality control in clinical chemistry.
Part 4: internal quality control", J. Clin. Chem. Clin. Biochem., 1980, 18,
534-541.
4
S Z Cekan, S B Sufi and E W Wilson, "Internal quality control for assays
of reproductive hormones: Guidelines for laboratories". WHO, Geneva,
1993.
5
M Thompson, "Control procedures in geochemical analysis", in R J
Howarth (Ed), "Statistics and data analysis in geochemical prospecting",
Elsevier, Amsterdam, 1983.
6
M Thompson, "Data quality in applied geochemistry: the requirements
and how to achieve them", J. Geochem. Explor., 1992, 44, 3-22.
7
Health and Safety Executive, "Analytical quality in workplace air
monitoring", London, 1991.
OIV-MA-AS1-11 : R2002
21
9
10
11
12
13
14
15
16
17
18
19
20
OIV-MA-AS1-11 : R2002
22
2 1/ 2
OIV-MA-AS1-11 : R2002
23
where n is the number of control measurements in a run from which the mean is
calculated. The value of n therefore must be constant from run to run, otherwise
control limits would be impossible to define. If a fixed number of repeats of a
control material per run cannot be guaranteed (e.g., if the run length were variable)
then charts of individual values must be used. Furthermore the equation indicates
that x or x must be estimated with care. An attempt to base an estimate on
repeat values from a single run would result in unduly narrow control limits.
Estimates must therefore include the between-run component of variance. If the
use of a particular value of n can be assumed at the outset, then x can be
estimated directly from the m means xi
x
j 1
/n
ij
xi / m
s
x
(x x )
m1
i
24
more than normally likely and will produce outlying values. Such values would
bias x and inflate s beyond its proper value. It is therefore advisable to recalculate
x and s after a further "settling down" period. One method of obviating the effects
of outliers in the calculation is to reject them after the application of Dixon's Q or
Grubbs'(11) test, and then use the classical statistics given above. Alternatively,
the methods of robust statistics could be applied to the data(12, 13).
(v) One of the charts shows nine successive plotting values falling on the
same side of the mean line.
A more thorough treatment of the control chart can be obtained by the application
of the full Westgard rules, illustrated in Figure 2.
OIV-MA-AS1-11 : R2002
25
4. REFERENCES
1
W A Shewhart, "Economic control of quality in manufactured product",
Van
Nostrand, New York, 1931.
2
ISO 8258:1991. "Shewhart control charts".
3
ISO 7873:1993 "Control charts for arithmetic means with warning limits".
4
ISO 7870:1993. "Control charts - general guide and introduction".
5
ISO 7966:1993. "Acceptance control charts".
6
S Levey and E R Jennings, Am. J. Clin. Pathol., 1950, 20, 1059-1066.
7
A B J Nix, R J Rowlands, K W Kemp, D W Wilson and K Griffiths, Stat.
Med., 1987,6,425-440.
8
J O Westgard, P L Barry and M R Hunt, Clin. Chem., 1981, 27, 493-501.
9
C A Parvin, Clin. Chem., 1992, 38, 358-363.
10
J Bishop and A B J Nix, Clin. Chem., 1993, 39, 1638-1649.
11
W Horwitz, Pure Appl. Chem., (in press).
12
Analytical Methods Committee, Analyst, 1989, 114, 1693-1697.
13
Analytical Methods Committee, Analyst, 1989, 114, 1699-1702.
---------------------------Technical report from the Symposium on the 'Harmonisation of quality assurance systems for Analysis
Laboratories, Washington DC, USA, 22-23 July 1993 sponsored by IUPAC, ISO et AOAC International
Prepared for publication by MICHAEL THOMPSON1 and ROGER WOOD2
1Department of Chemistry, Birkbeck College (University of London), London WC1H OPP, UK
2MAFF Food Science Laboratory, Norwich Research Park, Colney, Norwich NR4 7UQ, UK
1991-95 work group :
Chairman : M. Parkany (Switzerland) ; Membres : T. Anglov (Denmark) ; K. Bergknut (Norway and
sweden) ; P. De Bive (Belgium) ; K.-G. von Boroviczny (Germany) ; J.M. Christensen (Denmark) ; T.D.
Geary (South Australia) ; R. Greenhalgh (Canada) ; A.J. Head (United Kingdom) ; P.T. Holland (New
Zealand) ; W. Horwitz (USA) . A. Kallner (Sweden; J. Kristiansen (Denmark) ; S.H.H. Olrichs
(Netherlands) ; N. Palmer (USA) . M. Thompson (United Kingdom) ; M.J. Vernengo (Argentina) ; R. Wood
(United Kingdom).
OIV-MA-AS1-11 : R2002
26
1.
PURPOSE ..................................................................................................................... 5
2.
3.
4.
5.
METHODOLOGY .................................................................................................. 12
DEFINITION OF MEASUREMENT ERROR ........................................................... 13
5.2.3
Robustness .................................................................................................... 24
5.2.3.1
Definition .............................................................................................................. 24
5.2.3.2
Determination ....................................................................................................... 24
5.3
SECTION TWO: SYSTEMATIC ERROR STUDY.................................................... 24
5.3.1
5.3.1.1
5.3.1.2
5.3.1.3
OIV-MA-AS1-12 : R2005
5.3.2
Specificity ..................................................................................................... 36
5.3.2.1
Normative definition ............................................................................................. 36
5.3.2.2
Application ........................................................................................................... 36
5.3.2.3
Procedures ............................................................................................................ 36
5.3.2.3.1 Standard addition test ..................................................................................... 36
5.3.2.3.1.1
Scope ...................................................................................................... 36
5.3.2.3.1.2
Basic protocol ......................................................................................... 36
5.3.2.3.1.3
Calculations and results .......................................................................... 37
5.3.2.3.1.3.1
Study of the regression line r = a + b.v ........................................... 37
5.3.2.3.1.3.2
Analysis of the results ..................................................................... 38
5.3.2.3.1.3.3
Overlap line graphics ...................................................................... 40
5.3.2.3.2 Study of the influence of other compounds on the measurement result ......... 40
5.3.2.3.2.1
Scope ...................................................................................................... 40
5.3.2.3.2.2
Basic protocol and calculations .............................................................. 40
5.3.2.3.2.3
Interpretation .......................................................................................... 41
5.3.3
5.3.3.1
Presentation of the step ......................................................................................... 43
5.3.3.1.1 Definition ....................................................................................................... 43
5.3.3.1.2 General principles .......................................................................................... 43
5.3.3.1.3 Reference documents ..................................................................................... 43
5.3.3.2
Comparison of the alternative method with the OIV reference method ................ 44
5.3.3.2.1 Scope .............................................................................................................. 44
5.3.3.2.2 Accuracy of the alternative method compared with the reference method ..... 44
5.3.3.2.2.1
Definition ............................................................................................... 44
5.3.3.2.2.2
Scope ...................................................................................................... 44
5.3.3.2.2.3
Basic protocol and calculations .............................................................. 44
5.3.3.2.2.4
Interpretation .......................................................................................... 46
5.3.3.3
Comparison by interlaboratory tests ..................................................................... 47
5.3.3.3.1 Scope .............................................................................................................. 47
5.3.3.3.2 Basic protocol and calculations ...................................................................... 48
5.3.3.3.3 Interpretation .................................................................................................. 49
5.3.3.4
Comparison with reference materials .................................................................... 50
5.3.3.4.1 Scope .............................................................................................................. 50
5.3.3.4.2 Basic protocol and calculations ...................................................................... 50
5.3.3.4.3 Interpretation .................................................................................................. 51
5.4
SECTION THREE: RANDOM ERROR STUDY ....................................................... 52
5.4.1
OIV-MA-AS1-12 : R2005
5.4.3.1
Definition .............................................................................................................. 53
5.4.3.2
Scope .................................................................................................................... 53
5.4.3.3
General theoretical case ........................................................................................ 54
5.4.3.3.1 Basic protocol and calculations ...................................................................... 54
5.4.3.3.1.1
Calculations with several test materials .................................................. 54
5.4.3.3.1.2
Calculations with 1 test material............................................................. 56
5.4.3.4
Repeatability ......................................................................................................... 57
5.4.3.4.1 Definitions ...................................................................................................... 57
5.4.3.4.2 Scope .............................................................................................................. 58
5.4.3.4.3 Basic protocol and calculations ...................................................................... 58
5.4.3.4.3.1
General case ........................................................................................... 58
5.4.3.4.3.2
Particular case applicable to only 1 repetition ........................................ 58
5.4.3.4.4 Comparison of repeatability ........................................................................... 60
5.4.3.4.4.1
Determination of the repeatability of each method ................................. 60
5.4.3.4.4.2
Fischer-Snedecor test ............................................................................. 61
5.4.3.5
Intralaboratory reproducibility .............................................................................. 62
5.4.3.5.1 Definition ....................................................................................................... 62
5.4.3.5.2 Scope .............................................................................................................. 62
5.4.3.5.3 Basic protocol and calculations ...................................................................... 62
6.
6.5.3
6.5.4
6.5.4.1
Analysis chain of interlaboratory comparisons ..................................................... 70
6.5.4.2
Comparison with external reference materials ...................................................... 70
6.5.4.2.1 Standard uncertainty of reference material ..................................................... 71
6.5.4.2.2 Defining the validity limits of measuring reference material .......................... 71
7.
DEFINITION ......................................................................................................... 72
REFERENCE DOCUMENTS .................................................................................. 73
SCOPE .................................................................................................................. 73
METHODOLOGY .................................................................................................. 74
OIV-MA-AS1-12 : R2005
7.4.3.1
7.4.3.2
7.4.3.3
Principle ................................................................................................................ 75
Calculating the standard deviation of intralaboratory reproducibility ................... 78
Estimating typical sources of systematic errors not taken into account under
reproducibility conditions ..................................................................................... 79
7.4.3.3.1 Gauging error (or calibration error) ................................................................ 79
7.4.3.3.1.1
Procedure................................................................................................ 79
7.4.3.3.1.2
Calculations and results .......................................................................... 80
7.4.3.3.1.3
Estimating the standard uncertainty associated the gauging line
(or calibration line) ................................................................................. 81
7.4.3.3.2 Bias error ........................................................................................................ 82
7.4.3.3.2.1
Methods adjusted with only one certified reference material ................. 82
7.4.3.3.2.2
Methods adjusted with several reference materials (gauging ranges etc) 82
7.4.3.3.3 Matrix effect ................................................................................................... 83
7.4.3.3.3.1
Definition ............................................................................................... 83
7.4.3.3.4 Sample effect .................................................................................................. 86
7.4.4
7.4.4.1
7.4.4.2
Principle ................................................................................................................ 86
Using the standard deviation of interlaboratory and intramethod
reproducibility SRinter (method)............................................................................. 87
7.4.4.3
Using the standard deviation of interlaboratory and intermethod
reproducibility SRinter ............................................................................................ 87
7.4.4.4
Other components in the uncertainty budget......................................................... 88
7.5
EXPRESSING EXPANDED UNCERTAINTY ......................................................... 88
OIV-MA-AS1-12 : R2005
1. Purpose
The purpose of this guide is to assist oenological laboratories carrying out serial
analysis as part of their validation, internal quality control and uncertainty
assessment initiatives concerning the standard methods they use.
2. Preamble and scope
International standard ISO 17025, defining the "General Requirements for the
Competence of Testing and Calibration Laboratories", states that the accredited
laboratories must, when implementing a alternative analytical method, make sure
of the quality of the results obtained. To do so, it indicates several steps. The first
step consists in defining the customers' requirements concerning the parameter in
question, in order to determine, thereafter, whether the method used meets those
requirements. The second step includes initial validation for non-standardized,
modified or laboratory-developed methods. Once the method is applied, the
laboratories must use inspection and traceability methods in order to monitor the
quality of the results obtained. Finally, they must assess the uncertainty of the
results obtained.
In order to meet these requirements, the laboratories have a significant reference
system at their disposal comprising a large number of international guides and
standards. However, in practice, the application of these texts is delicate since,
because they address every category of calibration and test laboratory, they remain
very general and presuppose, on behalf of the reader, in-depth knowledge of the
mathematical rules applicable to statistical data processing.
This guide is based on this international reference system, taking into account the
specific characteristics of oenology laboratories routinely carrying out analyses on
series of must or wine samples. Defining the scope of application in this way
enabled a relevant choice of suitable tools to be made, in order to retain only those
methods most suitable for that scope. Since it is based on the international
reference system, this guide is therefore strictly compliant with it. Readers,
however, wishing to study certain points of the guide in greater detail can do so by
referring to the international standards and guides, the references for which are
given in each chapter.
The authors have chosen to combine the various tools meeting the requirements of
the ISO 17025 standard since there is an obvious solution of continuity in their
OIV-MA-AS1-12 : R2005
application, and the data obtained with certain tools can often be used with the
others. In addition, the mathematical resources used are often similar.
The various chapters include application examples, taken from oenology
laboratories using these tools.
It is important to point out that that this guide does not pretend to be exhaustive. It
is only designed to present, in as clear and applicable a way as possible, the
contents of the requirements of the ISO 17025 standard and the basic resources
that can be implemented in a routine laboratory to meet them. Each laboratory
remains perfectly free to supplement these tools or to replace them by others that
they consider to be more efficient or more suitable.
Finally, the readers attention should be drawn to the fact that the tools presented
do not constitute an end in themselves and that their use, as well as the
interpretation of the results to which they lead, must always be subject to critical
analysis. It is only under these conditions that their relevance can be guaranteed,
and laboratories will be able to use them as tools to improve the quality of the
analyses they carry out.
3. General vocabulary
The definitions indicated below used in this document result from the normative
references given in the bibliography.
Analyte
Object of the analysis method
Blank
Test carried out in the absence of a matrix (reagent blank) or on a matrix which
does not contain the analyte (matrix blank).
Bias
Difference between the expected test results and an accepted reference value.
Uncertainty budget
The list of uncertainty sources and their associated standard uncertainties,
established in order to assess the compound standard uncertainty associated with a
measurement result.
OIV-MA-AS1-12 : R2005
( xi x )
i 1
n 1
xi being the result of the measurement ith and x the arithmetic mean of the n
results considered.
Repeatability standard deviation
Standard deviation of many repetitions obtained in a single laboratory by the same
operator on the same instrument, i.e. under repeatable conditions.
Internal reproducibility standard deviation (or total intralaboratory
variability)
Standard deviation of repetitions obtained in a single laboratory with the same
method, using several operators or instruments and, in particular, by taking
measurements on different dates, i.e. under reproducibility conditions.
Random error
Result of a measurement minus the mean that would result from an infinite number
of measurements of the same measurand carried out under reproducibility
conditions.
OIV-MA-AS1-12 : R2005
Measurement error
Result of a measurement minus a true value of the measurand.
Systematic error
Mean error that would result from an infinite number of measurements of the same
measurand carried out under reproducibility conditions minus a true value of the
measurand.
NOTE Error is a highly theoretical concept in that it calls upon values that are not
accessible in practice, in particular the true values of measurands. On principle, the
error is unknown.
Mathematical expectation
For a series of n measurements of the same measurand, if n tends towards the
infinite, the mean x tends towards the expectation E(x).
n
lim
E ( x) n
xi
i 1
Calibration
Series of operations establishing under specified conditions the relation between
the values of the quantity indicated by a measuring instrument or system, or the
values represented by a materialized measurement or a reference material, and the
corresponding values of the quantity measured by standards.
Intralaboratory evaluation of an analysis method
Action which consists in submitting an analysis method to an intralaboratory
statistical study, based on a standardized and/or recognized protocol,
demonstrating that within its scope, the analysis method meets pre-established
performance criteria.
Within the framework of this document, the evaluation of a method is based on an
intralaboratory study, which includes the comparison with a reference method.
Precision
Closeness of agreement between independent test results obtained under
prescribed conditions
NOTE 1
Precision depends only on the distribution of random errors and
does not have any relationship with the true or specified value.
NOTE 2
The measurement of precision is expressed on the basis of the
standard deviation of the test results.
NOTE 3
The expression "independent test results" refers to results obtained
such that they are not influenced by a previous result on the same or a similar test
OIV-MA-AS1-12 : R2005
OIV-MA-AS1-12 : R2005
The linearity limits are the experimental limits of concentrations between which a
linear calibration model can be applied with a known confidence level (generally
taken to be equal to 1%).
Test material
Material or substance to which a measuring can be applied with the analysis
method under consideration.
Reference material
Material or substance one or more of whose property values are sufficiently
homogeneous and well established to be used for the calibration of an apparatus,
the assessment of a measurement method, or for assigning values to materials.
Certified reference material
Reference material, accompanied by a certificate, one or more whose property
values are certified by a procedure which establishes its traceability to an accurate
realization of the unit in which the property values are expressed, and for which
each certified value is accompanied by an uncertainty at a stated level of
confidence.
Matrix
All the constituents of the test material other than the analyte.
Analysis method
Written procedure describing all the means and procedures required to carry out
the analysis of the analyte, i.e.: scope, principle and/or reactions, definitions,
reagents, apparatus, procedures, expression of results, precision, test report.
WARNING The expressions "titration method" and "determination method" are
sometimes used as synonyms for the expression "analysis method". These two
expressions should not be used in this way.
Quantitative analysis method
Analysis method making it possible to measure the analyte quantity present in the
laboratory test material.
Reference analysis method (Type I or Type II methods)
Method, which gives the accepted reference value for the quantity of the analyte to
be measured.
Non-classified alternative method of analysis
A routine analysis method used by the laboratory and not considered to be a
reference method.
OIV-MA-AS1-12 : R2005
10
xi
i 1
OIV-MA-AS1-12 : R2005
11
Tolerance
Deviation from the reference value, as defined by the laboratory for a given level,
within which a measured value of a reference material can be accepted.
Value of a quantity
Magnitude of a particular quantity generally expressed as a unit of measurement
multiplied by a number.
12
steps are used to evaluate the measurement uncertainty. The latter, which is
regularly assessed, is an indicator of the quality of the results obtained by the
method under consideration.
Development or adoption of a
method
All these steps are inter-connected and constitute a global approach that can be
used to assess and control measurement errors.
Measurement error
OIV-MA-AS1-12 : R2005
13
In practice, the systematic error results in a bias in relation to the true value, the
random error being all the errors associated with the application of the method.
These errors can be graphically represented in the following way:
Precision
Error
Systematic error
True value
Random error
Mean value of an
infinite number of
results
Result of an analysis
The validation and quality control tools are used to evaluate the systematic errors
and the random errors, and to monitor their changes over time.
5. Validating a method
5.1 Methodology
Implementing the validation comprises 3 steps, each with objectives. To meet
these objectives, the laboratory has validation tools. Sometimes there are many
tools for a given objective, and are suitable for various situations. It is up to the
laboratory to correctly choose the most suitable tools for the method to be
validated.
OIV-MA-AS1-12 : R2005
14
Steps
Objectives
Scope of
application
- To define the analyzable matrices
- To define the analyzable range
Systematic
error
or bias
Linearity study
Repeatability study
Intralaboratory reproducibility
study
Specificity study
Comparison with a reference
method
Comparison with reference
materials
Interlaboratory comparison
Random error
15
5.2.2.3 Application
In practice, the quantification limit is generally more relevant than the detection
limit, the latter being by convention 1/3 of the first.
There are several approaches for assessing the detection and quantification limits:
- Determination on blank
- Approach by the linearity study
- Graphic approach
These methods are suitable for various situations, but in every case they are
mathematical approaches giving results of informative value only. It seems crucial,
whenever possible, to introduce a check of the value obtained, whether by one of
these approaches or estimated empirically, using the checking protocol for a
predetermined quantification limit.
5.2.2.4 Procedure
5.2.2.4.1 Determination on blank
5.2.2.4.1.1 Scope
OIV-MA-AS1-12 : R2005
16
This method can be applied when the blank analysis gives results with a non-zero
standard deviation. The operator will judge the advisability of using reagent
blanks, or matrix blanks.
If the blank, for reasons related to uncontrolled signal preprocessing, is sometimes
not measurable or does not offer a recordable variation (standard deviation of 0),
the operation can be carried out on a very low concentration in analyte, close to the
blank.
5.2.2.4.1.2 Basic protocol and calculations
Carry out the analysis of n test materials assimilated to blanks, n being equal to or
higher than 10.
- Calculate the mean of the xi results obtained:
n
xblank
xi
i 1
Sblank
( xi xblank )
i 1
n 1
- From these results the detection limit is conventionally defined by the formula:
Ld xblank (3.Sblank)
Example: The table below gives some of the results obtained when assessing the
detection limit for the usual determination of free sulfur dioxide.
OIV-MA-AS1-12 : R2005
17
Test material #
1
2
3
4
5
6
7
8
9
10
11
12
X
( mg/l)
0
1
0
1.5
0
1
0.5
0
0
0.5
0
0
18
( xi M x )( yi M y )
i 1
n
( xi M x )
i 1
S res
( yi, j y i, j )
i 1 j 1
pn 2
np
Mx
n
p ( xi M x )
i 1
The estimates of the detection limit DL and the quantification limit QL are
calculated using following formulae:
DL
QL
3 Sa
b
10 S a
OIV-MA-AS1-12 : R2005
19
X (ref)
Y1
Y2
Y3
Y4
1
2
3
4
5
10
15
20
1.9
2.4
4
5.3
5.3
11.6
16
19.7
0.8
2
2.8
4.5
5.3
10.88
15.2
20.4
0.5
2.5
3.5
4.7
5.2
12.1
15.5
19.5
1.5
2.1
4
4.5
5.3
10.5
16.1
20.1
OIV-MA-AS1-12 : R2005
DL = 0.48 mg.L-1
QL = 1.6 mg.L-1
20
QL = 10 hmax R
OIV-MA-AS1-12 : R2005
21
In all other cases, wines (or musts) shall be used whose measurand value as
obtained by the reference method is equal to the limit to be studied. Of course, in
this case the quantification limit of the reference method must be lower than this
value.
5.2.2.4.4.2 Basic protocol and calculation
Analyze n independent test materials whose accepted value is equal to the
quantification limit to be checked; n must at least be equal to 10.
- Calculate the mean of n measurements:
n
xi
x LQ
i 1
( xi xLQ )
n
S LQ
i 1
n 1
S QL
n
22
This is equivalent to checking that the coefficient of variation for QL is lower than
20%.
NOTE1
Remember that the detection limit is obtained by dividing the
quantification limit by 3.
NOTE2
A check should be made to ensure that the value of SLQ is not too
large (which would produce an artificially positive test), and effectively
corresponds to a reasonable standard deviation of the variability of the results for
the level under consideration. It is up to the laboratory to make this critical
evaluation of the value of SLQ.
Example: Checking the quantification limit of the
determination of malic acid by the enzymatic method.
Estimated quantification limit: 0.1 g.L-1
Wine
1
2
3
4
5
6
7
8
9
10
Values
0.1
0.1
0.09
0.1
0.09
0.08
0.08
0.09
0.09
0.08
Mean: 0.090
Standard deviation: 0.008
First condition:
LQ xQL
3.8710
The quantification
QL
OIV-MA-AS1-12 : R2005
23
5.2.3 Robustness
5.2.3.1 Definition
Robustness is the capacity of a method to give close results in the presence of
slight changes in the experimental conditions likely to occur during the use of the
procedure.
5.2.3.2 Determination
If there is any doubt about the influence of the variation of operational parameters,
the laboratory can use the scientific application of experiment schedules, enabling
these critical operating parameters to be tested within the variation range likely to
occur under practical conditions. In practice, these tests are difficult to implement.
5.3.1.3 Application
The linearity study can be used to define and validate a linear dynamic range.
This study is possible when the laboratory has stable reference materials whose
accepted values have been acquired with certainty (in theory these values should
have an uncertainty equal to 0). These could therefore be internal reference
materials titrated with calibrated material, wines or musts whose value is given by
OIV-MA-AS1-12 : R2005
24
25
Accepted
reference value
material
x1
...
xi
...
xn
Replica
1
y11
...
yi1
...
yn1
Measured values
Replica
...
j
...
y1j
...
...
...
...
...
yij
...
...
...
...
...
ynj
...
...
Replica
p
y1p
...
yip
...
ynp
yij a b.xi ij
where
yij
xi
b
a
ab.xi
ij
is the difference between yij and the expectation of the measurement value
of the ith reference material.
OIV-MA-AS1-12 : R2005
26
1
p
yij
j 1
1
My
n
1
n
Mx
xi
i 1
yi
i 1
( xi M x )( yi M y )
- estimated slope b
i 1
( xi M x )
i 1
a M y b Mx
yi
y i a b xi
- residual eij
eij yij y i
5.3.1.4.2.3 Charts
The results can be presented and analyzed in graphic form. Two types of charts are
used.
OIV-MA-AS1-12 : R2005
27
- The first type of graph is the representation of the values measured against the
accepted values of reference materials. The calculated overlap line is also plotted.
Overlap line
10,00
8, 00
s
e
lu
a
v6, 00
d
e
ru
s
a
e4, 00
M
y=x
yi^(rgression)
repli que
replica
11
repli que2
replica
2
repli que3
replica
3
repli que4
replica
4
2, 00
0, 00
0, 00
2,00
4,00
6,00
8,00
10,00
- The second graph is the representation of the residual values against the
estimated values of the reference materials ( y ) indicated by the overlap line.
The graph is a good indicator of the deviation in relation to the linearity
assumption: the linear dynamic range is valid if the residual values are fairly
distributed between the positive and negative values.
OIV-MA-AS1-12 : R2005
28
0,6
0,5
0,4
0,3
0,2
replica 1 1
replique
replica 2
replique2
0,1
replica 3
replique3
replique4
replica 4
0
0
10
12
-0,1
-0,2
-0,3
-0,4
Adjusted values y^
In case of doubt about the linearity of the regression, a Fischer-Snedecor test can
be carried out in order to test the assumption: "the linear dynamic range is not
valid", in addition to the graphic analysis.
5.3.1.4.2.4 Test of the linearity assumption
Several error values linked to calibration should be defined first of all: these can
be estimated using the data collected during the experiment. A statistical test is
then performed on the basis of these results, making it possible to test the
assumption of non-validity of the linear dynamic range: this is the FischerSnedecor test.
5.3.1.4.2.4.1 Definitions of errors linked to calibration
These errors are given as a standard deviation, resulting from the square root of the
ratio between a sum of squares and a degree of freedom.
Residual error
The residual error corresponds to the error between the measured values and the
value given by the regression line.
The sum of the squares of the residual error is as follows:
n
Qres
( yij y i )
i 1 j 1
OIV-MA-AS1-12 : R2005
29
( yij y i )
i 1 j 1
S res
np 2
Experimental error
The experimental error corresponds to the reproducibility standard deviation of the
experimentation.
The sum of the squares of the experimental error is as follows:
p
( yij yi )
n
Qexp
i 1 j 1
( yij yi )
n
i 1 j 1
Sexp
np n
or
p
( yij y i ) ( yij yi )
n
Qdef
i 1 j 1
i 1 j 1
OIV-MA-AS1-12 : R2005
30
Qres Qexp
n2
or
p
( yij y i ) ( yij yi )
n
S def
i 1 j 1
i 1 j 1
n2
Fobs
S def
S exp
OIV-MA-AS1-12 : R2005
Y1
Y2
Y3
Y4
0.41
1.15
1.72
2.45
2.95
4.09
6.07
8.12
10.2
0.37
1.12
1.63
2.37
2.83
3.86
5.95
8.01
10
0.4
1.16
1.76
2.45
2.99
4.04
6.04
8.05
10.09
0.41
1.17
1.71
2.45
2.95
4.04
6.04
7.9
9.87
31
Regression line
Line ( y = a + b*x)
b = 1.01565
a = - 0.00798
Errors related to calibration
Residual standard deviation Sres = 0.07161
Standard deviation of experimental reproducibility Sexp =
0.07536
Standard deviation of the adjustment error Sdef = 0.0548
Interpretation, Fischer-Snedecor test
Fobs = 0.53 < F1- = 2.37
The assumption of the non-validity of the linear
dynamic range is rejected
5.3.1.5 ISO 8466-type approach
5.3.1.5.1 Basic protocol
It is advisable to use a number n of reference materials. The number must be
higher than 3, but there is no need, however, to exceed 10. The reference materials
should be measured several times, under reproducibility conditions. The number
of measurements may be small at the center of the range studied (minimum = 2)
and must be greater at both ends of the range, for which a minimum number of 4 is
generally recommended. The accepted values of reference materials must be
regularly distributed over the studied range of values.
NOTE It is vital that the reproducibility conditions use the maximum number of
potential sources of variability.
The results are reported in a table presented as follows:
OIV-MA-AS1-12 : R2005
Replica
p
y1p
ynp
32
ya
b c
N
i
i
4
N
i
3
2
2
xi y xi xi xi N xi yi xi yi xi xi yi xi yi xi
i
i
i
i
i
i
i
i
i i i
2
3
3
2
2
3
x xi xi xi N xi xi xi xi xi xi x i2
i
i
i
i
i
i
i
i i i
i
xi N xi yi xi yi xi yi N xi xi xi xi yi xi x i yi xi
i
4
N
i
2
i
i
2
2
3
3
2
3
x xi xi xi N xi xi xi x xi xi x i2
i
i
i
i
i
i
i
i i i
i
OIV-MA-AS1-12 : R2005
33
y
y
y
y
y
xi xi i x i xi
xi xi i xi xi xi xi xi x i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
4
3
3
2
2
3
xi N xi xi xi N xi xi xi xi xi xi x i2
i
i
i
i
i
i
i
i i i
i
Once the model has been established, the following values are to be calculated:
- regression value associated with the ith reference material
y i a
-
y i
b c
eij yij yi
residual eij
( yij y i )
n
S res
i 1 j 1
np 2
DS
( N 2)
S res ( N 3) S res
2
Then
2
PG DS
2
S res
The value PG is compared with the limit value F1- given by the Fischer-Snedecor
table for a confidence level 1- and a degree of freedom 1 and (N-3).
NOTE In general the risk used is 5%. In some cases the test may be optimistic
and a risk of 10% will prove more realistic.
If PG F1-: the nonlinear calibration function does not result in an improved
adjustment; for example, the calibration function is linear.
If PG > F1-: the work scope must be as narrow as possible to obtain a linear
calibration function: otherwise, the information values from the analyzed samples
must be evaluated using a nonlinear calibration function.
OIV-MA-AS1-12 : R2005
34
Y1
Y2
Y3
Y4
35
22.6
19.6
21.6
62
49.6
49.8
53
90
105.2
103.5
130
149
149.8
205
203.1
202.5
197.3
330
297.5
298.6
307.1
18.4
294.2
300
Mea
n of
me
ase
250
ure
d
val
ues
200
y=x
yi^(lin reg)
yi'^(poly reg)
150
yi (mean)
100
50
0
0
50
100
150
200
250
300
350
Linear regression
y = 1.48.x 0.0015
Sres = 13.625
Polynomial regression
y = - 0.0015x + 1.485x 27.2701
S'res = 7.407
Fischer's test
PG = 10.534 > F(5%) = 10.128
PG>F the linear calibration function cannot be retained
OIV-MA-AS1-12 : R2005
35
5.3.2 Specificity
5.3.2.1 Normative definition
The specificity of a method is its ability to measure only the compound being
searched for.
5.3.2.2 Application
In case of doubt about the specificity of the tested method, the laboratory can use
experiment schedules designed to check its specificity. Two types of
complementary experiments are proposed here that can be used in a large number
of cases encountered in the field of oenology.
- The first test is the standard addition test. It can be used to check that
the method measures all the analyte.
- The second test can be used to check the influence of other compounds
on the result of the measurement.
5.3.2.3 Procedures
5.3.2.3.1 Standard addition test
5.3.2.3.1.1 Scope
This test can be used to check that the method measures all the analyte.
The experiment schedule is based on standard additions of the compound being
searched for. It can only be applied to methods that are not sensitive to matrix
effects.
5.3.2.3.1.2 Basic protocol
This consists in finding a significant degree of added quantities on test materials
analyzed before and after the additions.
Carry out variable standard additions on n test materials. The initial concentration
in analyte of test materials, and the standard additions are selected in order to
cover the scope of the method. These test materials must consist of the types of
matrices called for routine analysis. It is advised to use at least 10 test materials.
The results are reported in a table presented as follows:
OIV-MA-AS1-12 : R2005
36
Test
material
1
...
i
...
n
NOTE 1
NOTE 2
NOTE 3
Quantity
before
addition
(x)
x1
...
xi
...
Xn
Quantity
added
(v)
Quantity
after addition
(w)
Quantity
found
(r)
v1
...
vi
...
Vn
w1
...
wi
...
wn
r1 = w1 x1
...
ri = wi xi
...
rp = wn xn
vi
i 1
n
n
OIV-MA-AS1-12 : R2005
ri
i 1
n
37
- estimated slope b
(vi v)(ri r )
i 1
n
(vi v)
i 1
a r b.v
yi
ri a b vi
n
S res
ri ri 2
i 1
n2
S b S res
(v i v ) 2
i 1
S a S res
1
n
(v i v ) 2
i 1
38
Tobs
b1
Sb
T'obs
a
Sa
OIV-MA-AS1-12 : R2005
39
Y = X
Estimated relation between the added content and the content found
9
8
Y: Content found
Y: Content found
7
6
5
4
3
7
6
5
4
Y = X
3
2
1
0
0
0
0
X : Content found
OIV-MA-AS1-12 : R2005
40
Samples
x: Before
addition
Rep1
Rep2
Means
y: After addition
Difference
Rep1
Rep2
x1
x1
y1
y1
Mx1
My1
...
...
...
...
...
...
...
xi
xi
yi
yi
Mxi
Myi
...
...
...
...
...
...
...
xn
xn
yn
yn
Mxn
Myn
d
d1 = Mx1My1
...
di = MxiMyi
...
dn = MxnMyn
1
Mx
n
Mxi
i 1
My
1
n
Myi
i 1
Md
ni My Mx
d
i 1
Sd
(di M d )
i 1
n 1
Md
Sd
5.3.2.3.2.3 Interpretation
OIV-MA-AS1-12 : R2005
41
vin
1
2
3
4
5
6
7
8
9
10
Differences
sorbate
diff
0.2
0.05
-0.05
-0.05
0.05
0.05
0.05
-0.1
0.05
-0.05
Potassium sorbate
Md =
Sd =
Zscore =
0.02
0.086
0.23 <2
Salicylic acid
Md =
Sd =
Zscore =
-0.725
0.282
2.57 >2
salicylic
diff
-0.8
-0.65
-0.3
-0.4
-1.1
-1.05
-1.05
-0.8
-0.55
-0.55
OIV-MA-AS1-12 : R2005
42
OIV-MA-AS1-12 : R2005
43
5.3.3.2 Comparison of the alternative method with the OIV reference method
5.3.3.2.1 Scope
This method can be applied if the laboratory uses the OIV reference method, or a
traced, validated method, whose performance quality is known and meets the
requirements of the laboratorys customers.
To study the comparative accuracy of the two methods, it is advisable first of all to
ensure the quality of the repeatability of the method to be validated, and to
compare it with the reference method. The method for carrying out the
repeatability comparison is described in the chapter on repeatability.
5.3.3.2.2 Accuracy of the alternative method compared with the reference method
5.3.3.2.2.1 Definition
Accuracy is defined as the closeness of agreement between the values obtained by
the reference method and that obtained by the alternative method, independent of
the errors of precision of the two methods.
5.3.3.2.2.2 Scope
The accuracy of the alternative method in relation to the reference method is
established for a field of application in which the repeatabilities of the two
methods are constant.
In practice, it is therefore often advisable to divide the analyzable range of values
into several sections or "range levels" (2 to 5), in which we may reasonably
consider that the repeatabilities of the methods are comparable to a constant.
5.3.3.2.2.3 Basic protocol and calculations
In each range level, accuracy is based on a series of n test materials with
concentration values in analyte covering the range level in question. A minimum
number of 10 test materials is required to obtain significant results.
Each test material is to be analyzed in duplicate by the two methods under
repeatable conditions.
OIV-MA-AS1-12 : R2005
44
Test
material
x: Alternative
method
Rep1 Rep2
y: Reference
method
Rep1 Rep2
Means
Difference
x1
x1
y1
y1
Mx1
My1
...
...
...
...
...
...
...
xi
xi
yi
yi
Mxi
Myi
...
...
...
...
...
...
...
xn
xn
yn
yn
Mxn
Myn
d
d1 = Mx1 My1
...
di = Mxi Myi
...
dn = Mxn Myn
1
Mx
n
Mxi
i 1
1
My
n
Myi
i 1
Md
ni
d
Mx My
i 1
OIV-MA-AS1-12 : R2005
45
Sd
(di M d )
i 1
n 1
Md
Sd
5.3.3.2.2.4 Interpretation
- If the Zscore is lower than or equal to 2.0, it can be concluded that the accuracy of
one method in relation to the other is satisfactory, in the range level under
consideration, with a risk of error = 5%.
- If the Zscore is higher than 2.0, it can be concluded that the alternative method is
not accurate in relation to the reference method, in the range level under
consideration, with a risk of error = 5%.
NOTE Interpreting the Zscore is possible given the assumption that the variations
obey a normal law with a 95% confidence rate.
Example: Study of the accuracy of FTIR gauging to determine glucose
and fructose in relation to the enzymatic method. The first range level
covers the scale from 0 to 5 g.L-1 and the second range level covers a scale
from 5 to 20 g.L-1.
Wine
1
2
3
4
5
6
7
8
9
10
11
12
FTIR 1
0
0.2
0.6
0.7
1.2
1.3
2.1
2.4
2.8
3.5
4.4
4.8
OIV-MA-AS1-12 : R2005
IRTF2
0.3
0.3
0.9
1
1.6
1.4
2
0
2.5
4.2
4.1
5.4
Enz 1
0.3
0.1
0.0
0.8
1.1
1.3
1.9
1.1
2.0
3.7
4.1
5.5
Enz 2
0.2
0.1
0.0
0.7
1.3
1.3
2.1
1.2
2.6
3.8
4.4
5.0
di
-0.1
0.2
0.7
0.1
0.2
0.0
0.0
0.1
0.3
0.1
0.0
-0.2
46
Md
Sd
Zscore
Wine
1
2
3
4
5
6
7
8
9
10
11
12
Md =
Sd =
Zscore =
0.13
0.23
0.55 < 2
FTIR 1
5.1
5.3
7.7
8.6
9.8
9.9
11.5
11.9
12.4
16
17.7
20.5
IRTF2
5.4
5.7
7.6
8.6
9.9
9.8
11.9
12.1
12.5
15.8
18.1
20.1
Enz 1
5.1
5.3
7.2
8.3
9.1
9.8
13.3
11.2
11.4
15.1
17.9
20.0
Enz 2
5.1
6.0
7.0
8.5
9.3
10.2
13.0
11.4
12.1
15.7
18.3
19.1
di
0.1
-0.2
0.6
0.2
0.6
-0.1
-1.4
0.7
0.7
0.5
-0.2
0.7
0.19
0.63
0.30 < 2
For the two range levels, the Zscore is lower than 2. The
FTIR gauging for the determination of fructose glucose
studied here, can be considered accurate in relation to the
enzymatic method.
OIV-MA-AS1-12 : R2005
47
m
SR-inter
The test materials are analyzed with p replicas by the laboratory, these replicas
being carried out under repeatable conditions. p must at least be equal to 2.
In addition, the laboratory must be able to check that the intralaboratory variability
(intralaboratory reproducibility) is lower than the interlaboratory variability
(interlaboratory reproducibility) given by the analysis chain.
For each test material, the laboratory calculates the Zscore, given by the following
formula:
Z score
OIV-MA-AS1-12 : R2005
mlab m
S R int er
48
Lab mean
Chain
mean
Standard
deviation
m1
SR-inter(1)
...
...
mi
SR-inter(i)
...
...
mn
SR-inte(n)
Zscore
1j
j 1
x11
...
x1j
...
x1p
mlab1
...
...
...
...
...
...
...
Z score1
mlab1 m1
SR inter(1)
...
ij
xi1
...
xij
...
xip
mlabi
...
...
...
...
...
...
...
j 1
Zscorei
mlabi mi
SR inter(i)
...
xn1
...
xnj
...
xnp
xnj
mlabn j 1
p
Zscoren
mlabn mn
SR inter(n)
5.3.3.3.3 Interpretation
If all the Zscore results are lower than 2, the results of the method being studied can
considered identical to those obtained by the laboratories having produced
significant results.
NOTE Interpreting the Zscore is possible given the assumption that the variations
obey a normal law with a 95% confidence rate.
Samples
x1
x2
x3
x4
Lab
mean
Chain
mean
Standard
deviation
Zscore
34
34
33
34
33.75
32
0.29 <2
26
27
26
26
26.25
24
0.56 <2
49
Reference
material
1
...
i
...
n
x: Alternative method
Rep1
Rep2
Mean x
x1
x1
xi
xi
xn
xn
OIV-MA-AS1-12 : R2005
Mx1
...
Mxi
...
Mxn
T: Accepted value
of the reference
material
T1
...
Ti
...
Tn
Difference
d
d1 = Mx1-T1
...
di = Mxi-Ti
...
dn = Mxn-Tn
50
Mx
Mxi
1
n
i 1
MT
1
n
Ti
i 1
ni Mx M T
Md
i 1
Sd
(d i M d )
i 1
n 1
Md
Sd
5.3.3.4.3 Interpretation
- If the Zscore is lower than or equal to 2.0, it can be concluded that the accuracy of
the alternative method in relation to the accepted values for the reference material
is good on the range level under consideration.
- If Zscore is higher than 2.0, it can be concluded that the alternative method is not
accurate in relation to the accepted values for the reference materials in the range
level under consideration.
NOTE Interpreting the Zscore is possible given the assumption that the variations
obey a normal law with a 95% confidence rate.
OIV-MA-AS1-12 : R2005
51
Test apparatus
1
2
3
4
5
6
7
8
9
10
Ti (ref)
4.62
12.3
24.6
46.2
77
92.4
123.2
246.4
385
462
Y1
6.2
15.1
24.5
48.2
80.72
97.6
126.6
254.1
375.8
467.5
Y2
6.56
10.94
18
52.95
81.36
89
129.9
250.9
366.9
454.5
Y3
4.9
12.3
25.7
46.8
83.2
94.5
119.6
243.9
380.4
433.3
Y4
5.7
11.6
27.8
35
74.5
99.5
126.9
240.4
386.9
457.3
My
di
5.8
12.5
24.0
45.7
79.9
95.2
125.8
247.3
377.5
453.2
1.2
0.2
-0.6
-0.5
2.9
2.8
2.6
0.9
-7.5
-8.9
Md = -0.7
Sd = . 4.16
Zscore = 0.16
Given these results, the values obtained by the analysis
method for 4-EP by GC-MS can be considered accurate
compared with the accepted values of reference
materials.
OIV-MA-AS1-12 : R2005
52
53
...
p1
pi
pn
...
...
x1j
x1j
x1p1
x1p1
...
...
xij
xij
...
...
xipi xipi
...
...
xnj
xnj
...
...
...
...
xnpn
xnpn
In this situation, the standard deviation of total variability (or standard deviation of
precision Sv) is given by the general expression:
1
S v Var( xij ) (1 )Var(rpet)
k
where:
Var( xij )
Var(rpet)
OIV-MA-AS1-12 : R2005
54
- If the test materials were analyzed in duplicate with each replica (K = 2), the
expression becomes:
S v Var( xij )
Var(repeat)
2
- When only one measurement of the test material has been carried out with
each replica (K = 1), the variance of repeatability is null, the expression
becomes:
Sv Var( xij )
- Calculation of Var(xij)
The mean of the two replicas xij and xij is:
xij
xij x'ij
2
xij
Mxi
j 1
pi
pi
i 1
OIV-MA-AS1-12 : R2005
55
( xij M xi )
n
Var( xij )
pi
i 1 j 1
N n
NOTE This variance can also be calculated using the variances of variability of
each test material: Vari (xj). The following relation is then used (it is strictly
equivalent to the previous one):
n
Var( xij )
( pi 1).Vari ( x j )
i 1
N n
ni
wij
Var(repeat)
i 1 j 1
2N
where
The value of precision v means that in 95% of the cases, the difference between
two values obtained by the method, under the conditions defined, will be lower
than or equal to v.
NOTE 1
The use and interpretation of these results is based on the
assumption that the variations obey a normal law with a 95% confidence rate.
NOTE 2
One can also define a precision of 99% with v2.58 2.Sv 3.65.Sv
OIV-MA-AS1-12 : R2005
56
Var( xij )
( xi M x )
i 1
p 1
where:
xi
p
Mx
Var(repeat)
wi2
i 1
2p
OIV-MA-AS1-12 : R2005
57
The repeatability standard deviation Sr is the standard deviation for the results
obtained under the conditions of repeatability. It is a parameter of the dispersion of
the results, obtained under conditions of repeatability.
5.4.3.4.2 Scope
A priori, the repeatability study can be applied without difficulty to every
quantitative method, insofar as the repeatability conditions can be observed.
In many cases, repeatability is not constant throughout the range of validity of the
method. It is therefore advisable to define several sections or "range levels", in
which we may reasonably consider that the repeatability is comparable to a
constant. The repeatability calculation is then to be reiterated for each range level.
5.4.3.4.3 Basic protocol and calculations
5.4.3.4.3.1 General case
The number of test materials may vary in relation to the NUMBER of replicas. In
practice, we consider that the number of measurements of all test materials must be
higher than 20. It is not necessary for the repeatability conditions to be maintained
from one test material to another, but all the replicas carried out on the same test
material must be carried out under these repeatability conditions.
Repeatability
remains
Var(repeat)
S v Var( xij )
2
special
case
of
the
precision
calculation
measurement with each replica), and the calculation is the same as the calculation
of Var( xij )
( xij M x )
n
S r Var( x ij )
pi
i 1 j 1
N n
The value r means that in 95% of the cases, the difference between two values
acquired under repeatable conditions will be lower than or equal to r.
5.4.3.4.3.2 Particular case applicable to only 1 repetition
OIV-MA-AS1-12 : R2005
58
In practice, the most current situation for automated systems is the analysis of test
material with only one repetition. It is advisable to use at least 10 materials in
order to reach the 20 measurements required. The two measurement replicas of the
same test material must be carried out under repeatable conditions.
In this precise case, the calculation of Sr is simplified and becomes:
q
Sr
wi2
i 1
2p
in which:
Sr = the repeatability standard deviation
p = the number of test materials analyzed in
duplicate
wi = the absolute differences between duplicates
Repeatability r is calculated according to the formula:
r = 2.8 Sr
OIV-MA-AS1-12 : R2005
59
Sample no.
xi
(in mg/l)
xi
(in mg/l)
1
2
3
4
5
6
7
8
9
10
11
12
14
25
10
2
35
19
23
27
44
30
8
48
14
24
10
3
35
19
23
27
45
30
8
46
Wi
(absolute
value)
0
1
0
1
0
0
0
0
1
0
0
2
OIV-MA-AS1-12 : R2005
60
Let Sr-alt be the repeatability standard deviation of the alternative method, and Sr-ref.
the repeatability standard deviation of the reference method.
The comparison is direct. If the value of repeatability of the alternative method is
lower than or equal to that of the reference method, the result is positive. If it is
higher, the laboratory must ensure that the result rests compliant with the
specification that it accepted for the method concerned. In the latter case, it may
also apply a Fischer-Snedecor test to know if the value found for the alternative
method is significantly higher than that of the reference method.
5.4.3.4.4.2 Fischer-Snedecor test
Calculate the ratio:
Fobs
Sr
Sr
alt
ref
Use the critical Snedecor value with a risk equal to 0.05 corresponding to the
Fischer variable with a confidence level 1 , in which 1 = n(x)-n, and 2 = n(z)-m
degrees of freedom: F(N(x)-n, N(y)-m, 1- ). In the case of a calculated
repeatability with only one repetition on p test materials for the alternative method,
and q test materials for the reference method, the Fischer variable will have as a
degree of freedom 1 = p, and 2 = Q, i.e.: F(p, Q, 1- ).
Interpreting the test:
1/ Fobs > F1- , the repeatability value of the alternative method is
significantly higher than that of the reference method.
2/ Fobs < F1- , we cannot state that the repeatability value of the
alternative method is significantly higher than that of the reference method.
Example: The value of the repeatability standard deviation
found for the determination method of free sulfur dioxide is:
Sr = 0.54 mg/l
The laboratory carried out the determination on the same test
materials using the OIV reference method. The value of the
repeatability standard deviation found in this case is:
OIV-MA-AS1-12 : R2005
61
Fobs
2 = 12
1 = 12
F1- = 2.69 > 1.93
The Fobs value obtained is lower than the value F1-; we cannot
state that the repeatability value of the alternative method is
significantly higher than that of the reference method.
62
Replicas
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Test material 1
x1
x2
122
125
123
120
132
130
121
115
130
135
135
142
137
135
130
125
123
130
112
115
131
128
Test material 2
x1
x2
140
139
138
137
139
141
143
142
139
139
135
138
139
139
145
145
138
137
135
134
146
146
137
138
146
147
145
148
130
128
n=2
p1 = 11
p2 = 15
n = 26
OIV-MA-AS1-12 : R2005
63
SR = 6.35
R = 17.8
6. Quality control of analysis methods (IQC)
6.1 Reference documents
- Resolution OIV no 19/2002: Harmonized recommendations for internal
quality control in analysis laboratories.
- CITAC/EURACHEM: Guide for quality in analytical chemistry, 2002
Edition
- Standard NF V03-115, Guide for the use of reference materials
6.2 General principles
It is recalled that an analysis result can be affected two types of error: systematic
error, which translates into bias, and random error. For series analyses, another
type of error can be defined, which can be due to both systematic error and random
error: this is the series effect, illustrated for example by the deviation of the
measuring system during a series.
The IQC is designed to monitor and control these three errors.
6.3 Reference materials
The IQC is primarily based on exploiting the measurement results for reference
materials. The choice and constitution of the materials are therefore essential steps
that it must be controlled in order to provide an efficient basis for the system.
A reference material is defined by two parameters:
- Its matrix
- The assignment of its reference value
Several cases are possible; the cases encountered in oenology are summarized in
the following two-dimensional table:
OIV-MA-AS1-12 : R2005
64
External
value to
the
laborato
ry
Value
obtained
Value
by the
obtained
method
by a
to be
referenc
checked
e method
The use
the
of
instrume
nt value
a
as
referenc
e value
does not
control
accuracy
An
.
alternati
ve
approac
h must
set
be
up.
Referen
ce value
Value
obtained
by
formulat
ion
Matrix
Synthetic solution
Synthetic solutions can
be used to constitute
materials
reference
be
mustquite
solution
The
not
easily. They are using
produced
methods
compatible
It is
rules.
metrologicalwith
with non-specific
the
that signals,
recalled
to
and that are sensitive
value
formulation
The
matrix effects.
prone to
is organization
obtained
supplying
uncertainty. the solution
provide guarantees
must
of such a
The application
and be
quality
its
about
to
case can be used
possible.
if
certified
of
monitor the precisionThe
be
will
reference
as its
as well
the method,values
an
accompanied
solution
the synthetic
If
in
point
in a by
accuracy
at a
uncertainty
obtained
not tobeen
has
calibrated
avalue
relation
level.
confidence
given
calibrated
a
with
reference.
used to
This case can
the bereference
material,
of
precision
the
monitor
value can be determined
and to check
a method,
the
analyzing
by
point
in a using
its accuracy
solution
synthetic
The reference value is
the
with
compared
reference
the
method
by the method.
measured
has
value. This
external
be
to
is
measurement
The
to be checked. The
this
in
value
traceability
least 3
carried
material outis at measured
the supplier
if selected
point The
valuea
times.
over 10 repetitions, and
approved
is of
organization
mean
the
the
is
check will be made that3
of
preparation
for the insofar
as they
results,
the differences between
in
material
reference
interval
an
within
remain
these values are lower
be
cannot the
question. Itthan
lower
than the repeatability
methods
to
applied
the
repeatability
value; the most ofextreme
matrix
to
sensitive If necessary,
the
method.
values can be withdrawn,
effects. can check the
operator
up to a limit of two
the results
consistency
the
values. To ofensure
the
with
obtained
consistency of the values
for the
valuethe
formulation
10
obtained over
solution.
repetitions, the series is
to
can be used
casechecked
This
using
to be
The external value has been
determined on the wine by an
interlaboratory analysis chain.
Certain organizations propose
conditioned wine samples whose
values have been determined in
in certain
way. However,
this measurement
out 3
is carried
The
presentedmethod,
in this
the wines
cases, with
the reference
times
and/or
beenis doped
may have
wayselected
of
the mean
value
the
chemically
they
insofar as which
3 results,stabilized,
the
be affected.
matrixanmay
means
lower
interval
within
remainthe
be used to monitor
This case
of the
repeatability
the can
than
the precision of a method, and to
method.
The reference value is measured
point
into amonitor
accuracy
its can
checkcase
used
be
This
The
checked.
to be
by the method
value.
external and
withofthe
compared
to
method,
a
precision
the
material is measured over 10
this
in
traceability
value
has
This
point
a to
in is
its accuracy
check
be
and a check
repetitions,
has
chain
analysis
point if thewith
reference
the
compared
made that the differences between
be applied
canapplied
Itbe
been accredited.
to
It can
method.
than the
are lower
these values
sensitive toto matrix
to methodssensitive
methods
repeatability value; the most
effects.
extreme values can be withdrawn,
up to a limit of two values. To
ensure the consistency of the
values obtained over the 10
repetitions, this series is to be
checked on the one hand by
control materials established
during a previous session, placed
at the start and end of the series.
The value obtained can also be
OIV-MA-AS1-12 : R2005
65
Doped wine
A doped wine is a wine with an
artificial addition of an analyte.
This method is applicable when the
base wine is completely free of
analyte. These types of materials are
suitable for oenological additives
that are not native to the wine. If
this involves
practice,
In
component
with a conditioned
is applied
doping
and/or
doped
wine
no
matrix can
the wine, the
native tosamples
by
as proposed
stabilized natural.
chemically
Doping
longer be considered
materials
These
organizations.
must be carried out according to
constitute
claim torules.
cannot
value
Thea natural
metrological
values are
reference
matrix.
to uncertainty.
is prone
obtained The
analysis
an
generated
generally
3
outthe
carried
isbyto
measurement
The
monitor
be used
can
This case
chain.
withofthethereference
times
as
as well the
method,method,
precision
the
can be
monitor
to
case
This
the
mean
retained
value
be3
It ofcan
point.
athe
inisused
its accuracy
as
well
as
method,
the
of
precision
within
remain
they
as
insofar
results,
applied to methods sensitive to
in a point
accuracy
its
the
than
lower
interval
an
non-native
forcompared
effects
matrix
has
standard.
the external
with
method.
the
repeatability
the
not in
but This
wine,
ofofthe
compounds
if the
point
in this
value
traceability
a
monitor
to
used
be
can
case
This
of
compounds
native
of
case
The reference value is measured
supplying
organization
the
to check
andthe
of a method,
precision
wine.
The
to be checked.
using the method
for the
approved
has been
samples
compared
point
a
in
accuracy
its
material is measured over 10
of the reference
preparation
can be
method.is Itmaterial
the reference
with
to
made
and a check
repetitions,
applied to to
cannot be sensitive
It methods
in question.
to
applied
ensure that the differences between
matrix effects.
methods sensitive
effectsareto for
matrix
than the
lower non-native
these values
wine,
the
of
in the
but not
compounds
extreme
repeatability value; the most
of
compounds
native
of
case
values can be withdrawn, up tothea
wine.
limit of two values withdrawn. To
66
67
/ 2.S R .
/ 3.S R .
The limit defined for the cumulated mean narrows as the number of measurements
increases.
- This limit is an action limit:
3.S R
OIV-MA-AS1-12 : R2005
2.S R
n
68
Shewhart chart
0,31
0,29
Individualindivuduels
results
rsultats
Cumulated
mean
Moyenne
cumule
Average
upper
limit
Limite
supaction
d'action
moyenne
Average
lower
limit
Limit
inf action
d'action
moyenne
0,27
1
10
11
12
13
14
Individual
upper
limit
Limite
supaction
d'action
indiv
Individual action lower limit
0,25
0,23
OIV-MA-AS1-12 : R2005
69
OIV-MA-AS1-12 : R2005
70
combination of the uncertainties of the controlled method and the reference value
of the reference material.
6.5.4.2.1 Standard uncertainty of reference material
The reference values of these materials are accompanied by confidence intervals.
The laboratory must determine the nature of this data, and deduce from them the
standard uncertainty value for the reference value Sref. A distinction must be made
between several cases:
- The case in which uncertainty a is given in the form of an interval confidence at
95% (expanded uncertainty). This means that a normal law has been adopted. a
therefore constitutes an "expanded uncertainty" and corresponds to 2 times the
standard deviation Sref of the standard uncertainty of the reference values of the
materials provided.
S ref
a
2
S ref
a
3
S ref
a
6
71
S method
uncertaint y
2
Sref Smethod
Example: A pH 7 buffer solution is used to check a pHmeter. The confidence interval given by the pH solution
is +/- 0.01. It is indicated that this confidence interval
corresponds to the expanded uncertainty with a
confidence level of 95%. In addition the expanded
uncertainty of the pH-meter is 0.024.
2
/ 2
0.01 0.024
(
) (
)
2
2
OIV-MA-AS1-12 : R2005
72
On the one hand, that intended for the customers of the laboratory,
indicating the potential variations to take into account in order to
interpret the result of an analysis. It must be indicated, however, that
this information cannot be used as an external means of evaluating the
laboratory.
In addition, it constitutes a dynamic in-house tool for evaluating the
quality of the laboratory analysis results. Insofar as its evaluation is
regular and based on a fixed, well-defined methodology, it can be used
to see whether the variations involved in a method change positively
or negatively (in the case of an estimate based exclusively on
intralaboratory data).
73
Laboratories can use the two approaches jointly. It will be interesting to see
whether the results obtained using the intralaboratory approach give values lower
than the values of the interlaboratory approach.
7.4 Methodology
The work of uncertainty assessment involves 3 fundamental steps.
- Definition of the measurand, and description of the quantitative
analysis method
- Critical analysis of the measurement process
- Uncertainty assessment.
74
7.4.3 Estimation
approach)
calculations
of
standard
uncertainty
(intralaboratory
7.4.3.1 Principle
In the case of laboratories using large series of samples with a limited number of
methods, a statistical approach based on intralaboratory reproducibility,
supplemented by the calculation of sources of errors not taken into account under
OIV-MA-AS1-12 : R2005
75
S erreurs_ systmatiques
2
u(systematic_ errors) S R
2
OIV-MA-AS1-12 : R2005
76
The following non-exhaustive table gives examples of typical sources of error and
proposes an estimation approach for each of them, using integration under
reproducibility conditions as much as possible.
Source of error
Sampling
(constitution of
the sample)
Sub-sampling
(sampling a
quantity of
sample in order
to carry out the
test)
Type of error
Random
Commentary
Sampling is one of the
"businesses" defined in the
ISO 17025 standard.
Laboratories stating they do
not perform sampling, do not
include this source of error in
the uncertainty assessment.
Estimation method
Can be including in
intralaboratory reproducibility
by including sampling in
handling.
Random
Included in the
intralaboratory reproducibility
conditions if the test material
used is similar to routine test
materials.
Stability of the
sample
Random
Gauging of the
apparatus
Systematic/Rando
m
This error is
systematic if
gauging is
established for a
Source of error to be taken
long period, and
into account in absolute
becomes random if
methods.
gauging is regularly
carried out over a
time-scale
integrated under
reproducibility
conditions
Effect of
contamination
or memory
Random
Precision of
automata
Random
OIV-MA-AS1-12 : R2005
The reproducibility
conditions take this effect into
account, as long as the
reference materials are
inserted at various positions
in the analysis series.
This applies to intraseries
The reproducibility
drift in particular. This can be conditions take this effect into
controlled in particular by
account, as long as the
positioning the control
reference materials are
materials within the
inserted at various positions
framework of the IQC
in the analysis series.
This effect will be minimized
by the proper design of
measuring instruments and
suitable rinsing operations
77
Purity of the
reagents
Random
Measurement
conditions
Random
Effects of temperature,
moisture etc.
Matrix effect
Gauging effect
Systematic if
gauging is constant
Random if gauging
is regularly
renewed
Operator effect
Random
Bias
Systematic
To be integrated under
reproducibility conditions
using various batches of
reagents.
OIV-MA-AS1-12 : R2005
78
7.4.3.3 Estimating typical sources of systematic errors not taken into account
under reproducibility conditions
7.4.3.3.1 Gauging error (or calibration error)
Whenever the gauging of an instrument (or the calibration of an absolute method)
is not regularly redone, its output cannot be integrated in the reproducibility
values. An experiment schedule must be carried out in order to estimate it using
the residual error of the regression.
7.4.3.3.1.1 Procedure
The approach is similar to that carried out in the linearity study of the method.
It is recommended to implement a number n of reference materials. The number
must be higher than 3, but it is not necessary to go beyond 10. The reference
materials are to be measured p times under intralaboratory precision conditions, p
must be higher than 3, a figure of 5 is generally recommended. The accepted
values of reference materials must be regularly distributed on the range of values
under study. The number of measurements must be the same for all the reference
materials.
The results are reported in a table presented as follows:
Reference
materials
1
Accepted value of
the reference
material
x1
xi
xn
OIV-MA-AS1-12 : R2005
Replica
1
y11
yi1
.
yn1
Measured values
Replica
j
y1j
yij
ynj
Replica
p
y1p
yip
ynp
79
yij a b.xi ij
where
yij
xi
b
a
ab.xi
ij
is the difference between yij and the expectation of the measurement value
of the i reference material.
th
The parameters of the regression line are obtained using the following formulae:
- mean of p measurements of the ith reference material
yi
1
p
yij
j 1
Mx
OIV-MA-AS1-12 : R2005
1
My
n
1
n
xi
i 1
yi
i 1
80
( xi M x )( yi M y )
- estimated slope b
i 1
( xi M x )
i 1
a M y b Mx
yi
y i a b xi
- residual eij
eij yij y i
7.4.3.3.1.3 Estimating the standard uncertainty associated the gauging line (or
calibration line)
If the errors due to the regression line are constant over the entire field, the
standard uncertainty is estimated in a global, single way by the overall residual
standard deviation.
( yij y i )
u( gauging) S res
i 1 j 1
np 2
If the errors due to the regression line are not constant over the entire field, the
standard uncertainty is estimated for a given level by the residual standard
deviation for this level.
u( gauging), i Sres,i
( yij y i )
j 1
p 1
NOTE These estimates of standard deviations can be used if the linear regression
model and the gauging (or calibration) domain have been validated (see 5.3.1)
OIV-MA-AS1-12 : R2005
81
uref ucomp
NOTE 1 The methodology is identical in the case of methods adjusted with the
results of an interlaboratory comparison chain.
NOTE 2 Note the difference between a CRM used to adjust the bias of a method,
in which the uncertainty of its reference value combines with that of the method,
and a CRM used to control a method adjusted by other means (cf. 6.5.4.2). In the
second case, the uncertainty of the CRM should not be used for the uncertainty
assessment of the method.
7.4.3.3.2.2 Methods adjusted with several reference materials (gauging ranges
etc.)
There is no particular adjustment of bias apart from gauging work.
It is clear that each calibrator introduces bias uncertainty. There is therefore an
overall theoretical uncertainty of bias, which is a combination of the uncertainties
OIV-MA-AS1-12 : R2005
82
83
OIV-MA-AS1-12 : R2005
84
Reference method
Materi
als
1
2
3
4
5
6
7
0.3
0.3
0
0.3
1
0.2
8
0.3
5
0.2
9
0.3
7
7
0.3
0.3
2
0.3
2
0.2
9
0.3
5
0.2
9
0.3
6
7
0.3
0.3
1
0.3
2
0.2
9
0.4
5
0.2
0
0.3
6
7
0.3
0.3
0
0.3
2
0.2
8
0.4
4
0.2
0
0.3
6
7
0.3
0.3
1
0.3
1
0.2
8
0.3
5
0.2
9
0.3
6
6
FTIR
Mea
n
Ref
0.30
0.31
8
0.38
6
0.24
4
0.39
8
0.26
4
0.36
2
8
0
0.
0
3.
0
3.
0
3
1.
0
2
7.
0
4
6.
2
3.
3
5
7
0.3
0.3
1
0.3
2
0.2
7
0.4
6
0.2
2
0.3
6
6
0.3
0.3
1
0.3
0
0.2
7
0.4
6
0.2
3
0.3
5
6
0.3
0.3
0
0.3
1
0.2
7
0.4
5
0.2
2
0.3
5
5
0.3
0.3
0
0.3
1
0.2
6
0.4
6
0.2
2
0.3
6
6
Mean
FTIR
Diff
0.305
0.315
0.37
0.26
0.425
0.255
0.365
-0.004
-0.006
-0.016
0.01
0.03
-0.008
-0.008
OIV-MA-AS1-12 : R2005
85
-1
2
2
0.015 0.017 or +/- 0.045 g.L
2.
Effects that are not observed as part of the collaborative study must be
obviously negligible or be explicitly taken into account.
OIV-MA-AS1-12 : R2005
86
OIV-MA-AS1-12 : R2005
87
2.u ( x)
.100
x
NOTE This expression of uncertainty is possible given the assumption that the
variations obey a normal law with a 95% confidence rate.
These expressions result in a given uncertainty value with a confidence level of
95%.
OIV-MA-AS1-12 : R2005
88
REFERENCES
(1) OIV, 2001 Recueil des methods internationales danalyse des vins and des
mots; OIV Ed., Paris.
(2) OIV, 2002 Recommandations harmonises pour le contrle interne de qualit
dans les laboratoires danalyse; OIV resolution no 19/2002., Paris.
(3) Standard ISO 5725: 1994 Exactitude (justesse and fidlit) des results and
methods de mesure, classification index X 06-041-1
(4) IUPAC, 2002 Harmonized guidelines for single-laboratory validation of
analysis methods; Pure Appl. Chem., Vol. 74; n5, pp. 835-855.
(5) Standard ISO 11095: 1996 Etalonnage linaire utilisant des materials de
rfrence, reference number ISO 11095:1996
(6) Standard ISO 21748: 2004 Lignes directrices relatives lutilisation
destimation de la rptabilit, de la reproductibilit and de la justesse dans
lvaluation de lincertitude de mesure, reference number ISO ISO/TS 21748:2004
(7) Standard AFNOR V03-110: 1998 Procdure de validation intralaboratory
dune method alternative par rapport une method de rfrence, classification
index V03-110
(8) Standard AFNOR V03-115: 1996 Guide pour lutilisation des materials de
rfrence, classification index V03-115
(9) Standard AFNOR X 07-001: 1994 Vocabulaire international des termes
fondamentaux and gnraux de mtrologie, classification index X07-001
(10) Standard AFNOR ENV 13005: 1999 Guide pour lexpression de
lincertitude de mesure
(11) AFNOR, 2003, - Mtrologie dans lentreprise, outil de la qualit 2me dition,
AFNOR 2003 edition
(12) EURACHEM, 2000. - Quantifying Uncertainty in Analytical Measurement,
EURACHEM second edition 2000
(13) CITAC / EURACHEM, 2000 - Guide pour la qualit en chimie analytique,
EURACHEM 2002 edition
OIV-MA-AS1-12 : R2005
89
(14) Bouvier J.C., 2002 - Calcul de lincertitude de mesure Guide pratique pour
les laboratoires danalyse nologique, Revue Franaise dnologie no.197, NovDec 2002, pp: 16-21
(15) Snakkers G. and Cantagrel R., 2004 - Utilisation des donnes des circuits
de comparaison interlaboratoires pour apprcier lexactitude des results dun
laboratoire Estimation dune incertitude de mesure - Bull OIV, Vol. 77 857-876,
Jan Feb 2004, pp: 48-83
(16) Perruchet C. and Priel M, 2000 - Estimer lincertitude, AFNOR Editions
(17) Neuilly (M.) and CETAMA, 1993 - Modlisation and estimation des errors
de mesures, Lavoisier Ed, Paris
OIV-MA-AS1-12 : R2005
90
Annex N1
Table A
Law of SNEDECOR
This table indicates values of F in function with 1 and 2 for a risk of 0,05
P=0,950
1
2
1
2
3
161,4
18,51
10,13
199,5
19,00
9,55
215,7
19,16
9,28
224,6
19,25
9,12
230,2
19,30
9,01
234,0
19,33
8,94
236,8
19,35
8,89
238,9
19,37
8,85
240,5
19,38
8,81
241,9
19,40
8,79
1
2
3
4
5
6
7,71
6,61
5,99
6,94
5,79
5,14
6,59
5,41
4,76
6,39
5,19
4,53
6,26
5,05
4,39
6,16
4,95
4,28
6,09
4,88
4,21
6,04
4,82
4,15
6,00
4,77
4,10
5,96
4,74
4,06
4
5
6
7
8
9
5,59
5,32
5,12
4,74
4,46
4,26
4,35
4,07
3,86
4,12
3,84
3,63
3,97
3,69
3,48
3,87
3,58
3,37
3,79
3,50
3,29
3,73
3,44
3,23
3,68
3,39
3,18
3,64
3,35
3,14
7
8
9
10
4,96
4,10
3,71
3,48
3,33
3,22
3,14
3,07
3,02
2,98
10
11
12
13
4,84
4,75
4,67
3,98
3,89
3,81
3,59
3,49
3,41
3,36
3,26
3,18
3,20
3,11
3,03
3,09
3,00
2,92
3,01
2,91
2,83
2,95
2,85
2,77
2,90
2,80
2,71
2,85
2,75
2,67
11
12
13
14
15
16
4,60
4,54
4,49
3,74
3,68
3,63
3,34
3,29
3,24
3,11
3,06
3,01
2,96
2,90
2,85
2,85
2,79
2,74
2,76
2,71
2,66
2,70
2,64
2,59
2,65
2,59
2,54
2,60
2,54
2,49
14
15
16
17
18
19
4,45
4,41
4,38
3,59
3,55
3,52
3,20
3,16
3,13
2,96
2,93
2,90
2,81
2,77
2,74
2,70
2,66
2,63
2,61
2,58
2,54
2,55
2,51
2,48
2,49
2,46
2,42
2,45
2,41
2,38
17
18
19
20
4,35
3,49
3,10
2,87
2,71
2,60
2,51
2,45
2,39
2,35
20
21
22
23
4,32
4,30
4,28
3,47
3,44
3,42
3,07
3,05
3,03
2,84
2,82
2,80
2,68
2,66
2,64
2,57
2,55
2,53
2,49
2,46
2,44
2,42
2,40
2,37
2,37
2,34
2,32
2,32
2,30
2,27
21
22
23
24
25
26
4,26
4,24
4,23
3,40
3,39
3,37
3,01
2,99
2,98
2,78
2,76
2,74
2,62
2,60
2,59
2,51
2,49
2,47
2,42
2,40
2,39
2,36
2,34
2,32
2,30
2,28
2,27
2,25
2,24
2,22
24
25
26
27
28
29
4,21
4,20
4,18
3,35
3,34
3,33
2,96
2,95
2,93
2,73
2,71
2,70
2,57
2,56
2,55
2,46
2,45
2,43
2,37
2,36
2,35
2,31
2,29
2,28
2,25
2,24
2,22
2,20
2,19
2,18
27
28
29
30
4,17
3,32
2,92
2,69
2,53
2,42
2,33
2,27
2,21
2,16
30
40
60
120
4,08
4,00
3,92
3,23
3,15
3,07
2,84
2,76
2,68
2,61
2,53
2,45
2,45
2,37
2,29
2,34
2,25
2,17
2,25
2,17
2,09
2,18
2,10
2,02
2,12
2,04
1,96
2,08 40
1,99 60
1,91 120
3,84
3,00
2,60
2,37
2,21
2,10
2,01
1,94
1,88
1,83
2
1
OIV-MA-AS1-12 : R2005
10
10
1
2
2
1
91
Annex N2
Table B - Law of STUDENT
This table indicates values of t in function with P and
P
P
0,55
0,60
7.5.1.1.1.1.1.1.1
0,65
0,70
0,75
0,80
0,85
0,90
0,95
0,975
1
2
3
0,158
0,142
0,137
0,325
0,289
0,277
0,510
0,445
0,424
0,727
0,617
0,584
1,000
0,816
0,765
1,376
1,061
0,978
1,963
1,386
1,250
3,078
1,886
1,638
6,314
2,920
2,353
4
5
6
0,134
0,132
0,131
0,271
0,267
0,265
0,414
0,408
0,404
0,569
0,559
0,553
0,741
0,727
0,718
0,941
0,920
0,906
1,190
1,156
1,134
1,533
1,476
1,440
2,132
2,015
1,943
2,776
2,571
2,447
3,747
3,365
3,143
4,604
4,032
3,707
8,610
6,869
5,959
4
5
6
7
8
9
0,130
0,130
0,129
0,263
0,262
0,261
0,402
0,399
0,398
0,549
0,546
0,543
0,711
0,706
0,703
0,896
0,889
0,883
1,119
1,108
1,100
1,415
1,397
1,383
1,895
1,860
1,833
2,365
2,306
2,262
2,998
2,896
2,821
3,499
3,355
3,250
5,408
5,041
4,781
7
8
9
10
0,129
0,260
0,397
0,542
0,700
0,879
1,093
1,372
1,812
2,228
2,764
3,169
4,587
10
11
12
13
0,129
0,128
0,128
0,260
0,259
0,259
0,396
0,395
0,394
0,540
0,539
0,538
0,697
0,695
0,694
0,876
0,873
0,870
1,088
1,083
1,079
1,363
1,356
1,350
1,796
1,782
1,771
2,201
2,179
2,160
2,718
2,681
2,650
3,106
3,055
3,012
4,437
4,318
4,221
11
12
13
14
15
16
0,128
0,128
0,128
0,258
0,258
0,258
0,393
0,393
0,392
0,537
0,536
0,535
0,692
0,691
0,690
0,868
0,866
0,865
1,076
1,074
1,071
1,345
1,341
1,337
1,761
1,753
1,746
2,145
2,131
2,120
2,624
2,602
2,583
2,977
2,947
2,921
4,140
4,073
4,015
14
15
16
17
18
19
0,128
0,127
0,127
0,257
0,257
0,257
0,392
0,392
0,391
0,534
0,534
0,533
0,689
0,688
0,688
0,863
0,862
0,861
1,069
1,067
1,066
1,333
1,330
1,328
1,740
1,734
1,729
2,110
2,101
2,093
2,567
2,552
2,539
2,898
2,878
2,861
3,965
3,922
3,883
17
18
19
20
0,127
0,257
0,391
0,533
0,687
0,860
1,064
1,325
1,725
2,086
2,528
2,845
3,850
20
21
22
23
0,127
0,127
0,127
0,257
0,256
0,256
0,391
0,390
0,390
0,532
0,532
0,532
0,686
0,686
0,685
0,859
0,858
0,858
1,063
1,061
1,060
1,323
1,321
1,319
1,721
1,717
1,714
2,080
2,074
2,069
2,518
2,508
2,500
2,831
2,819
2,807
3,819
3,792
3,767
21
22
23
24
25
26
0,127
0,127
0,127
0,256
0,256
0,256
0,390
0,390
0,390
0,531
0,531
0,531
0,685
0,684
0,884
0,857
0,856
0,856
1,059
1,058
1,058
1,318
1,316
1,315
1,711
1,708
1,706
2,064
2,060
2,056
2,492
2,485
2,479
2,797
2,787
2,779
3,745
3,725
3,707
24
27
28
29
0,127
0,127
0,127
0,256
0,256
0,256
0,389
0,389
0,389
0,531
0,530
0,530
0,684
0,683
0,683
0,855
0,855
0,854
1,057
1,056
1,055
1,314
1,313
1,311
1,703
1,701
1,699
2,052
2,048
2,045
2,473
2,467
2,462
2,771
2,763
2,756
3,690
3,674
3,659
27
28
29
30
0,127
0,256
0,389
0,530
0,683
0,854
1,055
1,310
1,697
2,042
2,457
2,750
3,646
30
40
60
120
0,126
0,126
0,126
0,255
0,254
0,254
0,388
0,387
0,386
0,529
0,527
0,526
0,681
0,679
0,677
0,851
0,848
0,845
1,050
1,046
1,041
1,303
1,296
1,289
1,684
1,671
1,658
2,021
2,000
1,980
2,423
2,390
2,358
2,704
2,660
2,617
3,551
3,460
3,373
40
60
120
0,126
0,253
0,385
0,524
0,674
0,842
1,036
1,282
1,645
1,960
2,326
2,576
3,291
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
0,975
0,990
0,995
0,9995
OIV-MA-AS1-12 : R2005
0,990
0,995
0,9995
25
26
92
OIV-MA-AS1-13 : R2005
CONTENTS
1
1.1
1.2
INTRODUCTION
Background
Existing protocols, standards and guides
2
2.1
2.2
4
4.1
4.2
4.3
4.4
6
6.1
6.2
used
6.3
method
6.4
6.6
6.7
6.8
The method has been published in the scientific literature together with
some analytical characteristics
The method has been published in the scientific literature with no
characteristics given or has been developed in-house
The method is empirical
The analysis is ad hoc
Changes in staff and equipment
RECOMMENDATIONS
BIBLIOGRAPHY
6.5
OIV-MA-AS1-13 : R2005
Applicability
A2
Selectivity
A3
A3.1
A3.2
A3.3
A4
A4.1
A4.2
A4.3
A4.3.1
A4.3.2
A4.3.3
A4.3.4
Trueness
Estimation of trueness
Conditions for trueness experiments
Reference values for trueness experiments
Certified reference materials (CRMs)
Reference materials
Use of a reference method
Use of spiking/recovery
A5
Accuracy
A6
Recovery
A7
Concentration range
A8
Detection Limit
A9
A10
Sensitivity
A11
Ruggedness
A12
A13
Matrix variation
A14.
Measurement Uncertainty
OIV-MA-AS1-13 : R2005
FOR
UNCERTAINTY
Sensitivity analysis
Judgement
OIV-MA-AS1-13 : R2005
1.
INTRODUCTION
1.1
Background
Reliable analytical methods are required for compliance with national and
international regulations in all areas of analysis. It is accordingly internationally
recognised that a laboratory must take appropriate measures to ensure that it is
capable of providing and does provide data of the required quality. Such measures
include:
OIV-MA-AS1-13 : R2005
to ensure the viability of the method before the costly exercise of a formal
collaborative trial;
to provide evidence of the reliability of analytical methods if collaborative trial
data are not available or where the conduct of a formal collaborative trial is
not practicable;
to ensure that off-the-shelf validated methods are being used correctly.
The ICH text15 and methodology,16 which prescribe minimum validation study
requirements for tests used to support drug approval submission.
The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method
Validation and Related Topics (1998)12
Quantifying Uncertainty in Analytical Measurement (2000)9
Method validation was also extensively discussed at a Joint FAO/IAEA Expert
Consultation, December 1997, on the Validation of Analytical Methods for Food
Controls, the Report of which is available19.
The present Guidelines bring together the essential scientific principles of the
above documents to provide information which has been subjected to international
acceptance and, more importantly, to point the way forward for best practice in
single-laboratory method validation.
2.1
Allgemein
Terms used in this document respect ISO and IUPAC definitions where available.
The following documents contain relevant definitions:
i) IUPAC: Compendium of chemical terminology, 1987
ii) International vocabulary of basic and general terms in metrology. ISO 1993
2.2
OIV-MA-AS1-13 : R2005
3
METHOD VALIDATION, UNCERTAINTY, AND QUALITY
ASSURANCE
Method validation makes use of a set of tests which both test any assumptions on
which the analytical method is based and establish and document the performance
characteristics of a method, thereby demonstrating whether the method is fit for a
particular analytical purpose. Typical performance characteristics of analytical
methods are: applicability; selectivity; calibration; trueness; precision; recovery;
operating range; limit of quantification; limit of detection; sensitivity; and
ruggedness. To these can be added measurement uncertainty and fitness-forpurpose.
Strictly speaking, validation should refer to an analytical system rather than an
analytical method, the analytical system comprising a defined method protocol, a
defined concentration range for the analyte, and a specified type of test material.
For the purposes of this document, a reference to method validation will be taken
as referring to an analytical system as a whole. Where the analytical procedure as
such is addressed, it will be referred to as the protocol.
In this document method validation is regarded as distinct from ongoing activities
such as internal quality control (IQC) or proficiency testing. Method validation is
carried out once, or at relatively infrequent intervals during the working lifetime of
a method; it tells us what performance we can expect the method to provide in the
future. Internal quality control tells us about how the method has performed in the
past. IQC is therefore treated as a separate activity in the IUPAC Harmonisation
Programme.3
In method validation the quantitative characteristics of interest relate to the
accuracy of the result likely to be obtained. Therefore it is generally true to say
that method validation is tantamount to the task of estimating uncertainty of
measurement. Over the years it has become traditional for validation purposes to
represent different aspects of method performance by reference to the separate
items listed above, and to a considerable extent these guidelines reflect that
pattern. However, with an increasing reliance on measurement uncertainty as a
key indicator of both fitness for purpose and reliability of results, analytical
chemists will increasingly undertake measurement validation to support
uncertainty estimation, and some practitioners will want to do so immediately.
Accordingly, measurement uncertainty is treated briefly in Annex A as a
performance characteristic of an analytical method, while Annex B provides
additional guidance on some procedures not otherwise covered.
OIV-MA-AS1-13 : R2005
4.1
Testing assumptions
practised in the technique of interest, and that the purpose of any significance tests
is to check that there is no strong evidence to discount the assumptions on which
the particular protocol relies. The reader should bear in mind that more stringent
checks may be necessary for unfamiliar or less established measurement
techniques.
4.3
Though these different sources may not necessarily be independent, this list
provides a useful way of checking the extent to which a given validation study
addresses the sources of error.
The repeatability (within-run) term includes contributions from any part of the
procedure that varies within a run, including contributions from the familiar
gravimetric and volumetric errors, heterogeneity of the test material, and variation
in the chemical treatment stages of the analysis, and is easily seen in the dispersion
of replicated analyses. The run effect accounts for additional day-to-day variations
in the analytical system, such as changes of analyst, batches of reagents,
recalibration of instruments, and the laboratory environment (e.g., temperature
changes). In single-laboratory validation, the run effect is typically estimated by
*
Sampling uncertainty in the strict sense of uncertainty due to the preparation of the
laboratory sample from the bulk target is excluded from consideration in this document.
Uncertainty associated with taking a test portion from the laboratory sample is an
inseparable part of measurement uncertainty and is automatically included at various levels
of the following analysis.
+
Many alternative groupings or partitions of error are possible and may be useful in
studying particular sources of error in more detail or across a different range of situations.
For example, the statistical model of ISO 5725 generally combines laboratory and run
effects, while the uncertainty estimation procedure in the ISO GUM is well suited to
assessing the effects of each separate and measurable influence on the result.
OIV-MA-AS1-13 : R2005
10
This may not be applicable at concentrations less than 10 times the detection limit.
OIV-MA-AS1-13 : R2005
11
OIV-MA-AS1-13 : R2005
12
13
14
The method has been studied in a collaborative trial and so the laboratory has to
verify that it is capable of achieving the published performance characteristics of
the method (or is otherwise able to fulfil the requirements of the analytical task).
The laboratory should undertake precision studies, bias studies (including matrix
variation studies), and possibly linearity studies, although some tests such as that
for ruggedness may be omitted.
6.2
The laboratory is to use a fully validated method, but new matrix is to
be used
The method has been studied in a collaborative trial and so the laboratory has to
verify that the new matrix introduces no new sources of error into the system. The
same range of validation as the previous is required.
6.3
The laboratory is to use a well-established, but not collaboratively
studied, method
OIV-MA-AS1-13 : R2005
15
6.6
An empirical method is one in which the quantity estimated is simply the result
found on following the stated procedure. This differs from measurements intended
to assess method-independent quantities such as the concentration of a particular
analyte in a sample, in that the method bias is conventionally zero, and matrix
variation (that is , within the defined class) is irrelevant. Laboratory bias cannot be
ignored, but is likely to be difficult to estimate by single-laboratory experiment.
Moreover, reference materials are unlikely to be available. In the absence of
collaborative trial data some estimate of interlaboratory precision could be
obtained from a specially designed ruggedness study or estimated by using the
Horwitz function.
6.7
16
single bias test; a before and after experiment on typical test materials or control
materials. In general, the tests carried out should reflect the possible impact of the
change on the analytical procedure.
7
RECOMMENDATIONS
REFERENCES
17
OIV-MA-AS1-13 : R2005
18
OIV-MA-AS1-13 : R2005
19
A2 Selectivity
Selectivity is the degree to which a method can quantify the analyte accurately in
the presence of interferents. Ideally, selectivity should be evaluated for any
important interferent likely to be present. It is particularly important to check
interferents which are likely, on chemical principles, to respond to the test. For
example, colorimetric tests for ammonia might reasonably be expected to respond
to primary aliphatic amines. It may be impracticable to consider or test every
potential interferent; where that is the case, it is recommended that the likely worst
cases are checked. As a general principle, selectivity should be sufficiently good
for any interferences to be ignored.
In many types of analysis, selectivity is essentially a qualitative assessment based
on the significance or otherwise of suitable tests for interference. However, there
are useful quantitative measures. In particular, one quantitative measure is the
selectivity index ban/bint, where ban is the sensitivity of the method (slope of the
OIV-MA-AS1-13 : R2005
20
calibration function) and bint the slope of the response independently produced by
a potential interferent, provides a quantitative measure of interference. bint can be
determined approximately by execution of the procedure on a matrix blank and the
same blank spiked with the potential interferent at one appropriate concentration.
If a matrix blank is unavailable, and a typical material used instead, bint can be
estimated from such a simple experiment only under the assumption that mutual
matrix effects are absent. Note that bint is more easily determined in the absence of
the analyte because the effect might be confused with another type of interference
when the sensitivity of the analyte is itself affected by the interferent (a matrix
effect).
A3 Calibration and linearity
With the exception of gross errors in preparation of calibration materials,
calibration errors are usually (but not always) a minor component of the total
uncertainty budget, and can usually be safely subsumed into various categories
estimated by top-down methods. For example random errors resulting from
calibration are part of the run bias, which is assessed as a whole, while systematic
errors from that source may appear as laboratory bias, likewise assessed as a
whole. Never-the-less, there are some characteristics of calibration that are useful
to know at the outset of method validation, because they affect the strategy for the
optimal development of the procedure. In this class are such questions as whether
the calibration function plausibly (a) is linear, (b) passes through the origin and (c)
is unaffected by the matrix of the test material. The procedures described here
relate to calibration studies in validation, which are necessarily more exacting than
calibration undertaken during routine analysis. For example, once it is established
at validation that a calibration function is linear and passes through the origin, a
much simpler calibration strategy can be used for routine use (for example, a two
point repeated design). Errors from this simpler calibration strategy will normally
be subsumed into higher level errors for validation purposes.
A3.1 Linearity and intercept
Linearity can be tested informally by examination of a plot of residuals produced
by linear regression of the responses on the concentrations in an appropriate
calibration set. Any curved pattern suggests lack of fit due to a non-linear
calibration function. A test of significance can be undertaken by comparing the
lack-of-fit variance with that due to pure error. However, there are causes of lack
of fit other than nonlinearity that can arise in certain types of analytical calibration,
so the significance test must be used in conjunction with a residual plot. Despite its
current widespread use as an indication of quality of fit, the correlation coefficient
OIV-MA-AS1-13 : R2005
21
is misleading and inappropriate as a test for linearity and should not be used.
Design is all-important in tests for lack of fit, because it is easy to confound
nonlinearity with drift. Replicate measurements are needed to provide an estimate
of pure error if there is no independent estimate. In the absence of specific
guidance, the following should apply:
After an exploratory fit with simple linear regression, the residuals should be
examined for obvious patterns. Heteroscedasticity is quite common in analytical
calibration and a pattern suggesting it means that the calibration data are best
treated by weighted regression. Failure to use weighted regression in these
circumstances could give rise to exaggerated errors at the low end of the
calibration function.
The test for lack of fit can be carried out with either simple or weighted regression.
A test for an intercept significantly different from zero can also be made on this
data if there is no significant lack of fit.
A3.2 Test for general matrix effect
It simplifies calibration enormously if the calibrators can be prepared as a simple
solution of the analyte. The effects of a possible general matrix mismatch must be
assessed in validation if this strategy is adopted. A test for general matrix effect
can be made by applying the method of analyte additions (also called standard
additions) to a test solution derived from a typical test material. The test should
be done in a way that provides the same final dilution as the normal procedure
produces, and the range of additions should encompass the same range as the
procedure-defined calibration validation. If the calibration is linear the slopes of
the usual calibration function and the analyte additions plot can be compared for
significant difference. A lack of significance means that there is no detectable
general matrix effect. If the calibration is not linear a more complex method is
needed for a significance test, but a visual comparison at equal concentrations will
usually suffice. A lack of significance in this test will often mean that the matrix
OIV-MA-AS1-13 : R2005
22
A4 Trueness
A4.1 Estimation of trueness
Trueness is the closeness of agreement between a test result and the accepted
reference value of the property being measured. Trueness is stated quantitatively in
terms of bias; with smaller bias indicating greater trueness. Bias is typically
determined by comparing the response of the method to a reference material with
the known value assigned to the material. Significance testing is recommended.
Where the uncertainty in the reference value is not negligible, evaluation of the
results should consider the reference material uncertainty as well as the statistical
variability.
A4.2 Conditions for trueness experiments
Bias can arise at different levels of organisation in an analytical system, for
example, run bias, laboratory bias and method bias. It is important to remember
which of these is being handled by the various methods of addressing bias. In
particular:
OIV-MA-AS1-13 : R2005
23
24
25
fundamental, but depends on the level at which the analytical system is viewed.
Thus from the viewpoint of a single determination, any deviation affecting the
calibration for the run would be seen as a bias. From the point of view of the
analyst reviewing a years work, the run bias will be different every day and act
like a random variable with an associated precision. The stipulated conditions for
the estimation of precision take account of this change in view point.
For single laboratory validation, two sets of conditions are relevant: (a) precision
under repeatability conditions, describing variations observed during a single run
as expectation 0 and standard deviation r , and (b) precision under run-to-run
conditions, describing variations in run bias run as expectation 0, standard
deviation run . Usually both of these sources of error are operating on individual
12
2
analytical results, which therefore have a combined precision tot r2 n run
,
where n is the number of repeat results averaged within a run for the reported
result. The two precision estimates can be obtained most simply by analysing the
selected test material in duplicate in a number of successive runs. The separate
variance components can then be calculated by the application of one-way analysis
of variance. Each duplicate analysis must be an independent execution of the
procedure applied to a separate test portion. Alternatively the combined precision
tot can be estimated directly by the analysis of the test material once in
successive runs, and estimating the standard deviation from the usual equation.
(Note that observed standard deviations are generally given the symbol s, to
distinguish them from standard deviations ).
It is important that the precision values are representative of likely test conditions.
First, the variation in conditions among the runs must represent what would
normally happen in the laboratory under routine use of the method. For instance,
variations in reagent batches, analysts and instruments should be representative.
Second, the test material used should be typical, in terms of matrix and (ideally)
the state of comminution, of the materials likely to encountered in routine
application. So actual test materials or, to a lesser degree, matrix-matched
reference materials would be suitable, but standard solutions of the analyte would
not. Note also that CRMs and prepared reference materials are frequently
homogenised to a greater extent than typical test materials, and precision obtained
from their analysis may accordingly under-estimate the variation that will be
observed for test materials.
Precision very often varies with analyte concentration. Typical assumptions are i)
that there is no change in precision with analyte level, or ii) that the standard
deviation is proportional to, or linearly dependent on, analyte level. In both cases,
OIV-MA-AS1-13 : R2005
26
27
where is the relative uncertainty estimated a some concentration well above the
detection limit. u0 is the standard uncertainty estimated for zero concentration and
in some circumstances could be estimated as c L / 3 . In these circumstances it
would be reasonable to regard the validated range as extending from zero to a
small integer multiple of the upper validation point. Again this would depend on
professional judgement.
A8 Detection Limit
In broad terms the detection limit (limit of detection) is the smallest amount or
concentration of analyte in the test sample that can be reliably distinguished from
zero.22,23 For analytical systems where the validation range does not include or
approach it, the detection limit does not need to be part of a validation.
Despite the apparent simplicity of the idea, the whole subject of the detection limit
is beset with problems outlined below:
For most practical purposes in method validation, it seems better to opt for a
OIV-MA-AS1-13 : R2005
28
OIV-MA-AS1-13 : R2005
29
A11 Ruggedness
The ruggedness of an analytical method is the resistance to change in the results
produced by an analytical method when minor deviations are made from the
experimental conditions described in the procedure. The limits for experimental
parameters should be prescribed in the method protocol (although this has not
always been done in the past), and such permissible deviations, separately or in
any combination, should produce no meaningful change in the results produced. (A
meaningful change here would imply that the method could not operate within
the agreed limits of uncertainty defining fitness for purpose.) The aspects of the
method which are likely to affect results should be identified, and their influence
on method performance evaluated by using ruggedness tests.
The ruggedness of a method is tested by deliberately introducing small changes to
the procedure and examining the effect on the results. A number of aspects of the
method may need to be considered, but because most of these will have a
negligible effect it will normally be possible to vary several at once. An
economical experiment based on fractional factorial designs has been described by
Youden13. For instance, it is possible to formulate an approach utilising 8
combinations of 7 variable factors, that is to look at the effects of seven parameters
with just eight analytical results. Univariate approaches are also feasible, where
only one variable at a time is changed.
Examples of the factors that a ruggedness test could address are: changes in the
instrument, operator, or brand of reagent; concentration of a reagent; pH of a
solution; temperature of a reaction; time allowed for completion of a process etc.
A12 Fitness for Purpose
Fitness for purpose is the extent to which the performance of a method matches the
criteria, agreed between the analyst and the end-user of the data, that describe the
end-users needs. For instance the errors in data should not be of a magnitude that
would give rise to incorrect decisions more often than a defined small probability,
but they should not be so small that the end-user is involved in unnecessary
expenditure. Fitness for purpose criteria could be based on some of the
characteristics described in this Annex, but ultimately will be expressed in terms
of acceptable total uncertainty.
A13 Matrix variation
Matrix variation is, in many sectors, one of the most important but least
acknowledged sources of error in analytical measurements. When we define the
OIV-MA-AS1-13 : R2005
30
ci2u( xi ) 2
i 1,n
31
12
2
previously) stot s r2 n s run
. Note that where the precision terms are shown to
vary with analyte level, the uncertainty estimate for a given result must employ the
precision term appropriate to that level. The basis for the uncertainty estimate
accordingly follows directly from the statistical model assumed and tested in
validation. To this estimate must be added any further terms as necessary to
account for (in particular) inhomogeneity and matrix effect (see section A13).
Finally, the calculated standard uncertainty is multiplied by a coverage factor, k,
to provide an expanded uncertainty, that is, an interval expected to encompass a
large fraction of the distribution of values that may be attributed to the
measurand8. Where the statistical model is well established, the distribution
known to be normal, and the number of degrees of freedom associated with the
estimate is high, k is generally chosen to be equal to 2. The expanded uncertainty
then corresponds approximately to a 95% confidence interval.
There is one important caveat to be added here. In testing the assumed statistical
model, imperfect tests are perforce used. It has already been noted that these tests
can not prove that any effect is identically zero; they can only show that an effect
is too small to detect within the uncertainty associated with the particular test for
significance. A particularly important example is the test for significant laboratory
bias. Clearly, if this is the only test performed to confirm trueness, there must be
some residual uncertainty as to whether the method is indeed unbiased or not. It
follows that where such uncertainties are significant with respect to the uncertainty
calculated so far, additional allowance should be made.
OIV-MA-AS1-13 : R2005
32
In the case of an uncertain reference value, the simplest allowance is the stated
uncertainty for the material, combined with the statistical uncertainty in the test
applied. A full discussion is beyond the scope of this text; reference 9 provides
further detail. It is, however, important to note that while the uncertainty estimated
directly from the assumed statistical model is the minimum uncertainty that can be
associated with an analytical result, it will almost certainly be an underestimate;
similarly, an expanded uncertainty based on the same considerations and using k=2
will not provide sufficient confidence.
The ISO Guide8 recommends that for increased confidence, rather than arbitrarily
adding terms, the value of k should be increased as required. Practical experience
suggests that for uncertainty estimates based on a validated statistical model, but
with no evidence beyond the validation studies to provide additional confidence in
the model, k should not be less than 3. Where there is strong reason to doubt that
the validation study is comprehensive, k should be increased further as required.
OIV-MA-AS1-13 : R2005
33
B1 Sensitivity analysis
The basic expression used in uncertainty estimation
u(y(x1,x2,...)) =
ci2u( xi ) 2
i 1,n
OIV-MA-AS1-13 : R2005
34
B2 Judgement
It is not uncommon to find that while an effect is recognised and may be
significant, it is not always possible to obtain a reliable estimate of uncertainty. In
such circumstances, the ISO Guide makes it quite clear that a professionally
considered estimate of the uncertainty is to be preferred to neglect of the
uncertainty. Thus, where no estimate of uncertainty is available for a potentially
important effect, the analyst should make their own best judgement of the likely
uncertainty and apply that in estimating the combined uncertainty. Reference 8
gives further guidance on the use of judgement in uncertainty estimation.
OIV-MA-AS1-13 : R2005
35
The parameter may be, for example, a standard deviation (or a given
multiple of it), or the half-width of an interval having a stated level of
confidence.
2.
3.
[It is recognised that the term measurement uncertainty is the most widely used
term by International Organisations and Accreditation Agencies. However The
Codex ALIMENTARIUS Committee on Methods of Analysis and Sampling has
OIV-MA-AS1-14 : R2005
Recommendations
The following recommendations are made to governments:
1.
2.
3.
OIV-MA-AS1-14 : R2005
REFERENCES
1.
2.
3.
4.
OIV-MA-AS1-14 : R2005
Recovery
The OIV recommends the following practice with regards to reporting recovery
of analytical results.
o Analytical results are to be expressed on a recovery corrected basis
where appropriate and relevant, and when corrected it has to be stated.
o If a result has been corrected for recovery, the method by which the
recovery was taken into account should be stated. The recovery rate is to
be quoted wherever possible.
o When laying down provisions for standards, it will be necessary to state
whether the result obtained by a method used for analysis within
conformity checks shall be expressed on a recovery-corrected basis or
not.
OIV-MA-AS1-15 : R2009
Annex F
Specific methods for the analysis of
grape sugar1
(rectified concentrated musts)
Grape sugar are defined in Part I-6.2 and in Part II-2.1.12 of the International Code
of Oenological Practices of the OIV.
Specifications of grape sugar are described in file COEI-1-SUCRAI of International
Oenological Codex of the OIV.
OIV-MA-ANNEX-F
Type IV method
Method OIV-MA-F1-01
Conductivity
(Oeno 419A-2011)
1. Principle
The electrical conductivity of a column of liquid defined by two parallel
platinum electrodes at its ends is measured by incorporating it in one arm
of a Wheatstone bridge.
The conductivity varies with temperature and it is therefore expressed at
20C.
2. Reagents
Use only reagent grade chemicals
2.1 Purified water for laboratories, with specific conductivity below 2 S
cm-1 at 20C, for example EN ISO 3696 type II water.
2.2 Reference solution of potassium chloride.
Dissolve 0.581 g of potassium chloride, KCl previously dried to
constant mass at a temperature of 105C, in demineralised water (2.1).
Make up to one litre with demineralised water (2.1). This solution has a
conductivity of 1 000 S cm-1 at 20C. It should not be kept for more
than three months.
A commercial preparation can be used.
3. Apparatus
3.1 Conductivity meter enabling measurements of conductivity to be made
over a range from 1 to 1 000 microsiemens per cm (S cm-1).
3.2 Water bath for bringing the temperature of samples to be analysed to
approximately 20C (20 2C).
OIV-MA-F1-01: R2011
4. Procedure
4.1 Preparation of the sample to be analysed
Use a solution with a total sugar concentration of 25 0.5 % (m/m) (25
Brix): weigh a mass equal to 2500/P and make up to 100 g with water
(2.1),
P = percentage (m/m) of total sugars in the rectified concentrated must.
4.2 Determination of conductivity
Bring the sample to be analysed to 20C by immersion in a water bath.
Maintain the temperature to within 0.1C.
Rinse the conductivity cell twice with the solution to be examined.
Measure the conductivity and express the result in S cm-1.
5. Expression of the Results
The result is expressed in microsiemens per cm (Scm1) at 20C to the
nearest whole number for the 25% (m/m) (25 Brix) solution of rectified
concentrated must.
5.1 Calculations
If the apparatus does not have temperature compensation, correct the
measured conductivity using Table I. If the temperature is below 20C,
add the correction; if the temperature is above 20C, subtract the
correction.
6. Characteristics of the method
Repeatability (r)
r = 3 S/cm
Reproducibility (R)
R = 16 S/cm
OIV-MA-F1-01: R2011
TABLE I
Corrections to be made to the conductivity for temperatures different from
20C (S cm1)
Temperature (C)
Conductivity
20.2
19.8
20.4
19.6
20.6
19.4
20.8
19.2
21.0
19.0
21.2
18.8
21.4
18.6
50
100
150
200
250
10
11
300
11
12
13
350
11
12
14
15
400
11
12
14
16
18
450
10
12
14
16
18
20
500
11
13
15
18
20
22
550
10
12
14
17
19
22
24
600
11
13
16
18
21
24
26
(1)
(2)
OIV-MA-F1-01: R2011
Method OIV-MA-F1-02
Type IV method
OIV-MA-F1-02: R2011
3.1.2
4. Procedure
4.1 Preparation of sample
Use the solution obtained by diluting the rectified concentrated must to 40%
(m/v) (introduce 200 g of accurately weighed rectified concentrated must
into a 500 ml volumetric flask. Make up to the mark with water and
homogenise) and filter it through a membrane filter (0.45 m).
4.2 Chromatographic determination
Inject 5 (or 10) l of the sample prepared as described in paragraph 4.1. and
5 (or 10) l of the reference hydroxymethylfurfural solution (2.5) into the
chromatograph. Record the chromatogram.
The retention time of hydroxymethylfurfural is approximately six to seven
minutes.
The volume injected and the sequence are given for guidance. The
chromatographic determination can also be done with a calibration curve
5. Expression of results
The hydroxymethylfurfural concentration in rectified concentrated musts is
expressed in milligrams per kilogram of total sugars.
5.1 Method of calculation
Let the hydroxymethylfurfural concentration in the 40% (m/v) solution of
the rectified concentrated must be C mg/l.
The hydroxymethylfurfural concentration in milligrams per kilogram of
total sugars is given by:
250 x C/P
P = percentage (m/m) concentration of total sugars in the rectified
concentrated must.
6. Characteristics of the method
Repeatability (r)
r = 0.5 mg/kg total sugars
Reproducibility (R)
R = 3.0 mg/kg total sugars
OIV-MA-F1-02: R2011
OIV-MA-F1-03
Type IV method
4. Reagents
- Suspension of 2M calcium hydroxide of analytical quality obtained by carefully
pouring one litre of hot water (60C to 70C) on to about 120 g of unslaked lime
(CaO).
- Antifoam solution obtained by dilution of 2 ml of concentrated silicone antifoam
in 100 ml of water.
OIV-MA-F1-03: R2011
5. Equipment
- Standard laboratory equipment including volumetric flasks
- Analytic balance capable of weighing to within 0.1 g.
- Any type of distillation or steam distillation apparatus may be used provided that
it satisfies the following test:
Distil an ethanol-water mixture with an alcoholic strength of 10% vol. five times
in succession. The distillate should have an alcoholic strength of at least 9.9% vol.
after the fifth distillation; i.e. the loss of alcohol during each distillation should not
be more than 0.02% vol.
- Electronic density meter or hydrostatic balance.
6. Procedure
- Homogenise the test sample by inverting the flask several times.
- In a 500 ml volumetric flask, weigh about 200 g of concentrated must or rectified
concentrated must (to within 0.1 g). Note the weight (TS) of this test sample. Fill
up to the mark with deionised water. This solution is about 40% m/v in must.
Obtaining the distillate
- Transfer 250 ml of the 40% solution to the distillation flask, add to the flask
about 10 ml of calcium hydroxide in suspension, about 5 ml of antifoam solution
and, where applicable, a boiling regulator (e.g. pieces of porcelain).
- Gently bring to the boil.
- Recover the distillate in a 100 ml volumetric flask (about 90 ml).
- Leave the distillate to return to ambient temperature, then fill up to the mark with
deionised water.
Measurement of ASV
This is performed by electronic densitometry or by hydrostatic balance.
7. Calculation
Acquired alcoholic strength by volume = ASV measured X 200 X MV
TS
OIV-MA-F1-03: R2011
response e 4,00
s
n
o 3,00
p
s
e
r 2,00
response
rponse
Linearity
Linaire
(rponse)
1,00
0,00
0,0
1,0
2,0
3,0
4,0
5,0
6,0
content (%vol.)
OIV-MA-F1-03: R2011
Table 1: Recovery rate for determination of the acquired ASV of CMs and
Grape Sugar
Test specimen
CM 1
CM 1
Grape Sugar
(RCM) 1
Grape Sugar
(RCM) 1
CM 2
Grape Sugar
(RCM)2
Initial content
(%vol.)
0.00
0.00
0.00
Added content
(%vol.)
0.25
1.00
1.00
Recovered
content (%vol.)
0.22
0.98
0.94
Recovery
rate (%)
88
98
94
0.00
2.00
1.97
99
0.00
0.00
0.50
5.00
0.44
4.94
88
99
recovered content
6,00
y = 0,9968x - 0,039
5,00
4,00
3,00
2,00
added content
recovered content (measured -initial)
Linaire (recovered content (measured -initial))
1,00
0,00
0,00
1,00
2,00
3,00
4,00
5,00
6,00
y = 0,9951x + 0,0161
3,00
2,50
2,00
1,50
added content
recovered content (measured -initial)
Linaire (recovered content (measured -initial))
1,00
0,50
0,00
0,00
0,50
1,00
1,50
2,00
added content (%vol.)
2,50
3,00
3,50
OIV-MA-F1-03: R2011
8.3 Repeatability
The repeatability of the method was determined using 20 test specimens of
CM or grape sugar supplemented with alcohol or not. Each CM or RCM test
specimen was analysed 3 times, in order to ensure identical conditions. The
repeatability limits obtained are as follows:
Table 2: Repeatability of determination of the acquired ASV of CMs and
Grape Sugar
Repeatability for electronic densitometry Calculated value
Standard deviation
0.009
CV or RSD as %
0.9%
r limit
0.024 %vol.
r limit as %
3%
Repeatability for Hydrostatic balance Calculated value
Standard deviation
0.013
CV or RSD as %
r limit
r limit as %
1.7%
0.038 %vol.
5,3%
8.4 Reproducibility
The reproducibility of the results is determined by analysing the same must
twice, at different dates during a given period of time. The results are given
in Table 3.
Table 3 - Reproducibility of determination of the acquired ASV of CMs and
grape sugar
Reproducibility for electronic densitometry Calculated value
Standard deviation
0.043
CV or RSD as %
3%
R limit
0.12%vol.
R limit as %
9%
Reproducibility for Hydrostatic balance Calculated value
Standard deviation
0.026
CV or RSD as %
3.4%
R limit
0.076%vol.
R limit as %
10.6%
OIV-MA-F1-03: R2011
OIV-MA-F1-03: R2011
Method OIV-MA-F1-03
Type IV method
OIV-MA-F1-04: R2011
Total acidity
Method OIV-MA-F1-05
Type IV method
TOTAL ACIDITY
(Oeno 419A-2011)
1. Definition
The total acidity of the rectified concentrated must is the sum of its titrable
acidities when it is titrated to pH 7 against a standard alkaline solution.
Carbon dioxide is not included in the total acidity.
2. Principle of the method
2.1 Potentiometric titration or titration with bromothymol blue as an
indicator and comparison with an end-point colour standard.
3. Reagents
3.1 Buffer solutions
3.1.1 pH 7.0:
monopotassium phosphate, (KH2PO4) : ...................................... 107.3 g
1 M sodium hydroxide (NaOH) solution: .................................... 500 ml
water to: ..................................................................................... 1 000 ml
3.1.2 pH 4.0
Solution of potassium hydrogen phthalate, 0.05 M, containing 10.211 g
of potassium hydrogen phthalate (C8H5KO4) per litre at 20 C.
Note: commercial reference buffer solutions traceable to the SI may be used.
For example: pH 1.679 0.01 at 25C
pH 4.005 0.01 at 25C
pH 7.000 0.01 at 25C
3.2. 0,1 M sodium hydroxide (NaOH) solution.
3.3. 4 g/l bromothymol blue indicator solution:
4g
Bromothymol blue (C27H28Br2O5S): ...
Neutral ethanol 96 % vol: . 200 ml
Dissolve and add:
Water free of CO2: .
OIV-MA-F1-05: R2011
200 ml
1
Total acidity
OIV-MA-F1-05: R2011
Total acidity
OIV-MA-F1-05: R2011
OIV-MA-F1-06
Type IV method
pH
(Oeno 419A-2011)
1. Principle
The difference in potential between two electrodes immersed in the liquid
under test is measured. One of these two electrodes has a potential which is
a function of the pH of the liquid, while the other has a fixed and known
potential and constitutes the reference electrode.
2. Reagents
2.1 Buffer solutions
2.1.1 Saturated solution of potassium hydrogen tartrate, containing at least
5.7 g of potassium hydrogen tartrate per litre (C4H5KO6) at 20 C. (This
solution may be kept for up to two months by adding 0.1 g of thymol per
200 ml.)
pH/temperature
___________
3.57 at 20 C
3.56 at 25 C
3.55 at 30 C
2.1.2 Solution of potassium hydrogen phthalate, 0.05 M, containing 10.211
g of potassium hydrogen phthalate (C8H5KO4) per litre at 20 C.
(Maximum keeping period, two months.)
pH/temperature
____________
3.999 at 15 C
4.003 at 20 C
4.008 at 25 C
4.015 at 30 C
4. Procedure
4.1 Preparation of the sample for analysis
Dilute the rectified concentrated must with water to produce a concentration
of 25 0.5 % (m/m) of total sugars (25 Brix).
OIV-MA-F1-06: R2011
Sulphur dioxide
Type IV method
Method OIV-MA-F1-07
Sulphur dioxide
(Oeno 419B-2011)
1. Definitions
Free sulphur dioxide is defined as the sulphur dioxide present in the must in
the following forms: : H2SO3, HSO3
The equilibrium between these forms is a function of pH and temperature:
H2SO3
H + HSO3
OIV-MA-F1-07: R2011
Sulphur dioxide
Fig. 1 The dimensions given are in millimetres. The internal diameters of the four
concentric tubes forming the condenser are 45, 34, 27 and 10 mm.
The gas feed tube to the bubbler B ends in a small sphere of 1 cm diameter
with 20 0.2-mm diameter holes around its largest horizontal circumference.
Alternatively, this tube may end in a frit glass plate which produces a large
number of very small bubbles and thus ensures good contact between the
liquid and gaseous phases.
The gas flow through the apparatus should be approximately 40 litres per
hour. The bottle on the right of the diagram is intended to restrict the
pressure reduction produced by the water pump to 20 to 30 cm of water. To
regulate the vacuum to its correct value, a flowmeter with a semi-capillary
tube should be installed between the bubbler and the bottle.
2.2.2 A microburette.
3. Procedure
3.1 For rectified concentrated musts, use the solution obtained by diluting
the sample to be analysed to 40 % (m/v) as indicated in the chapter 'Total
acidity', section 5.1. Introduce 50 ml of this solution and 5 ml of phosphoric
acid (2.2.1) into the 250 ml flask A of the entrainment apparatus. Connect
the flask to the apparatus.
OIV-MA-F1-07: R2011
Sulphur dioxide
OIV-MA-F1-07: R2011
Chromatic Properties
Method OIV-MA-F1-08
Type IV method
Chromatic Properties
(Oeno 419A-2011)
OIV-MA-F1-08: R2011
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