Application and enantiomeric residue determination of diniconazole in tea and grape and apple by supercritical fluid chromatography coupled with quadrupole-time-of-flight mass spectrometry

A chiral separation and residue determination method for diniconazole enantiomers in tea, apple, and grape was developed and validated by supercritical fluid chromatography coupled with quadrupole-time-of-flight mass spectrometry (SFC-Q-TOF/MS). The two diniconazole enantiomers were separated on a Chiral CCA column, and the chromatographic conditions (mobile phase proportion and modifier, column temperature, backpressure, and auxiliary solvent) were optimized. The optimal SFC-Q-TOF/MS conditions were selected as a mobile phase of CO₂/isopropanol (IPA) (v/v, 96/4), flow rate at 2.0 mL/min, automated back pressure regulator (ABPR) at 2000 psi, column temperature at 25 ℃ and under electrospray ionization positive mode with the best auxiliary solvent of 2 mmol/L ammonium acetate in methanol/water (v/v, 1/1) at 0.20 mL/min flow rate. Residues in tea and fruit samples were extracted by acetonitrile/water (v/v, 4/1 for fruit and 2/1 for tea), purified by Cleanert TPT or Pesti-Carb solid phase extraction column, then analyzed by SFC-Q-TOF/MS with matrix-matched external standard quantification method. The elution order of diniconazole enantiomers on CCA column was R-(-)-diniconazole at first, and S-(+)-diniconazole at second. The standard curve concentration levels of R-(-)-diniconazole and S-(+)-diniconazole in samples ranged from 0.01 mg/L to 1.00 mg/L with the correlation coefficients greater than 0.99. The spiked recoveries of R-(-)-diniconazole and S-(+)-diniconazole in apple and grape at three levels of 0.005, 0.05 and 0.25 mg/kg were in the range of 69.8% to 102.1%, with relative standard deviations (RSDs) (n = 6) between 3.5% and 10.4%, and the limits of quantitation (LOQs) below 0.005 mg/kg. The spiked recoveries in black tea at three levels of 0.01, 0.10, and 0.50 mg/kg were in the range of 85.6% to 90.6%, with the RSDs (n = 6) ranging from 3.9% to 9.5%, and LOQ of 0.01 mg/kg. This residue analysis and determination method for diniconazole enantiomers in apple, grape and tea samples is convenient, reliable, and meets the residue analysis requirement. Also it is applicatied for the residue fates of R-(-)-diniconazole and S-(+)-diniconazole during the fresh tea leaves growing, green tea processing and black tea processing. The degradation half-times (DT₅₀) between R-(-)-diniconazole and S-(+)-diniconazole in the fresh tea leaves growing were 2.9 d and 3.1 d, respectively. The concentrations of R-(-)-diniconazole and S-(+)-diniconazole decreased gradually with time and on the 14th day after application were lower than 10% of the initial concentration. The average enantiomer fractions (EFs) of R-(-)-diniconazole and S-(+)-diniconazole at 2 h, 2, 5, 7, 10 and 14 d after application in fresh tea leaves were 0.505, 0.526, 0.523, 0.558, 0.453 and 0.489, respectively. This result is similar to the result of our last research for the enantiomers of cis-epoxiconazole-another triazole fungicide residues in fresh tea leaves. And in the whole black tea processing, 37.1%-49.3% and 35.9%-57.9% of R-(-)-diniconazole and S-(+)-diniconazole decreased, respectively. The total processing factors (PFs) of R-(-)-diniconazole and S-(+)-diniconazole for the black tea procedure were 0.507-0.629 and 0.421-0.641, respectively. The EFs of R-(-)-diniconazole and S-(+)-diniconazole in black tea processing ranged from 0.432 to 0.532. However, in the whole green tea processing, 22.3%-32.6% and 21.7%-40.3% of R-(-)-diniconazole and S-(+)-diniconazole decreased, respectively. The difference between black tea and green tea is nearly 15%, and in green tea is less decreased than in black tea. The total PFs of R-(-)-diniconazole and S-(+)-diniconazole for the green tea procedure were 0.674-0.777 and 0.597-0.783, respectively. The EFs of R-(-)-diniconazole and S-(+)-diniconazole in green tea processing ranged from 0.473 to 0.504. The PFs illustrated that for R-(-)-diniconazole and S-(+)-diniconazole decrease, the rolling and fermentation were the critical steps in black tea processing, and the rolling was the critical step in green tea processing, respectively.