Abstract
A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 10⁸ RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
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Validation Study
MeSH terms
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Adult
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Analytic Sample Preparation Methods
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Antimetabolites, Antineoplastic / blood
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Antimetabolites, Antineoplastic / pharmacokinetics*
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Antimetabolites, Antineoplastic / therapeutic use
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Azathioprine / blood
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Azathioprine / pharmacokinetics
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Azathioprine / therapeutic use
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Biotransformation
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Chromatography, High Pressure Liquid
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Drug Monitoring / methods*
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Erythrocytes / chemistry*
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Erythrocytes / metabolism
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Female
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Humans
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Indicators and Reagents / chemistry
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Male
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Mercaptopurine / blood
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Mercaptopurine / pharmacokinetics
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Mercaptopurine / therapeutic use
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Nucleic Acid Synthesis Inhibitors / blood
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Nucleic Acid Synthesis Inhibitors / pharmacokinetics*
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Nucleic Acid Synthesis Inhibitors / therapeutic use
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Pilot Projects
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Potassium Permanganate / chemistry
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / blood
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / drug therapy
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Purines / blood*
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Purines / metabolism
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Quaternary Ammonium Compounds / chemistry
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Thioguanine / blood
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Thioguanine / pharmacokinetics
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Thioguanine / therapeutic use
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Thionucleotides / blood*
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Thionucleotides / metabolism
Substances
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Antimetabolites, Antineoplastic
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Indicators and Reagents
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Nucleic Acid Synthesis Inhibitors
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Purines
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Quaternary Ammonium Compounds
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Thionucleotides
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Potassium Permanganate
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tetrabutylammonium
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Mercaptopurine
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Thioguanine
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Azathioprine