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Development and validation of a sensitive and specific LC–MS/MS cocktail assay for CYP450 enzymes: Application to study the effect of catechin on rat hepatic CYP activity
Author(s) -
Nguyen Van,
Espiritu Michael,
Elbarbry Fawzy
Publication year - 2020
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.4789
Subject(s) - chemistry , chromatography , dextrorphan , cyp2a6 , formic acid , cyp1a2 , tandem mass spectrometry , selected reaction monitoring , dextromethorphan , chlorzoxazone , protein precipitation , liquid chromatography–mass spectrometry , mephenytoin , cytochrome p450 , enzyme , catechin , cyp3a4 , analyte , mass spectrometry , cyp2c19 , biochemistry , cyp2e1 , pharmacology , medicine , polyphenol , receptor , antioxidant
A sensitive and specific liquid chromatography tandem mass spectrometric (LC–MS/MS) method that enables the simultaneous quantification of probe substrates and metabolites of cytochrome P450 (CYP) enzymes was developed and validated. These substrates (metabolites)—coumarin (7‐hydroxycoumarin), tolbutamide (4‐hydroxytolbutamide), S ‐mephenytoin (4‐hydroxymephenytoin), dextromethorphan (dextrorphan), and testosterone (6 β ‐hydroxytestosterone)—were utilized as markers for the activities of the major human CYP enzymes CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. Analytes were separated on Kinetex C 18 column (2.1 × 50 mm, 5 μm) using a binary gradient mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Metabolites were detected and quantified by MS using multiple reaction monitoring at m/z 163 → 107.2 for 7‐hydroxycoumarin, m/z 235 → 150.1 for 4‐hydroxymephenytoin, m/z 287 → 171 for 4‐hydroxytolbutamide, m/z 258 → 157.1 for dextrorphan, m/z 305 → 269 for 6 β ‐hydroxytestosterone, and m/z 237 → 194 for the internal standard. The assay exhibited good linearity over a range of 10–500 ng/mL with acceptable accuracy and precision criteria. As a proof of concept, the developed cocktail assay was successfully used to examine the potential impact of catechin on the activity of the major rat liver CYP enzymes.