Asunto(s)
Anticoagulantes/farmacocinética , Hidrocarburo de Aril Hidroxilasas/genética , Warfarina/farmacocinética , Factores de Edad , Anciano , Anticoagulantes/química , Pueblo Asiatico/genética , Estudios de Cohortes , Citocromo P-450 CYP2C9 , Femenino , Variación Genética , Hong Kong , Humanos , Masculino , Tasa de Depuración Metabólica/genética , Persona de Mediana Edad , Estereoisomerismo , Warfarina/químicaRESUMEN
Clinically used anticoagulant warfarin is usually available as a racemic mixture of S- and R-warfarin that are both metabolized mainly by cytochrome P450 isoenzymes. Determination of warfarin enantiomers and their enantiomeric 7-hydroxywarfarin (7-OH-warfarin) metabolites in the human plasma sample allows probing of cytochrome P450 isoenzyme activities and detection of ingestion of warfarin-containing products for the investigation of unexplained bleeding. The present study aims to develop a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of S-warfarin, R-warfarin, S-7-OH-warfarin and R-7-OH-warfarin in human plasma. Plasma samples were extracted with mixed-mode cation-exchange (MCX) cartridge with recoveries of greater than 87.0% for all four analytes. The selectivity of 7-OH-warfarin from other monohydroxylated warfarin metabolites such as 4'-, 6-, 8- and 10-hydroxywarfarins using a Chirobiotic V chiral column and multiple reaction monitoring (MRM) was addressed. The developed LC-MS/MS method is simple, specific and has been fully validated with satisfactory accuracy and adequate reproducibility with limit of quantification (LOQ) of 5 ng/ml for all four analytes. The method was successfully applied to analyze the steady state plasma concentrations of the four analytes in 30 patients.
Asunto(s)
Anticoagulantes/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Warfarina/análogos & derivados , Warfarina/sangre , Anticoagulantes/metabolismo , Calibración , Cromatografía Liquida/instrumentación , Estabilidad de Medicamentos , Congelación , Humanos , Estructura Molecular , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida , Estereoisomerismo , Espectrometría de Masas en Tándem/instrumentación , Temperatura , Factores de Tiempo , Warfarina/metabolismoRESUMEN
A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.