Quantitative Analysis Of R-and S-warfarin In Human Plasma And Its Application

OBJECTIVE To develop a sensitive and rapid liquid chromatography tandemmass spectrometric （LC MS/MS） method for the determination of R warfarin andS warfarin in human plasma; and its application in a drug drug interaction study ofmorinidazole and warfarin.METHOD R warfarin, S warfarin and methyclothiazide were extracted withethylether: methylenechloride（3:2，v/v）, then separated on a Astec Chirobiotic^TM V （150mm×4.6mm I.D.，5μm） column using5mmol·L^-1ammonium acetate （pH4.0）acetonitrile as mobile phase at a flow-rate of1.5mL·min^-1，and the mobile phase wassplitted and0.5mL·min^-1was introduced into MS. The API4000triple quadrupolemass spectrometer was operated in multiple reaction monitoring mode （MRM） vianegtive electrospray ionization （ESI） source using the transitions m/z307â†'m/z161forwarfarin, and m/z358â†'m/z322for the internal standard, respectively. In a random,two period crossover study,12healthy volunteers received a single oral dose of5mgracemic warfarin in the absence and presence of morinidazole. Blood samples werecollected according to a time schedule and determined by the method above.RESULTS The retention times of R warfarin and S warfarin both were less than5minutes, and the resolution of them was1.56. The linear calibration curves forR warfarin and S warfarin both were obtained in the concentration range of51000ng·mL^-1. Intra and inter day relative standard deviation （RSD） for R warfarin andS-warfarin over the entire concentration range across three validation runs were bothless than10%, and relative error （RE） ranged from4.9%to0.7%, respectively. The90%CI around the geometric mean ratio （the absence/presence of chronic morinidazole）of R and S warfarin for AUC0â†'168and Cmaxvalues both fall within the range of80%¹25%. CONCLUSIONS The method herein described was effective, convenient andsuitable for the study of metabolic interaction between morinidazole and warfarin. Theresults showed that coadministration of warfarin with morinidazole did not affect thepharmacokinetics of either R-warfarin or S-warfarin.