Enantioselective determinination of R-warfarin/S-warfarin in human plasma using liquid chromatography-tandem mass spectrometry and its application in a drug-drug interaction study

To study the drug-drug interaction of morinidazole and warfarin and its application, a sensitive and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of R-warfarin/S-warfarin in human plasma.  In a random, two-period crossover study, 12 healthy volunteers received a single oral dose of 5 mg racemic warfarin in the absence and presence of morinidazole.  Blood samples were collected according to a pre-designed time schedule.  R-warfarin, S-warfarin and methyclothiazide were extracted with ethylether∶methylenechloride (3∶2), then separated on a Astec Chirobiotic^TM V (150 mm × 4.6 mm ID, 5 μm) column using 5 mmol·L⁻¹ ammonium acetate (pH 4.0) – acetonitrile as mobile phase at a flow-rate of 1.5 mL?min⁻¹.  The mobile phase was splitted and 0.5 mL·min⁻¹ was introduced into MS.  A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the negative ion mode.  Quantification was performed using multiple reaction monitoring (MRM).  The resolution of warfarin enantiomers is 1.56.  The linear calibration curves for R-warfarin and S-warfarin both were obtained in the concentration range of 5 − 1 000 ng·mL⁻¹.  Intra- and inter-day relative standard deviation (RSD) for R-warfarin and S-warfarin over the entire concentration range across three validation runs was both less than 10%, and relative error (RE) ranged from −4.9% to 0.7%, separately.  The method herein described is effective and convenient, and suitable for the study of metabolic interaction between morinidazole and warfarin.  The results showed that coadministration of warfarin with morinidazole did not affect the pharmacokinetics of either R-warfarin or S-warfarin.