Simultaneous liquid chromatography tandem mass spectrometric determination of warfarin enantiomers in human plasma and its application to a bio-equivalence study in Indian population.

Author(s): Varad Ravindra Pradhan*, Ashutosh Pudage, Siddheshwar Patankar, M. V. Rathnam

Abstract

Warfarin (WAR), one of the most commonly used oral anticoagulant in the US and across the globe, is a drug of choice for millions. It’s a unique drug of its kind due to the multiple pharmacological and pharmacokinetic properties. Although highly efficacious, positive clinical outcomes during WAR therapy depend on maintaining a narrow therapeutic range for the drug. This goal is challenging due to large inter-individual variability in patient response, which has been attributed to diversity in drug metabolism. WAR is given as a racemic mixture and evidence suggest differences of R and S-WAR in their therapeutic activities and metabolism. Previous investigation of WAR metabolism has been hampered by the inability to quantify the individual enantiomers with reliable accuracy and within short time period. To overcome these limitations a single, simple, selective and rapid LC–MS/MS method is reported. The reported method separated individual R and S enantiomers of WAR with a short run time of just 4 min. per sample using R WAR d5 and S WAR d5 as internal standards. WAR enantiomers were resolved to reveal unique insight into the stereo-specific metabolism of WAR. The mean recovery was 98.9 % and 84.6 % for R WAR and S WAR respectively. The coefficient of variation of the assay was less than 5.5 % and 7.2 % for R WAR and S WAR with an accuracy of 93.1 % to 105.8 % and 94.5 % to 102.6 % for R WAR and S WAR respectively. Stability was evaluated under different conditions including bench top, freeze and thaw, auto sampler and long term. The validated method was applied for the determination of individual WAR enantiomers in human plasma samples from a bioequivalence study of 5mg fixed dose formulation in 10 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 10% incurred samples.

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