Simultaneous Determination of Dabigatran, Rivaroxaban, and Apixaban in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Background: Pharmacokinetic studies and therapeutic drug monitoring of anticoagulants require a simple, rapid, and reliable analytical method for monitoring plasma concentrations. The aims of the current work were to develop and validate a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 3 direct oral anticoagulants (dabigatran, rivaroxaban, and apixaban) in human plasma that is suitable for pharmacokinetic studies and routine therapeutic drug monitoring in busy hospital laboratories. Methods: This method included a hydrolysis step to account for the active acylglucuronide metabolites of dabigatran that demonstrate an equivalent anticoagulant effect as dabigatran. After hydrolysis, a simple one-step protein precipitation was used for sample preparation. Total dabigatran (the sum of free dabigatran and the contribution from dabigatran acylglucuronides), rivaroxaban, and apixaban, and their corresponding isotopically labeled internal standards were resolved on a C18(2) column. All compounds were detected using electrospray ionization liquid chromatography/tandem mass spectrometry in the positive mode. Results: For all 3 anticoagulants, standard curves were linear over the concentration range of 1.0-1000 mcg/L (r > 0.99), bias was < +/- 10%, and intraday and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 1.0 mcg/L. For all 3 anticoagulants and corresponding isotopically labeled internal standards, the absolute recoveries were similar and consistent, with mean values of 93%-102%. No significant matrix effects were observed. Conclusions: This method is simple, rapid, robust, and reliable and can be used to analyze the plasma concentrations of the drugs in patients on dabigatran or rivaroxaban therapy.