Development and validation of LC–MS/MS method for simultaneous determination of dabigatran etexilate and its active metabolites in human plasma, and its application in a pharmacokinetic study

• LC–MS/MS method for simultaneous quantification of dabigatran, dabigatran etexilate, and dabigatran acylglucuronide was validated. • Our results demonstrated this method as accurate, precise, and sensitive with wide calibration ranges. • The method was successfully applied in a pharmacokinetic study of the collected samples. Dabigatran is a direct thrombin inhibitor widely used for preventing various thrombotic events. Although there are several established LC–MS/MS based methods for quantification of plasma dabigatran etexilate and its active metabolites (dabigatran and dabigatran acylglucuronide), so far, there are no studies for simultaneous quantification of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. In the present study, a novel and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for assessment of dabigatran pharmacokinetics in human plasma samples according to FDA guidelines. We used the new method to simultaneously quantify dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. After deproteinization using acetonitrile, the supernatants were evaporated, dissolved in a mobile phase, and finally injected onto an HPLC system with a silica-based C 18 reverse-phase column. Mass spectrometer system was operated in multiple reaction monitoring mode (MRM) (dabigatran etexilate: m/z 629.464→290.100; dabigatran: m/z 472.300→289.100, and dabigatran acylglucuronide: m/z 648.382→289.100) and all the components were confirmed using positive electrospray ionization (ESI). Correlation coefficients > 0.999 were achieved for all the calibration curves with linear regression. The intra-day and inter-day accuracies of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide were 95.84–109.44 %, 99.4–103.42 %, and 98.37–104.42 %, respectively, while their corresponding precisions were 3.84–9.79, 1.07–8.76 %, and 2.56–4.51 %, respectively. We successfully applied the new method to determine the pharmacokinetic profiles of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in humans. Our findings demonstrated the method as robust, reliable, and sensitive.