Determination of rivaroxaban, apixaban and edoxaban in rat plasma by UPLC-MS/MS method.

To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of rivaroxaban, apixaban and edoxaban in rat plasma. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1 % formic acid in water at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 436.1 → 145.1 for rivaroxaban, m/z 460.0 → 443.1 for apixaban, m/z 548.2 → 366.1 for edoxaban and m/z 285.2 → 193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 1.0-200 ng/mL for rivaroxaban, 1.0-100 ng/mL for apixaban and 1.0-500 ng/mL for edoxaban. Total time for each chromatograph was 3.5 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations <10.5 % and the accuracy values ranged from -9.9 to 11.3 %. The method was successfully applied to a pharmacokinetic study of rivaroxaban, apixaban and edoxaban in rats.