Determination of dabigatran, rivaroxaban and apixaban by ultra‐performance liquid chromatography – tandem mass spectrometry (UPLC‐MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants

Abstract

BackgroundThree novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. ObjectiveTo develop and validate a reference ultra‐performance liquid chromatography – tandem mass spectrometry (UPLC‐MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. MethodsThe developed UPLC‐MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry‐over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory‐developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti‐Xa, Biophen Heparin (LRT), and Biophen DiXal anti‐Xa. Agreement between the various coagulation assays and UPLC‐MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. ResultsThe UPLC‐MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC‐MS/MS method was the LD dTT test. For rivaroxaban, the anti‐factor Xa assays were superior to the PiCT‐Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti‐Xa assay was superior to the PiCT‐Xa assay. ConclusionsStatistically significant differences were observed between the various coagulation assays as compared with the UPLC‐MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.