Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry

BACKGROUND: The non-vitamin K antagonist oral anticoagulants (NOACs) apixaban, dabigatran, and rivaroxaban are being administered in fixed doses without routine monitoring of anticoagulant activities. Despite this key advantage over vitamin K antagonists (VKAs), assessment of anticoagulant intensities is required in various clinical circumstances. We developed a multi-analyte approach for mass spectrometric analysis of NOACs in human plasma. METHODS: Plasma samples were precipitated with acetonitrile. Separation was achieved by liquid chromatography using a C18 column and a gradient elution within a run time of 2.5 min. Positive electrospray ionization was used and ion transitions monitored by a triple quadrupole mass spectrometer. Stable-isotope-labeled analogues of analytes were employed as internal standards for quantitative analysis. Certified external quality control samples were obtained for external validation. RESULTS: For all analytes, linearity could be demonstrated over the concentration range of 1-500 μg/L (R2>0.999), and the calculated limits of quantification were <1 μg/L. Results for inter- and intra-day assay precision and trueness were obtained using internal quality control samples and remained within the acceptance criterion of ±15%. External quality control samples were measured at the specified nominal values with inter- and intra-day precisions <14%. Matrix effects were fully compensated by co-eluting internal standards, which in turn did not relevantly influence ionization efficiency. CONCLUSIONS: The method enables rapid and reliable simultaneous determination of NOAC concentrations in human plasma. It was successfully introduced into clinical practice; a case with rivaroxaban overdose is presented to exemplify the method's applicability.