Dabigatran, rivaroxaban, apixaban, argatroban and fondaparinux and their effects on coagulation POC and platelet function tests.

BACKGROUND: In recent years, several selectively acting anticoagulants, including the direct thrombin inhibitors (DTI; argatroban, dabigatran) and the factor Xa inhibitors (rivaroxaban, apixaban, fondaparinux), have been developed. With their clinical application increasing, it is of interest to evaluate their interference with classical haemostaseological point-of-care tests. Additionally, the effect of the investigated anticoagulants on platelet function tests will come increasingly more into focus for monitoring not only hereditary platelet dysfunction, but also antiplatelet therapy. METHODS: Blood samples from healthy volunteers were spiked with therapeutic and supratherapeutic concentrations of the drugs listed above and investigated with regard to their effects on the following POCTs: activated clotting time (ACT), thromboelastometry with ROTEM, PFA and Multiplate. Light-transmission aggregometry (LTA) was used for a platelet function assay. RESULTS: At supratherapeutic concentrations, ACT and ROTEM analysis were always influenced after administration of the drugs listed above (except fondaparinux in EXTEM-CT). Therapeutic concentrations showed differential effects on these assays. LTA measurements revealed a distinct decrease in α-thrombin-induced platelet aggregation for both DTIs (therapeutic and supratherapeutic concentrations), while argatroban reduced platelet function in supratherapeutic concentrations. None of the drugs seemed to have any influence on PFA or Multiplate. CONCLUSIONS: Selective thrombin and factor Xa inhibitors exhibit distinct effects on POCTs and platelet function tests. This must be considered in assessing assay results when taking medical decisions.

UPLC-MRM Mass Spectrometry Method for Measurement of the Coagulation Inhibitors Dabigatran and Rivaroxaban in Human Plasma and Its Comparison with Functional Assays.

INTRODUCTION: The fast, precise, and accurate measurement of the new generation of oral anticoagulants such as dabigatran and rivaroxaban in patients' plasma my provide important information in different clinical circumstances such as in the case of suspicion of overdose, when patients switch from existing oral anticoagulant, in patients with hepatic or renal impairment, by concomitant use of interaction drugs, or to assess anticoagulant concentration in patients' blood before major surgery. METHODS: Here, we describe a quick and precise method to measure the coagulation inhibitors dabigatran and rivaroxaban using ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS). Internal standards (ISs) were added to the sample and after protein precipitation; the sample was separated on a reverse phase column. After ionization of the analytes the ions were detected using electrospray ionization-tandem mass spectrometry. Run time was 2.5 minutes per injection. Ion suppression was characterized by means of post-column infusion. RESULTS: The calibration curves of dabigatran and rivaroxaban were linear over the working range between 0.8 and 800 µg/L (r >0.99). Limits of detection (LOD) in the plasma matrix were 0.21 µg/L for dabigatran and 0.34 µg/L for rivaroxaban, and lower limits of quantification (LLOQ) in the plasma matrix were 0.46 µg/L for dabigatran and 0.54 µg/L for rivaroxaban. The intraassay coefficients of variation (CVs) for dabigatran and rivaroxaban were < 4% and 6%; respectively, the interassay CVs were < 6% for dabigatran and < 9% for rivaroxaban. Inaccuracy was < 5% for both substances. The mean recovery was 104.5% (range 83.8-113.0%) for dabigatran and 87.0% (range 73.6-105.4%) for rivaroxaban. No significant ion suppressions were detected at the elution times of dabigatran or rivaroxaban. Both coagulation inhibitors were stable in citrate plasma at -20°C, 4°C and even at RT for at least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay) for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs. CONCLUSIONS: Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma.