Multianalyte determination of NOAC using LC-MS/MS and comparison with functional coagulation assays

L. Slavik, J. Lukes, M. Zhanelova, M. Nemcova, J. Ulehlova, J. Prochazkova, A. Hlusi, M. Palova, J. Vaclavik (Olomouc, Czech Republic)

Date: 16.02.2017,
Time: 08:00 - 09:15

Objective: New oral anticoagulants, also called NOAC, are direct inhibitors of coagulation factors which meant improvement in anticoagulation therapy. Currently, three NOAC are used in anticoagulation therapy: dabigatran, direct thrombin inhibitor and rivaroxaban and apixaban, direct factor Xa inhibitors. Even though routine monitoring of NOAC is not needed, there are certain situations in which it is necessary to know the plasma level of anticoagulants, eventually their coagulation effect. Monitoring of NOACs can be addressed by measurement of anticoagulant activities, measurements of trombin production or by quantification of drug levels. Compared to functional tests such as trombin generation for all NOACs, calibrated chromogenic anti-Xa assays for rivaroxaban and apixaban or a calibrated diluted thrombin time assay for dabigatran, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) remains the gold standard in quantitative drug analysis.

Methods: This study was conducted on a set of blood samples from 116 patients with therapy by NOACs. Our LC-MS/MS method enables simultaneous determination of apixaban, dabigatran and rivaroxaban. The aim of our study was comparison of this method with functional tests for determination of NOACs as aPTT, dTT assay, PT, anti-Xa activity assai and TGA assay.

Results: The results of dabigatran levels showed statistically significant relationship between LC-MS/MS and aPTT (r = 0,82; p < 0,001), dTT (Hyphen: r = 0,817, p < 0,001, HemosIL: r = 0,819, p < 0,001) and one TGA parameter - tLag (r = 0,44, p = 0,0045). Correlation of remaining TGA parameters (Peak, AUC) was not significant as their initial values show high variability. The results of rivaroxaban shows better relationship between LC-MS/MS and anti-Xa assay (r=0,7025) than TGA. TGA assay did not show statistically significant relationship of its parameters (tLag, tPeak, Peak, AUC) and rivaroxaban concentration in plasma.

Conclusion: The results of all methods for dabigratran determination were compared using HPLC-MS as reference. dTT HemosIL showed better results for low concentrations when compared to HPLC-MS than dTT Hyphen as HemosIL uses non-linear calibration curve and therefore gives more accurate results. The determination of rivaroxaban and apixaban by Anti-Xa assay shows better results than TGA. Supported by grant LF-2016 and MH CZ – DRO (FNOl, 00098892)