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

J Thromb Haemost. 2014 Oct;12(10):1636-46. doi: 10.1111/jth.12702.


Background: Three 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.

Objective: To 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.

Methods: The 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.

Results: The 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.

Conclusions: Statistically 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.

Keywords: apixaban; dabigatran; drug monitoring; mass spectrometry; rivaroxaban.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Oral
  • Anticoagulants / administration & dosage*
  • Benzimidazoles / administration & dosage*
  • Blood Coagulation / drug effects
  • Blood Coagulation Tests*
  • Calibration
  • Chromatography, High Pressure Liquid*
  • Dabigatran
  • Factor Xa Inhibitors / chemistry
  • Humans
  • Morpholines / administration & dosage*
  • Pyrazoles / administration & dosage*
  • Pyridones / administration & dosage*
  • Quality Control
  • Reference Values
  • Reproducibility of Results
  • Rivaroxaban
  • Tandem Mass Spectrometry*
  • Thiophenes / administration & dosage*
  • beta-Alanine / administration & dosage
  • beta-Alanine / analogs & derivatives*


  • Anticoagulants
  • Benzimidazoles
  • Factor Xa Inhibitors
  • Morpholines
  • Pyrazoles
  • Pyridones
  • Thiophenes
  • beta-Alanine
  • apixaban
  • Rivaroxaban
  • Dabigatran