title: 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: 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.
authors.affiliation: Clinical Laboratory, Catharina Hospital, Eindhoven, The Netherlands; Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands; Expert Center Clinical Chemistry Eindhoven, Eindhoven, The Netherlands.
authors.forename: E M H
authors.initials: EM
authors.lastname: Schmitz
authors.forename: K
authors.initials: K
authors.lastname: Boonen
authors.forename: D J A
authors.initials: DJ
authors.lastname: van den Heuvel
authors.forename: J L J
authors.initials: JL
authors.lastname: van Dongen
authors.forename: M W M
authors.initials: MW
authors.lastname: Schellings
authors.forename: J M A
authors.initials: JM
authors.lastname: Emmen
authors.forename: F
authors.initials: F
authors.lastname: van der Graaf
authors.forename: L
authors.initials: L
authors.lastname: Brunsveld
authors.forename: D
authors.initials: D
authors.lastname: van de Kerkhof
mesh_terms: D000284:Administration, Oral; D000925:Anticoagulants / Q000008:administration & dosage*; D001562:Benzimidazoles / Q000008:administration & dosage*; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D000069604:Dabigatran; D065427:Factor Xa Inhibitors / Q000737:chemistry; D006801:Humans; D009025:Morpholines / Q000008:administration & dosage*; D011720:Pyrazoles / Q000008:administration & dosage*; D011728:Pyridones / Q000008:administration & dosage*; D011786:Quality Control; D012016:Reference Values; D015203:Reproducibility of Results; D000069552:Rivaroxaban; D053719:Tandem Mass Spectrometry; D013876:Thiophenes / Q000008:administration & dosage*; D015091:beta-Alanine / Q000008:administration & dosage / Q000031:analogs & derivatives*
chemical_list: D000925:Anticoagulants; D001562:Benzimidazoles; D065427:Factor Xa Inhibitors; D009025:Morpholines; D011720:Pyrazoles; D011728:Pyridones; D013876:Thiophenes; D015091:beta-Alanine; C522181:apixaban; D000069552:Rivaroxaban; D000069604:Dabigatran
keywords: apixaban; dabigatran; drug monitoring; mass spectrometry; rivaroxaban