title: UPLC-MRM Mass Spectrometry Method for Measurement of the Coagulation Inhibitors Dabigatran and Rivaroxaban in Human Plasma and Its Comparison with Functional Assays.
abstract: BACKGROUND
  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.
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Kuhn
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Gripp
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Flieder
authors.affiliation: Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Dittrich
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Hendig
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Busse
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Cornelius
authors.initials: C
authors.lastname: Knabbe
authors.affiliation: Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Bad Oeynhausen, Germany.
authors.forename: Ingvild
authors.initials: I
authors.lastname: Birschmann
mesh_terms: D000991:Antithrombins / Q000097:blood; D001681:Biological Assay / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D000069604:Dabigatran / Q000097:blood*; D065427:Factor Xa Inhibitors / Q000097:blood; D006801:Humans; D057230:Limit of Detection; D000069552:Rivaroxaban / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D054928:Validation Studies as Topic
chemical_list: D000991:Antithrombins; D065427:Factor Xa Inhibitors; D000069552:Rivaroxaban; D000069604:Dabigatran