Quantification of Apixaban, Dabigatran, Edoxaban, and Rivaroxaban in Human Serum by UHPLC-MS/MS—Method Development, Validation, and Application

BACKGROUND: Direct oral anticoagulants (DOACs) are prescribed for anticoagulation in patients with atrial fibrillation and venous thromboembolic disease. Fixed doses are recommended, but measuring their serum drug concentrations as a basis for dose adjustments may be useful in some clinical settings. METHODS: An ultra–high-performance liquid chromatography-tandem mass spectrometry method for the analysis of the DOACs apixaban, dabigatran, edoxaban, and rivaroxaban in human serum was developed and validated. A 100-µL serum sample was handled using a pipetting robot. Protein precipitation was performed with 375 µL of 1% formic acid in acetonitrile (vol/vol), and phospholipid removal was performed using a Waters Ostro 96-well plate. The injection volume was 1 µL, and run time was 3.0 minutes. RESULTS: The calibration range was 5–800 nmol/L. The between-day precision relative SDs were in the range of 3.3%–10%. Recoveries ranged from 85% to 105%, and matrix effects from 88% to 102%, when corrected with internal standard. Edoxaban was, in contrast to the other DOACs, unstable when stored for more than 6 hours at 30°C. The suitability of the method was demonstrated by analyzing routine samples from 345 patients undergoing anticoagulation treatment. CONCLUSIONS: The developed method fulfilled the set validation criteria, and its suitability was demonstrated in a routine setting. The instability of edoxaban may complicate the transport of routine samples to the laboratory.