LC-MS Quantification of Anticoagulants

LC-MS analysis was performed using the liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled with a triple quadrupole 6500 tandem mass spectrometer (AB Sciex, Foster City, CA, USA) according to the previously published method [8 (link),9 (link)]. Dabigatran, rivaroxaban, and stable isotopically labelled D3-Dabigatran (Toronto Research Chemicals Inc, Toronto, ON, Canada) and apixaban (Pfizer Inc., New York, NY, USA) were dissolved in methanol (LC-MS quality, Sigma, St. Louis, MO, USA) at a final concentration of 1 mg/mL expressed as free substances. These stock solutions were used for the preparation of all other standards. For quantification, a series of calibration standards in methanol were prepared (concentrations 0, 10, 50, 100, and 500 ng/mL of DABI, API, and RIVA). The calibration standards were prepared in addition to drug-free plasma from healthy volunteers. All solutions were stored at −20 °C.