Apixaban

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.

Apixaban (provided by Pfizer Inc., United States; material no. 1151519, batch no. ABA8622) was initially dissolved in dimethyl sulfoxide, and subsequently diluted with PBS to a concentration of 2.5 mg ml⁻¹. Diluted Apixaban was stored aliquoted at −20°C and was not re-frozen once thawed. Concentration of Apixaban was selected to be in line with clinically relevant dosing for the secondary prevention of thrombotic events in patients with atrial fibrillation, which is 250 ng ml⁻¹ (Artang et al., 2017 (link)).
Alteplase (Actilyse, provided by Boehringer-Ingelheim International GmbH, Germany; Z. Nr. 1-24,717) was dissolved in distilled water to a concentration of 1 mg ml⁻¹ and was stored aliquoted at -20°C (not re-frozen once thawed); after which it was further diluted with PBS. The final concentration of alteplase was also selected to be in line with clinically relevant dosing indicated for patients with ischemic stroke (1.3 mg L⁻¹), according to the manufacturer’s instructions and supporting pharmacokinetic data (Acheampong and Ford, 2012 (link)).