Edoxaban

Acetonitrile and water, both of HPLC-grade, ammonium formate, ethanol and formic acid were purchased from VWR International (Fontenay-sous-Bois, France). Apixaban, edoxaban and rivaroxaban were supplied by the Cayman Chemical Company (Ann Arbor, USA). Dabigatran etexilate and argatroban monohydrate were purchased from Sigma Aldrich (St. Louis, USA) and dabigatran from biosynth/carbosynth (Thal, Switzerland). Hydrochloric acid was supplied from Merck (Darmstadt, Germany) and acetone was supplied from Fisher Scientific (Loughborough, UK). Methanol was obtained from PanReac AppliChem (Darmstadt, Germany). Apixaban-13C-d₃ (Cayman Chemical Company, Ann Arbor, USA) was used as internal standard (IS). The blank serum for method validation was obtained from the German Red Cross blood donation in Dresden, Germany.
Quantification by functional testing was performed applying STA Liquid Anti-Xa Kit with drug specific calibration on a STA R Max3 analyzer (reagents, calibrators & analyzer: STAGO Deutschland GmbH, Düsseldorf, Germany).

FXa activity assay has been developed to test specificity of the selected inhibitors. 10 µM one-point screen and IC₅₀ measurement have been carried out as follows:
Human isolated Factor Xa protein was purchased from ThermoFisher Scientific (Waltham, MA, USA). An end-point assay was carried out using the fluorescent amide substrate Boc-Leu-Gly-Arg-AMC used for C1s 10 µM screen; assay conditions: 20 mM HEPES (pH 7,8) 100 mM NaCl, 10 mM CaCl₂ 30 °C, 0.05% TWEEN-20, 1% DMSO; low enzyme concentration (about 1 nM); in a 96 well microplate format (in 100 µL), using non-binding black plates (Greiner). 40 µL enzyme + 10 µL inhibitor compounds were incubated (10 min, RT), and 50 µL substrate solution (500 µM final concentration) was added; incubated for 30 min and finally the fluorescence has been read. Edoxaban (Cayman Chemical, MI, USA), a specific and highly potent FXa inhibitor has been used for inhibition control and for validation of the assay with a measured IC₅₀ = 0.649 nM value, according to the published data (Ki of 0.561 nM [50 (link)]).