Ticagrelor Measurement

Ticagrelor plasma concentrations were determined based on a method previously described.19 In brief, blood collected from the tail-vein was placed on ice and plasma prepared within 30 minutes of blood sampling by centrifugation at 1500g for 10 minutes at approximately 4°C. The plasma was transferred into tubes stored at or below −20°C within 1 hour of sample collection. Plasma concentration of ticagrelor was determined by a protein precipitation and liquid chromatographic-mass spectrometric method. Chromatographic separation was performed using an ACQUITY UPLC I-class system (Waters Corporation, Milford, MA, USA). Analytical column ACQUITY UPLC HSS T3, 2.1 × 30 mm was used with a 1.8-μm particle size. The mass detector was a Waters Quattro Premier XE triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA) using electrospray ionization. The lower limit of quantification was 0.05 μM. Plasma was obtained from mice in groups A and D; no significant difference in plasma levels of ticagrelor was detected between groups.