3.5. Plasma Stability Studies

The pooled plasma used for both plasma stability and plasma binding studies originated from 50 donors, collected as whole blood from donors in the United States at an FDA-approved collection center, processed into plasma by centrifugation and immediately frozen (Innovative Research; Novi, MI, USA) and purchased from Patricell (Nottingham, UK). Plasma was tested by the manufacturers for a range of FDA-required viral markers. Pooled human plasma, was centrifuged (3750 rpm, 10 min, 22 °C) and the supernatant buffered to pH 7.4 by diluting to 70:30 plasma to potassium phosphate buffer (50 mM, pH 7.4). Stock solutions were prepared in DMSO. For inhibitor incubations, esterase inhibitors BNPP and PMSF were added to plasma mixture to give final concentrations of 500 µM for each. For incubations without inhibitor an equivalent volume of DMSO was included. Plasma mixture was pre-incubated at 37 °C for 5 min. Test compounds (5 µM) and procaine (positive control, 50 µM) were added to pre-warmed plasma mixture containing inhibitors. Incubation volume was 1 mL. Samples were mixed and 80 µL was immediately sampled and quenched into 200 µL ACN spiked with donepezil (internal standard (IS), 50 ng mL⁻¹). Plates were incubated at 37 °C with shaking (100 rpm) and additional aliquots were sampled at 10, 30, 60, 120 and 180 min. (S)-AMB-FUBINACA (1) was further sampled at 240 and 300 min as instability in plasma has been reported previously [63]. Samples were centrifuged (3750 rpm, 10 min, 22 °C) and the supernatant (150 µL) diluted with 50 µL deionised water before analysis by UPLC-MS/MS. Peak area ratio of analyte to IS was used to determine half-lives of test compounds in plasma, both with and without the presence of esterase inhibitors.