The effects of AB-PINACA on the UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 activities were measured in ultrapooled human liver microsomes [18 (link)]. Human liver microsomal mixture (100 μL) containing 50 mM Tris buffer (pH 7.4), ultrapooled human liver microsomes (0.2 mg/mL), 5 mM UDPGA, alamethicin (25 μg/mL), 10 mM MgCl2, AB-PINACA (0.1–100 μM), and two sets of a cocktail of six UGT probes (set A: 2 μM chenodeoxycholic acid, 0.5 μM SN-38, and 0.5 μM trifluoperazine; set B: 1 μM N-acetylserotonin, 0.2 μM mycophenolic acid, and 1 μM naloxone). The incubation was continued for 60 min at 37 °C in a shaking water bath after addition of UDPGA, and the reactions were terminated by adding 50 μL of ice-cold propofol glucuronide and meloxicam in acetonitrile (ISs). After centrifugation, aliquots from supernatant from set A and set B (50 μL each) were mixed and 5 μL of the mixed supernatants were analyzed by SRM mode of LC-MS/MS as described in our previous report [19 (link)] and supplementary Table S1 and Figure S2. The linear ranges were 1–300 pmol for N-acetylserotonin β-d-glucuronide, chenodeoxycholic acid 24-acyl-β-glucuronide, mycophenolic acid glucuronide, naloxone 3-β-d-glucuronide, and SN-38 glucuronide, and 4–1200 pmol for trifluoperazine glucuronide. The accuracy and relative standard deviation values for six metabolites were 98.0–102.8% and 6.4–9.7%.
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