Ugt reaction mix solution a

Corning UltraPool human liver microsomes (HLM) 150 (20 mg/mL), human intestinal microsomes (HIM) pool (10 mg/mL) as well as recombinant human CYP enzymes (with P450 oxidoreductase, OR) including CYP1A2+OR (0.5 nmol), CYP2B6+OR (0.5 nmol), CYP2C8+OR (1.0 nmol), CYP2C9*1+OR (1 nmol), CYP2C19+OR (0.5 nmol), CYP2D6*1+OR (0.5 nmol), CYP2E1+OR + cytochrome b₅ (1 nmol), CYP3A4+OR + cytochrome b₅ (0.5 nmol), MAO-A (5 mg/mL), MAO-B (5 mg/mL), and UGT1A10 (5 mg/mL) were purchased from Corning Life Sciences B.V (Amsterdam, Netherlands). All microsomes and recombinant enzyme solutions were aliquoted and stored at −80 °C until further use. Co-factors including NADPH regenerating system solution A (25 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM MgCl₂ in water), NADPH regenerating system solution B (40 U/mL glucose-6-phosphate dehydrogenase in 5 mM sodium citrate), UGT reaction mix solution A (25 mM uridine 5′-diphospho-glucuronic acid in water), and UGT reaction mix solution B (250 mM Tris-HCl, 40 mM MgCl₂, and 0.125 mg/mL alamethicin in water) were also obtained from Corning Life Sciences B.V.

Methoxyacetylfentanyl hydrochloride (purity ≥ 98%), U-51754 hydrochloride (purity 98%), and U-47931E were purchased from LGC Standards (Wesel, Germany). Isocitrate, Isocitrate dehydrogenase, superoxide dismutase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), S-(5′-adenosyl)-L-methionine (SAM), dithiothreitol (DTT), reduced glutathione (GSH), acetyl carnitine, and acetyl coenzyme A (AcCoA) were all purchased from Sigma (Taufkirchen, Germany), NADP⁺ from Biomol (Hamburg, Germany), and acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid (LC-MS grade), methanol (LC-MS grade), glucuronidase (EC No. 3.2.1.32)/arylsulfatase (EC No. 3.1.6.1) from Helix pomatia L, and all other chemicals and reagents (analytical grade) from VWR (Darmstadt, Germany). phS9 (20 mg protein/mL, from 30 individual donors), UGT reaction mix solution A (25 mM UDP-glucuronic acid), and UGT reaction mix solution B (250 mM Tris–HCl, 40 mM MgCl₂, and 0.125 mg/mL alamethicin) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the enzyme preparations were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.