Pooled human liver microsomes hlms

The standard for AB-CHMINACA was obtained from Cayman Chemical Company (Ann Arbor, Michigan, USA) in neat powder. The standards for theophylline, benzamide, tramadol, 4-nitrophenol, 2,6-uridine-diphosphate glucuronic acid (UDPGA), alamethicin and NADPH (all neat, purity >99%) were obtained from Sigma-Aldrich (Diegem, Belgium).
Pooled human liver microsomes (HLMs; mix gender, n=200) were purchased from Tebu-bio (Boechout, Belgium). Baculovirus-insect cell microsomes containing expressed human CYP enzyme (CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 or 3A4) co-expressed with human CYP oxidoreductase and human cytochrome b5 were purchased from BD Biosciences (Erembodegem, Belgium) and Tebu-Bio. Ultrapure water was prepared using a Purelab flex water system from Elga (Tienen, Belgium). Methanol, acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). All organic solvents were HPLC grade or higher.

Ethylphenidate was obtained from LGC Standards (Molsheim, France) as neat powder (purity>99%) and it was dissolved in methanol (purity ≥ 99.9%) purchased from Merck (Darmstadt, Germany). The internal standard, theophylline, was obtained as powder (anhydrous, purity>99%) from Sigma-Aldrich (Diegem, Belgium). Pooled human liver microsomes (HLMs, mix gender, n=200) were purchased from Tebu-Bio (Boechout, Belgium). Pooled human liver cytosol (HLCYT, mix gender, n=50), chemical standards for 2,6-uridinediphosphate glucuronic acid (UDPGA), alamethicin (neat, purity>99%), adenosine 3′-phosphate 5′-phosphosulfate (PAPS; neat, purity>60%) lithium salt hydrate, 4-nitrophenol (4-NP), 4-nitrophenolglucuronide (4-NP-Gluc; neat, purity>99%), 4-nitrophenolsulfate (4-NP-Sulf; neat, purity>99%) and NADPH (neat, purity>99%) were purchased from Sigma-Aldrich.
Baculovirus-insect cell microsomes containing expressed human recombinant CYP enzyme (rCYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 or 3A4) co-expressed with human CYP oxidoreductase and human cytochrome b5 were purchased from BD Biosciences (Erembodegem, Belgium) and Tebu-Bio. Ultrapure water was prepared using a Purelab flex water system by Elga (Tienen, Belgium). Acetonitrile and formic acid were purchased from Merck. All organic solvents were HPLC grade.