Glutathione gsh

Hydrogenated soybean phospholipids (HSPC), cholesterol (Chol), and distearoyl phosphoethanolamine-PEG₂₀₀₀-Acrylamide (DSPE-PEG₂₀₀₀-ACA) were purchased from Shanghai Ponsure Biotech, Inc. Four-arm poly(ethylene glycol) amine (Tetra-PEG-amine) was provided by Xiamen Sinopeg company. Dibenzocyclooctyne-acid (DBCO acid, ≥95%) was supplied by Xi’an ruixi Biological Technology Co., Ltd. O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU, 99%) and glutathione (GSH, 99%) were purchased from J&K Scientific Ltd. N,N-diisopropylethylamine (≥99%, DIEA), N,N’-methylenebisAcrylamide (99%, MBAA), N,N,N’,N’-tetramethylethylenediamine (99%, TEMED), and anhydrous N,N-dimethylformamide (99.8%, DMF) were provided by Sigma. Ammonium persulfate (99.99%, APS) and 1,11-diazido-3,6,9-trioxaundecane (≥95%, N₃-PEG₃-N₃) were supplied by Aladdin. Acrylamide (99%, AM) was purchased from Adamas. Ethyl ether (≥99.7%, Et₂O) and chloroform (≥99%, CHCl₃) were provided by Sinopharm chemical reagent Co., Ltd. Ammonium sulfate (≥99%, (NH₄)₂SO₄) and PBS buffer (10×, pH 7.4) were supplied by Sangon Biotech company. The PBS buffer used in all experiments was diluted ten times. Deionized water was used to prepare all aqueous solutions. Other reagents and solvents were used as received.

Pooled human liver S9 fraction (H0610.S9) and NADPH regenerating system (K5100-5) were purchased from Sekisui Xenotech (Kansas City, KS, USA). The cofactors adenosine 3′-phosphate 5′-phosphosulfate lithium salt hydrate (PAPS), acetyl-coenzyme A (acetyl-CoA), uridine diphosphate glucuronic acid (UDPGA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The glutathione (GSH) was purchased from J&K (Shanghai, China). The NIST urine (SRM 3667) and NIST plasma (SRM 1950) samples were purchased from Ango Biotechnology Co. (Shanghai, China). LC–MS grade methanol (MeOH) and water (H₂O) were purchased from Honeywell (Muskegon, MI, USA). LC–MS grade acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). Ammonium hydroxide (NH₄OH) and ammonium acetate (NH₄OAc) were purchased from Sigma (St. Louis, MO, USA). Other chemical standards were purchased from Sigma-Aldrich (St. Louis, MO), J&K (Shanghai, China), and TopScience (Shanghai, China).

Vicagrel (free base, 98% pure), 2-oxo-clopidogrel (99% pure) were kindly supplied by the State Key Laboratory of Natural Medicines and the Center of Drug Discovery, China Pharmaceutical University, respectively. MP-AM (AM derivatized with 3-methoxyphenacyl bromide) and CAM of 2-oxo-clopidogrel was synthesized by the Center of Drug Discovery, China Pharmaceutical University. Prasugrel thiolactone metabolite (R-95913, 98% pure) was donated by Chia-Tai Tian Qing Pharmaceutical Co. Ltd (Jiangsu, China), and used as internal standard (ISTD) in the present experiment. The derivatizing agent 3-methoxyphenacyl bromide (MPBr) was purchased from TCI (Shanghai) Development Co., Ltd. Glutathione (GSH) was bought from J&K Scientific (Shanghai, China). Sodium carboxymethyl cellulose (CMC-Na) was purchased from Dai-Ichi Kogyo Seiyaku Co., Ltd (Shanghai, China). Nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system was bought from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA). All other chemicals and agents were of analytical grade.