Ethyl acetate

GA (assay 97.0%) was supplied by the China Resources Sanjiu Medical & Pharmaceutical (Shanghai, China). Ursolic acid (assay 99.2%, the internal standard [IS]) was purchased from the National Institutes for Food and Drug Control ([NIFDC], Guangzhou, China). Gal was synthesized by a non-aqueous enzymatic reaction in our laboratory. Egg phosphatidylcholine ([EPC], assay 98.0%) was purchased from Lipoid Co., Ltd. (Ludwigshafen, Germany). Cholesterol ([CH], assay 98%) was purchased from Advanced Vehicle Technology Pharmaceuticala. (Shanghai, China). Roswell Park Memorial Institute (RPMI)-1640 medium and trypsin solution (0.25%) were supplied by Thermo (Boston, MA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). The hepatocellular carcinoma (HepG2) cell line was provided by the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). Methanol, ethanol, ammonium acetate, and ethyl acetate were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China) and were of at least analytical grade. HPLC or LC-MC/MS reagents were filtered through a 0.22μm filter before analysis. Methanol, ethanol, ethyl acetate, and the other reagents in liquid-liquid extraction were of analytical grade and used without further purification. Watsons’ water was used in all experiments.

The standards of ENR, CIP, SAR, SMM, SM2, SQ, TIM, TYL, KIT, and TAM were supplied by the China Institute of Veterinary Drugs Control (Beijing, China). HPLC-grade ACN, MeOH, and formic acid were provided by Fisher Scientific (Fair Lawn, NJ, USA). Analytically pure ACN, MeOH, ethyl acetate, acetic acid, hexane, and TCA were obtained from the Guangzhou Chemical Reagent Company (Guangzhou, China). Ultrapure water was obtained by a Millipore system Milli Q (Molsheim, France).
Stock standard solutions (1 mg mL⁻¹) were prepared by dissolving each compound in HPLC-grade methanol or 2% ammonia in methanol (v/v) and were then stored at −20 °C in the dark until use. The working standard solutions of compounds at appropriate concentrations were prepared by diluting the individual stock standard solutions in HPLC-grade methanol on the day of use.

HT standard (purity >98%, molecular weight 154) was purchased from Sigma (St. Louis, MO, USA). PL (from soybean, >98%) and CH were purchased from Aladdin Chemicals (Shanghai, China). Disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween-80, ethyl alcohol and ethyl acetate were analytical reagent (Guangzhou Chemical Reagent Company, Guangzhou, China).

Sibutramine hydrochloride monohydrate (National Institutes for Food and Drug Control, China; Lot. no. 100624–200401, desiccated for two hours at 105°C before use). Ethyl acetate (AR, Guangzhou Chemical Reagent Co.), phosphoric acid (analytical grade, Guangzhou Chemical Reagent Co.), ammonium reineckate (analytical grade, Shanghai Hengxin Chemical reagent Co., Ltd.), methanol (HPLC grade, Guangzhou Chemical Reagent Co.), triethylamine (analytical grade, Guangzhou Chemical Reagent Co.), acetonitrile (HPLC grade, Merck & Co., Inc., USA), and sodium heptanesulfonate (analytical grade, Shandong Yuwang Industrial Co., Ltd.) were used in this study.