Hplc grade acetonitrile

Caffeine, theobromine, and theophylline were purchased from Sigma (St. Louis, MO, USA). A total of 30 tea samples of different brands (provided in Table 1) were purchased from local supermarkets (Riyadh, Saudi Arabia) as powder and stored in vacuum packages at a temperature below 0°C. HPLC grade acetonitrile and formic acid were purchased from BDH (Lutterworth, UK). Water used as the mobile phase solvent was prepared using a Millipore system (Milli-Q Advantage Elix, Millipore S.A.S. 67120 Molsheim, France), and then filtered on 0.2 μm nylon membrane filter from Whatman (Maidstone, UK).
Stock solutions of Caffeine, theobromine, and theophylline were 100 μg/mL, and they were prepared by dissolving the standard of each compound with an appropriate amount of water and subsequently stored at 4°C. The working standards were prepared by diluting the concentrated stock standard solutions in water before experiment. A calibration curve was constructed each day before analysis of the samples. HPLC grade water was injected as the blank.

Human cervical cancer cell (HeLa) was purchased from the American Type Culture Collection. Fetal bovine serum was obtained from Life Technologies Inc. (Waltham, MA, USA) and Minimal Essential Medium was obtained from LONZA (Walkersville, MD, USA). The Sephadex LH-20 was obtained from GE Healthcare (Uppsala, Sweden). Linoleic acid (18:2, >98% pure), oleic acid (18:1, 99% pure), 3-(4,5-dimethylthaizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), deuterium oxide (D₂O), doxorubicin, and lipopolysaccharide were purchased from Sigma (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol, as well as ACS-grade ethanol and hexane, were obtained from BDH (Mississauga, ON, Canada). ACS-grade dichloromethane and diethyl ether were obtained from Macron Fine Chemicals (Avantor, PA, USA). ACS-grade chloroform and LCMS-grade formic acid were obtained from Fisher Scientific (Ottawa, ON, Canada).

6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was from Fluka Chemie AG (Buchs, Switzerland). 2,2′-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), ferric chloride anhydrous, ferrous sulfate heptahydrate, potassium persulphate, sodium acetate, sodium carbonate and sodium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO). Acetic acid, hydrochloric acid, Folin-Ciocalteu reagent and HPLC-grade acetonitrile were from BDH (Dorset, UK). Ethanol and n-hexane for extraction was from Merck KGaA (Darmstadt, Germany). Deionized water was used for all the experiments.