Formic acid

BMAA hydrochloride, L-2,4-diaminobutyric acid (DAB) dihydrochloride, trichloroacetic acid (TCA), 5-dimethylamino-1-naphthalenesulfonyl chloride/dansyl chloride (DNS), formic acid, acetonitrile, methanol, hydrochloric acid (HCl) [37%], chloroform and Corning^® Costar^® Spin-X^© centrifuge filters were purchased from Sigma-Aldrich Company Ltd (Gillingham, United Kingdom). All chemicals used were of analytical grade or higher. The water was purified using a Milli-Q purification system from Merck Millipore (Merck KGaA, Darmstadt, Germany). Deuterium labelled BMAA (D₃-BMAA) was a gift from Johan Rosén at The National Food Agency in Sweden, and was synthesized as described in the publication by Rosén and Hellenäs12 (link). Stock solutions of BMAA (1 mg/ml) were prepared in Milli-Q water and stored at −20˚C.

Human liver microsomes (mixed gender, pool of 50 donors) were procured from XenoTechLLC (Kansas, USA; protein content: 20 mg/mL; catalogue number: H0610). Standard substrates and inhibitors were procured from Sigma-Aldrich, India, US Biologicals and Acros Organics. All the stable labeled internal standard(s) (IS) were from Toronto Research Chemicals, Canada. NADPH, formic acid, ammonium formate, sodium dihydrogen phosphate and disodium hydrogen phosphate, 12-well Corning Transwell filters, HBSS, HEPES, glucose, dimethyl sulfoxide (DMSO) and sodium bicarbonate were purchased from Sigma Aldrich, India. Acetonitrile (ACN) was from Merck, India. Milli-Q water (Millipore Corporation, India) was used for preparation of buffer. 96-well plates of 1 mL capacity were purchased from Axygen Scientific, USA. All gingerols and GE were isolated as previously described [27] . Caco-2, human colon carcinoma epithelial cell line was obtained from ATCC (HTB-37, Manassas, USA) and cells were used at passage number 40. Milli-cell apparatus from Millipore was used to measure the TEER.

6-shogaol (98+% purity) was purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). Reduced L-Glutathione, NaHCO₃, ethanol (200 proof), methanol, and dichloromethane were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (98+%, LC-MS grade), phosphate-buffered saline (Corning TM PBS (Corning, NY, USA), 1×), and DiL Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (DiIC18(3)) were obtained from Fisher Scientific (Hampton, NH, USA). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (R&D Flowery Branch, GA, USA). Ultrapure deionized water was supplied by a Milli-Q water system (Millipore, Bedford, MA, USA). M13 was synthesized and purified using a method that has been previously published [19 (link)].