Anhydrous na2so4

All reagents used were of analytical grade. All solutions were prepared using ultra-high pure (UHP) 18 Ω water. All laboratory ware was soaked for 24 h in an acid bath containing 10% (v/v) nitric acid and then rinsed with UHP water. Concentrated hydrochloric acid (12 mol L⁻¹), HNO₃ 69%, NaCl, NH₄Cl, anhydrous Na₂SO₄, CaCl₂.2H₂O, NaHCO₃, anhydrous D ( + )-glucose, sodium arsenate dibasic heptahydrate, cadmium acetate and lead acetate trihydrate were obtained from Fisher Scientific. d-glucuronic acid, Pancreatin (pig), pepsin (pig), Bovine serum albumin (BSA), KSCN, NaH₂PO₄, mucin (pig), D-glucosamine hydrochloride, lipase (pig), α-amylase (Bacillus species), urea and bile salts (bovine), modified eagle medium (DMEM) containing 4.5 g/L glucose, penicillin-streptomycin solution (10, 000 units penicillin and 10 mg streptomycin per mL), and Hank’s Balanced Salt Solution (HBSS) and glycine were obtained from Sigma–Aldrich (St. Louis, MO, USA). KH₂PO₄, MgCl₂·6H₂O, KCl, and uric acid were obtained from VWR. Caco-2 cells (HTB-37™) were purchased at passage 18 from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum and trypsin-EDTA were purchased from Invitrogen (Burlington, ON, Canada).

All glassware was prepared as described previously (Johnson et al., 2011). The internal standard was 4‐phenylbutanoic acid (Acros Organics) (10 mg) dissolved in 0.1 M NaOH (final concentration 2 mg l⁻¹) and added to each culture immediately prior to extraction. Samples were acidified to pH 2 using concentrated HCl, and the diamondoid carboxylic acids were extracted from the supernatants three times using 15 ml of ethyl acetate (HPLC grade; Fisher Scientific, Loughborough, UK) as previously described (Johnson et al., 2011). Solvent extracts were pooled, dried with 5–10 g anhydrous Na₂SO₄ (Fisher Scientific), and concentrated by rotary evaporation (Buchi) at 40°C. Samples were transferred to a gas chromatography vial (Chromacol) and stored at −20°C. Samples were injected with a 1 µl splitless injection (injector temperature 250°C) onto a 30 m x 250 µm x 0.25 µm Rtx – 1MS column using helium as the carrier gas at a constant flow of 1 ml min^‐1. Oven temperatures were as follows: an increase from 40 to 250°C at 10°C min^‐1 followed by a final hold at 250°C for 10 min. The transfer line was held at 230°C onto a source for the MS which was in full‐scan mode (scan range 50–650 Da). Data were analysed and integrated using ChemStation for GC‐MS (Agilent, Stockport, UK).