Hplc grade acetonitrile

Pure compounds of Fluoxetine, Quetiapine, Olanzapine, Levetiracetam, Aripiprazole and Alprazolam, and internal standard (IS) dansyl-norvaline were purchased from Sigma Aldrich (St Louis, MO). HPLC-grade acetonitrile was purchased from Carlo Erba reagents (Milan, Italy) and formic acid was from Merck (Darmstadt, Germany). Water was deionized and filtered by means of a Milli-Q Plus equipment (Millipore Corporation, Bedford, MA).

Analytical grade chloroform and methanol were from Dorwil (Buenos Aires, Argentina), HPLC grade acetonitrile was obtained from Carlo Erba (Val-de-Reuil, France), and formic acid, choline (> 98%), deuterochloroform (CDCl₃, 99.8%), and deuterium oxide (D₂O, 99.9%) were purchased from Merck (Kenilworth, NJ, USA). Betaine (> 98%) was from Thermo Fisher Scientific (Geel, Belgium). Diazepam (> 99.9%) and standard solutions of CBDA, CBD, cannabichromene (CBC), cannabinol (CBN), cannabigerol (CBG), THCA, THC, and Δ⁸-THC at 1 mg/mL were purchased from Lipomed (Cambridge, MA, USA). Standard solutions of Δ⁹-tetrahydrocannabivarinic acid (THCVA), Δ⁹-tetrahydrocannabivarin (THCV), cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV) and cannabichromenic acid (CBCA) at 1 mg/mL were purchased from Cerilliant (Round Rock, TX, USA).

All the detected compounds used in this paper, including the internal standard, were ordered from Sigma-Aldrich (Milano, Italy) with a purity > 99%. An individual stock solution of each compound was prepared by the dissolution of 0.5 mg of every single molecule in 0.5 mL of HPLC-grade methanol (Carlo Erba, Milano, Italy). Other solutions were obtained from the dilution of the stock solutions in methanol. HPLC-grade acetonitrile was purchased from Carlo Erba (Milano, Italy). HPLC-grade formic acid (99%) was obtained from Merck (Darmstadt, Germany). Deionized water (>18 MΩ cm resistivity) was obtained by purification of water with a Milli-Q SP system (Millipore, Bedford, MA, USA). Sterile test tubes were purchased from Becton–Dickinson (Franklin Lakes, NJ, USA). The analytical procedures were carried out in polypropylene vials, test tubes and plastic centrifuge tubes in order to preserve the concentration and stability of the hormones. The glass has demonstrated that it could interfere with those molecules as reported in the section “Evaluation of the stability of steroids in glass and plastic” (Results and Discussion, Table 3).

Each faecal sample was lyophilised for 72 hours, and AHLs were extracted from one gram of lyophilised faeces according to the following steps: First, solid-liquid extraction was conducted using a Soxhlet apparatus with 100 mL of high performance liquid chromatography (HPLC)-grade ethyl acetate (Carlo Erba). Two μL of the internal standard N-hexanoyl-L-homoserine lactone-d3 (C6d3-HSL, Cayman Chemical) at 20 mM was added, and the process was left to run for 4 hours. Second, the extraction product was evaporated using a rotatory evaporator and resuspended in 5 mL of HPLC-grade acetonitrile (Carlo Erba). Then, 45 mL of water was added (90% water/10% acetonitrile). Third, solid phase extraction was performed: The sample was loaded onto a reverse-phase C18 cartridge (Sep-Pak Waters 12cc 2g) after pre-conditioning with 15 mL of methanol and 30 mL of water. The cartridge was rinsed successively with 50 mL of 90% water/10% acetonitrile and with 10 mL of hexane. The AHLs were then eluted with 20 mL methanol. Fourth, the elution product was evaporated under a nitrogen stream at 50°C. The residue was dissolved in 200 μL of acetonitrile. All solvents were supplemented with 0.1% formic acid.

Ketoprofen, naproxen, diclofenac, ibuprofen, and mefenamic acid (> 98% purity in each cases) as the model analytes were purchased from Sigma-Aldrich (Steinheim, Germany). A stock solution of 100 mg L⁻¹ of each drug was prepared in the HPLC grade ethanol and then stored in the refrigerator at 4 °C. Working standard solutions were prepared by the stepwise dilution of the stock with double distilled water (DDW) before utilizing. HPLC grade acetonitrile (> 99.9%) and methanol (> 99.9%) were supplied from Carlo Erba (Val-de Reuil, France). DDW was obtained from Shahid Ghazi Pharmaceutical Co. (Tabriz, Iran). All other analytical grade chemicals and reagents were obtained from Merck (Darmstadt, Germany). Epoxy glue was purchased from a Betonartoom building materials market in Karaj (Iran). Stainless steel wire (0.3 mm in diameter) was bought from BS Stainless Company (United Kingdom).

(-)-Epicatechin (EC), 2´-7´dichlorofluorescein diacetate (DCFH-DA), ampicillin sodium salt, nistatine, agar, yeast extract, fluorodeoxyuridine (FUdR), phosphate-buffered saline (PBS), cholesterol, Bradford reagent, guanidine hydrochloride (GuHCl), 2,4-dinitrophenylhydrazine (DNPH), malondialdehyde, hexanal, hexenal and 4-HNE were purchased from Sigma-Aldrich (Madrid, Spain). Dimethyl sulfoxide (DMSO) was obtained from Panreac (Barcelona, Spain) and trichloroAcetic acid from Fluka Analytical (Madrid, Spain). HPLC grade acetonitrile was from Carlo Erba (Rodano, Italy). Acetic acid was from Merck (Darmstadt, Germany). Fluorescein thiosemicarbazide was from Carbosynth (Berkshire. UK)