Lcquan software

Chemical composition was determined using Ultra High Performance Liquid Chromatography (UHPLC) modified as previously described [31 (link),43 ]. UHPLC analysis was performed using a Thermo Scientific UHPLC UltiMate 3000 (Waltham, MA, USA) with a Hypersil GOLD™ a Q column (100 × 2.1 mm i.d., 1.9 µm, Thermo Scientific™) and results were analyzed with Thermo Scientific™ LCQUAN™ software. Crude extracts of Al and Gg and standard solutions (oxyresveratrol, resveratrol, gallic acid and glabridin) were dissolved in methanol at concentrations of 1 and 10 mg/mL. Samples were filtered (0.20 mm, Millipore) and 1 μL was directly injected. Solvents for HPLC analysis were formic acid (0.1% v/v) in water as solvent A and formic acid (0.1% v/v) in methanol as solvent B, at a flow rate of 0.5 mL/min. These experiments used the following gradient: 30% B linear (0–4 min), 30–50% B linear (4–5 min), 50–70% B linear (5–8 min), 70–100% B linear (8–12 min), 100% B (12–15 min), and 30% B linear (15–18 min). An equilibrium period of 5 min was performed prior to the injection of subsequent samples. Chromatograms were recorded at 280 nm (glabridin and gallic acid), 305 nm (resveratrol), and 326 nm (oxyresveratrol), using the photodiode detector. Quantitative determination of compounds was performed using peak area with an external standard.

SK-ChA-1 cells were seeded in 6-wells plates and cultured until confluence. Cells were treated using the PDT protocol as described in “PDT protocol” (n = 3 per group). After 90 min, the cells were washed with 1 mL cold PBS and the cells were lysed in 1 mL lysis buffer (40% acetonitrile, 40% methanol, 20% water). The cells were scraped and transferred to 2-mL centrifuge tubes that were shaken for 10 min at 4 °C. Next, the samples were centrifuged for 15 min at 20,000×g (4 °C), after which the supernatant was aspirated and stored at −80 °C. LC-MS analysis was performed on an Exactive mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 autosampler and pump (Thermo Scientific). The MS operated in polarity-switching mode with spray voltages of 4.5  and −3.5  kV. Metabolites were separated using a Sequant ZIC-pHILIC column [2.1 × 150 mm, 5 µm, guard column 2.1 × 20 mm, 5 µm (Merck)] using a linear gradient of acetonitrile and eluent A (20 mM (NH₄)₂CO₃, 0.1% NH₄OH in ULC/MS grade water [Biosolve, Valkenswaard, the Netherlands)]. The flow rate was set to 150 µL/min. Metabolites were identified and quantified using LCquan software (Thermo Scientific) on the basis of exact mass within 5 ppm and further validated in accordance with the retention times of standards. Peak intensities were normalized based on total ion count.

Bile acids were determined using high-performance liquid chromatography (HPLC) with mass spectrometry (MS). Calibration standards were prepared for individual deuterated bile acids at concentrations of 0.005–40 μM. Samples were prepared as previously described.[50 (link)] HPLC–MS was performed using a Dionex UltiMate 3000 HPLC system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Breda, the Netherlands) and an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters, Etten-Leur, the Netherlands) at 40°C. The ratio of mobile phase A (1 mM ammonium formate in water [pH 4.4]) to mobile phase B (acetonitrile:water [95:5 v/v] containing 1 mM ammonium formate) was varied over 14 minutes, with a flow rate of 600 μL/minute. The injection volume was 3 μL and the autosampler temperature was 20°C. The mass spectrometer, equipped with a heated electrospray ionization source, was operated in negative mode and full-scan spectra were recorded. The spray voltage was 3 kV, and capillary and probe heater temperatures were 350°C and 320°C, respectively. Nitrogen was used as the sheath and auxiliary gas, set at 60 and 20 (arbitrary units), respectively. The resolution was 100,000 at m/z 200. The data were analyzed using LCQUAN software (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA).