Sequant zic philic column

The liquid chromatography separation was performed with an Accela ultra-high-performance LC (UHPLC) system. A polymeric SeQuant^® ZIC^®-pHILIC column (5 μm, 150 mm × 2.1 mm) (150,460, Merck) and a SeQuant^® ZIC^®-pHILIC Guard Kit (20 × 2.1 mm) (50,438, Merck) were used operating at 45 °C. The injection mode was set at 5 μL, and the mobile phase flow rate was set at 0.3 mL/min. Mobile phase solvents were A (95% H₂O, 5% methanol, 0.1% formic acid) and B (100% methanol). The eluting gradient program in both positive and negative ion mode was the following: 0–1.0 min (95% A, 5% B), 1.0–4.0 min (45% A, 55% B), 4.0–9.0 min (45% A, 55% B), 9.0–10.0 min (20% A, 80% B), 10.0–10.1 min (20% A, 80% B), 10.1–15.0 min (0% A, 100% B), 15.0–15.1min (0% A, 100% B), and 15.1–20.0 min (95% A, 5% B).

The samples were analysed using a Thermo Scientific Ultimate 3000 RSLCnano system (Thermo Scientific, CA, USA). The pHILIC separation was performed with a SeQuant ZIC-pHILIC column (150 × 4.6 mm, 5 µm) equipped with the corresponding pre-column (Merck KGaA, Darmstadt, Germany)—the column temperature was maintained at 25 °C. A linear biphasic LC gradient was conducted from 80% B to 20% B over 15 min, followed by a 2 min wash with 5% B, and 8 min re-equilibration with 80% B, where solvent B is acetonitrile and solvent A is 20 mM ammonium carbonate in water. The flow rate was 300 µl/min, column temperature was held at 25 °C, injection volume was 10 µl, and samples were maintained at 4 °C in the autosampler (Creek et al. 2011 (link)).

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.