Hypersil column

HPLC separation of metabolites was carried out using a Thermo Hypersil column (C18, 150 cm × 0.2 cm, 1.9 uL) on a Vanquish UHPLC system (Thermo Scientific, Waltham, MA, USA) equipped with a chilled autosampler (15 °C), heated column compartment (30 °C) and PDA (spectra collected from 190 nm to 680 nm). The mobile phase was comprised of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The injection volume was 3 μL. The flow rate was 0.3 mL min⁻¹ with a gradient elution of 2–100% acetonitrile over 18 min with a re-equilibration time of 2 min. Analytes were detected by mass spectrometry using Q Exactive Plus (Thermo Scientific, Bremen, Germany), with a heated electrospray ionisation (ESI) source. The capillary temperature was 300 °C and the sheath, auxiliary and spare gases set at 28, 15 and 4 units, respectively. Source voltage was 3.6 kV for positive mode and 3.3 kV for negative mode. The mass spectrometer was operated in positive negative switching mode with full scan (90–1350 m/z) at 35,000 resolutions in both modes.

The samples were analyzed on a Thermo UPLC system (Accela 1250, Thermo). UPLC separation was accomplished on an octadecylsilane (ODS) Hypersil column (2.1 mm × 250 mm, 1.9 μm, Thermo). The mobile phase, consisting of ACN and 0.1% formic acid in water, was used at a flow rate of 0.44 mL/min. The gradient elution program was: 5%–15% ACN (B) (0–1 min), 15% B (1–4 min), 15%–25% B (4–5 min), 25%–40% B (5–6 min), 40%–55% B (6–8 min), 55%–100% B (8–9 min), 100% B (9–11 min), 100%–5% B (11–12 min), and 5% B (12–14 min). The injection volume was 5 μL, and the detection wavelength was 254 nm. The column temperature was maintained at 45 °C using a temperature controller.