LC-HRMS Analysis of Phytochemicals

All prepared sample extracts were analysed by LC-HRMS as described earlier [13 (link)]. In brief, a UHPLC system (Accela, Thermo Fisher Scientific, San Jose, CA, USA) equipped with a reversed-phase XBridge C₁₈, 150 × 2.1 mm i.d., 3.5 µm particle size (Waters, Milford, MA, USA) analytical column as well as water containing 0. % FA (v/v) (eluent A) and MeOH containing 0.1% FA (v/v) (eluent B) were used for linear gradient elution starting with 90% A and continuous increase of B up to 100% in 30 min after an initial hold time of 2 min. The UHPLC system was coupled to an LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with an ESI source. All measurements were performed in the positive ionisation mode with a scan range of m/z 100–1000 and a resolving power setting of 60,000 full width at half maximum (FWHM) at m/z 400.
For quality control (QC), a standard solution consisting of 15 authentic reference standards (N-methylanthranilate, ferulic acid, 2.5-dihydroxybenoic acid, syringic acid, methyl-indole-3-carboxylate, indole-3-acetonitrile, kaempferol, 4-triacetate lactone, l-tryptophan, alpha linolenic acid, galangin, 3′,4′,5′-O-trimethyltricetin, orientin, schaftoside and reserpine) each in a concentration of 1 mg/L, dissolved in MeOH + H₂O 1 + 1 (v/v) + 0.1% formic acid was prepared and measured every eighth injection throughout each LC-HRMS sequence. Retention time stability, peak area precision as well as mass accuracy were determined for all QC samples to verify proper measurement performance throughout the whole sequence with the help of QCScreen [35 (link)] (data not shown).