LC-MS Metabolite Profiling of Methanolic Extracts

An Agilent LC-MS system composed of an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6545 ESI-Q-TOF-MS in positive mode was used to obtain Ultra-high-performance liquid chromatograms. Aliquots (1 μL) of methanolic extracts (5 mg mL⁻¹ in MeOH) were analyzed on a Kinetex phenyl-hexyl (1.7 μm, 2.1 × 50 mm) column. Isocratic elution of 90% A (A: 100% H₂O + 0.1% formic acid) for 1 min followed by 6 min linear gradient elution to 100% B (95% MeCN + 5% H₂O + 0.1% formic acid) with a flow rate of 0.4 mL min⁻¹. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV and a sheath gas flow rate of 11 L min⁻¹. Ions detected in the full scan at an intensity above 1000 counts at 6 scans per s, with an isolation width of 1.3 ∼ m/z, a maximum of 9 selected precursors per cycle and using ramped collision energy (5× m/z/100 + 10 eV). Purine C₅H₄N₄ [M + H]⁺ ion (m/z 121.050873) and hexakis (1H,1H,3H-tetrafluoropropoxy)-phosphazene C₁₈H₁₈F₂₄N₃O₆P₃ [M + H]⁺ ion (m/z 922.009798) were used as internal lock masses.^{23 (link)} Metabolites were identified based on their retention times, molecular formulae and their fragmentation patterns, compared to earlier reported data aided with GNPS spectral library search and Sirius.