LC-MS Analysis of Metabolites

LC-MS analysis was done by using HPLC-ESI-MS-NEG-PHENOMENEX in negative ionization mode. System was equipped with binary pump, auto sampler, thermostated column compartment and iFunnel quadrapole time-of-flight spectrometer (Q-TOF). Zorbax eclipse C18 column (4.6 × 250 mm, 5 μm particle size) was used for compounds separation at 25°C temperature. In the present study, 0.1% (v/v) formic acid (A) and acetonitrile (B) was used in gradient elution. Gradient was initiated at 80% A and 20% B to 30% B (after 10 min), followed by 40% B (40 min), 60% B (60 min) and 90% B (80 min) and finally returned to the initial conditions. Solvent system B was injected with flow rate of 0.8 ml/min. Mass spectrometer was operated in range of 100–1,000 m/z. N₂ gas was used as a nebulizer. Drying gas flow rate was 8 L/min at 325°C and nebulizer gas at 25 psi with fragmentor voltage 150 V (Patel and Ghane, 2021 (link)). For data analysis, mass hunter qualitative analysis software package (Agilent Technologies) was used. Detected compounds were validated on the basis of molecular formula, molecular mass, retention time and m/z ratio. For the authentication of compounds, details were compared with available literature and Metline personal metabolites database.