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Total lipids were extracted according to t … Total lipids were extracted according to the method of Bligh and Dyer (1959). Crude lipid extracts were dissolved in chloroform and subjected to LC-MS analysis using a Shimadzu LCMS-IT-TOF mass spectrometer combined with a Shimadzu LC-20AD HPLC system. A two-solvent system was used to generate the mobile phase: solvent A, methanol-water (95:5, v/v) containing 0.2% ammonium formate, pH 5.9; solvent B, acetonitrile-methanol-water (95:2:3, v/v/v) containing 0.2% ammonium formate, pH 5.9. The pH of both solvents A and B was adjusted by adding 30% NH4OH to the mixtures of solvents containing 0.2% (v/v) formic acid. At the beginning of the gradient, the mobile phase was 100% solvent B for 3.33 min. Solvent B was linearly decreased to 60% over 6.67 min and successively decreased to 30% over 1.33 min. Solvent B was held at 30% for 3.33 min and then increased to 100% for reequilibration. The flow rate was held 0.18 mL min−1 for 3.33 min at the beginning of the gradient and linearly increased to 0.2 mL min−1 over 11.33 min. The flow rate was then increased to 0.4 mL min−1 at 14.66 min after the beginning of the gradient, maintained for 13.33 min, and then decreased to 0.18 mL min−1. Total elution time was 40 min.
High-resolution ESI-MS were acquired in both positive and negative ion modes by switching the polarity during individual analyses. Conditions for measurement of ESI-MS were as follows: mass range, m/z 150 to 1600; interface voltage, 4.5 V; curved desolvation line temperature, 200°C; heat block temperature, 200°C; ion accumulation time, 10 ms; detector voltage, 1.80 kV; nebulizer gas, N2 (15 L·min−1). The collision-induced dissociation experiment was performed using Ar as the collision gas, with a relative collision energy of 50% and a relative collision gas flow of 50%. and a relative collision gas flow of 50%.
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