2.8 Chromatography-mass spectrometry
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Samples were separated on Agilent 1290 Infinity LC Ultra Performance Liquid Chromatography (UHPLC) HILIC and C18 columns; HILIC column temperature 35°C; flow rate 0.3 mL/min; injection volume 2 μL; mobile phase composition A: 90% water + 2 mM ammonium formate + 10% acetonitrile, B; methanol + 0.4% formic acid; gradient elution program was as follows: 0-1.0 min, 85% B; 1.0-3.0 min, B changed linearly from 85% to 80%; 3.0-4.0 min, 80% B; 4.0-6.0 min, B changed linearly from 80% to 70%; 6.0-10.0 min, B changed linearly from 70% to 50%; 10-12.5 min, B maintained at 50%; 12.5-12.6 min, B changed linearly from 50% to 85%; 12.6-18 min, B maintained at 85%. C18 column temperature 40°C; flow rate 0.4mL/min; injection volume 2μL; mobile phase composition A: water + 5 mM ammonium acetate + 0.2% ammonia, B: 99.5%; acetonitrile + 0.5% ammonia; gradient elution program was as follows: 0-5 min, B changed linearly from 5% to 60%; 5-11 min, B changed linearly from 60% to 100%; 11-13 min, B maintained at 100%; 13-13.1 min, B changed linearly from 100% to 5%; 13.1-16 min, B maintained at 5%; samples were placed in an autosampler at 4°C throughout the analysis. In order to avoid the influence caused by the fluctuation of signal detected by the instrument, continuous analysis of samples was performed in random order. QC samples were inserted into the sample cohort to monitor and evaluate the stability of the system and the reliability of the experimental data. Mass spectrometry was performed using an AB 6500 QTRAP mass spectrometer (AB SCIEX). The ESI source conditions were as follows: sheath gas temperature, 350°C; dry gas temperature, 350°C; sheath gas flow, 11 L/min; dry gas flow, 10L/min; capillary voltage, 4000 V or -3500 V in positive or negative voltage, modes; nozzle, 500 V; and nebulizer pressure, 30 psi, monitored using MRM mode.