Detection of Metabolic Profiling by LC-MS
LC-MS analysis uses Ultra-High Performance Liquid chromatography
(Ultimate 3000, USA) combined with the Thermo-Orbitrap Elite mass
spectrometer. Metabolic profiling was performed in electrospray
ionization (ESI) positive and ESI negative ion mode using an ACQUITY
UPLC I-Class system (Waters Corporation, USA) coupled with an AB SCIEX
Triple TOF 5600 System (AB SCIEX, USA). The binary gradient elution
systems consisted of water containing 0.1% formic acid (v/v, A) and
acetonitrile containing 0.1% formic acid (v/v, B). Separation was
achieved using the following conditions: 5% B for 0-2min; 5-95% B for
2-13min; and afinal 95% B for 13-15min; post time was 5min. The
chromatographic conditions were as follows: injection volume was 3μl;
column temperature was 25 °C; flowrate was 0.4ml/ min; post time was
5min. Mass spectrometry uses positive ion mode combined with negative
ion mode with the following parameters: Positive mode: Heater Temp 300
°C, Sheath Gas Flow rate, 45arb, Aux Gas Flow Rate , 15 arb, Sweep Gas
Flow Rate, 1arb, spray voltage, 3.0KV, Capillary Temp, 350 °C, S-Lens RF
Level, 30%. Scan ranges: 200-1500. Negative mode: Heater Temp 300 °C,
Sheath Gas Flow rate, 45arb, Aux Gas Flow Rate, 15arb, Sweep Gas Flow
Rate, 1arb, spray voltage, 2.5KV, Capillary Temp, 350 °C, S-Lens RF
Level, 60%. Scan ranges: 200-1500.