2.4 UHPLC-MS/MS analytical conditions
The method to analyze TGC in bio-samples has been described in previous review[11]. Relative method was optimized in our study. The chromatographic system was consisted of an AB SCIEX QTRAP® 4500 triple-quadrupole mass-spectrometric system (AB Sciex, MA, USA) coupled with an ultrafast liquid chromatography (AB Sciex, MA, USA). Separation was performed on a Waters XBridge® BEH Shield RP18 column (3.0×50mm, 2.5μm) from Waters Corporation, USA. The temperatures of auto sampler and column oven were 15°C and 40°C respectively. The mobile phase was composed of aqueous mixed with 10 mM ammonium formate (pH=2.5) and acetonitrile according a gradient volume ratio (100:0→10:90→100:0), and pumped at 0.3 mL/min. Quantitative analysis of TGC was processed utilizing positive electron spray ionization in multiple reaction monitoring mode at m/z 586.3→513.2 and 586.3→456.2. TGC-d9 was monitoring in the channel of 595.1→514.2. Mass spectrometer parameters for identification of TGC and TGC-d9 were summarized in Table 1.