Ultra performance liquid chromatography 1 class

The cecal contents (50 mg) were vortexed in methanol/water solution (1 : 1 v/v, 1 ml) and centrifuged at 12,000 × g and 4°C for 5 min to collect the supernatant. Then the supernatant (50 μl) was derivatized in 50 μl derivatizing reagent and analyzed with UPLC-MS/MS. UPLC-MS/MS analysis was performed using an Ultra Performance Liquid Chromatography I-Class (Waters Co., Ltd., USA) coupled with a Tandem Mass Spectrometry (Waters Co., Ltd., USA). A 13-min linear gradient flow rate of 0.3 ml/min was used to inject samples into a UPLC RP Column (1.6 μm, 2.1 mm × 100 mm). Eluents A (0.1% formic acid in water) and B (methanol : isopropyl alcohol is 4 : 1) were used for the system. As follows, the solvent gradient: 5% B, 0 min; 5–15% B, 2.0 min; 15–55% B, 9.0 min; 55–100% B, 11.0 min; 100–5% B, 11.1 min; 5% B, 13 min. The Ultra Performance Liquid Chromatography operated with 150°C source temperature, 2.0 kV capillary voltage, 30.0 V cone voltage, 450°C desolvation temperature, 150 l/h cone gas flow, 1000 l/h desolvation gas flow, 0.15 ml/min collision gas flow rate. The collision gas used high-purity argon, and the rest of the gas paths used high-purity nitrogen. The data were analyzed by MassLynx software (Waters Co., Ltd., USA).