Instrumentation and Conditions for UHPLC-Q-TOF/MS Analysis

The separation was performed on an Agilent^®1290 Infinity II (Agilent Technologies Inc., USA) using a Waters ACQUITY HSS T3 column (100 × 2.1 mm, i.d. 1.8 µm) maintained at 30°C. AB SCIEX^® TripleTOF 5600+ Plus ultra-performance liquid chromatography-tandem mass spectrometer (UHPLC-Q-TOF/MS) was used to acquire the MS/MS spectra on information-dependent basis during the LC/MS experiment. In this mode, acquisition software (Analyst TF1.7 software) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria (20). The mobile phase containing 0.1% formic acid was composed of water (A) and acetonitrile (B) at a flow rate of 0.3 ml/min. A gradient program was used as follows (time, min/A%): 0.5/99, 1.5/99, 7/1, 15/1, 15.5/99, 20/99. The injection volume was 5 μl in partial loop mode. Electrospray ionization mass spectrometry (ESI-MS) was operated in negative/positive ion mode under the following operating parameters: curtain gas, 35; ion source gas 1, 55; ion source gas 2, 55; temperature, 550; declustering potential, ± 80; collision energy, ± 40; accumulation time, 0.16 s. The pooled QC represented the sample matrix and metabolite composition of the samples, which were used to construct the calibration curves and to judge precision, stability and recovery are within the acceptable range.