Lc 20ad uflc system

LC-MS was used to identify the major markers of bioactive substances. The system for analysis consisted of a LC-20AD UFLC system (Shimadzu Corporation) linked to a LCMS-2020 mass spectrometer. The running conditions were designed as follows: Gradient elution by the mixture of mobile phases A (0.1% formic acid in water) and B (Acetonitrile) at minutes 0–65, with the ratio of 90–0% in A and 10–100% in B; at minutes 65–80 with the ratio of 0–90% in A and 100–10% in B. The flow rate was fixed at 1 ml/min and the column temperature was maintained at 35°C. The injection volume was adjusted to 20 µl and the analytical column used was a Cosmosil 5C18-MS-II (5 µm, 4.6×250 mm; Nacalai Tesque, Inc.). Dual ion modes [electrospray ionization, (ESI)(+) and (−)] were used in MS detection and the transmission of (M+H)+ and (M-H)-was set as the optimum condition. The MS detection was set to full scan range (50-1,200 m/z). The interface voltages were set at 4.5 kV for ESI(+) and −4.5 kV for ESI(−). With nitrogen as the nebulizing and drying gas, the flow rate was set at 1.5 and 15 l/min, respectively. Nitrogen gas temperature was set at 350°C. Desolvation line temperature was set at 300°C and heat block temperature was maintained at 500°C. Chromatographic fingerprint analysis was performed using 3D HPLC, which was primarily performed as described previously (45 (link),46 (link)).