4.4.2. Liquid Chromatographic Mass Spectrometry Analysis

The sample extracts were analyzed using a UPLC-ESI-MS/MS system, which mainly includes UPLC (Shim-pack UFLC SHIMADZU CBM30A, SHIMADZU, Kyoto, Japan) and MS/MS (Applied Biosystems 6500 QTRAP, AB SCIEX, Foster City, CA, USA). The UPLC separation was completed on a Waters Acquity UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 μm) (Waters Corp., Milford, MA, USA). The mobile phase A solvent was water containing 0.04% acetic acid, and the mobile phase B solvent was acetonitrile containing 0.04% acetic acid. The elution gradient was shown as follows: 0–11 min 95–5% A, 11–12 min 5–5% A, 12–12.1 min 5–95% A and 12–15 min 95–95% A. The flow was 0.4 mL/min and the injection volume was 2 μL. The column temperature was set to 40 ◦C.

The effluents were alternatively connected to an electrospray ionization-triple quadrupole-linear ion trap MS/MS (ESI-QTRAP-MS/MS). Linear ion trap (LIT) and triple quadrupole (QQQ) scans were carried out using QTRAP. The mass spectrometry conditions were as follows: the ESI temperature was set to 500 ◦C, the mass spectrometry voltage was 5500 V, the GS 1 and GS 2 were set to 55 psi and 60 psi, respectively, and the CUR was set to 25 psi. QQQ scans were obtained as MRM experiments with collision gas (nitrogen) set to 5 psi. The DP and CE for individual MRM transitions were optimized in the QQQ. The data were analyzed by the mass spectrometry software (Version 1.6.1 Applied Biosystems Company, Framingham, MA, USA).
