ABSTRACT
Objective: An ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UPLC- QqQ-MS/MS) method has been established to simultaneously determine the contents of six bioactive constituents [japonicaside A (JA), L-phenylalanion secologanin B (PSB), luteolin, scopolamine, (1S,6R,7R,10R)-6-carboxy-10-methyl-α-methylene-1-(1- oxobutyl)-cyclohexane acrylic acid (CMCA), and 3α,5α-tetrahydrodeoxycordifoline lactam (TL)] from Reduning Injection. Methods: This chromatographic separation was performed on an Agilent Zorbax SB-Aq C18 (150 mm × 2.1 mm, 3.5 μm) column with the mobile phase consisting of 0.1% formic acid water (A) and methanol (B) in a gradient elution (0.01-2.00 min, 5% B; 2.00-4.00 min, 5%-40% B; 4.00-11.00 min, 40%-95% B; 11.00-13.00 min, 95% B; 13.00-13.10 min, 95%-5% B; 13.10-14.00 min, 5% B) at a flow rate of 0.5 mL/min and the colunm temperature was set at 20℃. The analytes were detected using electrospray ionization (ESI) source by positive and negative ion monitoring mode. A triple quadrupole mass spectrometer was operated by ESI source in positive and negative ionization mode with multiple reaction monitoring for the detection of the six compounds. In the positive ion monitoring mode, the flow rate of Collision Gas (CAD) was 8 mL/min, the flow rate of Curtain Gas (CUR) was 20 mL/min, the temperature (TEM) was 500℃ and the Ion Spray Voltage (IS) was 4 500 V. In the negative ion monitoring mode, the flow rate of Collision Gas (CAD) was 8 mL/min, the flow rate of Curtain Gas (CUR) was 20 mL/min, the temperature (TEM) was 500℃ and the Ion Spray Voltage (IS) was -4 500 V. Results: All calibration curves of the six components showed excellent linear regressions (r ≥ 0.999 0) within the test range. The average recoveries were 78.93%, 114.65%, 101.99%, 90.98%, 98.08%, and 115.58%, and the average contents of six bioactive constituents in 16 batches of Reduning Injection were 2.00, 26.63, 52.63, 5.29, 34.64, and 9.69 μg/mL, respectively. Conclusion: The established method is rapid, accurate, and has high repeatability, which could provide scientific evidences for the quality control of Reduning Injection.