HPLC-DAD conbined with TOF/MS technique in determination of saikosaponin a, baicalin and glycyrrhizic acid in Xiaochaihu Decoction / 第二军医大学学报

ABSTRACT

Objective: To establish a method for determination of saikosaponin a, baicalin and glycyrrhizic acid in Xiaochaihu Decoction. Methods: A combined method of HPLC-DAD and time of flight mass spectrometry (TOF/MS) was used; the chromatography condition was as following Agilent Zorbax XDB C18 column (25°C, 2.1 mm × 50 mm, 1.8 μm); mobile phase methanol (0.025%, V/V, formic acid)-water 0.025%, V/ V, formic acid) with gradient elution at a flow rate of 0.15 ml · min-1. The injection volume was 0.25 μl. Atomospheric pressure electronic spray ionization was used to quantify saikosaponin a at the capillary voltage of 4 000 V, the flow and temperature of drying gas were 10.0 L · min-1 and 350°C, respectively; nebulizer pressure was 40 psi (275 792 Pa), and the fragment voltage was 200 V; the m/z of saikosaponin a was 803.420 0-803.490 0 for quantitative analysis. The wavelengths for detection of baicalin and glycyrrhizic acid were 275 nm and 250 nm, respectively. Results: The calibration curves were Y=461 182X + 2 000 000(r=0.999 0),Y=0.949 9X-1.046 4(r=0.999 5) and Y=5.978 9X-27.418(r=0.999 5) for saikosaponin a (0.211 6-127.3 μg · ml-1), baicalin (0.758-455 μg · ml-1) and ammonium glycyrrhizinate (2.268-136.8 μg · ml-1), respectively. The recovery rates of the low, medium and high concentrations were (95.54 ± 1.60)%, (99.39 ± 3.97)% and (103.8 ± 1.97)% for saikosaponin a, (102.3 ± 0.47)%, (100.9 ± 1.32)% and (97.15 ± 2.10)% for baicalin, and (102.6 ± 1.96)%, (100.3 ± 3.12)% and (97.75 ± 1.25)% for ammonium glycyrrhizinate, respectively. The intra-day and inter-day RSD was both less than 5% and the RSD was less than 5% for 21 days. Conclusion: The method in the present study is simple, sensitive, rapid, and accurate for quality control of Xiaochaihu Decoction.