葛根芩连片特征图谱及含量测定方法研究

目的：建立葛根芩连片的特征图谱，并对葛根素和盐酸小檗碱进行定量研究。方法：采用HPLC法建立特征图谱和含量测定方法，并以液质联用对特征峰进行指认。采用TC-C₁₈色谱柱(4.6 mm×250 mm，5 μm)，以甲醇为流动相A，以0.15%三氟乙酸溶液为流动相B，梯度洗脱(0~25 min，23%A→30%A;25~26 min，30%A→35%A;26~39 min，35%A→42%A;39~40 min，42%A→45%A;40~55 min，45%A)，流速1.0 mL·min^-1，检测波长250 nm(用于特征图谱建立和葛根素含量测定)、348 nm(用于盐酸小檗碱怾含量测定)；采用电喷雾离子源进行正离子模式扫描，对葛根芩连片特征图谱中8个特征峰进行指认。结果：建立了以葛根素和小檗碱为参照峰的葛根芩连片HPLC特征图谱，并指认了葛根素、盐酸小檗碱、3-羟基葛根素、3-甲氧基葛根素、葛根素木糖苷、大豆苷、盐酸巴马汀和黄芩苷8个特征峰；葛根素含量在8.8~12.9 mg·片^-1之间，盐酸小檗碱含量在2.5~7.0 mg·片^-1之间。 结论：本法为葛根芩连片的质量控制提供了有效的技术方法；提出的以双标峰进行特征峰相对保留时间定位，为中药特征图谱评价系统提供新的思路。

Objective: To establish the characteristic fingerprint of Gegen Qinlian tablets by HPLC method,and simultaneously perform the quantitative analysis of puerarin and berberine hydrochloride. Methods: T characteristic fingerprint and quantitative analysis was performed by HPLC.The characteristic peaks were identified by LC-MS.The samples were separated on the TC-C₁₈ column(4.6 mm×250 mm,5 μm),and eluted with a gradient elution(0-25 min,23%A→30%A;25-26 min,30%A→35%A;26-39 min,35%A→42%A;39-40 min,42%A→45%A;40-55 min,45%A)of methanol as the mobile phase A and the solution containing 0.15% trifluoroacetic acid as the mobile phase B at a flow rate of 1.0 mL·min^-1.The detection wavelengths were 250 nm for characteristic fingerprint establishment and puerarin assay and 348 nm for berberine hydrochloride assay.Eight characteristic peaks in the fingerprints were identified with applying electrospray ionization(ESI)source in the positive ion mode. Results: The characteristic fingerprint of Gegen Qinlian tablets was established with puerarin and berberine as the reference peaks.Eight characteristic peaks in the fingerprints were identified as puerarin,berberine hydrochloride,3'-hydroxypuerarin,3'-methoxypuerarin,puerarinxyloside,daidzin,palmatine hydrochloride,and baicalin.The content of puerarin and berberine hydrochloride in different batches ranged from 8.8 to 12.9 mg per tablet and from 2.5 to 7.0 mg per tablet.Conclusion: The established HPLC can be served as a scientific and effective way for quality control of Gegen Qinlian tablets and eight characteristic peaks were identified by LC-MS.Relative retention time calculated by two reference substances provides an effective method for overcoming peak drift,which is a new evaluation model of characteristic fingerprint analysis for traditional Chinese medicine.