双标线性校正法用于延胡索中5个生物碱的定性分析

Simultaneous qualitative analysis of five alkaloids in Corydalis Rhizoma by the method of liner calibration with two reference substances

目的:建立延胡索中巴马汀、小檗碱、脱氢延胡索碱、延胡索乙素和延胡索甲素5个生物碱的高效液相色谱(HPLC)方法,考察双标线性校正(LCTRS)法对其色谱峰定性分析的可行性。方法:采用HPLC法,以乙腈为流动相A,0.1%磷酸水溶液(用三乙胺调pH 6.0)为流动相B,梯度洗脱(0~23 min, 20%A;23~35 min, 20%A→30%A;35~50 min, 30%A→80%A),流速为1.0 mL·min^(-1),柱温为35℃,检测波长为280 nm,进样量为5μL;测定延胡索中5个生物碱在20根不同品牌和型号C_(18)色谱柱的实际保留时间,以巴马汀和延胡索甲素2个成分作为双标化合物,使用双标线性校正法定位各成分色谱峰,并用3根未知色谱柱进行方法验证。同时以中间色谱峰脱氢延胡索碱为参照物,采用相对保留时间法对其他4个成分色谱峰的保留时间进行预测。比较这2种方法的预测准确性和色谱柱的符合率。结果:在10批次样品中,5个生物碱成分均有较好的分离。其中采用双标线性校正法的色谱峰预测准确率和色谱柱符合率均为100%,均显著高于相对保留时间法,能够更好地预测延胡索中待测成分在未知色谱柱上的保留时间。结论:建立的双标线性校正法预测色谱峰保留时间准确度高,可用于延胡索中生物碱类成分定性分析,值得进一步推广及应用。

Objective:To establish a high performance liquid chromatographic method for the qualitative analysis of five alkaloids, namely, palmatine, berberine, dehydrocorydaline, tetrahydropalmatine and corydaline in Corydalis Rhizoma, and to investigate the feasibility of liner calibration with two reference substances(LCTRS) method for the qualitative analysis of the peaks. Methods: HPLC method was used. The mobile phase consisted of solventA(acetonitrile) and solvent B(water containing 0.1% phosphoric acid, adjusted to pH 6.0 with triethylamine). The gradient elution(0-23 min, 20%A;23-35 min, 20%A→30%A;35-50 min, 30%A→80%A) was performed by HPLC at a flow rate of 1.0 mL·min^(-1). The column temperature was 35 ℃ and the detection wavelength was set at 280 nm. The injection volume was 5 μL. The actual retention time of five alkaloids in Corydalis Rhizoma was determined on 20 C_(18)columns of different brands or types. Two components, palmatine and corydaline, was selected as the two reference compounds. LCTRS method was used for accurately locating chromatographic peaks, and the method was verified on another three unknown columns. The retention time of the other four components were also predicted by the relative retention time method using the intermediate chromatographic peak of dehydrocorydaline as the single reference. The prediction accuracy and column compliance of these two methods were compared. Results: The five alkaloids were well separated in the 10 batches of samples. Among them, the peak prediction accuracy and column compliance of the LCTRS method were both 100%,which were significantly higher than those of the relative retention time method, and could better predict the retention time of the components in Corydalis Rhizoma on the unknown column. Conclusion: The established LCTRS method for the high-accuracy prediction of peak retention time can be used for the qualitative analysis of five alkaloids in Corydalis Rhizoma and is worthy of further promotion and application.