建立2D-HPLC同时检测钩藤碱、异钩藤碱、去氢钩藤碱、异去氢钩藤碱的方法

Development of a 2D-HPLC for simultaneous determination of rhynchophylline,isorhynchophylline,corynoxeine and isocorynoxeine

目的建立二维高效液相色谱法(2D-HPLC)同时检测钩藤碱、异钩藤碱、去氢钩藤碱、异去氢钩藤碱四种钩藤生物碱含量的方法。方法采用Aston SC2(3.5 mm×25 mm,5μm)一维色谱柱,Aston SH C_(18)(3.5 mm×10 mm,5μm)中间色谱柱,Aston SCB(4.6 mm×125 mm,5μm)分析色谱柱,一维流动相为CAA-1,分析流动相为BPI-1∶MPI-1∶OPI=45∶14∶41,流速1.0 mL/min,柱温40℃,检测波长254 nm,进样量500μL,分析时间9.5 min。结果钩藤碱、异钩藤碱、去氢钩藤碱、异去氢钩藤碱线性范围分别为9.77~10000.00 ng/mL(r=0.9996)、10.74~11000.00 ng/mL(r=0.9997)、10.74~11000.00 ng/mL(r=0.9997)、10.74~11000.00 ng/mL(r=0.9996),精密度、稳定性和重复性相对标准偏差(RSD)<5.00%,准确度为95.20%~104.01%,方法回收率为93.63%~101.38%。结论建立的2D-HPLC同时检测钩藤中四种生物碱含量的方法简单准确,可作为钩藤药材质量控制的新方法。

Objective To develop a two-dimensional high-performance liquid chromatography(2D-HPLC)for simultaneous determination of the contents of four kinds of Uncaria alkaloids:rhynchophylline,isorhynchophylline,corynoxeine and isocorynoxeine.Methods The 2D-HPLC apparatus was comprised of a first chromatographic column in version Aston SC2(3.5 mm×25 mm,5μm),an intermediate column in version Aston SH C_(18)(3.5 mm×10 mm,5μm),and an analytical column in version Aston SCB(4.6 mm×125 mm,5μm).The mobile phase of the first and second liquid chromatography system were CAA-1 and mixed mobile phase(V_(BPI-1 basic mobile phase)∶V_(MPI-1 mobile phase)∶V_(OPI-1 organic mobile phase)=45∶14∶41).The chromatographic parameters included a flow rate of 1.0 mL/min,a column temperature of 40℃,a wavelength of 254 nm,an injection volume of 500μL and a detection time of 9.5 min.Results The linear ranges of rhynchophylline,isorhynchophylline,corynoxeine and isocorynoxeine were 9.77~10000.00 ng/mL(r=0.9996),10.74~11000.00 ng/mL(r=0.9997),10.74~11000.00 ng/mL(r=0.9997),10.74~11000.00 ng/mL(r=0.9996),respectively.The relative standard deviation(RSD)of precision,stability and repeatability were all less than 5.00%.The accuracy was 95.20%~104.01%,and the recovery rate was 93.63%~101.38%.Conclusion The 2D-HPLC developed for simultaneous determination of four kinds of alkaloids in Uncaria is simple and accurate,which can be used as a new method for quality control of Uncaria.