基于HPLC-QAMS法的清肝降压胶囊中10个成分同时定量测定研究

Simultaneous determination of 10 components in Qinggan Jiangya capsules by HPLC-QAMS

目的:建立同时测定清肝降压胶囊中10个成分的高效液相色谱一测多评(HPLC-QAMS)方法,并验证其准确性和可行性。方法:采用Phenomsil C18色谱柱(4.6 mm×250 mm,5μm),以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱;流速:1.0 mL·min^(-1);柱温:30℃;检测波长:320 nm(检测2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷)、250 nm(检测3'-羟基葛根素、葛根素和3'-甲氧基葛根素)、208 nm(检测泽泻醇F、泽泻醇A、24-乙酰泽泻醇A和23-乙酰泽泻醇B)和270 nm(检测隐丹参酮和丹参酮ⅡA)。以葛根素为内参物,建立其与其他9种成分的相对校正因子(RCF),并计算含量,实现一测多评;同时采用外标法(ESM)测定清肝降压胶囊中10个成分的含量,比较一测多评法计算值与外标法实测值的差异。结果:10个成分的线性范围分别为8.136~162.7、7.488~149.8、30.54~610.9、14.11~282.2、0.3680~7.360、0.7040~14.08、0.4320~8.640、3.056~61.12、0.5120~10.24、0.8480~16.96μg·mL^(-1),r为0.9992~0.9999;平均回收率分别为98.5%、98.0%、100.0%、100.0%、97.8%、98.5%、97.0%、99.1%、99.3%和100.1%,RSD为1.3%、1.1%、0.96%、0.64%、1.6%、1.5%、1.2%、0.83%、1.1%和0.72%;葛根素与2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、3'-羟基葛根素、3'-甲氧基葛根素、泽泻醇F、泽泻醇A、24-乙酰泽泻醇A、23-乙酰泽泻醇B、隐丹参酮、丹参酮ⅡA的相对校正因子分别为1.1551、1.1262、1.2760、0.9643、0.8959、0.8593、1.3662、0.8399和0.9663;一测多评法计算值和外标法实测值之间无明显差异。结论:所建立的方法简便、准确,可用于清肝降压胶囊的质量评价研究。

Objective:To establish an HPLC-QAMS method for simultaneous determination of 10 components in Qinggan Jiangya capsules,and validate the accuracy and feasibility of the method.Methods:A Phenomsil C18(4.6 mm×250 mm,5μm)column was used in the HPLC assay.Acetonitrile-0.1%phosphoric acid aqueous solution with gradient elution was employed as the mobile phase.The flow rate was 1.0 mL·min^(-1),the column temperature was maintained at 30℃ and the detection wavelength were set at 320 nm for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside,250 nm for 3'-hydroxypuerarin,puerarin and 3'-methoxypuerarin,208 nm for alisol F,alisol A,alisol A-24-acetate and alisol B 23-acetate,and 270 nm for cryptotanshinone and tanshinoneⅡA.Puerarin was used as the internal reference substance to calculate the relative correction factors(RCFs)of others nine components,and evaluate their contents accordingly.At the same time,the external standard method(ESM)was used to quantify the ten components in Qinggan Jiangya capsules.The results of the ESM and QAMS method were compared.Results:The linear range of 10 components were 8.136^(-1)62.7,7.488^(-1)49.8,30.54-610.9,14.11-282.2,0.3680-7.360,0.7040^(-1)4.08,0.4320-8.640,3.056-61.12,0.5120^(-1)0.24 and 0.8480^(-1)6.96μg·mL^(-1)(r=0.9992-0.9999),respectively.The average recoveries were 98.5%,98.0%,99.8%,100.0%,97.8%,98.5%,97.0%,99.1%,99.3% and 100.1% with RSDs of 1.3%,1.1%,0.96%,0.64%,1.6%,1.5%,1.2%,0.83%,1.1% and 0.72%,respectively.RCFs of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside,3'-hydroxypuerarin,3'-methoxypuerarin,alisol F,alisol A,alisol A-24-acetate,alisol B 23-acetate,cryptotanshinone and tanshinone ⅡA to puerarin were 1.1551,1.1262,1.2760,0.9643,0.8959,0.8593,1.3662,0.8399 and 0.9663,respectively.Significant difference was not found between the calculated values of QAMS method and the measured values of ESM.Conclusion:The method is simple and accurate,and can be used for quality evaluation of Qinggan Jiangya capsules.