一测多评法测定银杏叶片中小分子有机酸含量

目的: 建立银杏叶片剂中5 种小分子有机酸［莽草酸、没食子酸、原儿茶酸、6-羟基犬尿喹啉酸(6-HKA)、对羟基苯甲酸］的一测多评含量测定方法。方法: 采用ZORBAX Eclipse XDB-C₈ 色谱柱(250 mm×4.6 mm,5 μm),以0.1% 磷酸水溶液(A)-乙腈(B)为流动相,梯度洗脱(0~25 min,3.5%B;25~26 min,3.5%B→70%B;26~35 min,70%B;35~36 min,70%B→3.5%B;36~60 min,3.5%B),流速1 mL·min^-1,波长切换测定(0~6 min:215 nm,检测莽草酸、没食子酸;6~60 min:254 nm,检测原儿茶酸、6-HKA、对羟基苯甲酸),柱温30 ℃。以原儿茶酸为内参物,分别建立莽草酸、没食子酸、6-HKA、对羟基苯甲酸的相对校正因子,并对相对校正因子进行重现性考察;采用外标法测定原儿茶酸的含量,利用相对校正因子计算其他4 种小分子有机酸含量,同时采用外标法测定15 批银杏叶片剂中5 种小分子有机酸含量,比较2 种方法的测定结果。结果: 莽草酸、原儿茶酸、没食子酸、6-HKA、对羟基苯甲酸的线性范围分别为43.9~1 097.5、13.2~330.0、141.6~3 540.0、11.3~282.5、12.9~322.5 ng;在以上线性范围内,原儿茶酸与莽草酸、没食子酸、6-HKA、对羟基苯甲酸的相对校正因子分别为1.147 4、0.416 8、0.504 3、0.552 2;且在不同实验条件下重现性良好。15批银杏叶片剂中5 种小分子有机酸含量的计算值(莽草酸0.11%~1.01%,没食子酸0.13%~0.26%,6-HKA0.15%~0.32%,对羟基苯甲酸0.07%~0.18%)与实测值无显著差异。结论: 经方法学验证,本文方法可用于银杏叶片剂中小分子有机酸莽草酸、没食子酸、原儿茶酸、6-羟基犬尿喹啉酸(6-HKA)、对羟基苯甲酸的含量测定。

Objective: To establish a method for determination of five kinds of small molecular organic acid ［shikimic acid,gallic acid,protocatechuic acid,6-hydroxykynurenic acid(6-HKA),p-hydroxy benzoic acid)in Yinxingye tablets by quantilative analysis of malticomponents by single market(QAMS). Methods: The analysis was performed at 30 ℃,on an ZORBAX Eclipse XDB-C₈ column(250 mm×4.6 mm,5 μm), eluted with a gradient program(0-25 min,3.5%B;25-26 min,3.5%B→70%B;26-35 min,70%B;35-36 min,70%B→3.5%B;36-60 min,3.5%B)using 0.1% phosphoric acid aqueous solution(A)-acetonitrile (B)as the mobile phase. The flow rate was 1 mL·min^-1,and the detection wavelength was switching(0-6 min:215 nm,detecting shikimic acid and gallic acid;6-60 min:254 nm,detecting protocatechuic acid, 6-HKA and p-hydroxy benzoic acid). Protocatechuic acid was used as the internal reference substance, respectively establishing the relative correction factor(RCF)of shikimic acid,gallic acid,6-HKA, p-hydroxy benzoic acid,and making the reproducibility inspection of the RCF;external standard method (ESM)was performed for the content determination of protocatechuic acid,and the contents of the other four small molecular organic acids were calculated according to the RCF,respectively. At the same time,the contents of these five small molecular organic acids in 15 batches of Yinxingye tablets were determined by ESM,and then,the results obtained from the two different methods were compared. Results: The linear range of shikimic acid,protocatechuic acid,gallic acid,6-HKA and p-hydroxy benzoic acid were 43.9-1 097.5 ng, 13.2-330.0 ng,141.6-3 540.0 ng,11.3-282.5 ng,and 12.9-322.5 ng,respectively. Within the linear range mentiond above,the RCFs of shikimic acid,gallic acid,6-HKA,p-hydroxy benzoic acid with reference to protocatechuic acid were 1.1474,0.416 8,0.504 3,and 0.552 2,respectively. The reproducibility was good under different experimental conditions. For the five small molecular organic acids,there was no significant difference between calculated value(shikimic acid 0.11%-1.01%,gallic acid 0.13%-0.26%,6-HKA 0.15%-0.32%,p-hydroxy benzoic acid 0.07%-0.18%)and the measured value in the 15 batches. Conclusion: With the validation of methodology,the method established in our study can be used for content determination of shikimic acid,protocatechuic acid,gallic acid,6-HKA and p-hydroxy benzoic acid in Yinxingye tablets.