目的:建立柏子养心丸中阿魏酸、桂皮醛、五味子醇甲、五味子甲素及五味子乙素5 个成分的双标多测法,验证该方法在柏子养心丸质量评价中应用的准确性和可行性。方法:采用HPLC 法,以乙腈为流动相A,0.1% 醋酸溶液为流动相B,梯度洗脱（0~70 min,20%A;70~85 min,20%A → 80%A）,流速为1 mL·min-1,检测波长335 nm（0~31 min）、285 nm（31~52.5 min）、215 nm（52.5~85 min）,柱温35 ℃。采用6 根不同型号的C18 色谱柱,测定柏子养心丸5 个成分的实际保留时间,计算各成分在各色谱柱保留时间的平均值,得到各成分的标准保留时间,选取阿魏酸（峰1）、五味子乙素（峰5）作为双标化合物,使用双标线性校正法准确定位各成分色谱峰,预测成分在未知色谱柱的保留时间并进行方法学验证。以阿魏酸为参照物,计算其余各成分的相对校正因子,对各成分进行定量。结果:双标线性校正法能够准确地预测待测组分在未知色谱柱上的保留时间;桂皮醛、五味子醇甲、五味子甲素、五味子乙素相对于阿魏酸的相对校正因子分别为0.2644、0.4062、0.3772、0.3994,其RSD 均小于2%;各成分含量计算值和实测值无显著差异。结论:双标多测法同时测定柏子养心丸中5 个成分可行且准确。
Objective: To establish a two reference substances for determination of multiple components method （TRSDMC） for the determination of five components（ferulic acid,cinnamaldehyde,schizandrin,schisandrin A, and γ-schizandrin B） of Baizi Yangxin pills,and to verify the accuracy and feasibility of the method in the quality evaluation of Baizi Yangxin pills. Methods: HPLC method was used with acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B in gradient elution mode（0-70 min,20%A;70-85 min,20%A → 80%A）, and the flow rate was 1 mL·min-1;the detection wavelength was 335 nm（0-31 min） for ferulic acid,285 nm （31-52.5 min） for cinnamaldehyde,and 215 nm（52.5-85 min） for schizandrin,schisandrin A and γ-schizandrin B;the column temperature was 35 ℃. The actual retention time of five components in Baiziyangxin pills was determined on six different types of C18 chromatographic columns. The average retention time of each component in each column was calculated to obtain the standard retention time of each component. Ferulic acid（peak 1） and schisandrin B（peak 5） were selected as the reference substances for the determination. The chromatographic peaks of each component were accurately located by linear calibration using two reference substances,and the position of each component in unknown chromatographic column could be predicted in the same method. The methodology was validated. Taking ferulic acid as reference,the content of each component was determinated. At the same time,the contents of cinnamaldehyde,schizandrin and schisandrin A in Baizi Yangxin pills were determined by external standard method,and the contents obtained by TRSDMC and external standard method were compared to verify the scientificity and feasibility of TRSDMC. Results: The linear calibration using two reference substances can accurately predict the retention time of the tested components on the unknown column. The relative correction factors of ferulic acid and cinnamaldehyde,schizandrin,schisandrin A,and γ-schizandrin B were 0.264 4, 0.406 2,0.377 2 and 0.399 4,respectively. With RSD values less than 2%,there was no significant difference between the contents obtained by TRSDMC and external standard method for each component. Conclusion: TRSDMC for simultaneous determination of 5 components in Baizi Yangxin pills is feasible and accurate.
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