目的: 建立陈皮中5个黄酮类成分的双标多测法。方法: 在3 台不同厂家生产的液相色谱仪、13根不同品牌和型号的 C18色谱柱上,以橙皮苷为参照物,测得其与芸香柚皮苷、川陈皮素、3,3’,4’,5,6,7,8-七甲氧基黄酮、橘皮素的相对校正因子;以5个组分在13根色谱柱上实际保留时间的平均值作为标准保留时间,再以橙皮苷和橘皮素为双标物,利用双标线性校正法对待测物进行定位,从而实现定性定量。同时采用外标法测定陈皮中芸香柚皮苷、川陈皮素、3,3’,4’,5,6,7,8-七甲氧基黄酮、橘皮素含量,比较两种方法测定结果的差异,以验证双标多测法在含量测定中的准确性和可行性。结果: 定量方面,相对校正因子可以耐受不同仪器和色谱柱,以及波长、柱温、流动相比例和流速在一定范围内的变动,测得的芸香柚皮苷、川陈皮素、3,3’,4’,5,6,7,8-七甲氧基黄酮、橘皮素相对校正因子分别为0.86、0.50、1.11、0.59,其RSD均不大于2.0%;定性方面,双标线性校正法预测值的绝对偏差均小于0.5 min,准确度优于相对保留时间法。双标多测法与外标法所测定的5个组分的含量相对平均偏差小于4%,表明结果无显著性差异。结论: 双标多测测定陈皮中5个黄酮类成分方法可行性好、耐用性好。
Objective: To establish two standards for determination of multiple components(TSDMC) in quantitative analysis of hesperidin, tangeretin, nobiletin, 3,3’, 4, 4’, 5, 6, 7-heptamethoxyflavone and narirutin in Citrus Reticulate Pericarpium. Methods: The peak area and retention time of the five flavonoids in Citrus Reticulate Pericarpium were measured for quantitative and qualitative analysis with three different brands of HPLC instruments and 13 C18 columns of different brands and types. For the quantitative analysis, hesperidin was used as the reference for determination of relative correction factors of tangeretin, nobiletin, 3, 3’, 4, 4’,5,6,7-heptamethoxyflavone and narirutin. For the qualitative analysis, the average retention time of the five components on each column was regarded as the standard retention time. Both hesperidin and tangeretin were used as linear calibration standards to calculate the retention time of the other three components. Meanwhile, the contents of hesperidin, tangeretin, nobiletin, 3, 3’, 4, 4’, 5, 6, 7-heptamethoxyflavone and narirutin were determined by external standard method,and the deviation of two methods was used to verify the accuracy and feasibility. Results: For quantitative results, the relative correction factors were reproducible with different HPLC instruments, different brands and types of columns, as well as a certain variation range of proportion of the wavelength, mobile phase, column temperature and flow rate. The relative correction factors of tangeretin, nobiletin, 3,3’, 4, 4’, 5,6,7-heptamethoxyflavone and narirutin were 0.86, 0.50, 1.11, 0.59, and their RSDs were less than 2.0%. For qualitative results, the absolute deviations of the predicted values of the linear calibration using two reference substances wereless than 0.5 min while those of the relative retention time method were more than 1.5 min, which indicated that the linear calibration using two reference substances was more accurate than the relative retention time method for chromatographic peak identification on 13 columns.The relative average deviations of the contents of the 5 components determined by TSDMC and the external standard method were less than 4%, which showed no significant difference. Conclusion: TSDMC for simultaneous determination of 5 components in Citrus Reticulate Pericarpium is feasible and durable.
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