HPLC相对保留时间法中参照物的优选

目的：提出并建立相对保留时间法中参照物的优选原则，以提高该法的准确度和重现性。方法：在2台高效液相色谱仪和15根不同品牌和型号的C₁₈色谱柱上，记录4种色谱条件下、3种中药中26个成分的保留时间，一方面对实测保留时间进行统计分析，另一方面基于推导的Δt_R（Δt_R是指保留时间实测值与保留时间计算值差的绝对值）公式进行推导分析。结果：参照物的选择对计算效果的影响较大，应选择保留时间覆盖率（保留时间覆盖率反映了参照物在整个保留时间段上的相对位置。当参照物为第一个成分时，保留时间覆盖率为0，当参照物为最后一个成分时，为100%）在50%~100%的成分为参照物，其中，60%~90%最优；选择非线性偏离较小的成分作为参照物。结论：该原则简单、准确，具有普适性，可以提高相对保留时间法的准确度和重现性。

Objective: In order to improve the accuracy and reproducibility of the relative retention time method,the optimal selection principle of reference compound is proposed and established. Methods: The retention times of 26 compounds in 3 traditional Chinese medicines were recorded by 2 HPLC instruments using 4 kinds of chromatographic conditions and 15 ODS columns with different manufacturers and brands. Firstly,statistical analysis of the measured retention time was carried out. Secondly,based on the deduced formulas of Δt_R (Δt_R was the absolute value of the difference between the measured retention time and the calculated retention time),the deduction and analysis were performed. Results: The selection of reference compound had a great influence on the calculation results. Based on above studies,the principle for selecting reference compound for HPLC relative retention was proposed,including:(1) Selecting those compounds with 50%-100% coverage of retention time (The coverage of retention time stands for the relative position of a compound between first eluted compound and last eluted compound. For example,coverage of first compound is zero and 100% for last compound.) and the optimal coverage was 60%~90%. (2) Choosing compounds with smaller linear deviation. Conclusion: The principle is simple,accurate and universal. It can improve the accuracy and reproducibility of the relative retention time method.

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