杂质类定量分析方法验证中的统计学评价

目的：通过马来酸依那普利原料药中杂质依那普利拉的定量检测方法，论证杂质类定量分析方法验证的科学性和严谨性，为今后验证研究提供评价参考依据。方法：对欧洲药典中原料药依那普利拉中杂质限度检查分析方法优化后，通过析因设计进行试验，在原有方法验证性能参数的基础上，引入更多的统计参数以评价方法是否满足预期应用。结果：专属性：马来酸与依那普利拉分离度19.0>1.5，依那普利拉与依那普利分离度为30.9>4.0，专属性符合要求。线性：0.5~12.0 μg·mL^-1的浓度范围内呈线性，线性方程为Y=139.86+38 982.17X（r=0.999 8，r>0.999）。检测下限（LOD）为0.29 μg·mL^-1；定量下限（LOQ）为0.76 μg·mL^-1。准确度：2.0 μg·mL^-1以上的各浓度水平的偏倚均不超过2.0%，95%的偏倚置信区间不超过3.3%。精密度：2.0 μg·mL^-1以上的各浓度水平的重复性精密度均不超过1.03%；中间精密度均不超过2.5%，中间精度的置信上限不超过5.0%。方法综合能力评价：方法在浓度范围为2.0~9.0 μg·mL^-1内，方法最大的总变异值不超过2.65%；95%预测区间在98.0%~106.0%范围内；95%/90%容忍区间在95.0%~108.0%范围内；相对于该杂质0.3%的质量标准限度，满足其定量检测用途。结论：新建立的方法经验证适于其预期用途：能满足对原料药马来酸依那普利中依那普利拉杂质在限度标准为0.3%的定量检测所需；新的验证试验设计可获得更多科学可靠的统计评价指标，这些指标使得方法验证的结论更加科学、可靠和直观，并且能够从整体把握方法的适用性；新提供的方法变异容忍区间和预测区间，对今后评估方法验证和转移是否成功提供了判断依据；本文所采用的方法验证试验设计和性能参数，可为今后更科学严谨地进行杂质类定量分析方法验证工作提供参考。

Objective: To demonstrate the accuracy and rigor of the quantitative analysis method of the impurities by using enalaprilat in the raw material of enalapril maleate,and to provide reference for future confirmation research. Methods: After optimizing the impurity limit analysis method of the drug substance enalapril maleate in the European Pharmacopoeia, the test was carried out by factorial design, and based on the validation of the performance parameters of the original method, more statistical parameter evaluation methods were introduced to evaluate whether they met the intended use. Results: Specificity:The separation degree of maleic acid and enalaprilat was 19.0>1.5, the separation of enalaprilat and enalapril was 30.9>4.0, and the specificity met the requirements. Linearity:The concentration range of 0.5-12.0 μg·mL^-1 was linear, and the linear equation was Y=139.86+38 982.17X(r=0.999 8, r>0.999). LOD was 0.29 μg·mL^-1;LOQ was 0.76 μg·mL^-1. Accuracy:The bias of each concentration level above 2.0 μg·mL^-1 was not more than 2.0%, and the 95% confidence interval was less than 3.3%. Precision:The repeatability precision of each concentration level above 2.0 μg·mL^-1 was not more than 1.03%;the intermediate precision was less than 2.5%, and the upper limit of the intermediate precision was not more than 5%. Method comprehensive ability evaluation:The method had a concentration range of 2.0-9.0 μg·mL^-1, and the maximum total variability value of the method was not more than 2.65%;95% prediction interval was in the range of 98.0%-106.0%;95%/90% tolerance range was in the range of 95.0%-108.0%; Relative to 0.3% quality standards of the impurity, the method met its quantitative detection purposes. Conclusion: The newly established method has been validated to be suitable for its intended use-it can meet the quantitative detection requirement of 0.3% of the enalaprilat impurity of the raw material drug enalapril maleate. The new validation experimental design can obtain more scientific and reliable statistical evaluation indicators,which make the conclusions of the method validation more scientific, reliable and intuitive,and can grasp the applicability of the method from the whole;the newly provided method variability tolerance interval and prediction interval provide a basis for judging whether the evaluation method verification and transfer are successful in the future;the experimental design and performance parameters of the method validation used in this paper can provide reference for the scientific and rigorous validation of the quantitative analysis method of impurities in the future.

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