Objective: To develop a validation method based on the uncertainty profile (UP) and β-content tolerance intervals (β-CTI), and apply the proposed method to validate the high performance liquid chromatography (HPLC) quantitative analysis for rhubarb. Methods: Taking the resolution of five chromatographic peaks of aloe-emodin, rhein, emodin, chrysophanol and physcion as the targets, the chromatographic parameters including the column temperature, and the mobile phase ratio were optimized by using the one-factor experimental design. Then the“3×5×4” full factorial design was used to obtain the validation data of the HPLC quantitative analysis. The experiment included 4 concentration levels, and each concentration level was performed in 5 replicates on 3 different days. The analysis of variance was performed on the validation data. The uncertainty of the HPLC quantitative analysis for aloe-emodin, rhein, emodin, chrysophanol and physcion at different concentrations was evaluated. Set±10% as the acceptable limits, the uncertainty profiles for five components in rhubarb were constructed to validate the HPLC quantitative analysis method. Results: With methanol and 0.1% phospho-ric acid solution (83∶17) as mobile phases, the resolution of the chromatographic peaks for the five components in rhubarb was greater than 1.5 at 25 ℃. The calibration curves of aloe-emodin, rhein, emodin, chrysophanol, and physcion displayed good linearity over the ranges of 0.013-50.65, 0.879-43.05, 1.016-50.8, 1.002-50.1, 1.023-51.15 μg·mL-1, respectively. The uncertainties of HPLC quantitative analysis for aloe-emodin, rhein, emodin, chrysophanol and physcion were within acceptable limits (±10%) over the ranges of 2.032-40.64,1.758-35.16, 2.032-40.26, 2.004-40.08 and 2.046-40.92 μg·mL-1, respectively. Conclusion: The UP method is easy to operate and can comprehensively and scientifically evaluate the accuracy of the analytical method. Application of the UP in the validation stage can control the risk of using the analytical method in routine phase.
DAI Sheng-yun, ZHAN Shu-yi, OU-YANG Xiao-jie, MA Shuang-cheng, ZHENG Jian, SUN Fei
. Uncertainty profile for validating the HPLC quantitative analysis method of rhubarb[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(12)
: 2202
-2210
.
DOI: 10.16155/j.0254-1793.2021.12.19
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