目的:本文借鉴准确度轮廓的研究思路,提出采用β-容度容许区间(β-CTI)建立基于不确定度轮廓(UP)的决策验证方法,并将该方法应用于大黄HPLC含量测定方法验证。方法:以芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚5个成分色谱峰的分离度为优化目标,采用单因素实验对影响色谱分离效果的柱温、流动相比例等因素进行优化,确定最佳色谱分离条件。采用“3×5×4”型全析因实验设计获取HPLC验证数据,即实验分为3 d进行,每天1次,每次实验包含4个浓度水平,每个浓度水平进行5次重复实验。对采集的验证数据进行方差分析,通过计算β-CTI评估不同浓度的芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚HPLC定量分析不确定度。以±10%为不确定度可接受限,建立UP对大黄5个成分的HPLC定量分析方法进行验证。结果:以甲醇-0.1%磷酸水溶液(83∶17)为流动相,在柱温25 ℃条件下,大黄5个化学成分色谱峰的分离度均大于1.5。芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚质量浓度分别在1.013~50.65、0.879~43.05、1.016~50.8、1.002~50.1、1.023~51.15 μg·mL-1范围内与峰面积呈良好的线性关系。芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚质量浓度分别在2.032~40.64、1.758~35.16、2.032~40.26、2.004~40.08、2.046~40.92 μg·mL-1浓度范围内定量不确定度均小于10%,说明所建立的HPLC定量分析方法能够准确测定大黄中总蒽醌的含量。结论:UP方法操作简便,能全面、科学地评价分析方法的准确性,对提高定量分析验证方法的可靠性、降低应用风险具有一定的参考价值。
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.
[1] The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Harmonized Tripartite Guideline, Q2 (R1), Validation of Analytical Procedures: Text and Methodology, Current Step 4 Version[EB/OL].https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf
[2] HUBERT P, NGUYEN-HUU JJ, BOULANGER B, et al. Harmonization of strategies for the validation of quantitative analytical procedures: a SFSTP proposal—part Ⅰ[J].J Pharm Biomed, 2004, 36(3): 579
[3] FEINBERG M, BOULANGER B, DEWE W, et al. New advances in method validation and measurement uncertainty aimed at improving the quality of chemical data[J].Anal Bioanal Chem, 2004, 380:502
[4] GONZALEZ AG, HERRADOR MA. Accuracy profiles from uncertainty measurements[J].Talanta, 2006, 70:896
[5] MARINI RD, CHIAP P, BOULANGER B, et al. LC method for the determination of R-timolol in S-timolol maleate: validation of its ability to quantify and uncertainty assessment[J].Talanta, 2006, 68:1166
[6] FEINBERG M. Validation of analytical methods based on accuracy profiles[J].J Chromatogr A, 2007, 1158:174
[7] DAI SY, XU B, ZHANG Y, et al. Establishment and reliability evaluation of the design space for HPLC analysis of six alkaloids in Coptis chinensis (Huanglian) using Bayesian approach[J].Chin J Nat Med, 2016, 9(14): 697
[8] DAY SY, XU B, ZHANG Y, et al. Robust design space development for HPLC analysis of five chemical components in Panax notoginseng saponins[J].J Liq Chromatogr RT, 2016, 39(10): 504
[9] GONZALEZ AG, HERRADOR MA. A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles[J].Trac-Trend Anal Chem, 2007, 26(3): 227
[10] SAFFAJ T, IHSSANE B. Uncertainty profiles for the validation of analytical methods[J].Talanta, 2011, 85(3): 1535
[11] HASNAA H, TAOUFIQ S, AIMIN T, et al. Full validation using β-content, γ-confidence tolerance interval: application for LC-MS/MS determination of doxycycline in human plasma[J].Chemometr Intell Lab, 2017, 168: 89
[12] BOUCHAFRA H, EI KARBANE M, AZOUGAGH M, et al. Application of a new strategy of validation based on "β,γ-content tolerance interval" for checking the chiral chromatography method for quantification of the chiral impurity of levofloxacin[J].J Brazil Chem Soc, 2015, 27(3): 500
[13] XUE Z, XU B, SHI X Y, et al. Overall uncertainty measurement for near infrared analysis of cryptotanshinone in tanshinone extract[J].Spectrochim Acta A, 2017, 170: 39
[14] RUDAZ S, FEINBERG M. From method validation to result assessment: established facts and pending questions[J].Trac-Trend Anal Chem, 2018, 105: 68
[15] 刘佳, 李莉.不同道地产区大黄功效组分含量的差异分析[J].中国现代中药, 2017, 19(5): 662
LIU J,LI L. Analysis on the difference of effective components of Rhubarb in different regions[J].Mod Chin Med,2017, 19(5): 662
[16] 吴丽, 徐春蕾, 季易彦, 等. 大黄及大黄蒽醌类有效成分对胰腺腺泡细胞损伤的保护作用[J].中药新药与临床药理, 2014, 25(5): 590
WU L, XU CL, JI YY,et al. Protective effects of rhubarb and its anthraquinones on pancreatic acinar cell injury[J].Tradit Chin Drug Res Clin Pharmacol, 2014, 25(5): 590
