Study On The Establishment And Reliability Of HPLC Analysis And Design Space Of Traditional Chinese Medicine

Quality by design (QbD) is a new and systematic approach for the development of drugs. This approach begins with predefined objectives, and uses science-based risk management to obtain the understanding of product and process. Although the QbD concept is used in the field of manufacturing process originally, the main idea is similar to the development of analytical methods. So the analytical quality by design (AQbD) came into being. Using the AQbD, the first step is to define the analytical target and then followed by the whole assessment about the critical method parameters aiming to the analytical target. Combine design of experiment and statistical analysis to build a robustness design space for the analytical method. The last is to set up the control strategy for the analytical method based on the design space. Establishing the analytical method by the means of structured, systematic and progressive layers is beneficial to improve the accuracy and reliability of method. There are more and more researches of QbD on the international while the research on the chromatographic analysis of traditional Chinese medicine is little. But with its progressive and the support of FDA and ICH, the wide application of QbD in traditiona Chinese medicine will be the trend.In this paper, QbD was made into the building of HPLC design space and its reliability of traditional Chinese medicine. Taking the HPLC analysis method of Panax Notoginseng Saponins and Coptis chinensis for example, a whole process of the building of the design space and it’s reliability of HPLC analysis method by QbD was introduced. The main contents of this paper are summarized as follows:Take Panax Notoginseng Saponins as the example. This study combines design of experiments and design space methodology to optimize the HPLC separation of PNS. Three common chromatographic parameters (i.e. the temperature, the initial proportion of acetonitrile and the gradient slope) were selected to construct a Box-Behnken design which consisted of 17 experiments. Take critical resolution and the analysis as the critical method attributes, and the results showed that the quadratic model was the best to construct the design space. The R2 for the two models are 0.9988 and 0.9989, repectively. And then choose the confidence interval to analyze the reliability of the design space. The optimal separation was predicted at temperature 20℃, with a gradient starting at 15% of acetonitrile and a gradient slope of 0.55%·min-1. Accuracy profile approach was employed to validate the established HPLC method. The results clearly showed that QbD methodology could be effectively applied to optimize the HPLC chromatographic conditions for the analysis of PNS.Take Coptis chinensis as the example. First, five chromatographic parameters were identified to construct a Plackett-Burman experimental design. The critical resolution, analysis time and peak width were responses modeled by multivariate linear regression. The results showed that the percentage of acetonitrile, concentration of sodium dodecyl sulfate and concentration of potassium phosphate monobasic were statistically significant (p-value<0.05) parameters. Then, the Box-Behnken experimental design was applied further to evaluate the interactions of the three parameters on selected responses. Full quadratic models were built and were used to establish the analytical design space. Moreover, the reliability of design space was estimated by the Bayesian posterior predictive distribution. The optimal separation was predicted at 40% acetonitrile,1.7 g·mL-1 sodium dodecyl sulfate and 0.03 mol·mL-1 potassium phosphate monobasic. Finally, the accuracy profile methodology was used to validate the established HPLC method.Take Panax Notoginseng Saponins as the example.The method was developed in two main phases:screening and optimization. During the screening phase, the most suitable stationary phase, column temperature and flow rate were identified. And the secondary influential parameters, such as the gradient slope, the initial concentration of acetonitrile and the initial isocratic hold of the gradient elution system were fine tuned in the optimization phase. In this phase, a seventeen-run experiment was used to examine multifactorial effects of these parameters on the critical resolution, the analysis time and the peak symmetry. The Monte Carlo simulation method was successfully used to build the chromatographic design space. And the process capability index Cp was introduced to evaluate the robustness of the design space. Compare the three methods, i.e. interval method, Bayesian method and Monte Carlo method. The comparative result showed that the separation conditions predicted in the design space was the same. The robust combination of separation conditions predicted in the design space was obtained with the gradient slop of 1.6%·min-1, the initial concentration of acetonitrile of 20% and the initial isocratic hold of 20 min and the total analysis time was no more than 40 min. At last, the verification experiment was performed within the working design space by the accuracy profile methodology and model was found to be accurate. The results demonstrated that rapid separation of 5 components of PNS herbal extract could be achieved on the chromatographic column packed with narrow size particles at backpressures available in ordinary HPLC instruments.