Robustness of liquid chromatographic method: How to achieve it and how to confirm it?
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The quality of liquid chromatographic methods developed for pharmaceutical applications is strongly dependent on their robustness. The robustness issue requires a studious strategy where many important aspects regarding both the optimization and validation phases must be considered; therefore, these aspects will be presented in this paper. The first step in evaluating the robustness is robustness screening during the method optimization. In this phase, important chromatographic factors (organic modifier content, pH of the mobile phase, column temperature, etc.) and their influence on the chromatographic parameters should be examined. A different type of response surface design could be utilized in this step, and its application allows the examination of the response surface to identify the robust regions, such as the maximum, minimum or inflection point. Total robustness is achieved if the response is robust for all of the investigated factors. However, if total robustness does not occ...ur, the quality of the chromatographic method can be expressed by partial robustness. Additionally, the robustness of the selected chromatographic optimum can be analyzed by estimating the robustness criterion. This approach is based on calculating the derivatives of selected chromatographic responses with respect to the investigated factors and then monitoring the rate of change of the response while the factors deviate within the expected range. The second level of the robustness evaluation is robustness testing at the beginning of the method validation process. Primarily, this phase includes defining the factors that are to be examined and their levels. It is desirable to select as many factors as possible (factors related to the mobile phase, stationary phase, sample preparation, equipment, etc.) and set their levels as narrow regions around the nominal level. This step is followed by selecting an adequate experimental design that allows the estimation of a relatively great number of factors through a relatively small number of experiments (usually fractional factorial or Plackett-Burman design). The final step of this level consists of analyzing the obtained results and interpreting the significance of the effects. The methods commonly applied for estimating the effects are graphical analysis (involving normal and half-normal probability plots) and statistical analysis (including the algorithm of Dong and error estimation based on a priori declared negligible effects). Lastly, the information extracted from the robustness testing enables relatively simple definition of system suitability tests, which is an integral part of routine application of any chromatographic method. The systematic robustness evaluation is applied to an experimental example for analyzing beta blockers and agonists in a hydrophilic interaction liquid chromatography experiment. It can be concluded that evaluating the robustness of the chromatographic method in the right way provides a lot of significant data, which enable safe method application and transfer.
Source:Liquid Chromatography: Principles, Technology and Applications, 2013, 57-98
- Nova Science Publishers, Inc.