Bioanalytical chromatographic method validation according to current regulations, with a special focus on the non-well defined parameters limit of quantification, robustness and matrix effect

Abstract

Method validation is a mandatory step in bioanalysis, to evaluate the ability of developed methods in providing reliable results for their routine application. Even if some organisations have developed guidelines to define the different parameters to be included in method validation (FDA, EMA); there are still some ambiguous concepts in validation criteria and methodology that need to be clarified. The methodology to calculate fundamental parameters such as the limit of quantification has been defined in several ways without reaching a harmonised definition, which can lead to very different values depending on the applied criterion. Other parameters such as robustness or ruggedness are usually omitted and when defined there is not an established approach to evaluate them. Especially significant is the case of the matrix effect evaluation which is one of the most critical points to be studied in LC-MS methods but has been traditionally overlooked. Due to the increasing importance of bioanalysis this scenario is no longer acceptable and harmonised criteria involving all the concerned parties should be arisen. The objective of this review is thus to discuss and highlight several essential aspects of method validation, focused in bioanalysis. The overall validation process including common validation parameters (selectivity, linearity range, precision, accuracy, stability…) will be reviewed. Furthermore, the most controversial parameters (limit of quantification, robustness and matrix effect) will be carefully studied and the definitions and methodology proposed by the different regulatory bodies will be compared. This review aims to clarify the methodology to be followed in bioanalytical method validation, facilitating this time consuming step.