RATIONAL EXPERIMENTAL-DESIGN FOR BIOANALYTICAL METHODS VALIDATION - ILLUSTRATION USING AN ASSAY-METHOD FOR TOTAL CAPTOPRIL IN PLASMA

Generally, bioanalytical chromatographic methods are validated accordi ng to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper , a rational, total performance evaluation programme for chromatograph ic methods is presented. The design was developed in particular for th e pre-validation and main validation phases. The entire experimental d esign can be performed within six analytical runs. The first run (pre- validation phase) is used to assess the validity of the expected conce ntration-response relationship (lack of fit, goodness of fit), to asse ss the specificity of the method and to assess the stability of proces sed samples in the autosampler for 30 h (benchtop stability). The latt er experiment is performed to justify overnight analyses. Following ap proval of the method after the pre-validation phase, the next five run s (main validation phase) are performed to evaluate method precision a nd accuracy, recovery, freezing and thawing stability and over-curve c ontrol/dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. A nalysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stab ility and over-curve control/dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/dilution, addition al to ANOVA, also a paired comparison is applied. As a consequence, th e recommended design combines the performance of as few independent va lidation experiments as possible with modern statistical methods, resu lting in optimum use of information. A demonstration of the entire val idation programme is given for an HPLC method for the determination of total captopril in human plasma.