Robustness testing ranges evaluated

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

In recent work within our laboratories, we have evaluated a number of unbonded silicas by CEC specifically for the separation of a range of pharmaceutically relevant basic analytes and mixtures. We purposefully chose strong basic analytes that comprised a wide range of lipophilicities, molecular weights and log P values to robustly test the separation systems. The basic analyte test mixture contained two AstraZeneca R D compounds, benzylamine, nortriptyline, diphenhydramine and procainamide. 

To determine the robustness of a method, several approaches exist. Basically, the situation for robustness testing is similar to that for screening during optimization, except for the range within which the factors are examined. The influence of small but deliberate changes in parameters on the response (s) is evaluated using either an OVAT or an experimental design approach (12). 

Sample preparation parameters should also be studied to establish acceptable ranges for the method. Sample solvent and volume, extraction time, filter type, and volume can be studied and modifications made to determine which are critical. For example, sample solvent composition and volume can be modified to assure that the organic solvent level is not on the edge of failure. Similarly the extraction time can be modified over a range (i.e., 20%) as can fhe settings or type of apparatus (e.g., mechanical shakers, sonic baths) used for this sample preparation step. Filter type and the volume discarded before collection of the final sample solution can be evaluated as part of robustness testing and alternate filter types qualified fo enhance fhe mefhod design space. 

Finally, the same questions should be asked in a critical manner of the in-process and final acceptance tests for raw materials, in-process intermediates, and final product. Imported and research tests often do not make good quality control laboratory tests. The quality control tests need to be robust, meaningful, validated, and reproducible. Many research assays are narrow in scope, unstable, user-dependent, and complex, and they also yield wide-ranging results. These assay characteristics are exactly the opposite of what is needed in quality control therefore, all analytical methods and assays must be critically evaluated for relevance and validated for use. 

Before phase III and process finalization, process robustness is evaluated via several scaled-down runs to quantify process variability [14]. These studies form the basis for the process validation program if they are well documented, use operational range extremes, and test different resin/raw material lots. Based on the resulting data, additional optimization to improve robustness is conducted. Fully developed analytical assays are employed during these pre-phase III scale-down runs to ensure the process meets the quality acceptance criteria. Then during phase III, any remaining assays are validated [14]. 

Defining the context of use for novel biomarkers in man represents an important area of collaborative research interest. Understanding reference ranges for novel DILI biomarkers in preclinical species and their evaluation in diverse healthy human populations and liver disease cohorts is an important area of investigation and question to address. Further areas of research focus should also be targeted toward the generation of robust cross species bioanalytical assays that are standardized or point-of-care tests in parallel with a comprehensive understanding of cross species differences in biomaiker expression, mechanisms of release, and clearance, distribution, and kinetics. 

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