Making Methods Rugged for Regulated Bioanalysis

Abstract

Methods started in discovery are optimized as they progress through preclinical and clinical development. Making a robust assay includes testing individual steps for consistency and points of failure. Assays may be transferred, optimized and revalidated several times. A rugged assay will not only meet regulatory requirements, but will execute with a low failure rate and confirm results under repeat analysis. Challenging aspects such as differential recovery, sample stabilization, resolution of isomers or conjugate analysis must be tackled and made routine. The proper selection of the IS can overcome limitations. It is best to know the potential points of failure before a study has started, but lessons learned from each study also provide invaluable insights to improve assay ruggedness.

So You Think Your Assay is Robust?

“…The use of an internal standard (stable isotope labeled analyte in particular) and the extraordinary specificity of mass spectrometric detection render bioanalytical assays very tolerant to small changes in experimental conditions. There are exceptions, many of which have been well described in the published literature, most recently by Unger et al [9], with examples of validated assays failing in routine sample analysis because a critical aspect had been overlooked. Good method development includes aspects of robustness testing and conscientious analysts complete method development with a good understanding of critical parameters.…”