The introduction of context of use to the vernacular of bioanalytical analysis has opened up both great possibilities and many questions around the flexibility and limitations of validation activities. In the circumstance that a protocol is introduced with a defined context of use and defined target analytes – the construction of a suitable validation plan might be straightforward. However, in the context of existing biomarker panels, developed and validated to meet a pharmacokinetic context of use, how does one redefine and revalidate to address new uses? This discussion will feature a reactive oxygen species panel originally designed to monitor levels of glutathione, cysteine, N-acetyl cysteine and homocysteine as free and total concentrations utilizing LC/MS/MS. The method splits sample pools into two processes, one of which measures free thiols and the other total thiols. It has been used to understand overall reactive oxygen species levels after treatment with redox modulators such as N-acetyl cysteine amide. In such a case, the context of use would only require that such a method be able to distinguish 2-fold or greater changes in each analyte relative to starting conditions across various tissues. Yet, a traditional pharmacokinetic validation would require +/- 15% variability for 4 analytes, a much more difficult hurdle to achieve. One could imagine that such a routine biomarker panel could be employed for other applications, maybe the analysis of reactive oxygen species in brain tissue, which could require more precision and the ability to detect 10% changes, or in a purely “exploratory” mode to see if there is any detectable change. Each of these instances could require a different set of validation parameters. This presentation will seek to address the various contexts of use and their validation requirements and compare those to the FDA guidelines for methods with a pharmacokinetic context of use. Data will be presented from reactive oxygen species monitoring from N-acetyl cysteine amide studies in ocular matrices.
Describe various contexts of use for which a single multiplex assay may be suitable.
Understand the differences between a validation to address a pharmacokinetic context of use compared to validation requirements for biomarker contexts of use.
Evaluate existing multiplex methods for their suitability to address multiple contexts of use.