Specificity Testing in Bioanalytical Assay Validations—Challenges and Opportunities

Specificity assessment is essential in bioanalytical method development to ensure accurate analyte quantification in the presence of endogenous matrix components and potential exogenous interferences. Regulatory expectations have evolved over time, culminating in the harmonized ICH M10 guideline, which establishes a globally aligned framework for bioanalytical method validation. Although LC-MS/MS provides high analytical specificity, challenges remain, particularly from isobaric interferences and increasingly complex multi-analyte assays. A common practical issue is trace contamination of authentic reference standards, such as parent drug in metabolite standards or vice versa, which can generate unacceptable cross-signal contributions and compromise specificity assessments. When such impurities preclude conventional specificity testing, this perspective discusses scientifically justified approaches, including predetermined baseline corrections to account for contaminated standards. It also highlights incurred sample reanalysis (ISR) as a more meaningful assessment of assay specificity under these constrained conditions because it reflects the true in vivo composition of parent drug and metabolites. The application of ISR has emerged as a robust, regulatory-endorsed tool for confirming assay reliability over time and, as such, could also serve as an alternative for some of the issues discussed.