Introduction
The bioanalytical assays are characterized and validated to ensure that the assay is sensitive, specific, and fit for the intended purpose.
The assay validations are laborious, expensive, and time-consuming and any failure during the validation process can push back your critical timeline. To prevent setbacks during the validation process, the following are the parameters you should assess during early development:
1) Matrix Interference
2) Specificity
3) Analyte Stability
4) Sensitivity
5) Suitability of critical reagents
1) Matrix interference/Selectivity:
Potential interfering substances in a biological matrix include endogenous matrix components such as metabolites, decomposition products – and from the actual study – concomitant medications and other xenobiotics. During method development, interference of the biological matrices must be evaluated. The selectivity of the method is evaluated by analyzing blank samples of the appropriate biological matrix (e.g., plasma) from multiple sources. In addition, the recovery at a high and low concentration of the positive controls spiked to the different sources of blank matrices are evaluated.
Depending on the intended use of the assay, the impact of hemolyzed samples, lipemic samples, or samples from special populations is included in the selectivity assessment. Usually, the matrix interference results in the variable spike recovery. Investigating these interference allows devising the mitigation strategy during the assay development stage.
2) Specificity:
The specificity assessment is conducted to ensure that detection reagents are specific to the analyte and does not cross-react with the matrix components. The specificity is evaluated by spiking homologous molecules that are closely related to the analyte with or without the positive control.
3) Analyte Stability:
Early determination of the chemical stability of the analyte in a given matrix, including the effects of sample collection, handling, and storage of the analyte are critical for successful assay validations.
4) Sensitivity:
The LLOQ defines the method sensitivity and should be determined during method development. The method should be developed and validated such that it will be able to meet the requirements necessary for the intended study samples.
5) Suitability and characterization of critical reagent:
Regulatory agency excepts an appropriate characterization (determine the identity, purity, and stability) of all reference standards and critical reagents, such as antibodies, labeled analytes, and matrices.
Source
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
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