Generic approach to validation of small-molecule LC-MS/MS biomarker assays.

While the regulatory guidelines that describe the validation requirements for small molecules are very comprehensive, they are written primarily for xenobiotic drug molecules. However, the presence of endogenous analyte in control matrix presents an added analytical challenge that must be overcome if small-molecule biomarker assays are to be developed and characterized, especially where downregulation of analyte concentrations is expected. A generic surrogate matrix calibration protocol has been successfully applied to the measurement of a number of small-molecule exploratory biomarkers using LC-MS/MS. The use of analyte-free matrix enables conventional calibration curves to be constructed across the anticipated range of sample concentrations. The evaluation of matrix effects is carried out using an experiment similar to the parallelism experiment used in ligand-binding assays. There is currently no published consensus approach to validation of small-molecule biomarker methods. This paper presents a generic approach to endogenous method validation for consideration as bioanalytical best practice for this type of assay.