Method development workflow for quantifying protein biomarkers by hybrid LC-MS/MS.

Industry-standard guidance on method development and validation of hybrid LC-MS/MS assays for protein biomarkers, particularly on evaluation of parallelism, is lacking. Using a protein endogenous to humans and mice as a model analyte, a quantitative hybrid LC-MS/MS workflow was developed using a surrogate matrix approach with a recombinant form of the protein as the calibrant. The developed workflow identified a surrogate matrix, established parallelism between the surrogate and authentic matrices and assessed parallelism between the recombinant and authentic forms of the protein. The final method was qualified using precision and accuracy with recovery assessments. The established workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for endogenous protein biomarkers.