Developmental Validation of a High-Specificity Multiplex Assay for Human Body Fluid Identification

Versatility, sensitivity, and ease of use make QQQ-MRM the "gold standard" for the analysis of complex samples. In the current project, QQQ-MRM assay development required characterization of individual precursor-product ion pairs to identify those best suited for use in a multiplex assay. Biomarker proteins, peptides, and transitions were evaluated to identify those that had a unique fragmentation pattern, were abundant, efficiently ionized, and had a mass-to-charge ratio greater than that of the peptide. The targeted-ion inclusion lists compiled for each individual fluid were then used to build a dynamic, retention time restricted QQQ-MRM assay for each body fluid. Based on the results of empirical testing, the optimized settings and controls resulted in the unambiguous detection of all targeted protein biomarkers for saliva, semen, peripheral blood, menstrual fluid, vaginal fluid, and urine. Standard Operating Procedures (SOPs) have been drafted consistent with ISO17025 and ASCLD/LAB-International standards. These SOPs include instructions on sample preparation and instrumental method parameters, along with data-interpretation guidelines. 34 tables, 17 figures, 26 references, and appended methodological materials