LAUSR

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with stable isotope labeled internal standards (SIL-IS) is the gold standard for quantitative analysis of drugs and metabolites in complex biological samples. Significant isotopic effects associated with deuterium labeling often causes the deuterated IS to elute at a different retention time from the target analyte, diminishing its capability to compensate for matrix effects. In this study, we systematically compared the analytical performance of deuterated (2H) SIL-IS to non-deuterated (13C and 15N) SIL-ISs for quantifying urinary 2-methylhippuric acid (2MHA) and 4-methylhippuric acid (4MHA), biomarkers of xylenes exposure, with an LC-ESI-MS/MS assay. Analytical method comparison between IS demonstrated a quantitative bias for urinary 2MHA results, with concentrations generated with 2MHA-[2H7] on average 59.2% lower than concentrations generated by 2MHA-[13C6]. Spike accuracy, measured by quantifying analyte-spiked urine matrix and comparing the result to the known spike concentration, determined that 2MHA-[2H7] generated negatively biased urinary results of -38.4% whereas no significant bias was observed for 2MHA-[13C6]. Post-column infusion demonstrated that ion suppression experienced by 2MHA and 2MHA-[13C6] was not equally experienced by 2MHA-[2H7], explaining the negatively biased 2MHA results. Quantitation of urinary 4MHA results between IS exhibited no significant quantitative bias. These results underscore the importance of careful selection of internal standards for targeted quantitative analysis in complex biological samples.