MSACL 2023 Abstract

Mitigating Matrix Effects in LC-ESI-MS/MS Analysis: Comparison of 2H- and 13C/13N-labeled Internal Standards

Brett A. Bowman, Benjamin C. Blount, Deepak Bhandari
Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention

Brett Bowman, B.S. Chemistry (Presenter) Centers for Disease Control and Prevention

Presenter Bio: Chemist specializing in LC-MS/MS for biomonitoring purposes

Relevant Financial Disclosures (within past 24 months, reported on Feb 17, 2023) Salary Centers for Disease Control and Prevention Conflict mitigation NOT REQUIRED.

INTRODUCTION:
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. Relatively significant isotopic effects associated with deuterium labeling often cause the deuterated IS to elute at a different retention time from the target analyte, diminishing its capability to compensate for matrix effects.

OBJECTIVES:
The primary objective of this study is to compare the effectiveness of 2H-labeled internal standards to 13C/15N-labeled internal standards for accounting matrix effects in urine specimens.
METHODS: The analytical performance of a dilute-and-shoot LC-ESI-MS/MS method with both 2H-labeled and 13C/15N-labeled ISs was examined for two biomarkers of xylene exposure (2MHA and 4MHA). Urinary 2MHA and 4MHA concentrations were assessed in 37 human urine specimens, and an analytical method comparison was performed. Due to significant differences in urinary results between internal standards, further validation experiments were performed. Spike accuracy across 14 urine specimens was performed to assess the accuracy of each internal standard. Post-column tee-infusion experiments were performed to identify the matrix effects.

RESULTS:
Analytical method comparison between ISs demonstrated that the 2H-labeled IS resulted in significantly lower urinary 2MHA concentrations compared to concentrations generated with the 13C-labeled IS. Accuracy through spiking experiments determined that the 2H-labeled IS exhibited a negative bias for 2MHA in urine matrix whereas the 13C-labeled IS generated accurate urinary results. Post-column infusion identified that non-equivalent matrix effects experienced by the 2H-labeled IS and the unlabeled 2MHA resulted in inaccurate urinary concentrations. Due to insignificant isotopic effects of 13C-labeling, coelution of the unlabeled analyte and the 13C-labeled IS occurred, and the internal standard experienced equivalent matrix effects. No bias was observed across ISs for 4MHA, and post-column infusion demonstrated that equivalent matrix effects were observed for 2H-labeled IS, 13C/15N-labeled IS, and unlabeled 4MHA.

CONCLUSION:
Selection and validation of a SIL-IS is a critical step in the development of an effective LC-MS/MS assay for biomonitoring applications. The relatively significant isotopic effects observed with 2H-labeling causes different retention times to the unlabeled analyte, and therefore may bias quantitation in complex biological matrices.

DISCLAIMER:
The views and opinions expressed in this report are those of the authors and do not necessarily represent the views, official policy or position of the U.S. Department of Health and Human Services or any of its affiliated institutions or agencies. The use of trade names is for identification purposes and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services.