MSACL 2025 Abstract

Targeted LC-MRM Method to Monitor Biomarkers of Liver Metabolic Activity in a Rat Model

Kahina Chabi (1), François-Xavier Cossette (2), Mélanie Tremblay (2), Mariana Oliveira (2), Christopher F. Rose (2), Lekha Sleno (1)
(1) University of Quebec in Montréal (UQAM) Chemistry Department, (2) Hepato-Neuro Laboratory, CRCHUM, Université de Montréal, Montreal, QC, Canada

Kahina Chabi (Presenter) Université du Québec à Montréal

Presenter Bio: Kahina is currently pursuing her Ph.D. in Biochemistry at the Université du Québec à Montréal (UQAM) under the supervision of Professor Lekha Sleno. She completed her bachelor’s and master’s degrees in biochemistry at UQAM, where she developed a strong interest in mass spectrometry and drug metabolism. Her doctoral research focuses on the development of advanced LC-MS/MS methods to investigate metabolic alterations associated with liver diseases and to identify potential biomarkers of liver function. Her work combines small molecule and protein analysis. She is involved in collaborative research projects and is passionate about translating analytical methods into clinically relevant applications.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Liver disease progresses silently and is often diagnosed and monitored with clinical biomarkers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST); enzymes released during hepatocyte cell death. However, these biomarkers not only lack specificity and sensitivity, but they do not reflect real-time liver metabolic capacity. A minimally invasive liver test to monitor function, progression as well as to adjust treatments remains an unmet clinical need. Acetaminophen (APAP), a widely used analgesic, is predominantly metabolized in the liver through three main pathways: glucuronidation, sulfation, and cytochrome P450–mediated oxidation into NAPQI, a toxic intermediate subsequently conjugated with glutathione and eliminated via the mercapturic acid pathway. The goal of this study is to develop a method to quantitatively measure circulating APAP metabolites following a low-dose administration in a rat model.

METHODS:
We developed a targeted LC-MRM method, on a Sciex QTRAP 5500 platform, to quantify six key APAP-derived metabolites, from glucuronidation, sulfation, oxidation, and conjugation with glutathione. Chromatographic separation was optimized using a ZORBAX Eclipse Plus C18 column with a gradient elution program. APAP was administered orally at 25 and 50 mg/kg to naïve male and female rats (~225 g) and blood samples were longitudinally collected. Serum proteins were precipitated using acetonitrile, and supernatants were dried under vacuum, reconstituted in aqueous solution, and analyzed by LC-MRM. Method performance (sensitivity, reproducibility, linearity) is being evaluated using both matrix-matched calibration curves and using stable-isotope-labeled internal standards.

PRELIMINARY RESULTS:
The developed method enables robust detection and quantification of six APAP metabolites in rat serum. The MRM transitions were optimized to ensure high selectivity and sensitivity, and the chromatographic conditions resulted in excellent peak shape and retention time reproducibility. Peak serum concentrations of metabolites were observed at approximately 30 minutes post-dose, suggesting this timepoint as optimal for capturing metabolic activity. The lower dose tested (25 mg/kg) was sufficient to produce a measurable metabolic profile, supporting its use in future low-dose longitudinal or clinical studies. Sex-based differences were also observed: female rats displayed a delayed clearance of certain conjugated metabolites, notably the glucuronide and cysteine derivatives, compared to their male counterparts. This trend, which suggests possible sex-specific differences in phase II conjugation or renal excretion, requires to be further investigated. These findings underscore the importance of including both sexes in preclinical biomarker development. The method is being applied to rats of a model of cholestatic liver injury, to assess the impact on APAP metabolism. Ultimately, metabolite ratios will be explored as composite biomarkers of liver enzymatic capacity and redox status.

CONCLUSION and PERSPECTIVES:
We present a robust and reproducible LC-MS/MS method to monitor low-dose APAP metabolism in rats as a potential tool for assessing liver health status in a rat model. Preliminary data in SHAM-operated animals confirm the method’s sensitivity and reveal metabolic differences between sexes. The use of low-dose APAP was adequate to capture relevant metabolic profiles, supporting its applicability in minimally invasive and translational contexts. This platform holds promise for the development of a functional liver biomarker strategy.