HILIC-UPLC-MS for exploratory urinary metabolic profiling in toxicological studies

Area of Science:

• Metabolomics
• Analytical Chemistry
• Toxicology

Background:

• Reversed-phase chromatography has limitations in analyzing highly polar metabolites.
• Global metabolic profiling is crucial for understanding toxicity and protective effects.
• Hepatotoxicity studies require robust analytical methods for complex biological samples.

Purpose of the Study:

• To investigate hydrophilic interaction ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (HILIC-UPLC-TOF-MS) for global metabolic profiling of rat urine.
• To assess the repeatability and stability of the HILIC-UPLC-TOF-MS method over an extended run time.
• To identify metabolic changes in rat urine during galactosamine-induced hepatotoxicity and evaluate the protective effect of glycine.

Main Methods:

• HILIC-UPLC-TOF-MS was employed for the analysis of rat urine samples.
• Quality control samples were repeatedly analyzed to assess within-run repeatability and batch stability.
• Metabolic profiles were analyzed to classify samples according to treatment groups (galactosamine, glycine, control).

Main Results:

• HILIC-UPLC-TOF-MS demonstrated excellent within-run repeatability (retention time CV < 1.7%, signal intensity CV < 14%) and mass accuracy (<0.005 amu).
• Significant metabolic profile alterations were observed in galactosamine-treated rats, including increased urocanic acid and N-acetylglucosamine, and decreased 2-oxoglutarate.
• A novel finding was the elevated urinary tyramine in galactosamine-exposed rats, indicating disturbed amino acid metabolism.

Conclusions:

• HILIC-UPLC-MS is a promising and reproducible method for global metabolic profiling of rat urine, even over extended run times.
• The method provides enhanced coverage of polar metabolites compared to reversed-phase chromatography, aiding biomarker discovery.
• This approach successfully identified key metabolic perturbations in galactosamine-induced hepatotoxicity.