HILIC-UPLC-MS for Exploratory Urinary Metabolic Profiling in Toxicological Studies

Abstract

Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) permits the analysis of highly polar metabolites, providing complementary information to reversed-phase (RP) chromatography. HILIC-UPLC-TOF-MS was investigated for the global metabolic profiling of rat urine samples generated in an experimental hepatotoxicity study of galactosamine (galN) and the concomitant investigation of the protective effect of glycine. Within-run repeatability and stability over a large sample batch (>200 samples, 60 h run-time) was assessed through the repeat analysis of a quality control sample. Following system equilibration, excellent repeatability was observed in terms of retention time (CV < 1.7%), signal intensity (CV < 14%), and mass variability (<0.005 amu), providing a good measure of reproducibility. Classification of urinary metabolic profiles according to treatment was observed, with significant changes in specific metabolites after galN exposure, including increased urocanic acid, N-acetylglucosamine, and decreased 2-oxoglutarate. A novel finding from this HILIC-UPLC-MS approach was elevated urinary tyramine in galN-treated rats, reflecting disturbed amino acid metabolism. These results show HILIC-UPLC-MS to be a promising method for global metabolic profiling, demonstrating high within-run repeatability, even over an extended run time. Retention of polar endogenous analytes and xenobiotic metabolites was improved compared with RP studies, including galN, N-acetylglucosamine, oxoglutarate, and urocanic acid, enhancing metabolome coverage and potentially improving biomarker discovery.

Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation

“…Retention occurs through solute partitioning between a pseudoimmobilised water layer and the bulk mobile phase, as well as by adsorption and ionic interactions. There are important applications of the technique in biomedical and biological science, including in pharmaceutical analysis [1], metabolomic fingerprinting [2,3], anti-doping investigations [4,5] and clinical applications [6]. The mobile phases used in HILIC are typically hydro-organic mixtures of an aprotic solvent such as acetonitrile (>70%ACN, v/v) in the presence of a soluble buffer (e.g.…”

Untargeted metabolomic analysis of human serum samples associated with different levels of red meat consumption: A possible indicator of type 2 diabetes?

“…The analyzed data was exported to Simca 14 (Umetrics, Umea, Sweden) for multivariate analysis. Data quality was evaluated in terms of reproducibility using an approach utilize in others works by Spagou et al, 2011. As a quality control estimate all the spectral data were mean centered, pareto scaled and analyzed with principal components analysis (PCA).…”

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…is is also because there has been no de nite and versatile inlet method that is able to simultaneously separate a wide variety of metabolites. In the case of LC/MS metabolomics, there are at least 5 groups of LC conditions, which include reverse phase analysis using octadecylsilyl (ODS) column for relatively hydrophobic metabolites, 45) normal phase analysis using columns for hydrophilic interaction chromatography (HILIC) of hydrophilic metabolites, [46][47][48] reverse phase analysis using pentafluorophenylpropyl stationary phase column for hydrophilic metabolites, 49) ion-paring methods using ODS column for anionic metabolites, 50) and lipidomics methods using ODS column for lipids. Recently, a comparison of the chromatographic performance of a traditional ODS column with various HILIC and mixed-mode columns showed that ODS and zwitterionic HILIC columns are the best combination for wide metabolite coverage.…”