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Poster Abstract
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Optimization of workflow for HILIC separations in LC-MS-based metabolomics
Debasish Ghosh1; Chris Beecher2; Marcel L Prokai1; Rachel Valentine1; Ron Benson3; Alex Schrum3; Tanya Napolitano3; Felice De Jong2; Vladimir Shulaev1
1University of North Texas, Denton, TX; 2IROA Technologies, Chapel Hill, NC; 3Shodex, New York, NY
Introduction
Metabolomics involves analysis of hundreds of metabolites in complex matrices. Most of the endogenous metabolites are polar in nature making their chromatographic separation and analysis challenging. In recent years hydrophilic interaction chromatography (HILIC) gained popularity for separation of polar metabolites. Despite being an excellent technique to retain polar metabolites HILIC requires extensive method development. For metabolomics applications the major challenge is the selection of chromatographic columns that can efficiently separate hundreds of cellular metabolites and are stable at the wide pH range to be useful in metabolomics applications. Here we present the development of optimized workflow for evaluation of LC-MS columns for HILIC separations in metabolomics using the IROA MSMLS library.
Methods
UPLC-ESI-MS was carried out on a Thermo 1250 Accela UPLC system coupled to the Thermo Orbitrap Velos Pro mass spectrometer operated in the ESI mode. We used a library composed of 600 authentic unlabeled metabolites belonging to different metabolite classes that was prepared from an accurately prepared mother liquor. All the compounds are distributed across six 96-well plates. From each plate 30 µL per well was pooled to prepare retention time library. Compounds were separated using different buffer systems to optimize the efficiency of separation and peak quality. Separations were performed using a Shodex HILICpak VG-25 2B (2.0 × 50 mm) polymer-based HILIC column with an amino functionalized polyvinyl alcohol stationary phase (2.5 µm particles).
Preliminary Data
The IROA MSMLS (Mass Spectrometry Metabolite Library of Standards) is a collection of high‑purity small biochemical compounds designed to support mass spectrometry‑based metabolomics. The library contains 600 metabolites covering different metabolite classes and provides a good representation of polar metabolome. It a broad coverage of metabolites involved in primary metabolism, like amino acids, organic acids, nucleotides, sugars, lipids, and vitamins. The IROA MSMLS library proved to be a very useful resource for evaluation of chromatographic columns and optimization of separation conditions for HILIC analysis in metabolomics. The selection of unique sets of metabolites can be used to build robust retention time and spectral libraries to improve metabolite identification and quantification in both untargeted and targeted studies. In our experiments Shodex HILICpak VG-25 column provided high efficiency and robust recovery of large number of polar metabolites and reducing sugars, making it well suited for LC–MS-based metabolomics analysis of highly polar compounds. To improve the chromatographic separation, we tested several buffer systems. The final method provided good chromatographic separation for polar and highly polar metabolites. The suitability of the method for reproducible analysis of polar metabolites in biological samples was successfully demonstrated by profiling polar extracts.
Novel Aspect
The improved method can be used for high-throughput evaluation of LC-MS columns for HILIC separation in metabolomics applications.
Conflict of Interest Disclosure
CB, and FDJ are employees of IROA Technologies; RB, TN and AS are employees of Resonac America Inc. (Shodex)