Critical assessment of chromatographic metadata in publicly available metabolomics data repositories.

INTRODUCTION: The structural identification of metabolites represents one of the current bottlenecks in non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics. The Metabolomics Standard Initiative has developed a multilevel system to report confidence in metabolite identification, which involves the use of MS, MS/MS and orthogonal data. Limitations due to similar or same fragmentation pattern (e.g. isomeric compounds) can be overcome by the additional orthogonal information of the retention time (RT), since it is a system property that is different for each chromatographic setup. OBJECTIVES: In contrast to MS data, sharing of RT data is not as widespread. The quality of data and its (re-)useability depend very much on the quality of the metadata. We aimed to evaluate the coverage and quality of this metadata from public metabolomics repositories. METHODS: We acquired an overview on the current reporting of chromatographic separation conditions. For this purpose, we defined the following information as important details that have to be provided: column name and dimension, flow rate, temperature, composition of eluents and gradient. RESULTS: We found that 70% of descriptions of the chromatographic setups are incomplete (according to our definition) and an additional 10% of the descriptions contained ambiguous and/or incorrect information. Accordingly, only about 20% of the descriptions allow further (re-)use of the data, e.g. for RT prediction. Therefore, we have started to develop a unified and standardized notation for chromatographic metadata with detailed and specific description of eluents, columns and gradients. CONCLUSION: Reporting of chromatographic metadata is currently not unified. Our recommended suggestions for metadata reporting will enable more standardization and automatization in future reporting.

Current state-of-the-art of separation methods used in LC-MS based metabolomics and lipidomics.

Metabolomics deals with the large-scale analysis of metabolites, belonging to numerous compound classes and showing an extremely high chemical diversity and complexity. Lipidomics, being a subcategory of metabolomics, analyzes the cellular lipid species. Both require state-of-the-art analytical methods capable of accessing the underlying chemical complexity. One of the major techniques used for the analysis of metabolites and lipids is Liquid Chromatography-Mass Spectrometry (LC-MS), offering both different selectivities in LC separation and high sensitivity in MS detection. Chromatography can be divided into different modes, based on the properties of the employed separation system. The most popular ones are Reversed-Phase (RP) separation for non- to mid-polar molecules and Hydrophilic Interaction Liquid Chromatography (HILIC) for polar molecules. So far, no single analysis method exists that can cover the entire range of metabolites or lipids, due to the huge chemical diversity. Consequently, different separation methods have been used for different applications and research questions. In this review, we explore the current use of LC-MS in metabolomics and lipidomics. As a proxy, we examined the use of chromatographic methods in the public repositories EBI MetaboLights and NIH Metabolomics Workbench. We extracted 1484 method descriptions, collected separation metadata and generated an overview on the current use of columns, eluents, etc. Based on this overview, we reviewed current practices and identified potential future trends as well as required improvements that may allow us to increase metabolite coverage, throughput or both simultaneously.

Development and application of a HILIC UHPLC-MS method for polar fecal metabolome profiling.

The fecal metabolome is a complex mixture of endogenous, microbial metabolites, and food derived compounds. Hydrophilic interaction liquid chromatography (HILIC) enables the analysis of polar compounds, which is a valuable alternative to reversed-phase liquid chromatography in the field of metabolomics due to its ability to retain a greater portion of the polar metabolome. The objective of the study was to find the optimal chromatographic solution to perform non-targeted metabolomics of feces by means of HILIC ultra-high-pressure liquid chromatography mass spectrometry (UHPLC-Q-TOF-MS). The performance was systematically investigated analyzing a pooled fecal sample, and mixtures of 150 metabolites from different families, including for example amino acids, amines, indole derivatives, fatty acids and carbohydrates. Three different stationary phases (zwitterionic, amide and unbonded silica) were operated at three pH values (4.6, 6.8 and 9.0), and three salt gradient conditions (5-5, 5-10 and 5-25 mM ammonium acetate). Amide and zwitterionic stationary phases performed similarly at low pH, with highest number of detected standards, which increased by increasing the salt gradient. The amide column showed slightly better performance in terms of separation of isomers and peak widths and remarkably good performance at basic pH, with highest number of metabolite features in the non-targeted analysis. The zwitterionic column operated best in terms of number of detected standards, retention time distribution of standards and metabolite feature across whole chromatographic run. Thus, the zwitterionic column was proven to suit for non-targeted analysis of fecal samples, resulting in good coverage of especially amino acids and carbohydrates.