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See detailAn exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices
Iturrospe, Elias; Da Silva, Katyeny Manuela; Talavera Andujar, Begona UL et al

in Journal of Chromatography. A (2020)

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad ... [more ▼]

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features ( < 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes. [less ▲]

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See detailIsotopologue ratio normalization for non-targeted metabolomics
Weindl, Daniel UL; Wegner, André UL; Jäger, Christian UL et al

in Journal of Chromatography. A (2015), 1389

Robust quantification of analytes is a prerequisite for meaningful metabolomics experiments. In non-targeted metabolomics it is still hard to compare measurements across multiple batches or instruments ... [more ▼]

Robust quantification of analytes is a prerequisite for meaningful metabolomics experiments. In non-targeted metabolomics it is still hard to compare measurements across multiple batches or instruments. For targeted analyses isotope dilution mass spectrometry is used to provide a robust normalization reference. Here, we present an approach that allows for the automated semi-quantification of metabolites relative to a fully stable isotope-labeled metabolite extract. Unlike many previous approaches, we include both identified and unidentified compounds in the data analysis. The internal standards are detected in an automated manner using the non-targeted tracer fate detection algorithm. The ratios of the light and heavy form of these compounds serve as a robust measure to compare metabolite levels across different mass spectrometric platforms. As opposed to other methods which require high resolution mass spectrometers, our methodology works with low resolution mass spectrometers as commonly used in gas chromatography electron impact mass spectrometry (GC–EI-MS)-based metabolomics. We demonstrate the validity of our method by analyzing compound levels in different samples and show that it outperforms conventional normalization approaches in terms of intra- and inter-instrument reproducibility. We show that a labeled yeast metabolite extract can also serve as a reference for mammalian metabolite extracts where complete stable isotope labeling is hard to achieve. [less ▲]

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