Elsevier

Journal of Chromatography A

Volume 1608, 20 December 2019, 460413
Journal of Chromatography A

LC–MS/MS analysis of the central energy and carbon metabolites in biological samples following derivatization by dimethylaminophenacyl bromide

https://doi.org/10.1016/j.chroma.2019.460413Get rights and content
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Highlights

  • Non-discriminant derivatization of biological samples using DmPABr reagent.

  • Single RPLC-MS/MS analysis in positive ionisation mode provides full coverage.

  • Absolute quantification utilising internal standards labelled by stable isotopes.

  • Proof of concept for amines, carboxylic acids and thiol-containing metabolites.

  • Application to a variety of biological matrices including urine and cell extracts.

Abstract

Recent advances in metabolomics have enabled larger proportions of the human metabolome to be analyzed quantitatively. However, this usually requires the use of several chromatographic methods coupled to mass spectrometry to cover the wide range of polarity, acidity/basicity and concentration of metabolites. Chemical derivatization allows in principle a wide coverage in a single method, as it affects both the separation and the detection of metabolites: it increases retention, stabilizes the analytes and improves the sensitivity of the analytes. The majority of quantitative derivatization techniques for LC–MS in metabolomics react with amines, phenols and thiols; however, there are unfortunately very few methods that can target carboxylic acids at the same time, which contribute to a large proportion of the human metabolome. Here, we describe a derivatization technique which simultaneously labels carboxylic acids, thiols and amines using the reagent dimethylaminophenacyl bromide (DmPABr). We further improve the quantitation by employing isotope-coded derivatization (ICD), which uses internal standards derivatized with an isotopically-labelled reagent (DmPABr-D6). We demonstrate the ability to measure and quantify 64 central carbon and energy-related metabolites including amino acids, N-acetylated amino acids, metabolites from the TCA cycle and pyruvate metabolism, acylcarnitines and medium-/long-chain fatty acids. To demonstrate the applicability of the analytical approach, we analyzed urine and SUIT-2 cells utilizing a 15-minute single UPLC-MS/MS method in positive ionization mode. SUIT-2 cells exposed to rotenone showed definitive changes in 28 out of the 64 metabolites, including metabolites from all 7 classes mentioned. By realizing the full potential of DmPABr to derivatize and quantify amines and thiols in addition to carboxylic acids, we extended the coverage of the metabolome, producing a strong platform that can be further applied to a variety of biological studies.

Keywords

Dimethylaminophenacyl bromide
Derivatization
Urine
LC–MS
N-Acetylated amino acids
TCA cycle

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