Metabolic profiling by reversed-phase/ion-exchange mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 15:1143:122072. doi: 10.1016/j.jchromb.2020.122072. Epub 2020 Mar 16.

Abstract

Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.

Keywords: Ion-exchange; Liquid chromatography; Mass spectrometry; Metabolomics; Reversed-phase.

MeSH terms

  • Amino Acids / chemistry
  • Bile Acids and Salts / chemistry
  • Carnitine / analogs & derivatives
  • Carnitine / chemistry
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Chromatography, Reverse-Phase
  • Hormones / chemistry
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Isoleucine / blood*
  • Isoleucine / urine*
  • Metabolome
  • Metabolomics / methods*
  • Methylmalonic Acid / blood*
  • Methylmalonic Acid / urine*
  • Nucleosides / chemistry
  • Small Molecule Libraries / chemistry
  • Succinic Acid / blood*
  • Succinic Acid / urine*
  • Tandem Mass Spectrometry
  • Vitamins / chemistry

Substances

  • Amino Acids
  • Bile Acids and Salts
  • Hormones
  • Nucleosides
  • Small Molecule Libraries
  • Vitamins
  • acylcarnitine
  • Isoleucine
  • Methylmalonic Acid
  • Succinic Acid
  • Carnitine