King's College London

Research portal

Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry

Research output: Contribution to journalArticle

Arundhuti Sen, Christopher Knappy, Matthew R. Lewis, Robert S. Plumb, Ian D. Wilson, Jeremy K. Nicholson, Norman W. Smith

Original languageEnglish
Pages (from-to)141–155
JournalJournal of Chromatography A
Volume1449
Early online date19 Apr 2016
DOIs
Accepted/In press14 Apr 2016
E-pub ahead of print19 Apr 2016
Published3 Jun 2016

Documents

  • 1-s2.0-S0021967316304630-main

    1_s2.0_S0021967316304630_main.pdf, 253 KB, application/pdf

    Uploaded date:20 Apr 2016

    Version:Accepted author manuscript

    Licence:CC BY-NC-ND

King's Authors

Abstract

Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7 ≤ cLogP ≤ 2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO2-methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO2-methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7 min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.

Download statistics

No data available

View graph of relations

© 2018 King's College London | Strand | London WC2R 2LS | England | United Kingdom | Tel +44 (0)20 7836 5454