title: Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry.
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 7min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.
authors.affiliation: Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, Franklin-Wilkins Building, King's College London, London SE1 9NH, United Kingdom; MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine, Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom.
authors.forename: Arundhuti
authors.initials: A
authors.lastname: Sen
authors.affiliation: Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, Franklin-Wilkins Building, King's College London, London SE1 9NH, United Kingdom; Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2DD, United Kingdom; MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine, Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: Knappy
authors.affiliation: MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine, Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Lewis
authors.affiliation: Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2DD, United Kingdom; Waters Corporation, Milford, MA, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Plumb
authors.affiliation: Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2DD, United Kingdom.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Wilson
authors.affiliation: Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2DD, United Kingdom; MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine, Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom.
authors.forename: Jeremy K
authors.initials: JK
authors.lastname: Nicholson
authors.affiliation: Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, Franklin-Wilkins Building, King's College London, London SE1 9NH, United Kingdom. Electronic address: norman.2.smith@kcl.ac.uk.
authors.forename: Norman W
authors.initials: NW
authors.lastname: Smith
mesh_terms: D000085:Acetates / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D064753:Ammonium Hydroxide / Q000737:chemistry; D002853:Chromatography, Liquid; D025924:Chromatography, Supercritical Fluid / Q000379:methods; D005561:Formates / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods; D055442:Metabolome; D000432:Methanol; D012997:Solvents; D014556:Urine; D014867:Water / Q000737:chemistry
chemical_list: D000085:Acetates; D000588:Amines; D005561:Formates; D012997:Solvents; D014867:Water; D064753:Ammonium Hydroxide; D000432:Methanol
keywords: Additive; Method development; Modifier; Polar; SFC; Stationary phase