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.
Highlights
Supercritical fluid chromatography (SFC) as a viable separation technique was first reported by Klesper et al in 1962 [1]; the decades since have seen a steady increase in its reported use [2] for an ever-expanding range of applications [3]
3.1 Screening of polar urinary metabolites for test compound identification For the planned SFC method development study, it was necessary to identify a set of representative polar compounds that eluted with reasonable peak shapes and retention times in a variety of routinely-used chromatographic conditions
The bridged ethylene hybrid (BEH) 2-EP column was chosen for compound screening since it has been widely used for SFC separations of polar analytes
Summary
Supercritical fluid chromatography (SFC) as a viable separation technique was first reported by Klesper et al in 1962 [1]; the decades since have seen a steady increase in its reported use [2] for an ever-expanding range of applications [3]. Acidic or basic additives has been shown to reduce peak tailing and retention for polar analytes to different extents, depending upon the additive type as well as analyte characteristics, including their octanol/water partition coefficients, relative acidity, molecular size and functional group topology [18,19,20] These findings have led to the suggestion that, with the appropriate combination of stationary phase, modifier and modifier additive, SFC methods can be developed to replace many RPLC or hydrophilic interaction liquid chromatography (HILIC) methods for polar compound analysis [8]. The lack of readily implementable LC-based technologies for the comprehensive and routine profiling of these compounds is a significant constraint on our ability to monitor some basic biochemical differences between normal and diseased (e.g. cancerous) cells New separation strategies such as SFC, which increase metabolome coverage while reducing solvent requirements, are of great interest to metabolic phenotyping studies. The utility of these findings was confirmed by separating a subset of polar analytes in human urine using the Torus Diol column with 20mM ammonium formate in methanol as an organic modifier, and comparing the results to conditions currently in general use for polar analyte separation on SFC
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