HPLC, UHPLC

Ion Chromatography Coupled to MS for Metabolomic Analysis

Mar 09 2010

Author: Ken Cook, Paul Dewsbury, Karl Burgess, Cees Bruggink on behalf of Thermo Fisher Scientific Chromatography and Mass Spectrometry

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The fields of metabolomics and metabonomics attempt to phenotype and quantify the vast array of metabolites present in biological samples.

Reverse phase HPLC coupled to mass spectroscopy is a valuable tool in the separation and identification of these compounds. Reverse phase HPLC techniques cover a wide range of compounds, however ionic and polar compounds such as organic acids, carbohydrates, nucleotides and amino acids are difficult to separate or even retain on traditional reverse phase columns. Ion exchange chromatography can prove to be a far better separation process for these compounds. The problem with the separation mode in this application relates to the salt eluents employed in ion exchange being incompatible with mass spectrometers. Here we describe an ion exchange system which can achieve good separation of these polar compounds with on-line desalting to allow MS detection. This technique allows studies of key metabolites which do not separate well on traditional reverse phase methods. Some of these ionic metabolites may merely be positional isomers adjacent to each other in a synthetic pathway and as such, isobaric. Mass Spectroscopy alone in these cases would not be enough to give a positive identification between them. There is clearly a requirement for good separation of isomeric polar metabolites.

Ion Chromatography (IC) was developed with eluent suppression techniques in 1978 by Small et al. [1]. It has grown to be the primary analysis technique for the analysis of small anions and a strong method choice for
inorganic cation analysis. It is also a very efficient method for the analysis of small polar compounds such as organic acids and amines [2] all detected by conductivity. Larger charged biomolecules, such as peptides, nucleotides and carbohydrates, are successfully separated with IC but normally without the use of suppression techniques [3,4]. Suppression is routinely linked to conductivity detection of the analyte. In the case of peptides, UV detection is used and carbohydrates can be detected electrochemically with pulsed amperometic detection. However, it is the eluent suppression techniques that are interesting to Mass Spectroscopy [MS] as it converts the high salt eluents from Ion Exchange Chromatography into MS compatible pure water. So the ion exchange separation of these metabolites can still be utilized and coupled to suppression to allow
detection by MS. The field of IC covers an impressively wide range of compounds with an array of column chemistries and detection techniques utilized in addition to suppressed conductivity [5]. The implications of making all these analytes accessible to a MS based metabolomics study, with the aid of an on-line desalting technique are quite evident.

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