Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and -ultra high performance liquid chromatography coupled to tandem mass spectrometry: applications to serum, urine and tissue extracts

Abstract

The effective analysis of polar ionic metabolites by LC-MS, such as those encountered in central carbon metabolism, represents a major problem for metabolic profiling that is not adequately addressed using strategies based on either reversed-phase or HILIC methods. Here we have compared analysis of central carbon metabolites on optimized methods using HILIC, porous graphitic carbon or ion pair chromatography (IPC) using tributyl ammonium as IP reagent. Of the 3 chromatographic approaches examined only IPC enabled us to obtain a robust analytical methodology. This system was used to profile more than a hundred endogenous metabolic intermediates in urine, serum and tissue samples. However, whilst we found IPC to be the best of the approaches examined considerable care was still needed to obtain robust data. Thus, in excess of 40 of representative biological samples were needed to "condition" a new analytical column and further 10 matrix injections were then required at the beginning of each analytical batch in order to obtain robust and reproducible chromatographic separations. An additional limitation that we have found was that, for a small number of phosphorylated and poly carboxylic acid metabolites, measurement was only possible if the analytes were present in relatively high concentrations. We also found that, whilst this methodology could be used for the analysis of both in vitro cell culture media, cell extracts, tissue, and biological fluids (blood, urine), for the best results columns should only be used to analyze a single matrix. However, despite the need for extensive column conditioning, and the manifold disadvantages resulting from the contamination of the separation system and mass spectrometer with the ion pair reagent, IPC-MS currently provides the best means of analyzing these polar, ionic and problematic metabolites.

Keywords: Central carbon metabolism; HILIC; Ion pair chromatography; Mass spectrometry; Metabolite profiling; Porous graphitic carbon.