Hydrophilic interaction chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectroscopy—A new approach for the separation and identification of extremely polar analytes in bodyfluids

Abstract

A method for the unambiguous identification of highly polar molecules based on the separation on a silica gel column run in hydrophilic interaction chromatography (HILIC) mode followed by mass spectroscopic (MS) analysis and subsequent measurement by nuclear magnetic resonance (NMR) spectroscopy is described. Polar neutral, acidic and basic compounds of small molecular size usually not retained on reversed phase stationary phases can be separated and unequivocally identified by means of MS and NMR spectroscopy. The method is applied to exemplify the identification of the endogenous metabolite trigonelline and the polar antibiotic amoxicilline in human urine.

Introduction

Multivariate statistical techniques such as principle compound analysis (PCA) or partial least squares (PLS) are used routinely to find differences in the composition of endogenous metabolites from body fluids or cell extracts in order to discriminate between different groups of living objects, e.g. in toxicology screening or clinical investigations. Spectroscopic techniques like NMR and MS are typically used for this purpose because of their high information content. Metabonomic investigations based on NMR techniques have been pioneered by Nicholson and Lindon et al. in the 90s [1]. The statistical evaluation of LC–MS data obtained from body fluids was first described by Plumb et al. [2]. The combination of both techniques, i.e. running the same sample with NMR and LC–MS should in principle help to identify an unknown marker. The combined statistical analysis of a large set of spectroscopic data should allow for the identification of compounds of interest as shown by Crockford et al. [3]. This approach, however, cannot distinguish between self-molecular correlations and statistical correlations to a compound experiencing the same changes in concentration. Therefore, the unambiguous identification of discriminating maker cannot be solely based on the combined statistical correlation results.

The only reliable way for unequivocal identification of unknowns involves the separation and isolation of the compound of interest followed by MS and NMR spectroscopic investigations. It has been shown in the past that direct coupling of LC–NMR/MS is a very efficient way for the identification of drug metabolites in body fluids and natural products from plant extracts [4], [5], [6], [7], [8], [9], [10]. In all these cases the analytes of interest were well retained on RP columns. Only Dear et al. [11] reported the use of ion exchange chromatography directly coupled to NMR for the structural elucidation of an extremely polar drug metabolite.

In body fluids, the highest concentrations for endogenous metabolites are found for very polar, small molecules [12], [13] showing only poor retention on reversed phase columns which results in coelution of chromatographic peaks. Due to the fact that these peaks are eluting at a highly aqueous mobile phase and without sufficient separation, the ionisation efficiency in electrospray ionisation might drastically decrease.

Hydrophilic interaction chromatography which was first introduced by Alpert [14] allows for the separation of highly polar molecules and can be coupled directly to the MS due to its use of an aqueous mobile phase [15].

In contrast to reversed phase separations, HILIC chromatography starts with a relatively low polar organic mobile phase on a highly polar stationary phase. Examples for stationary phases used in hydrophilic interaction chromatography are unmodified silica gel or silica whose surface has been modified by polar, covalently bound groups, e.g. aminopropyl- or cyanopropylgroups or three-dimensional crosslinked co-polymers whose surface has been modified by carbamoyl groups [16]. Idborg et al. [17] used a silica-based stationary phase which has been modified with zwitterionic hydrocarbon chains operated in HILIC mode to separate polar endogenous metabolites from urine samples followed by MS detection. Under these conditions, polar compounds are effectively retained on such columns operated in HILIC mode. As solvent systems used for this type of chromatography are the same as used in RP separations the eluent can be directly introduced into atmospheric pressure (AP) ion sources and NMR flow probes or tubes. The current paper describes a method for the identification of very polar endogenous metabolites or xenobiotics in body fluids like urine on an unmodified silica gel column run in HILIC mode directly coupled to a time-of-flight (TOF) mass spectrometer and a peak sampling unit which allows for subsequent transfer into NMR tubes or NMR flow probes. It is demonstrated that sufficient amounts of highly polar compounds can be separated and identified by the combined use of mass and NMR spectroscopic techniques even when originating from complex body fluids like urine.