Liquid chromatography coupled with high-field proton NMR for profiling human urine for endogenous compounds and drug metabolites

Abstract

A number of applications of high-field NMR spectroscopy have previously been described for the analysis of biological fluids (e.g. blood plasma, urine, bile, cerebrospinal fluid) both for endogenous compounds and for xenobiotics and their metabolites [l-4]. In the field of xenobiotic metabolism it has been amply demonstrated that multinuclear NMR spectroscopy of such biological fluids can provide a rapid and convenient approach to the detection of metabolites [l-8]. Similarly ‘H NMR spectroscopy has proved to be of value in the study of clinical samples and in studies on the mechanisms and time course of toxicity [9-111. However, the complexity of the biological fluid spectra obtained has often been such that identification of sample components from the single-pulse ‘H NMR spectrum has not been straightforward. In such circumstances two orthogonal approaches to the identification of unknowns can be used, singly, or in combination. In the first, sophisticated multipulse (and multidimensional) NMR methods can be used to extract structural information directly. In the second, the problem can be simplified by the selective removal of endogenous contaminants followed, if necessary, by the separation and isolation of a particular analyte. To this end strategies have been devised based on solidphase extraction-chromatography monitored by NMR spectroscopy (SPEC-NMR) [12-141. The extraction and purification of target compounds is achieved by stepwise gradient elution from a suitable SPE phase (e.g. octadecylsilane bonded silica). This methodology has proved to be robust, convenient, inexpensive and rapid. However, despite the practical success of the SPEC-NMR approach it remains a ‘low resolution’ technique (in terms of chromatographic separation), the deficiencies of which are compensated for, to a significant extent, by the NMR spectrometer which serves as the ‘detector’. For very complex mixtures, such as intact biological fluids, the hyphenation of NMR with liquid chromatography might be expected to offer clear benefits. Such systems have already been described [15-201 but, due to the technical difficulties involved, such as insufficient dynamic range and sensitivity and problems with solvent suppression, LC-NMR has not been widely exploited. However, given the significant recent technical advances in this field including increases in NMR spectrometer field strengths, methods for ensuring adequate solvent suppression together with the design of dedicated LC-NMR probes, the authors have