Combined HPLC, NMR Spectroscopy, and Ion-Trap Mass Spectrometry with Application to the Detection and Characterization of Xenobiotic and Endogenous Metabolites in Human Urine

Abstract

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.

Application of Directly Coupled High Performance Liquid Chromatography-NMR-Mass Spectometry and 1H NMR Spectroscopic Studies to the Investigation of 2,3-Benzofuran Metabolism in Sprague-Dawley Rats

“…However, NMR spectroscopic methods are not always sufficient for metabolite structure elucidation, for example in the determination of sulfate conjugates, because the introduced moiety does not contain NMR-detectable nuclei. In such cases, directly coupled HPLC-NMR with parallel MS has the potential to allow full structural characterization of xenobiotic metabolites in a complex biological matrix in a single chromatographic run (Shockcor et al, 1996;Abel et al, 1999;Bailey et al, 2000). By combining the NMR and MS data acquisition in a directly coupled system, the problems of synchronizing LC-MS and HPLC-NMR data are overcome.…”

The application of NMR-based metabonomics in neurological disorders

“…59,60 Once a potential biomarker candidate of disease class or response has been identified, then the NMR spectroscopic detection can be physically integrated with HPLC-MS to effect simultaneous molecular characterization of separated biomolecules from biofluid samples. 61 The recent development of ultra high-pressure liquid chromatography (UPLC) has further enhanced the capabilities for metabolite isolation and detection. The advances offered by UPLC include higher metabolite concentration per band, more rapid separation, and improved resolution.…”

Peer Reviewed: Advancing Hyphenated Chromatographic Systems.

“…This was soon followed by the first application of gradient reversed-phase HPLC/NMR/MS using the well-known metabolites of acetaminophen, which were present in the solid-phase extracts of human urine following a normal therapeutic dose of the drug (6). Acetaminophen provides a useful "proof of concept" test because a major metabolite is acetaminophen sulfate.…”