COMBINED HPLC, NMR-SPECTROSCOPY, AND ION-TRAP MASS-SPECTROMETRY WITH APPLICATION TO THE DETECTION AND CHARACTERIZATION OF XENOBIOTIC AND ENDOGENOUS METABOLITES IN HUMAN URINE

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 ''triple-hyphenated'' HPLC-NMR-MS system. Combined UV, IH NMR, an d positive-ion electrospray MS detection was achieved in the continuou s-flow mode using whole human urine from a subject dosed with acetamin ophen. By means of HPLC-NMCR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirm ation of the identity of the acetaminophen glucuronide and sulfate met abolites, together with a number of endogenous metabolites. In particu lar, the HPLC-NMR-MS approach allowed the unequivocal identification o f phenylacetylglutamine in human urine, an endogenous metabolite not p reviously observed in IH NMR spectra of urine because of extensive ove rlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation wi th HPLC are discussed. The new technique of HPLC-NMR-MS will provide t he scope for more comprehensive and fully automated analysis of bioflu ids and other complex mixtures than was previously available from sing le hyphenation of these instruments.