Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very pow erful tool for the structural identification of xenobiotic metabolites ill complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous meta bolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the expe riment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha -methyl -2-naphthyl acetic acid) is administered as the S-enantiomer and is metabol ised in vivo to form its demethylated metabolite which is subsequently conj ugated with beta -D-glucuronic acid as well as with sulfate. Naproxen is al so metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the presen t investigation, the metabolism of naproxen was investigated in urine sampl es with a very simple sample preparation using a combination of directly-co upled HPLC-H-1 NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer s ystem was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra prov ide evidence to distinguish isomers such as the type of glucuronides formed , and the HPLC-MS data allow identification of molecules containing NMR-sil ent fragments such as occur in the sulfate ester. (C) 2001 Elsevier Science B.V. All rights reserved.