Application of directly coupled LC–NMR–MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2′,3′-dideoxy-3′-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC–NMR and LC–NMR–MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC–NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC–NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC–NMR–MS which furnished the molecular weight, and through the use of MS–MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.