Hplc-nmr identification of the human urinary metabolites of (—)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV)

Abstract

1. Human urine samples from a clinical trial of the anti-HIV compound (—)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-cytosine (BW524W91) have been analysed using ¹⁹F-nmr and ¹H-hplc-nmr spectroscopy.

2. The identities and relative levels of the xenobiotic species in the urine have been determined by 470-MHZ ¹⁹F-nmr spectroscopy and by directly coupled 600-MHz ¹H-hplc-nmr in the stop-flow mode with confirmation of the metabolite identities being made by comparison with nmr spectra of synthetic standard compounds.

3. The principal urinary xenobiotic was the unchanged drug, but the glucuronide ether conjugate at the 5' position of BW524W91, one of the two diastereomeric sulphoxides and the deaminated metabolite were also characterized.

4. The detection limit of directly coupled hplc-600-MHz ¹H-nmr spectroscopy was evaluated by measuring two-dimensional nmr spectra of the glucuronide conjugate of BW524W91 and shown to be approximately 1 μg material for ¹H-¹H-TOCSY and 20 μg metabolite for ¹H-¹³C-HMQC spectra for overnight (16 h) acquisition.