DIRECTLY COUPLED 880 MHZ HPLC-NMR SPECTROSCOPY OF URINE AND ITS APPLICATION TO THE IDENTIFICATION OF THE MAJOR PHASE-II METABOLITES OF TOLFENAMIC ACID

Tolfenamic acid (N-(2-methyl-3-chlorophenyl)anthranilic add) is metabo lized in vivo to form several oxidized metabolites which are subsequen tly conjugated with beta-D-glucuronic acid. In the present investigati on, the metabolism of tolfenamic acid was investigated using H-1 NMR s pectroscopy of urine samples obtained from a human volunteer after ora l administration of 300 mg of the drug. Both one- and two-dimensional NMR experiments were performed in order to identify the phase II metab olites of tolfenamic acid in the stepwise-eluted solid phase extractio n fractions of urine. To identify the metabolites unambiguously, direc tly coupled 800 MHz HPLC-NMR spectroscopy was applied to selected samp les. The beta-1-O-acyl glucuronides of the parent compounds N-(2-methy l-3-chlorophenyl)anthranilic acid, N-(2-hydroxymethyl-3-chlorophenyl)a nthranilic acid, hydroxymethyl-3-chloro-4-hydroxyphenyl)anthranilic ac id, N-(2-formyl-3-chlorophenyl)anthranilic acid, N-(2-methyl-3-chloro- 4-hydroxyphenyl)anthranilic acid, and N-(2-methyl-3-chloro-4-hydroxyph enyl)anthranilic acid were identified. None of these phase II metaboli tes has previously been identified directly in biofluids. The first pr actical demonstration of directly coupled 800 MHz HPLC-NMR to a proble m in drug metabolism is shown here. By the use of this ultrahigh-field NMR spectrometer, the gain in spectral dispersion allowed the determi nation of the exact position of hydroxylation on the aromatic rings of tolfenamic acid.