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 metabolized in vivo to form several oxidized metabolites which are subsequently conjugated with beta-D-glucuronic acid. In the present investigation, the metabolism of tolfenamic acid was investigated using H-1 NMR spectroscopy of urine samples obtained from a human volunteer after oral administration of 300 mg of the drug. Both one- and two-dimensional NMR experiments were performed in order to identify the phase II metabolites of tolfenamic acid in the stepwise-eluted solid phase extraction fractions of urine. To identify the metabolites unambiguously, directly coupled 800 MHz HPLC-NMR spectroscopy was applied to selected samples. The beta-1-O-acyl glucuronides of the parent compounds N-(2-methyl-3-chlorophenyl)anthranilic acid, N-(2-hydroxymethyl-3-chlorophenyl)anthranilic acid, N-(2-hydroxymethyl-3-chloro-4-hydroxyphenyl)anthranilic acid, N-(2-formyl-3-chlorophenyl)anthranilic acid, N-(2-methyl-3-chloro-4-hydroxyphenyl)anthranilic acid, and N-(2-methyl-3-chloro-4-hydroxyphenyl)anthranilic acid were identified. None of these phase II metabolites has previously been identified directly in biofluids. The first practical demonstration of directly coupled 800 MHz HPLC-NMR to a problem in drug metabolism is shown here. By the use of this ultrahigh-field NMR spectrometer, the gain in spectral dispersion allowed the determination of the exact position of hydroxylation on the aromatic rings of tolfenamic acid.