Abstract
Tolfenamic acid, an anti-inflammatory drug (NSAID), is metabolized in vivo to form several oxidative metabolites which are all conjugated with β-d-glucuronic acid. In this study, the metabolites of tolfenamic acid were identified by 1H nuclear magnetic resonance (NMR) spectroscopy in urine samples obtained on days 7 to 10 from a human volunteer after oral administration of 200 mg of the drug three times per day (steady-state plasma concentration). The metabolites of tolfenamic acid were initially concentrated by preparative solid phase extraction (PSPE) chromatography, thereby removing the endogenous polar compounds that are present in the urine. The individual metabolites were purified by preparative high performance liquid chromatography (HPLC) and then identified using 1H NMR. Both one- and two-dimensional NMR experiments were performed to identify the phase II metabolites of tolfenamic acid; the study shows the applicability of 1H NMR for the identification of drug metabolites in biological fluids. In addition to NMR analysis, two metabolites were also identified by mass spectrometry (MS). The glucuronides of the following parent compounds,N-(2-methyl-3-chlorophenyl)-anthranilic acid (T),N-(2-hydroxymethyl-3-chlorophenyl)-anthranilic acid (1),N-(2-hydroxymethyl-3-chloro-4-hydroxyphenyl)-anthranilic acid (2), N-(2-formyl-3-chlorophenyl) anthranilic acid (3),N-(2-methyl-3-chloro-4-hydroxyphenyl)-anthranilic acid (4),N-(2-methyl-3-chloro-5-hydroxyphenyl)-anthranilic acid (5), N-(2-carboxy-3-chlorophenyl)-anthranilic acid (6),N-(2-hydroxymethyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (7),N-(2-methyl-3-chlorophenyl)-5-hydroxy-anthranilic acid (8),N-(2-methyl-3-chloro-4-metoxyphenyl)-anthranilic acid (9),N-(2-methyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (10), andN-(2-methyl-4-hydroxyphenyl)-anthranilic acid (11) were identified. The phase II metabolites (5–11) had not previously been identified in urine from humans administered tolfenamic acid. The phase I metabolites of the glucuronides 7, 8, 10, and 11 were identified here for the first time. An HPLC method was developed that simultaneously separates all the phase II metabolites identified as well as some phase I metabolites in urine samples obtained after intake of tolfenamic acid.
Footnotes
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Send reprint requests to: Ulla Grove Sidelmann, Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, Dk-2100 Copenhagen, Dennmark.
- Abbreviations used are::
- NSAID
- nonsteroidal anti-inflammatory drug
- TFA
- trifluoroacetic acid
- PSPE
- preparative solid phase extraction
- Received December 4, 1996.
- Accepted March 18, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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