Purification and H-1 NMR spectroscopic characterization of phase II metabolites of tolfenamic acid

TY - JOUR

T1 - Purification and H-1 NMR spectroscopic characterization of phase II metabolites of tolfenamic acid

AU - Sidelmann, U. G.

AU - Christiansen, E.

AU - Krogh, L.

AU - Cornett, Claus

AU - Tjornelund, J.

AU - Hansen, S. H.

PY - 1997

Y1 - 1997

N2 - Tolfenamic acid, an anti-inflammatory drug (NSAID), is metabolized in vivo to form several oxidative metabolites which are all conjugated with beta-D-glucuronic acid, In this study, the metabolites of tolfenamic acid were identified by H-1 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 H-1 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 H-1 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-methoxyphenyl)-anthranilic acid (9), N-(2-methyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (10), and N-(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.

AB - Tolfenamic acid, an anti-inflammatory drug (NSAID), is metabolized in vivo to form several oxidative metabolites which are all conjugated with beta-D-glucuronic acid, In this study, the metabolites of tolfenamic acid were identified by H-1 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 H-1 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 H-1 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-methoxyphenyl)-anthranilic acid (9), N-(2-methyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (10), and N-(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.

KW - chromatography identification

M3 - Tidsskriftartikel

SN - 0090-9556

VL - 25

SP - 725

EP - 731

JO - Drug Metabolism and Disposition

JF - Drug Metabolism and Disposition

IS - 6

ER -