Direct Nuclear Magnetic Resonance Spectroscopic Analysis of C-labeled Antipyrine Metabolites in Human Urine

Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe an approach using C labeling and NMR spectroscopy for the direct and simultaneous analysis of major metabolites of antipyrine in human urine. [C-Methyl-C]antipyrine (500 mg) was dosed orally to human volunteers, and the post-dose urine was analyzed by 100-MHz C NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer (DEPT) without any pretreatments such as deconjugation, chromatographic separation, or solvent extraction. Consequently, all the major metabolites in urine were successfully detected with favorable signal-tonoise ratios in the limited acquisition time (30 min). The reproducibility of the NMR detection was sufficient for the quantitative evaluation of the metabolic profile. A quantitative method is proposed using a combination of inverse gated decoupling and DEPT experiments with [2-C]sodium acetate as an internal standard. The present approach is useful and practical to evaluate variation of in vivo activities of the conjugation enzymes as well as oxidative enzymes responsible for the formation of antipyrine metabolites in humans. This direct approach would enhance the value of the antipyrine test because of its simplicity and convenience. Antipyrine, an antipyretic and analgesic, has been extensively used as a probe to study the influence of age, diseases, drugs, heredity, and environmental factors on oxidative hepatic drug-metabolizing capacity (Hartleb, 1991; St. Peter and Awni, 1991). In humans, antipyrine is metabolized by several forms of cytochrome P450 (Sharer and Wrighton, 1996), and the resulting oxidative metabolites are extensively conjugated and excreted in urine (Bassmann et al., 1985; Palette et al., 1991; Moreau et al., 1992). The main oxidative metabolites are 3-hydroxymethylantipyrine (HMA), 4-hydroxyantipyrine (OHA), and norantipyrine (NORA). Glucuronide conjugation is the major phase II pathway (Fig. 1) (Bottcher et al., 1982b). The quantitation of antipyrine and its metabolites excreted in urine has been used to understand variation in oxidative hepatic drugmetabolizing capacity (Ohashi et al., 1991; Robertz Vaupel et al., 1992; Ali et al., 1995; Yang et al., 1996). Several methods using high-performance liquid chromatography (HPLC) have been reported for the determination of antipyrine and its metabolites (Danhof et al., 1979; Teunissen et al., 1983; Bassmann et al., 1985; Mikati et al., 1988; Palette et al., 1991; Velic et al., 1995). However, the direct and simultaneous determination of all phase I and phase II metabolites in biological materials has not been reported using HPLC, except for a radio-HPLC method (Velic et al., 1995). However, the method is troublesome to use and unsuitable for application to humans because of the radiation hazard. Although enzymic or chemical deconjugation followed by HPLC analysis is still the standard approach, analytical problems arise from the diversity of metabolites and the instability of NORA and OHA liberated after the deconjugation (Danhof et al., 1979; Bottcher et al., 1982a, 1984; Teunissen et al., 1983; Palette et al., 1991), which varies according to the conditions of hydrolysis and the nature of the conjugates (Bottcher et al., 1982a,b, 1984; Teunissen et al., 1983; Moreau et al., 1992). A stable isotope tracer technique using C labeling of substrates (approximately 100% enrichment) followed by NMR spectroscopy of biofluids has been demonstrated to be useful for pharmacokinetic research (Baba et al., 1990, 1995; Malet-Martino and Martino, 1992; Akira et al., 1993, 1997, 1998; Akira and Shinohara, 1996). Because of the high specificity of detection, the application of the tracer technique enables analysis of biological fluids without resorting to extraction and chromatographic separations. Therefore, the technique saves much time and analytical effort, and the decomposition and loss of compounds can be minimized. In a previous study, antipyrine metabolites in rat urine were successfully detected using the NMR approach with C labeling (Akira et al., 1999). In the present study, the NMR approach with C labeling has been used for the direct analysis of major metabolites that occur in human urine after oral dosing with C-labeled antipyrine. Materials and Methods Chemicals and Reagents. [C-Methyl-C]antipyrine (.99 atom% C), [C-methyl-C]OHA (.99 atom% C), HMA, and OHA sulfate (OHA-S) were synthesized as previously described (Akira et al., 1999). [2-C]Sodium 1 Abbreviations used are: HMA, 3-hydroxymethylantipyrine; HMA-G, HMA glucuronide; DEPT, distortionless enhancement by polarization transfer; HPLC, high-performance liquid chromatography; NORA, norantipyrine; NORA-G, NORA glucuronide; OHA, 4-hydroxyantipyrine; OHA-S, OHA sulfate; OHA-G, OHA glucuronide; T1, spin-lattice relaxation time; IS, internal standard. Send reprint requests to: Kazuki Akira, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail: [email protected] 0090-9556/01/2906-903–907$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 6 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 330/907704 DMD 29:903–907, 2001 Printed in U.S.A. 903 at A PE T Jornals on O cber 3, 2017 dm d.aspurnals.org D ow nladed from