Abstract

Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe a new approach using ¹³C labeling and NMR spectroscopy for the direct and simultaneous detection of all phase I and phase II metabolites of antipyrine in rat urine. [C-methyl-¹³C]Antipyrine was synthesized and administered orally to rats (100 mg/kg), and the 0- to 24-h postdose urine was analyzed by 100-MHz ¹³C NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer without any pretreatments such as deconjugation, chromatographic separation, and solvent extraction. Consequently, all the major metabolites in urine were successfully detected with favorable signal-to-noise ratios in the limited acquisition time (30 min). The assignments of the resonances were performed by enzymic modification and spiking authentic samples. The reproducibility of the NMR detection was sufficient for the quantitative evaluation of the metabolic profile. Effects of 3-methylcholanthrene on antipyrine metabolism were examined by this approach to evaluate variation of in vivo phase I and phase II metabolism of antipyrine in rats. The present approach is useful and practical to evaluate variation of in vivo activities of conjugation enzymes as well as oxidation enzymes responsible for the formation of antipyrine metabolites in rats. This direct approach would enhance the value of the antipyrine test because of the simplicity and convenience.