Direct detection of antipyrine metabolites in rat urine by C-13 labeling and NMR spectroscopy

Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe a new approa ch using C-13 labeling and NMR spectroscopy for the direct and simultaneous detection of all phase I and phase II metabolites of antipyrine in rat uri ne. [C-methyl-C-13]Antipyrine was synthesized and administered orally to ra ts (100 mg/kg), and the 0- to 24-h postdose urine was analyzed by 100-MHz C -13 NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer without any pretreatments such as deconjugation, chro matographic 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 t he resonances were performed by enzymic modification and spiking authentic samples. The reproducibility of the NMR detection was sufficient for the qu antitative evaluation of the metabolic profile. Effects of 3-methylcholanth rene on antipyrine metabolism were examined by this approach to evaluate va riation of in vivo phase I and phase II metabolism of antipyrine in rats. T he present approach is useful and practical to evaluate variation of in viv o activities of conjugation enzymes as well as oxidation enzymes responsibl e for the formation of antipyrine metabolites in rats. This direct approach would enhance the value of the antipyrine test because of the simplicity a nd convenience.