Direct nuclear magnetic resonance spectroscopic analysis of (13)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 (13)C labeling and NMR spectroscopy for the direct and simultaneous analysis of major metabolites of antipyrine in human urine. [C-Methyl-(13)C]antipyrine (500 mg) was dosed orally to human volunteers, and the post-dose urine was analyzed by 100-MHz (13)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-to-noise 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-(13)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.