1. An h.p.l.c. method for the simultaneous determination of naphthol, naphthyl glucuronide and naphthyl sulphate in urine is described. This procedure is based on the selective formation of an ion pair between the sulphate conjugate and tetrabutyl lammonium, which allows its resolution from the glucuronide on a reverse phase column. 2. The h.p.l.c. method is sed to assess the selective enzyme hydrolysis method which is frequently employed in drug conjugate assays. Results suggest that the use of sulphatase and beta-glucuronidase may give data which are quantitatively and qualitatively erroneous.

The effect of four inhibitors of cytochrome P-450-mediated drug oxidations (SKF 525A, cimetidine, metyrapone and alpha-naphthoflavone) on the urinary metabolite pattern and 14CO2 exhalation rate (CER)-time profile following [N-methyl-14C]antipyrine administration has been investigated. The CER-time profiles indicated that inhibition of antipyrine metabolism was in the rank order SKF 525A greater than cimetidine greater than metyrapone greater than ANF. The urinary metabolite patterns showed selectively in action towards particular pathways, 3-hydroxylation being primarily decreased by SKF 525A and cimetidine, and N-demethylation by ANF. The results provide further evidence for involvement of multiple forms of cytochrome P-450 in antipyrine metabolism.

Antipyrine metabolite kinetics have been investigated in the rat with respect to dose and time dependence. The metabolic pathways, 4-hydroxylation, benzylic oxidation, and N-demethylation, are of equal quantitative importance (approximately 20 per cent of dose) and show no dose dependence over the range 25-500 mg kg-1. By using [N-methyl-14C]-antipyrine, the single carbon fragment lost by N-demethylation may be monitored as 14CO2. Serial sampling of 14CO2 exhalation rate provides a half-life estimate which, according to theoretical principles, reflects the antipyrine plasma half-life. When both half-lives were measured in the same animals a statistically significant correlation was evident. At doses of 250 mg kg-1 and 500 mg kg-1 there is an increase in CER half-life (218 and 303 min respectively) when compared to a dose below 100 mg kg-1 (152 min). The metabolite formation rate constants are decreased accordingly at the high doses but are invariant over the dose range 25-100 mg kg-1. Although inter-rat variation in antipyrine metabolite kinetics was substantial, intra-rat variability was small. The noninvasive nature of determining antipyrine metabolite kinetics via breath and urine analysis provides a potentially useful animal model system to investigate the factors influencing hepatic mixed function oxidase activity in vivo.

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