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Two assay procedures are described for the quantitation of norantipyrine sulphate and norantipyrine glucuronide present in biological material. One, a selective acid hydrolytic assay procedure that affords the discriminative determination of both conjugates without prior separation, measures free norantipyrine by tlc-uv. The other is a tlc separation procedure for intact norantipyrine conjugates, which, in conjunction with radiolabelled material, derived from [3-14C]antipyrine, enables the direct quantitation of both conjugates independently. In man, about 25% of dose of antipyrine (1200 mg) was excreted as norantipyrine glucuronide within 48 h. The amount of norantipyrine sulphate was small. In the rat, norantipyrine sulphate represented about 15-20% of the dose of antipyrine (40-50 mg kg-1); no norantipyrine glucuronide was formed. Free norantipyrine has not been detected in urine of either species after antipyrine dosage. Pretreatment with 3-methylcholanthrene in the rat substantially enhanced excretion of norantipyrine sulphate, whereas induction with phenobarbitone was without effect on the microsomal N-demethylation of antipyrine. The lability of free norantipyrine was examined under different conditions and contrasted with the relative stability of norantipyrine conjugates. Two of the main degradation products of norantipyrine were identified as 4-phenylazo-norantipyrine and a 4,4'-bipyrazole-derivative.

A t.l.c.-assay has been developed for the simultaneous determination from the urine of man and animals of three major hydroxylated metabolites of antipyrine (4,4'-dihydroxy-antipyrine, 4-hydroxy-antipyrine, 3-hydroxymethyl-antipyrine). The methodology is also applicable to bile fluid, liver perfusate and liver homogenate. Genuine conjugates are cleaved by acid hydrolysis and free, acid stable metabolites are extracted. Extracts are subjected to t.l.c. and the chromatograms analysed quantitatively by u.v.-reflectance measurements using authentic materials as standards. Calibration curves are linear with a correlation coefficient r greater than 0.990. Recovery for each metabolite is greater than 95%. Reproducibility of the method is good, with variation coefficients in the range of 3-7%, depending on concentration. The sensitivity of the method is sufficient for practical needs. The specificity of the procedure was confirmed using radio-labelled antipyrine. In man, 4-hydroxy-antipyrine is the principal hydroxylation product in this series, accounting for about 35-40% of the dose. 3-Hydroxymethyl-antipyrine makes up for about 13-17% and 4,4'-dihydroxy-antipyrine represents 3-6% of the dose of antipyrine. In the rat, 4-hydroxy-antipyrine accounts for about 15-31%, 3-hydroxymethyl-antipyrine for 22-28% and 4,4'-dihydroxy-antipyrine for up to 11-18% of the dose. Variation of this pattern in different strains is moderate. In both species, the major portion of phase-I metabolites is excreted as conjugates. Part of them appears in a free form.

A large-scale procedure for the isolation of four urinary glucuronides in antipyrine metabolism is described; the isolated compounds are used as standards in a direct h.p.l.c. assay. The four glucuronides were characterized by u.v. and 1H-n.m.r. spectroscopy, and after hydrolysis by a t.l.c. assay of the corresponding aglycones. A reversed-phase h.p.l.c. assay procedure has been developed for the direct quantification of the four antipyrine glucuronides; this separates 3-hydroxymethyl-antipyrine glucuronide, 4,4'-dihydroxy-antipyrine glucuronide, norantipyrine glucuronide and 4-hydroxy-antipyrine glucuronide in a single run. Urinary elimination patterns of these glucuronides have been determined in five female and five male volunteers after antipyrine (1200 mg) administration. The direct assay of urinary glucuronides enables the simultaneous determination of glucuronidation activities and four different phase-I metabolites of antipyrine in vivo.