ISOLATION AND PURIFICATION OF 3 GLUCURONIDES OF ANTIPYRINE - PROPOSALFOR AN ORIGINAL ANALYTICAL METHOD FOR QUANTITATION OF SULFOCONJUGATEDAND GLUCUROCONJUGATED METABOLITES

The determination of the concentrations of antipyrine metabolites in b iological fluids is hampered by the difficulty in obtaining pure conju gated compounds to be used as standards. Most authors have proposed de termination of total forms by high performance liquid chromatography ( HPLC) after deconjugation of these metabolites using chemical or enzym atic hydrolysis. Up to now there is no satisfactory hydrolysis method for the study of all antipyrine metabolites. The situation is further complicated by the fact that the deconjugated metabolites are highly u nstable whichever technique is being used. Because of the lack of stab ility of all these molecules it has been necessary to isolate the gluc uroconjugated compounds from urine. We describe a method which allows us to obtain highly purified glucuroconjugated metabolites of antipyri ne. Sulphoconjugated compounds have been synthesized previously. We ar e thus able to propose a chromatographic procedure which allows us to determine simultaneously all stable phase I and phase II metabolites o f antipyrine in biological fluids without any step of extraction. This analytical technique allows us to study the activity of the different isoenzymes implicated in the metabolism of antipyrine.