Preparative isolation and purification of urinary conjugates in antipyrine metabolism in man and rat.

A series of six major urinary conjugates in the metabolism of antipyrine in man and rat has been investigated. A preparative isolation procedure has been developed using chromatography of methanolic extracts from urine on silica gel. In a two-step chromatographic procedure, methanolic extracts are first separated in 1) a "free fraction", containing unconjugated phase-I-metabolites and unchanged antipyrine, 2) a sulfate fraction and 3) a glucuronide fraction. Sulfate and glucuronide fraction, respectively, are each subjected to a second run for separation into their three components. Thus, the following conjugates have been prepared: 4-hydroxy-antipyrine sulfate, norantipyrine sulfate, 4,4'-dihydroxy-antipyrine sulfate, and 4-hydroxy-antipyrine glucuronide, norantipyrine glucuronide, 3-hydroxymethyl-antipyrine glucuronide. Methodology is also applicable to bile fluid and liver perfusate. Stability of isolated conjugates against acid hydrolysis has been studied to show that strongly marked differences exist in this series of conjugates. Field desorption mass spectrometry has been used for the direct identification of intact conjugates in an underivatized form. Using 13C-NMR, the structure of norantipyrine glucuronide has been established as an 5-enol glucuronide. By analogy, a structure of 5-enol sulfate is proposed for norantipyrine sulfate. From a semiquantitative examination by TLC of urine extracts from man and rat, it becomes apparent, that in the rat, at the dose level studied, sulfate formation is the predominant conjugation pathway. In man, glucuronides are the most prominent type of conjugates. Formation of sulfates is minimal up to a dose of 15 mg/kg antipyrine.