Isolation and characterization of the four antipyrine glucuronides and determination of their urinary excretion pattern in man by a reversed-phase h.p.l.c. assay

Abstract

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.

Synthesis of the metabolites of a free radical scavenger edaravone (MCI-186, Radicut™)

“…13,14 The [ 1 H]-NMR data on a metabolite from bile were identical to those on the synthesized glucuronide in both D 2 O and DMSO-d 6 . The result that the synthesized glucuronide was identical to the metabolite suggested that the metabolite was not a mixture of isomers but was only one isomer, b-O-glucuronide.…”

“…As 3-methyl-1-phenyl-2-pyrazolin-5-one has three possible tautomers [9][10][11], three isomeric glucuronides might be possibly formed by the glycosylation reaction; the relevant isomeric glucuronides are possibly connected with glucuronic acid at the 5-position oxygen atom (O-glucuronide [12]), 4-position carbon atom (C-glucuronide [13]), or 2-position nitrogen atom (N-glucuronide [14]), respectively, as shown in Figure 2. X-ray analysis of the sodium salt of the glucuronate [8] revealed that the synthesized glucuronate was b-O-glucuronide, and neither C-glucuronide nor N-glucuronide was produced (Fig.…”

Determination of antipyrine sulphoconjugates in isolated cultured rat hepatocytes

“…This method, developed without preliminary extraction or hydrolysis, allows the analysis of phases I and I1 metabolites on a single assay, by direct injection of culture medium. This approach, without hydrolysis, has already been considered by Baessman et al (1985), who proposed a direct determination of the major urinary glucuronide conjugates in man.…”

“…T o evaluate the pattern of conjugates of a given xenobiotic is not simple, and the lack of conjugate standards often complicates the problem. Hydrolysis of conjugates, either enzymically (Boettcher et al 1982c, Danhof et al 1979, Eichelbaum et al 1981, Nakagawa and Nakamura 1982, Teunissen et al 1983 or chemically (Baessman et al 1985, Boettcher et al 1984, Buppodom et al 1986), remains the standard approach, but the known instability of phase I metabolites of antipyrine makes the results questionable. This instability must be taken into account in the analytical measurements of phase I metabolites released from their conjugates by hydrolysis, especially with 4-OH-antipyrine and nor-antipyrine which are very labile (Baess-///I- , Teunissen et al 1983).…”

Separation of phenolic compounds and corresponding glucuronides by coupled-column micellar reversed-phase liquid chromatography

“…lengthy incubation times, the introduction of impurities, variation in the extent of hydrolysis, and poor discrimination between the aglycones of different conjugates. The conventional processes to sample clean-up are liquid-liquid extraction [2][3][4] or offline solid phase extraction [5][6][7][8][9][10] and preparative LC [11,12]. These pretreatments could also be timeconsuming and would cause impairments in sample information as well as in precision of results.…”