A radiometric high-pressure liquid chromatography assay for the simultaneous determination of the three main oxidative metabolites of antipyrine in studies in vitro

✍ Scribed by G. Clare Kahn; Alan R. Boobis; Ian A. Blair; Martin J. Brodie; Donald S. Davies

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 663 KB
• Volume: 113
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90080-4

✦ Synopsis

A quantitative radiometric high-pressure liquid chromatography assay for the estimation of the three main oxidative metabolites of antipyrine in vitro using [3-'*C]antipyrine as substrate is described. Baseline separation of antipyrine, 3-hydroxymethylantipyrine, 4-hydroxyantipyrine, and norphenazone was achieved, after methylation, using a reverse-phase FBondapak CIa column with a mobile phase of 17% acetonitrile in water. The metabolites could be estimated free of interference as confirmed by gas chromatography-mass spectrometry. Unlabeled metabolites were used as recovery standards. Activity could be determined with as little as 100 pg human liver microsomal protein. Maximum velocities for the formation of the three metabolites ranged from 1 to 3.5 nmol product mg-r min-' with rat liver and from 0.3 to 0.6 nmol product mg-' min-' with human liver.