Colorimetric determination of N-hydroxylated metabolites in microsomal studies

✍ Scribed by Pierre M. Bélanger; Odette Grech-Bélanger; Michel Blouin

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 545 KB
• Volume: 118
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90154-8

✦ Synopsis

A calorimetric method is described which can be used for the routine determination of primary and secondary IV-hydroxylamino compounds in drug metabolism studies using microsomal preparations. The assay is carried out on the supernatant obtained after protein precipitation of the incubation mixture. The method is based on the reduction of ferric ion by the hydroxylamino moiety with the resulting ferrous ion being quantitated by coupling with 2,4,6-tripyridyl-s-triazine to form a purple color with a maximum absorbance at 595 nm. The method is specific to primary and secondary hydroxylamino compounds and calibration curves can be obtained in the range of concentrations of 1 to 20 pg/ml. Hydroxamic acid, amido, amino, phenolic, nitro, nitroso, oxime, nitrone, aldehyde, and ketone compounds do not produce any color reaction when analyzed by the same method. The method is simple, rapid, and many samples can be analyzed in a short period of time.