High-performance liquid chromatographic screening for unknown sunset yellow fcf and orange ggn metabolites in rat faeces using a continous gradient elution system

Abstract

The water-soluble azo dyes, Sunset Yellow FCF and Orange GGN were administered in different doses to male Wistar rats by stomach intubation. The 24-h faeces were collected and analysed both for the parent dyes and those metabolic aromatic aminosulphonic acids which are also excreted in urine and are assumed to be potential metabolites in faeces: sulphanilic acid and N-acetylsulphanilic acid for FCF, metanilic acid and N-acetylmetanilic acid for GGN and the common metabolites 1-amino-2-naphthol-6-sulphonic acid (ANSA) and N-acetyl-ANSA. Aqueous faeces suspensions were centrifuged liquid chromatographic (HPLC) system after filtration, and were also submitted to an ion-pair extraction procedure followed by HPLC analysis of the isolated purified metabolites. Screening for several metabolic aromatic sulphonic acids of different polar characters could be performed in a single run by means of a reversed-phase ion-pair HPLC system with continous linear gradient elution. A method for differentiation between metabolite peaks and endogenous peaks has been worked out by comparing continous gradient chromatograms of faeces extracts, recorded before and after administration of dye under similar conditions. These chromatograms clearly demonstrate that in addition to the metabolites mentioned, many new unknown dye metabolites are formed or excreted in rat faeces which are not present in urine. By selecting the detector wavelength in the visible spectrum we also were able to prove that some of these unknown metabolites obviously are coloured. This could be confirmed by a recently developed thin-layer chromatographic method.