The allergen in commercial Red No. 225 (Sudan III), which has been used as a coloring matter for cosmetics or topical medications, was identified. Sudan I (0.057%), 2-naphthol (0.0048%) and azobenzene (0.0080%) were determined by HPLC, but commercial Red No. 225 comprised most unknown compounds. Therefore, to identify allergens, impurities were isolated, respectively. After solvent precipitation of most Sudan III, impurities in the filtrate were separated into five fractions by preparative thin-layer chromatography. Fraction (Fr) 1, Fr 2 and Fr 3 consisted of predominantly single compound, respectively. The three fractions, Fr 2, Fr 3 and Fr 4, showed positive responses in guinea pig skin sensitization test. As a result of analayses with nuclear magnetic resonance spectrometry and mass spectrometry, the structure of Fr 3 was clarified as an isomer of Sudan III; 1-(o-phenylazophenylazo)-2-naphthol. The identification of impurities in the Fr 2 and Fr 4 has been under investigation.
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