2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings.
A quick and selective analytical method was developed for the simultaneous quantitation of 2-methylimidazole, 4-methylimidazole, and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole, which are known to be formed by Maillard reactions. The methodology reported here employs stable-isotope dilution analysis (SIDA) using 4-methylimidazole-d6 and [(13)C6]-2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole as internal standards. It was successfully applied in a model assay to show that the addition of ammonium chloride during the manufacture of licorice promotes imidazole formation depending on the added amount of ammonium chloride without the well-known impact of present caramel food colorings. Furthermore, a monitoring assay of 29 caramel coloring-free licorice products showed that both 4-methylimidazole and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole are endogenously generated in detectable quantities. None of the samples showed 2-methylimidazole levels above the limit of detection, 50 μg/kg.
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