Determination of 2‑Methylimidazole, 4‑Methylimidazole, and 2‑Acetyl-4-(1,2,3,4-tetra­hydroxy­butyl)­imidazole in Licorice Using High-Performance Liquid Chromatography–Tandem Mass Spectrometry Stable-Isotope Dilution Analysis

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-<i>d</i>₆ and [¹³C₆]-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