Analysis of Methylimidazoles in Caramel-Colored Carbonated Beverages

June 1, 2013

The Application Notebook

The Application Notebook, The Application Notebook-06-01-2013, Volume 0, Issue 0

Caramel colorings are used as additives in a broad range of food and beverage products, but have no nutritional or preservative function. Recently, the potential hazard to humans of ammonia- and ammonia-sulfite-process caramel colorings was raised, because they contain the by-product 4-methylimidazole, which is a potential carcinogen (1).

Caramel colorings are used as additives in a broad range of food and beverage products, but have no nutritional or preservative function. Recently, the potential hazard to humans of ammonia- and ammonia-sulfite-process caramel colorings was raised, because they contain the by-product 4-methylimidazole, which is a potential carcinogen (1). The methylimidazole compounds are difficult to analyze due to their polar nature and low molecular weight. Traditional reversed phase techniques are unsuccessful in retaining these small polar compounds. Therefore, most HPLC methods utilize ion-exchange resins for analysis. Another common method involves GC analysis after the analytes first undergo a derivatization step. The purpose of the work shown in this article was to develop a simple and fast analytical method to determine the levels of 2-methylimidazole and 4-methylimidazole in caramel colored carbonated beverages.

Experimental

A 16 oz individual plastic container of three popular diet colas were obtained. 10 mL aliquots of each sample were then placed into separate 40 mL glass vials and shaken vigorously to expunge the dissolved carbon dioxide. 1 mL aliquots of each degassed sample were then placed directly into separate 2 mL HPLC autosampler vials. Samples were analyzed directly with no further sample treatment. For quantitative purposes, a calibration range was developed for each analyte at levels of 50, 100, 200, and 300 ng/mL. Standards were diluted in acetonitrile.

Figure 1: Standard, each analyte at 200 ng/mL in acetonitrile, column: Ascentis Express HILIC, 10 cm × 2.1 mm i.d., 2.7 µm particles (53939-U), mobile phase: 2 mM ammonium formate (95:5 acetonitrile:water) pH 4.4 titrated with formic acid, flow rate: 0.6 mL/min, pressure: 130 bar, column temp.: 50 °C, detector: MS, ESI(+), m/z 50–800, injection:1 µL. Peaks: 1. 4-Methylimidazole, 2. 2-Methylimidazole.

Due to the ionic nature of the methylimidazoles, peak tailing is an issue on silica based materials, and low pH conditions are necessary to help minimize excessive ionic interactions. In this study, the use of the HILIC chromatographic technique was selected due to its ability to retain and separate hydrophilic compounds by differences in polarity, whether the analytes are acidic, basic, charged, or neutral. In fact, HILIC often provides retention and selectivity that reversed phase and normal phase techniques lack. Chromatograms of a calibration standard and one sample extract analyzed on an Ascentis® Express HILIC column are shown in Figures 1 and 2.

Figure 2: Diet cola 1. Y-axis enlarged to show detail. Peak IDs and conditions are the same as Figure 1.

Reference

(1) Petition to Bar the Use of Caramel Colorings Produced With Ammonia and Containing the Carcinogens 2-Methylimidazole and 4-Methylimidazole, Center for Science in the Public Interest, February 16, 2011.

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