Analysis of Methylimidazoles in Caramel Colored Carbonated Beverages

By: Craig Aurand and Michael D. Buchanan, US Reporter Volume 30.3


Caramel colorings are used as additives in a broad range of food and beverage products to impart a desired color, 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 carcinogen1. 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 this 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.


Three 16 oz plastic bottles of different popular diet colas were obtained. Then, 10 mL aliquots from each bottle were placed into separate 40 mL glass vials and shaken vigorously to expunge the dissolved carbon dioxide. One milliliter 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.

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 the three sample extracts analyzed on an Ascentis® Express HILIC column are shown in Figures 1 & 2.

Standard, Each Analyte at 200 ng/mL in Acetonitrile

Figure 1. Standard, Each Analyte at 200 ng/mL in Acetonitrile

HPLC Conditions
Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 µm particles (Product No. 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


Diet Cola 1, 2, 3 Y-axis enlarged to show detail

Figure 2. Diet Cola 1, 2, 3 Y-axis enlarged to show detail. Peak IDs and conditions are the same as Figure 1.

Results and Discussion

The Ascentis Express HILIC phase was shown to be a suitable stationary phase for the analysis of 2-methylimidazole and 4-methylimidazole providing sufficient retention, plus good resolution and peak shape. Even with limited sample preparation (degassing), no co-eluting interference was observed in the elution range of either methylimidazole compound under full scan MS conditions. Interestingly, only 4-methylimidazole was observed in the three tested diet colas, with each having a slightly different concentration. Since each manufacturer uses a unique secret formulation for their diet cola, it was expected that each would contain a distinctive quantity of methylimidazole.


The use of an Ascentis Express HILIC column offers a unique approach for analysis of the methylimidazole compounds. This direct analysis technique simplifies the sample workflow, and combined with HILIC chromatographic conditions, results in a simple and fast analytical method to determine the levels of 2-methylimidazole and 4-methylimidazole in caramel colored carbonated beverages.

Legal Information

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC




  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.


Related Links