Analysis of Methylimidazoles in Caramel Colored Carbonated Beverages

sigma-aldrich.com/analytical reporter 30.3 |22 Food and Beverage AnalysisAnalysis of Methylimidazoles in Caramel Colored Carbonated BeveragesCraig Aurand and Michael D. Buchananmike.buchanan@sial.comIntroductionCaramel 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 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.experimentalA 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.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 1-4.Figure 2. diet cola 1Y-axis enlarged to show detail. Peak IDs and conditions are the same as Figure 1.Figure 3. diet cola 2Y-axis enlarged to show detail. Peak IDs and conditions are the same as Figure 1.Figure 4. diet cola 3Y-axis enlarged to show detail. Peak IDs and conditions are the same as Figure 1.0 2 4 6Min121. 4-Methylimidazole2. 2-MethylimidazoleMin0 2 41Min0 214Min0 214Figure 1. Standard, each analyte at 200 ng/ml in acetonitrile column: Ascentis Express HILIC, 10 cm x 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 ºC detector: MS, ESI(+), m/z 50-800 injection: 1 µL23Order: 800-325-3010 (U.S.) 814-359-3441 (Global) Did you know . . .Our Ascentis Express HILIC Guide (T412061, OMI) is a valuable tool to learn how to achieve faster analysis of polar compounds. Information about ES-Cyano, F5, HILIC, and OH5 phases are included.results and discussionThe Ascentis® Express HILIC phase was shown to be a suitable stationary phase, able to provide sufficient retention, plus good resolution and peak shapes, of 2-methylimidazole and 4-methylimidazole. 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. Because each manufacturer uses a unique secret formulation for their cola with a different amount of caramel coloring from the others, it was expected that each would contain a distinctive quantity of methylimidazole.conclusionThe use of an Ascentis Express HILIC column offers a unique approach for analysis of the methylimidazole compounds. The 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.reference1. 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. Featured ProductsDescription Cat. No. Ascentis Express HPLC Column (2.7 µm particles)HILIC, 10 cm x 2.1 mm I.D. 53939-U Related ProductsDescription Cat. No. Ascentis Express HPLC Columns, 10 cm x 4.6 mm I.D., 2.7 µm particlesES-Cyano 53491-UF5 53590-UHILIC 53979-UOH5 53776-UAnalytical Reagents and SolventsAmmonium formate, eluent additive for LC-MS 55674Formic acid, eluent additive for LC-MS 56302Acetonitrile, LC-MS CHROMASOLV®, >99.9% 34967 Related InformationTo learn more about Sigma-Aldrich solutions for food analysts, visit sigma-aldrich.com/foodNEW Food and Beverage Analysis BrochureThis 32-page application guide illustrates innovative technologies that can be leveraged to provide viable customer solutions to simplify sample preparation and increase speed and sensitivity for many food and beverage applications, including:• Food Chemistry (carbohydrates, dietary fiber, fats, proteins, antioxidants, vitamins, organic acids, minerals, flavors and fragrances, sweeteners, preservatives, water content, and physical characteristics)• Food Biology (genetically modified organisms and microbial growth)• Food Safety (pesticides, veterinary drugs, mycotoxins/aflatoxins, allergens, dioxins, phytochemicals, heavy metals, phthalates, PAHs, and acrylamides)• Adulterants / Product Dilution• BeveragesTo request your copy, visit sigma-aldrich.com/lit-requestFood and Beverage AnalysisIncrease speed and sensitivity with proven solutionsFood ChemistryFood BiologyFood SafetyAdulterantsBeveragesFood & BeverageTo learn more about Sigma-Aldrich solutions for food analysts, visit sigma-aldrich.com/food