Caramel coloring is often used to darken food products such as carbonated beverages and soy sauces. Recently, two byproducts of its manufacture—2- and 4-methylimidazoles (2-MI and 4-MI)—have come under scrutiny. Studies from the National Toxicology Program (NTP) and other researchers concluded that there is clear evidence of the carcinogenicity of both chemicals.1–3 California’s Office of Environmental Health Hazard Assessment (OEHHA) listed 4-MI as a carcinogen in January with a calculated no significant risk level (NSRL) of 16 µg per person per day.4 In February, a group of scientists from the Center for Science in the Public Interest filed a petition with the U.S. Food and Drug Administration (FDA) to bar the use of caramel coloring containing 2-MI and 4-MI, which “serve purely cosmetic purposes.”
Conventional methods for identification of 2-MI and 4-MI in caramel color include gas chromatography methods that involve labor-intensive procedures such as hot solvent extraction and acetyl derivatization and are thus not suitable for high-throughput analysis.5–6 Several liquid chromatography methods were also reported for 4-MI analysis.7–8 However, to the authors’ best knowledge, no previous study has reported the simultaneous quantification of 2-MI and 4-MI in beverages and other food products.
This study describes an ultrahigh-performance liquid chromatography tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantification of 2-MI and 4-MI in various soda products. In this method, soda samples were degassed by sonication, diluted, and directly analyzed. Chromatography was performed on an UltiMate 3000 Rapid Separation (RS) UHPLC system (Dionex Corporation, Sunnyvale, Calif.), and separation was achieved using an Acclaim Trinity P1 Mixed-Mode column. The TSQ Quantum MS/MS instrument (Thermo Fisher Scientific, San Jose, Calif.) was operated in selected-reaction-monitoring (SRM) mode for the best sensitivity and selectivity. In the dark soda drinks selected in this study, 4-MI was quantified at hundreds of parts-per-billion (ppb) levels. For comparison, a colorless lemon-lime flavored soda drink was also analyzed by this method, and no quantifiable levels of target analytes were found.
Chemical standards used in this study were purchased from Sigma-Aldrich (2-MI, M50850; 4-MI, 199885, structures shown in Figure 1). Methanol and acetonitrile were obtained from Burdick & Jackson (HPLC/UV grade). Ammonium formate and formic acid were purchased from Sigma-Aldrich for mobile phase preparation. Stock solutions of 2-MI and 4-MI were prepared by dissolving each pure chemical in deionized (DI) water to the concentration of 1 mg/mL (1000 parts-per-million, ppm). Working solutions were prepared by mixing 2-MI and 4-MI in DI water at 10 ppm and then diluting to 1 ppm and 100 ppb. Calibration standards were prepared from working solutions at seven levels: 1 ppb, 5 ppb, 10 ppb, 50 ppb, 100 ppb, 200 ppb, and 500 ppb.
Bottled dark-colored soda drink samples were purchased from a local retail grocery store, including regular, diet, and zero calorie varieties where possible. A bottled lemon-lime flavored colorless soda sample was also purchased for comparison. All samples were stored at room temperature until opened for analysis. A 20 mL aliquot of each sample was poured into a 150 mL specimen cup and degassed for 30 seconds in a sonication bath. A 100 µL aliquot of each degassed sample was then pipetted into a 1.5 mL autosampler vial and vortex mixed with 900 µL DI water; 10 µL of each prepared sample was injected for UHPLC-MS/MS analysis.
Chromatography was performed on the UltiMate 3000 RS UHPLC system, with separation of 2-MI and 4-MI achieved on an Acclaim Trinity P1 column with isocratic elution. Mobile phase consisted of 10% methanol, 5% acetate buffer (100 mM, pH 5.7), and 85% DI water and was delivered at a flow rate of 0.5 mL/min. The column temperature was set at 15 degrees C. A TSQ Quantum Access MS/MS instrument was selected as the detector and was operated in SRM mode to achieve the best selectivity and sensitivity. A heated electrospray ionization (HESI) source was used to interface the UHPLC and MS/MS systems. The detailed source parameters are listed in Figure 2. Two SRM transitions were used for the quantitation (Q-SRM) and confirmation (C-SRM) for each analyte, with collision energy set at 22 V. Details of the SRM transitions are listed in Figure 2.
The objective of this study was to develop a quantitative method for simultaneous determination of 2-MI and 4-MI in soda products. These two target analytes are positional isomers with identical molecular weight.
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