Simultaneous Quantitation of 2- and 4-Methylimidazole in Carbonated Drinks

The chemicals 2- and 4-methylimidazole (2-MI and 4-MI) are by-products produced during the manufacturing of caramel coloring ingredients used to darken food products such as carbonated beverages and soy sauce. These two chemicals were revealed as probable carcinogens in studies performed by the National Toxicology Program (NTP) and other researchers (1,2). In addition, 4-MI is listed as a carcinogen by the California Office of Environmental Health Hazard Assessment (OEHHA) in January 2011 with a calculated No Significant Risk Level (NSRL) of 16 μg per person per day (3). Thus, the quantitation of these two carcinogens in foods is of particular importance for food safety and human health.

Conventional methods for identification of 2-MI and 4-MI in caramel color include gas chromatography methods which involve labor intensive procedures (4). Liquid chromatographic methods have also been reported for 4-MI analysis (5).

This study demonstrates simultaneous quantitation of 2- and 4-methylimidazoles in carbonated drinks by ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC MS-MS).

Experimental

The analyses were performed on a Dionex UltiMate® 3000 Rapid Separation Liquid Chromatography UHPLC system. Separation was achieved on an Acclaim® Trinity™ P1 mixed-mode column (2.1 × 50 mm) with isocratic elution at 0.5 mL/min. The mobile phase consisted of 10% methanol in pH 5.7 ammonium acetate buffer (total buffer concentration at 5 mM). Column temperature was maintained at 15 °C. A Thermo Scientific TSQ Quantum Access™ with heated electrospray ionization source (HESI) was operated in selected reaction monitoring (SRM) mode with the settings listed with the chromatogram. Vaporizer temperature was set at 350 °C and capillary temperature was set at 200 °C. Sheath gas and auxiliary gas were both set at 60 arbitrary units.

Figure 1: SRM chromatograms of real samples. The chemical 4-MI is quantified at 396 ppb in original dark carbonated drink.

Results

As shown in Figure 1 (right), two target analytes were baseline separated on the Trinity P1 column. Both analytes were observed with the same two SRM transitions with different responses; the SRM with stronger response was used for quantitation (Q-SRM) and the other was used for confirmation (C-SRM). The ratio of the two SRM responses was used as additional confirmation to avoid possible false positives. Figure 1 (left) also demonstrates the direct analysis of a diluted dark carbonated drink after ten-fold dilution with positive detection of 4-MI. This diluted sample was re-analyzed after spiking with 100 ppb of each 2-MI and 4-MI, with the result showing in Figure 1 (right).

UltiMate and Acclaim are registered trademarks and Trinity is a trademark of Dionex Corporation. TSQ Quantum Access is a trademark of Thermo Fisher Scientific.

References

(1) http://ntp.niehs.nih.gov/files/535_Web_Final.pdf (Accessed May, 2011).

(2) http://ntp.niehs.nih.gov/files/516final_web.pdf (Accessed May, 2011).

(3) http://oehha.ca.gov/prop65/law/pdf_zip/010711NSRLrisk4EI.pdf (Accessed May, 2011).

(4) R.A. Wilks, M.W. Johnson, A.J. Shingler, J. Agric. Food Chem. 25(3), 1077 (1977).

(5) C. Moretten, G. Crétier, H. Nigay, J. Rocca, J. Agric. Food Chem. (2011) (published online March 7, 2011, available at http://pubs.acs.org/doi/full/10.1021/jf104464f, accessed March, 2011).

Dionex Corporation

1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408) 737-0700, fax (408) 730-9403

Website: www.dionex.com