Cancer-causing cola compounds caught by GC-MS/MS

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  • Published: Apr 2, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Cancer-causing cola compounds caught by GC-MS/MS

Quantification of methylimidazoles in drink products is challenging

4-Methylimidazole and (to a lesser extent) 2-methylimidazole are compounds that occur in dark beers, such as stout and root beer, and colas. In the latter they arise from the caramel colouring. Smaller amounts occur in grilled meat and some roasted foodstuffs, such as coffee. Studies in rodents have found these compounds to be carcinogenic at high doses, so they have been classed by the IARC (the International Agency for Research on Cancer) as possibly carcinogenic in humans (group 2B). Californian regulators have indicated that 29 μg/day of 4-methylimidazole would be considered as the ‘No significant risk level’, while the EU has introduced limits of 200 and 250 mg/kg for 4-methylimidazole in different classes of caramel colouring agents, although there are no specific EU limits in beverages.

Analysis of the methyl imidazoles in drink products has been accomplished by LC-MS, GC-MS and capillary isotachophoresis. GC-MS is a quick method, but generally requires derivatisation owing to the lack of volatility of the methyl imidazoles. Isobutylchloroformate (IBCF) is the most common derivatisation agent, but most current derivatisation methods are either lengthy, involving many extractions, or lack sensitivity. The Korean researchers aimed to devise a speedy method of derivatisation and GC-MS/MS, which would enable the rapid screening of multiple samples.

Improved derivatisation and GC-MS/MS method for methylimidazole assay

The new derivatisation method involved direct treatment with IBCF in an acetonitrile/isobutanol/pyridine 50:30:20 by volume solvent mixture. Aqueous sodium bicarbonate (1.0 M) and hexane were then added, giving a two-phase mixture; GC-MS/MS was carried out on the organic phase.

The GC instrument was a Shimadzu GC-2010 plus fitted with a DB-17 50% phenyl-methylpolysiloxane capillary column (J&W Scientific). The temperature gradient ran from 80 to 250 °C, while the total run time was 11.5 minutes. A Shimadzu TQ8030 triple quadrupole mass spectrometer was used, giving appropriate quantification ions for both species.

Matrix effects were a problem with the analysis: samples of drinks spiked with the methyl imidazoles gave different response factors to standards in pure solvents, despite using d6-4-methylimidazole and 2-ethylimidazole as internal standards. It is surprising that the use of a deuterated standard failed to fully compensate for the matrix effects. A standard addition calibration method was used to compensate for these inaccuracies, where known amounts of methylimidazoles were added to each analysed worked-up sample and the GC repeated. The modified method showed good repeatability and sensitivity, with limits of quantification (LOQ) of 4.3 μg/L for 2-methylimidazole and 5.5 μg/L for 4-methylimidazole. The authors note that the addition of standards to solutions can be automated, cutting down on the extra time required for the standard addition method.

Varying amounts of the methylimidazoles were found in different brands of colas and dark beers. Cola drinks from fast food outlets tended to contain less of the compounds of interest than canned products, presumably because the former are produced from concentrates mixed with carbonated water, where there is always a temptation to dilute to a greater extent than recommended.

Quick assay for methylimidazoles in drink products now possible

The new method avoids lengthy sample work-up procedures, allowing for the quick analysis of multiple beverage samples. The main disadvantage is the need for spiking in the sample addition method, although automation can reduce this burden. It is very unusual for a deuterated standard to be affected by the matrix to a different extent than the analogous undeuterated compound of interest, and it would be interesting to investigate this phenomenon in more depth.

More generally, while there may be some justification for the investigation of methylimidazole levels in cola, it seems strange to be concerned about the levels of these compounds in alcoholic drinks, bearing in mind that alcohol itself is established as a major cause of cancer, while the methylimidazoles have not been shown to have adverse effects in humans and only affect rodents at high doses.

Related Links

Journal of Food Science, 2017, Early View paper. Wang et al. Simple and fast sample preparation followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) for the analysis of 2- and 4-methylimidazole in cola and dark beer.

Journal of Chromatography A, 2009, 1216, 4798-4808. Frenich et al. Compensation for matrix effects in gas chromatography–tandem mass spectrometry using a single point standard addition.

Wikipedia, 4-Methylimidazole

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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