Analysis of 2- and 4-Methylimidazole

Introduction

2- and 4-methylimidazole (2-MI and 4-MI), figure 1, are heterocyclic compounds derived from imidazole through substitution of the hydrogen in either position 2 or 4 by a methyl group. Both 2-MI and 4-MI are known animal carcinogens and they may form when carbohydrates and amino-containing compounds reacts during browning of food, but also in certain fermentation processes, and in types of caramel coloring produced with ammonia or ammonia and sulfites based processes. High levels of 4-MI has been found in many cola soft drinks including Coca-Cola, Pepsi-Cola, Diet Coke, and Diet Pepsi. Additionally, it has been found in roasted foods, grilled meats, and coffee. Dark beers and common cola soft drinks may contain more than 100 μg of 4-MI per 12-ounce canister.

2- and 4-methylimidazole (2-MI and 4-MI), figure 1, are heterocyclic compounds derived from imidazole through substitution of the hydrogen in either position 2 or 4 by a methyl group. Both 2-MI and 4-MI are known animal carcinogens and they may form when carbohydrates and amino-containing compounds reacts during browning of food, but also in certain fermentation processes, and in types of caramel coloring produced with ammonia or ammonia and sulfites based processes. High levels of 4-MI has been found in many cola soft drinks including Coca-Cola, Pepsi-Cola, Diet Coke, and Diet Pepsi. Additionally, it has been found in roasted foods, grilled meats, and coffee. Dark beers and common cola soft drinks may contain more than 100 μg of 4-MI per 12-ounce canister. In February 2011, the Center for Science in the Public Interest (CSPI) petitioned United States Food and Drug Administration (US FDA) to withdraw its authorization for caramel colorings that contain 4-MI, and in the interim to change the name of the additive to “ammonia-sulfite process caramel coloring” or “chemically modified caramel coloring” for labeling purposes. California has added 4-MI to its list of probable carcinogens, and of this reason leading manufacturers of cola soft drinks declared in March 2012 to change their recipes to avoid a cancer warning label in compliance with Californian law. In the spring of 2013, a large quantity of soy sauce products was seized in Taiwan containing excessive amounts of heterocyclic organic chemicals including 4-methylimidazole. The European Food Safety Authority has concluded that exposure to 4-MI from caramel coloring is not of concern. Although the issue is controversial, it is sensible to take precautions, especially for excessive exposure of heterocyclic organic chemicals. Therefore, quantitative analytical methods are needed that can be used for exposure assessment.

Figure 1. Chromatogram showing the separation of a standard solution containing 0.5 ppb (0.5µg/mL) of each 2- and 4-methylimidazole in diluent (acetonitrile and Milli-Q water 70:30 v/v) on a SeQuant® ZIC®-HILIC, 5 µm/200 Å, 150 x 4.6 mm column using a mobile phase containing (75:25 v/v) of acetonitrile and potassium phosphate 20 mM pH 6.0, hence 5 mM total ionic strength in mobile phase. 20 µL samples were injected and analysed at 1.0 mL/min on an Agilent 1260 HPLC system equipped with UV detector set at 214 nm.

HILIC has proven as an attractive technique for separation of small polar molecules but it has mainly been considered as a MS friendly technique using volatile acetate or formate buffers in the mobile phase, conditions preventing analysis at low UV wavelength. Bonded zwitterionic stationary phases such as the SeQuant ZIC-HILIC columns can, however, be used with inorganic buffers like phosphate despite the limited solubility of potassium phosphate in high acetonitrile eluents. Use a premixed mobile phase, thus avoid separate bottles for acetonitrile and buffer to minimize risk with precipitation. Keep also in mind that precipitation of salt generally occur when using over 80 volume-% acetonitrile in the mobile phase, though at low buffer strengths 85% is the absolute maximum. This application note present a simple yet rugged analytical procedure for direct analysis of 2- and 4-MI using hydrophilic interaction liquid chromatography (HILIC) and UV detection.

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