An analytical method to determine 4 (5)-methylimidazole (4-MI) in food was developed.4-MI was extracted with chloroform-ethyl ether-ethyl alcohol (2:2:1) mixture under alkaline conditions, followed by back extraction with 0.2N hydrochloric acid. The acidic aqueous phase was condensed to about 3ml in a rotary vacuum evaporator at 60°C. The extraction with chloroform and back extraction with 0.05N hydrochloric acid was carried out once more for the concentrate. Diazo reagent (sulfamine, sodium nitrite mixed solution) and sodium carbonate were added to the acidic aqueous phase and the resulting coloring substances were subjected to thin layer chromatography after transfer to ethyl acetate. A chromatoscanner (dual wavelength) was used for quantitative analysis of 4-MI. The recoveries of 4-MI were 93-103% in cola drinks, 73-83% in worcestershire sauces and 70-78% in tsukudanis of laver (cooked and seasoned laver).4-MI was found in various kinds of foods by this method, and the concentration of 4-MI was especially high in worcestershire sauces (average 2.5ppm) and tsukudanis of laver (average 1.8ppm).
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YOSHIKAWA et al. (1981) studied this question.