2-Acetyl-4(5)-tetrahydroxybutylimidazole (THI) is a compound formed during the production of ammonia type caramel which is used as a colouring agent for beers. The substance has been found to cause lymphocyte depression in rats fed vitamin Be-deficient diets'. As a result, international specifications now include a maximum concentration of 40 pg/g of THI in ammonia caramels (based on total solids content of the sample).
Two publications on the determination of THI in caramel have appeared in the past three years 2*3 Both of these employ high-performance liquid chromatography .
(HPLC) either directly' or after derivatization of THI with 2,4_dinitrophenyl-hydrazine3. These methods were concerned with mainly caramel itself and not with determination in beers. Since caramel colour can be diluted by up to 1000 times in beer, methodology should be capable of quantitating 40 ng/ml THI in commercial samples. The present work includes method development for THI in beers that enables the detection of as low as 10 ng/ml THI with confirmation by chemical derivatization followed by analysis of the product under different HPLC conditions.
EXPERIMENTAL
Reagents
THI was dissolved in deionized water and diluted to yield a concentration of 10 pg/ml. Aliquots of this were used for direct HPLC analysis, for sample spiking and for derivatization studies. Pyridine "plus" (Applied Science Labs., State College, PA, U.S.A.) and acetic anhydride (J. T. Baker, Phillipsburgh, NJ, U.S.A.) were used as received. All other solvents and chemicals were analytical reagent-grade materials.
Cation-exchange columns
About 100 g each of Amberlite CG-SO(H) type 1 (BDH, Toronto, Canada) and Dowex 5OW-X8 (100-200 mesh) (Bio-Rad, Richmond, CA, U.S.A.) were allowed to soak in deionized water overnight. Each batch was rinsed several times with water, then a slurry poured to a bed height of 10 cm into separate 1 cm I.D. glass