Separation and Determination of Intermediates and Side Reaction Products in Food Red No. 3 (Erythrosine) and Stabilities of Side Reaction Products

Abstract

In the previous paper, the preparation of three kinds of iodoresorcinols and the separation and identification of the reaction products of iodine with resorcinol were reported. In this work, an analytical method for the intermediate and side reaction products of Food Red No. 3 (Erythrosine, Colour Index No. 45430, FD & C Red No. 3) and the reaction products of iodine with 2-(2′, 4′-dihydroxybenzoyl)benzoic acid (2′, 4′-diOHBBA) was developed.The intermediate resorcinol and the side reaction products, 2, 4, 6-triiodoresorcinol, 2, 4-diiodoresorcinol and 2′, 4′-diOHBBA, were determined by a combination of thin layer chromatography (TLC) and densitometry (J. Food Hyg. Soc. Japan 26, 243-252 (1985)). The relationship between the integrated value on the densitogram and the concentration of each compound was found to be linear in the range of 25-150ppm (0.05-0.3μg/spot) for resorcinol, 500-10, 000ppm (1-20μg/spot) for 2, 4, 6-triiodoresorcinol, 100-400ppm (0.2-0.8μg/spot) for 2, 4-diiodoresorcinol, 200-2, 000ppm(0.4-4μg/spot)for 4, 6-diiodoresorcinol and 250-1500ppm (0.5-3μg/spot) for 2′, 4′-diOHBBA (Fig. 1). The detection limit was 0.008μg for resorcinol, 0.8μg for 2, 4, 6-triiodoresorcinol, 0.08μg for 2, 4-diiodoresorcinol, 0.16μg for 4, 6-diiodoresorcinol and 0.06μg for 2′, 4′-diOHBBA (Table 1).Reaction of iodine (3 g-atom) with 2′, 4′-diOHBBA (1mol) yielded three compounds, which could be separated by TLC (Fig. 2). Spots A (Rf 0.73) and B (Rf 0.52) are thought to be 2-(3′, 5′-diiodo-2′, 4′-diOHB)BA and 2-(3′-iodo-2′, 4′-diOHB)BA, respectively (Table 2).The intermediate and the side reaction products in Food Red No. 3 were extracted with a mixture of 10% hydrochloric acid-ethanol-water (1:1:1) from this color and filtered. The filtrate was extracted with ethyl ether. The ethyl ether extract was analyzed by TLC-densitometry. The average recoveries for resorcinol and 2′, 4′-diOHBBA added to Erythrosine standard (free from the intermediate and side reaction products) at the level of 0.2% were 82.0 and 74.8%, respectively, but 2, 4, 6-triiodoresorcinol was not recovered. The poor recovery results from degradation of 2, 4, 6-triiodoresorcinol itself (Fig. 4). The degradation products were 2, 4-di, 2- and 4-mono-iodoresorcinols and two other unidentified compounds (Figs. 5 and 6).Ten commercial samples of Food Red No. 3 were analyzed by this method. The intermediate resorcinol and above side reaction products were not found in any of the samples. One of the side reaction products of Food Red No. 3, 2, 4, 6-triiodoresorcinol might coexist with the degradation products, di- and mono-iodoresorcinols in this color.