| Compared with natural pigments, synthetic pigments have strong color stability, and can be mixed with various pigments, which are widely used in cosmetics, foods and medical supplies. Azo dyes are the largest group of synthetic colorants (60-70%). Sunset Yellow FCF and New Coccine are kind of synthetic azo pigment and can be used to color a large number of different substrates such as pharmaceuticals, cosmetics and foods. Azo synthetic pigment and its metabolites have variety of potential toxicological effects such as carcinogenicity, mutagenicity, liver and kidney toxicity as increasingly studies have shown. So azo synthetic pigment used as food additive attracts more and more attention by researchers around the world. The national food administration organizations have stringent regulations to the usage of synthetic pigment. The usage of Sunset Yellow FCF and New Coccine has also been clearly defined in Chinese National Standard GB2760-2011. But the events of illegal use of food additives still occur frequently, a number of serious incidents even have a direct threat to the safety of people’s lives. Therefore, we need to develop a practical method to strictly control the amount of synthetic pigment added, in order to protect human health.There are several methods commonly used in the determination of Sunset yellow and New Coccine including high performance liquid chromatography, spectrophotometry and capillary electrophoresis. High performance liquid chromatography and capillary electrophoresis methods are simple and high detection speed, but also need high cost of detection and are not conducive to on-site detection. Spectrophotometric method is an earlier analysis method which can be applied to a wide range of simple equipment and have low-cost, but the disadvantage is that the accuracy is limited. This experiment is committed to develop a sensitive, accurate, rapid and simple analytical method to detect the amount of Sunset Yellow and New Coccine.The most important feature of immunoassay is its high specificity. And it also has wide range of applications with a simple, easy-to-site operation and high sensitivity. So this method is very suitable for the detection of trace substances in food additives, pesticide residues and drug impurities. There has been reported that the immunoassay method had successfully applied to the detection of Sudan red, lemon yellow and other synthetic pigments. In this study, two indirect competitive enzyme-linked immunosorbent assays (ELISA) were developed for the determination of Sunset Yellow and New Coccine.According to the structure of Sunset Yellow and New Coccine, the analogues were designed and synthetised as the hapten to avoid tautomerism. EDC method and mixed acid anhydride method were used to synthetize the immunogen and coating antigen. The polyclonal antibody was obtained by immunizing New Zealand white rabbits. Then the titer, sensitivity, specificity of the antibodies were determined using the indirect ELISA method, simultaneously the limit of detection, accuracy and precision of the established methods in food samples were also investigated.The results for Sunset Yellow are as follows, the sensitivity (IC50) was0.52ng/mL, and the linear range was in0.01～10ng/mL. In specificity, the cross reactivity were less than1.5%. The detection limit of the immunoassay was0.12ng/mL in carbonated drinks,0.04ng/mL in beancurd and1.11ng/mL in braised pork, respectively. The analytical recoveries were93.21%～106.25%. The coefficient of variation was less than15.47%.The results for New Coccine are as follows, the sensitivity was36.82ng/mL, and the linear range was in1～10000ng/mL in PBS buffer. The cross reactivity were less than0.3%. The intra-assay variability was ranged from1.7%to6.5%and the inter-assay variability was less than17.7%.Additionally, the proposed method was also compared with the HPLC method. The linear range of HPLC method was40-300μg/mL and was narrower than the linear range of the ELISA method, two methods had been very good recoveries. However, the above results were carried out in buffer, further research was needed in the food system.In this study, we successfully synthesized the immunogen of Sunset Yellow FCF and New Coccine and prepared their polyclonal antibodies respectively, and developed an indirect enzyme-linked immunosorbent assay for rapid and convenient detection of Sunset Yellow FCF and New Coccine. This research is helpful for the development of high quality ELISA kits, and opened a way for antibody preparation and ELISA methods development of other synthetic pigments. |
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