Establish And Optimization Of Simultaneous Detect Acid Orange Ⅱ And Auramine O In Food By HPLC

Compared with other water-soluble dyes, Acid Orange Ⅱ and Auramine O, as the inedible pigments, are more easily dyeing and harder to fade. However, the unscrupulous traders often added the two non-foods colorings to foods illegally for there are no national standard recommended analytical methods of these two at present. This study is aimed to establish and optimize HPLC to detect these two kinds pigments, research the pre-treatment conditions of Acid Orange Ⅱ and Auramine O according to analysis the recoveries through their changes, and check different kinds samples in the market by using the above methods. The main findings are as follows:1. Establish an equal concentration and fixed wavelength HPLC to detect these two kinds of inedible pigments based on the structure and properties of the Acid Orange II and the Auramine O. After studying and optimizing the factors of column, column temperature, the type and proportion of the mobile phase, flow rate, etc., the HPLC detection conditions are obtained:The column:Kromasil C18 (4.6×150mm, 5um); mobile phase:methanol-50mmol/L ammonium acetate (V:V)= 50:50; flow rate:1.0mL/min; detection wavelength:450nm; column temperature:35℃. Under these conditions, the retention time of the Auramine O’s and the Acid Orange Ⅱ are 7.632min and 11.548min respectively, the theoretical plate number are 3021 and 9084, the peak base width are 0.447 and 0.435, the separation are 5.62 and 7.11, respectively, and the peak was normal and no phenomenon of smearing.2. Use ultrasound centrifugation to extract the Acid Orange Ⅱ and the Auramine O of different kinds of foods, and apply the established HPLC to determine the two substances quantitatively. Taking the two kinds of inedible pigments total recoveries as an index, investigate five extraction parameters of pretreatment which are the extraction agent types, extraction time, ultrasonic frequency, solid-liquid ratio, ultrasonic temperature, optimized extraction parameters and optimize them by response surface methodology finally. The best extraction parameters of pretreatment are extraction agent-Methanol, ultrasonic time-30min, ultrasonic temperature-20℃, solid-liquid ratio-1:3, ultrasonic frequency-3. Under these conditions, the actual test results are the recovery of the Acid Orange II is 90.03%, while the Auramine O is 87.26%.3. Evaluate methodologically the methods how the established and optimized HPLC detect the Acid Orange II and the Auramine 0 simultaneously. The results showed that the Acid Orange II in the range of 0.025-0.125ug has a good linear relationship, and the linear equation is Y= 2*106X-1240.1 (r=0.9999, while the detection limit is 0.120ug/kg, and the precision is 1.19%. Furthermore, the reproducible results in five kinds of foods like yuba、croaker、pickle、chicken、 paprika are respectively 1.11%,1.98%,1.78%,1.93% and 1.80%, with the intraday stability RSD is 1.18%. And the recovery results in these five kinds of foods are respectively 90.47%(RSD=1.2%),80.53%(RSD=1.3%),88.93%(RSD=0.8%), 80.38%(RSD=2.0%),83.10%(RSD=1.8%)The Auramine O in the range of 0.025-0.125ug has a good linear relationship, and the linear equation is Y=7*106X-5284.2 (r=0.9996), while the detection limit is 0.032ug/kg, and the precision is 1.10%, Furthermore, the reproducible results in five kinds of foods, like yuba、croaker、pickle、chicke、paprika, are respectively 1.47%、1.88%、1.87%、1.85%、1.93%, with the intraday stability RSD is 0.87%, The recovery results in these five kinds of foods are respectively 102.64% (RSD=1.8%),84.76(RSD=1.7%),89.12%(RSD=1.4%),98.42%(RSD=1.6%), 86.8%(RSD=2.2%).4. Apply the HPLC established in this study to detect the content of the Acid Orange Ⅱ and the Auramine O which are illegally added in different kinds of foods on the market. The 40 samples are which sold in the market and the supermarket, which are bean products (including Yuba, tofu, etc.), meat (chicken, vacuum-packed chicken, etc.), fresh fish (fish, vacuum-packed croaker, etc.), spices (including paprika, bean paste, etc.). The content of the Acid Orange Ⅱ within the chili powder in the market detected is 5.34 mg/kg. The content of the Auramine O within the croake is 1.92 mg/kg, while both non-food colorings were not found in the remaining samples.