Development Of IAC-HPLC Method To Determine Sudan Ⅰ

In this study, Sepharose 4FF was activated by ECH to be Sepharse 4FF-epoxy, and further bound with 6-Aminocaproic acid (AHA). Then Sepharse 4FF-epoxy-AHA was coupled with the anti-Sudan I monoclonal antibody. The Sudan I immunoafFinity column was prepared, and a method (IAC-HPLC) for determination of spiked Sudan I in chilli powder was developed. The results of this study are descripted as follows:1 Activation and modification of Sepharose 4FFSepharose 4FF was activated by ECH, and explored the conditions of Sepharose 4FF activation. The optimum conditions were obtained as follows:the reaction temperature was 40℃, the reaction time was 2 h 20 min, percentage of DMSO and ECH was 46.8% and 30%, respectively. The initial concentration of sodium hydroxide was 8 mol/L, and its final concentration was 0.8 mol/L. Under this optimum condition, epoxy density was 80μmol/g. Sepharose 4FF-epoxy coupled with AHA spacer then optimized the coupled conditions of Sepharose 4FF-epoxy and NH2-Sudan I. The optimum conditions were obtained as follows:coupled buffer was pH 8.30.1 mol/L NaHCO3 with 0.5 mol/L NaCl, the reaction temperature was 25℃, the reaction time was 8.5 h.2 Sepharose 4FF-epoxy-AHA coupled with ligandsSepharose 4FF-epoxy-AHA coupled with NH2-Sudan I, and the coupled conditions was optimized. The optimum conditions were obtained as follows:the concentration of ED AC was 19 mg/mL, the reaction temperature was 27℃, the reaction time was 16 h. Under above optimal conditions, high temperature would damage the antibody activity. This conditions were further optimized, the results showed that the concentration of ED AC was 19 mg/mL, the reaction time was 72 h in 4℃.3 Preparation of Sudan I immunoaffinity columnSepharose 4FF-epoxy-AHA was coupled with the anti-Sudan I monoclonal antibody which was purified by the caprylic acid ammonium sulfate. Immunoaffinity column capacity of 0.3 g medium coupled with 1 mg antibody was 1.6μg by the detection of HPLC.4 Development of IAC-HPLC to determine Sudan IA reversed-phase high performance liquid chromatographic method was used to determine the samples elution, the mobile phase consisted 0.1% formic water-acetonitrile-0.1% formic acetonitrile-acetone (21.25:3.75:60:15, v/v/v/v), and the detection wavelength was 478 nm. And optimized IAC operating parameters, the results showed that loading buffer was 20% acetonitrile-PBS, sampled on one times, loading the volume of acetonitrile elution was 3 mL. Average recoveries of Sudan I from chili powder spiked at levels of 0.25-3 mg/kg ranged from 44.52% to 77.40%. The detection limit was 125 ng/mL, the coefficient of variation was 4.6% to 8.3%. The immunoaffinity chromatoghraphic column was prepared successfully.