Development Of IAC-HPLC Method To Determine Zearelenone And Ochratoxin A In Cereal

Zearelenone (ZEN) is a nonsteroidal estrogenic mycotoxin which is mainlyproduced by numerous species and subspecies of Fusarium including: Gibberellazeae, Fusarium graminearum and Fusarium tricinctum.Oehratoxins are products of several moulds of the genuses: Aspergillusochraceus and Penicillium verrucosum. They have been shown to be nephrotoxic,hepatotoxic, teratogenic, carcinogenic and immunosuppressive under diverse toxic tokidney and liver of several animal species and human beings. In several forms ofochratoxins: A, B, C and D, OTA occurs in various plant products commonly withhighly toxic and harmful to people's health. In 1993, the International Agency forResearch on Cancer (IARC) classified OTA as possibly carcinogenic for humans.A method (IAC-HPLC) for determination of ZEN and OTA in cereal wasestablished respectively. 49 samples of cereal were detected by the anti-ZEN IAC and20 samples of cereal were detected by the anti-OTA IAC which were produced by ourlaboratory, the results were compared by ELISA kit. The agreement between the twomethods was excellent.All the results are deseripted as follow:1 Development of High Performance Liquid Chromatography method to determineZearelenone in cereal1.1 Preparation of Zearalenone immunoaffinity columnThe Zearalenone immunoaffinity column (IAC) was prepared by couplingCNBr-activated Sepharose 4Fast Flow(4FF) with the anti-ZEN monoclonal antibodywhich was purified by the caprylic acid ammonium sulfate method. The couplingreaction was identified by UV-absorbance measurements and the IAC to be prepared was evaluated by indirect-competition ELISA and HPLC. The column capacity wasdetermined to be 0.40μg when using 0.5 mL CNBr activated Sepharose 4FF and 350μg purified anti-ZEN monoclonal antibody.1.2 IAC-HPLC method to quantify zearalenone in cereal is eatablishedAverage recoveries of ZEN from wheat and maize spiked at levels of 60μg/kg～300μg/kg ranged from 76.33％to 90.10％and 78.36％～99.64％, withrelative standard deviations from 6.68％to 10.93％and 7.28％～10.33％,respectively. The detection limit was 10μg/kg based on a signal-to-noise ratio of 3:1.1.3 The cereal samples purchased from market were detectedComparative analysis of 49 cereal samples using ELISA kit and HPLC method,the result showed non-significant lack (P＞0.05).2 Development of High Performance Liquid Chromatography method to determineOchratoxin A in cereal2.1 Preparation of Ochratoxin A immunoaffinity columnThe OTA immunoaffinity column (IAC) was prepared by couplingCNBr-activated Sepharose 4Fast Flow(4FF) with the anti-OTA monoclonal antibodywhich was purified by the caprylic acid ammonium sulfate method. The couplingreaction was identified by UV-absorbance measurements and the IAC to be preparedwas evaluated by indirect-competition ELISA and HPLC. The column capacity wasdetermined to be 0.15μg when using 0.5 mL CNBr activated Sepharose 4FF and 100μg purified anti-OTA monoclonal antibody.2.2 IAC-HPLC method to quantify OTA in cereal is eatablishedAverage recoveries of OTA from wheat and maize spiked at levels of 5μg/kg～100μg/kg ranged from 90.72％to 102.87％and 75.60％～105.28％, with relativestandard deviations from 0.59％to 12.83％and 10.13％～12.72％, respectively.The detection limit was 0.2μg/kg based on a signal-to-noise ratio of 3:1. 2.3 The cereal samples purchased from market were detectedComparative analysis of 20 cereal samples using ELISA kit and HPLC method,the result showed non-significant lack (P＞0.05).