Development Of A Gas Chromatograhic Method To Determine Deoxynivalenol In Wheat And Corn And Preparation Of Monoclonal Antibody Immunoaffinity Column For Ochratoxin A

Deoxynivalenol (DON) and ochratoxin A (OTA) are mycotoxins that usually contaminate cereals and their products. DON is a teratogenic toxic metabolite produced mainly by Fusarium spp. DON can cause nausea, feed refusal, weight loss diarrhea, immunosuppression and renal toxicity. OTA is a nephrotoxic, teratogenic and imunotoxic compound produced by Aspergillus spp and Penicillium spp. Kidneys and livers are the main target organs of OTA.IACs for OTA were prepared and a gas chromatographic method to detect DON in wheat and corn was developed in this study. The results of the study are concluded as below.1. Development of a gas chromatographic method to determination DON in wheatand corn.Samples were extracted with acetronile-water (84:16,V/V). The wheat extract was then purified by a Florisil column, and the corn extract purified by a two-step procedure using Florisil and active carbon columns. The final extract was then derivatized with N-heptafluorobutyrylimidazole (HFBI) and quantitated by GC with electron capture detector (GC-ECD).The limit of detection (LOD) of this method was 0.01mg/kg for DON. Samples with DON levels from 0.075 to 40mg/kg could be quantitated by this method. Recveries of DON spiked at levels of 0.5-1.5mg/kg ranged from 82.2% to 98.53% for wheat and from 86.0% to 103.4% for corn, with coefficients of variation (CVs) less than 8%.22 wheat samples and 20 corn samples were quantitated for DON by both GC-ECD and ELISA kit. According to t-pair test with the software of Sigmaplot, the results showed that the two methods have no obvious difference (P=0.09>0.05). And the GC-ECD method developed was more sensitive than the ELISA kit produced by r-Biopharm, of which LOD was 0.050mg/kg , and linearity was 0.2-6 mg/kg.2. Development of IAC for OTAThe performances of IAC for OTA were analyzed by indirected competitive enzyme-linked immunosobent assay (IC-ELISA) and high-performance liquid chromatography (HPLC). The IAC has a column capacity (the capability of binding OTA) of 200ng, with the recovery ranged from 90.38% to 100.1%, and could be used up to 3 times. The IAC developed in our lab had no significant difference from the products fromabord as far as their identical performances was concerned.OTA in cereal samples were determined by HPLC linked with IAC prepared in our lab. Cereal samples were extracted with 60% acetronile. The extracts were diluted 1:10 by 0.01M PBS (pH7.4) and then purified by the IAC, and the eluate was detected by HPLC with fluorescence detector.LOD of the method was 0.2ju,g/kg. Samples with OTA levels from 0.6 to 400 ug/kg could be quantitated by this method. Recoveries of OTA spiked at levels of l-10ug/kg ranged from 78.7% to 87.1% with coefficients of variation (CVs) less than 6.5%. Total of 15 cereal samples were determined by this method, the positive detective rate was 46.7%, the highest OTA concentration detected was 0.785|j.g/kg.