Immunoaffinity column clean up for the analysis of mycotoxins

Abstract

Immunoaffinity column chromatography was identified as the best method for mycotoxin analysis due to great sensitivity, wide application, ease of use, and operator safety. Very recently, manufacturers and testing laboratories have begun to investigate ways to improve the efficiency of testing with Immunoaffinity columns by performing analyses with columns capable of isolating multiple mycotoxins, which are then all detected in a single HPLC run. These improvements make testing easier while still keeping all the advantages of Immunoaffinity analysis. Also, trichothecene testing methods have been developed that broaden the number of trichothecenes that can be easily analyzed from a wide variety of samples. Groups of scientists from the European Union and the United States have been experimenting with immunoaffinity methods capable of purifying up to ten different mycotoxins with a single column. The work of Lusky and Gobel demonstrated that aflatoxins, ochratoxin A and zearalenone could be analyzed effectively in samples of rice, barley, rye and feed, with recovery of toxins averaging in the 90 % range with the exception of aflatoxin G₂. Recovery of aflatoxin G₂ was subsequently boosted as high as 93.4 % with a modification of the immunoaffinity column. Using gradient and wavelength switching for detection, all toxins could be measured with baseline resolution in a single run of 31 minutes. Recoveries were similar across matrices, a typical advantage of immunoaffinity chromatography. In a complex analysis involving isolating and detection of ten mycotoxins from beer and sake, scientists from the United States Alcohol and Tobacco Tax and Trade Bureau and their industry collaborators found recovery of the following toxins using multiple mycotoxin column analyses of beer and sake: aflatoxins B₁, B₂, G₁, and G₂; fumonisin B₁, B₂, B₃; ochratoxin A; zearalenone and, deoxynivalenol. Another recent development in mycotoxin testing has increased interest in analysis of different trichothecenes. Pascale and coworkers recently developed an HPLC method for T-2 toxin (T-2), which is generally difficult to analyze because it lacks a chemical signature. In the improved method, T-2 is isolated from wheat, corn, oats, sorghum or rice with an immunoaffinity column. This renders the T-2 sufficiently pure that it can be derivatized with a fluorophore and detected by HPLC analysis, where it elutes at about 10 min with baseline resolution from other peaks. Recoveries from spiked cereals were from 80 to 99 %, and the relative standard deviation with these samples is excellent, being below 6 % in all cases. For deoxynivalenol, an earlier method by Cahill and coworkers produced recoveries from wheat of 90 % at relative standard deviations of 8.3 %. Detection was through absorbance during an isocratic HPLC run of 5.5 min. Both of these methods for trichothecenes provide the ease and accuracy needed for analysis of this class of toxins.