Develoment, evaluation and application of methods for mycotoxin analysis.

Mycotoxins are toxic secondary metabolites produced by fungi. Due to their high toxicity and the widespread presence of these compounds in various food commodities and agricultural products, mycotoxins are still a worldwide problem and it is important to establish maximum permissible levels in diverse types of food. Consequently, the development of a mycotoxin detection method that is rapid, sensitive, and specific is essential. Different analytical methods have a variety of performance factors and are thus suited for different purposes. Nowadays, over 300 mycotoxins are known and they show great diversity in their chemical and physicochemical properties. The different mycotoxins in different food matrices require specific methods of extraction, clean-up and determination, which affect the performance of analysis.We investigated the suitability of acidified acetone/water mixtures as a substitute for acetonitrile/water for simultaneous mycotoxin analysis for three kinds of matrices: those of wheat, maize, and rice. Thirteen extraction solvents based on acetone, acetonitrile, and methanol were compared for deoxynivalenol, zearalenone, fumonisins B1 and B2, and beauvericin analysis by LC-MS/MS without sample dilution. In further analysis, methanol-based and acetone-based solvents were selected for 27 mycotoxins including aflatoxins, beauvericin, citrinin, enniatins, fumonisins, gliotoxin, ochratoxin A, patulin, sterigmatocystin, trichothecenes type A and B, verrucarol, and zearalenone. Extraction efficiency was determined by comparing LC-MS/MS signals with the signals of spiked matrix extracts, and this revealed that acetone/water/acetic acid (80:19:1, v/v/v) was the best extraction solvent. We propose this solvent as a replacement for acetonitrile-based solvents for mycotoxin extraction for multi-toxin methods. ELISA is a rapid mycotoxin screening method and has become a favourite for routine analysis, but it is still important to validate this method. Therefore, we investigated the performance of the ELISA when compared with the hyphenated method, LC-MS/MS by measuring deoxynivalenol, fumonisin B and zearalenone in maize samples inoculated with Fusarium verticillioides and Fusarium graminearum in experimental fields in Germany. This was done from 2006 to 2009. To investigate the fluctuation of ELISA, three inter-laboratory results were also compared with one LC-MS/MS laboratory. Good correlations and good agreement between methods were found upon analysis by linear regression and Bland-Altman plot. However, the performance of ELISA depended on the skill of the technician and the cross-reactivity of the ELISA test kits with similar compounds. Furthermore, we found that the ELISA is valuable to use as a screening method for samples with a high level of mycotoxin contamination, in which case it is rapid and easy to use. In cases of a low level of mycotoxin contamination, the sample results should be confirmed by LC-MS/MS. Finally, we investigated the occurrence of fumonisin in unpolished rice or Red cargo rice, which is a staple human food. Red cargo rice retains its bran layer, and hence may be contaminated by mycotoxins such as fumonisins produced by Fusarium spp. However, little information on the determination and detection of fumonisins in rice has been reported. Therefore, we optimized a detection method for fumonisins and surveyed the occurrence of fumonisins, particularly the major toxicant (FB1), in Thai red cargo rice by LC-MS/MS. This method provides a sensitive detection limit at 1.0 ng g-1. The limit of quantification was 5.0 ng g-1. An accuracy showed high yield of mean recovery after fortification sample. Of the 58 Thai red cargo rice samples from the retail markets, two samples were found to be naturally contaminated with fumonisin B1 at a trace level (lower than 5.0 ng g-1). No fumonisin B2 was found in any of the samples.