Wageningen Academic World Mycotoxin Journal, 2016; 9 (5): 847-861 Publishers SPECIAL ISSUE: Mycotoxins in a changing world Challenges in the analysis of multiple mycotoxins J. Stroka1* and C.M. Maragos2 1Joint Research Centre, European Commission, Retieseweg 111, 2440 Geel, Belgium; 2Agricultural Research Service, National Center for Agricultural Utilization Research, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA; [email protected] Received: 17 January 2016 / Accepted: 15 June 2016 © 2016 Wageningen Academic Publishers OPEN ACCESS REVIEW ARTICLE Abstract The problems associated with different groups or ‘families’ of mycotoxins have been known for some time, and for many years certain groups of mycotoxins have been known to co-occur in commodities and foods. Until fairly recently commodities and foods were analysed for individual toxins or groups of related toxins and attempts to measure multiple groups of toxins required significant investments in terms of time, effort, and expense. Analytical technologies using both the instrument-intensive techniques, such as mass spectrometry, and screening techniques, such as immunoassays, have progressed significantly in recent years. This has led to the proliferation of techniques capable of detecting multiple groups of mycotoxins using a variety of approaches. Despite considerable progress, the challenges for routine monitoring of multiple toxins continue. Certain of these challenges, such as the need for co-extraction of multiple analytes with widely different polarities and the potential for carry-over of matrix components that can influence the results, are independent of the analytical technique (MS or immunoassay) used. Because of the wide variety of analytical platforms used for multi-toxin analysis, there are also specific challenges that arise amongst the analytical platforms. We showed that chromatographic methods with optical detection for aflatoxins maintain stable response factors over rather long periods. This offers the potential to reduce the analytical burden, provided the use of a single signal receives general acceptance once shown in practise as working approach. This must however be verified by a larger community of laboratories. For immunosensors the arising challenges include the reusability of sensors and, for chromatography-based assays they include the selection of appropriate calibration systems. In this article we seek to further describe the challenges associated with multi-toxin analysis and articulate how such challenges have recently been addressed. Keywords: mycotoxins, plant toxins, liquid chromatography, mass spectrometry, determination, biosensors 1. Introduction The mycotoxins generally accepted to be of the greatest importance to human and animal health, and to have the While mycotoxins likely pre-date human agriculture, it greatest economic impact are those belonging to several is only fairly recently that the nature of these compounds groups of toxins (‘families’): the aflatoxins, trichothecenes, and their prevalence in agricultural products have become fumonisins, ochratoxins, and zearalenones. known. Recent history has seen dramatic changes in agronomic practices, and further changes are expected Until fairly recently, the analysis of such toxins was done as climates change throughout the world. As previous individually or as families. The decision of which toxins contributions to this issue have noted, this will likely to test for was done based upon the fungi that might be lead to changes in the distribution, if not the type, of expected to be present in the commodity and the toxins mycotoxin contamination present in our food supply. Since which they might produce. This targeted approach is logical, the discovery of aflatoxins, mycotoxins of many different particularly because the costs for monitoring can be high, types have been discovered and analytical methods for especially in relation to the value of the commodity being their detection and quantification have been developed. tested. However, new analytical technologies such as high ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2016.2038 847 J. Stroka and C.M. Maragos resolution mass spectrometry and biosensors now facilitate maize, in which most the above mycotoxins have been detection of multiple toxins in a relatively simultaneous reported to occur. Consideration should also be given to fashion. This can permit monitoring for larger numbers differences in consumption patterns in different regions of of potential contaminants, permitting a less targeted the world, which when combined with exposure estimates, approach for monitoring and revealing heretofore unknown may suggest a ‘one size fits all’ approach to regulation may potential hazards. The promise of the newer technologies not be appropriate for every region or climate. is greater efficiency in detection of hazards. Increasing such efficiency and reducing monitoring costs have the In parallel to regulatory developments, progress on the potential to permit increased monitoring, further reducing availability of technologies that allow for the simultaneous the potential for exposure- a positive feedback loop. An determination of mycotoxins has occurred. On one hand additional benefit of testing for many compounds is that new immunochemical tools have progressed, allowing they provide prevalence data that may be serve as criteria the application of easy-to-use systems for end-users. for putting a new compound on the list of those for which On the other hand improved mass selective detectors targeted analysis is appropriate. have become available in many analytical laboratories, replacing to a certain extent the classical (optical) detectors The potential benefits of multi-toxin detection are clear. for mycotoxin identification and quantification by liquid There are, however, challenges associated with multi-toxin chromatography. While immunochemical methods are analysis. These include resolving conceptional and societal rather specific for a certain mycotoxin or a group of closely issues as well as the inherent technical challenges. Detecting related mycotoxins, modern mass-spectrometers can be large numbers of potential metabolites and toxins was applied to target structurally different mycotoxins in a once limited by technical constraints, but increasingly single method. Antibody-based techniques and mass the technologies to detect greater and greater numbers spectrometry are not mutually exclusive, and using them of compounds in complex commodities and foods are together (e.g. immunoaffinity column clean-up followed becoming more widely available. This has created a by LC-MS/MS) is a potent combination for assuring very central philosophical challenge to analysts: how many (and high selectivity. which) toxins to include in the test panel. In this case, social considerations both help and hinder the process for As noted, some guidance in selecting targets is obtained target selection. In a very real sense, societies declare which through the need to meet regulatory requirements. problems they regard as significant by exercising control However, the application of broader methods has the over them (regulation). Hence the presence of regulatory potential to allow for the identification of previously levels helps to determine which toxins are important targets. unknown food safety risks (Sulyok et al., 2010). This is particularly true for commodities having established A fundamental issue in multi-toxin detection is which toxins problems with known toxins. If the food supply were a should be the focus in multi-toxin tests. Certainly, the static system, limiting targets to regulated toxins might be main drivers for mycotoxin testing in commercial routine sufficient for adequate control and protection. However, the testing as well as food or feed law enforcement laboratories food supply is a very dynamic system with a tremendous are the legal obligations that apply, or trade specifications. number of variables including the environment, agronomic As a matter of fact, until the first half of the 1990’s it was practices, post-harvest treatment, processing, and shipment primarily aflatoxins, and little more, that were routinely – often to destinations far from the source of production. screened for. In 1998 the first EU legislation on aflatoxins As discussed elsewhere in this special issue of World and ochratoxin A (OTA) came into force with Commission Mycotoxin Journal, climate change may also be a factor Regulation (EC) No 1525/98 (EC, 1998) and until then the leading to a shift of fungal populations and consequently main detection techniques for liquid chromatographic a shift of mycotoxin patterns. Furthermore, new toxins methods were based on the determination of the natural emerge, and precursors and metabolites of established fluorescence of both, the aflatoxins and OTA. Now, about toxins (e.g. the ‘masked’ respectively ‘modified’ mycotoxins) two decades later, the legal framework of mycotoxin are increasingly found. The result is that the number of regulations in the EU has widened, to include those mycotoxins regarded as relevant has increased over time. previously mentioned, as well as deoxynivalenol (DON), Nonetheless, the process of looking for new possible patulin, citrinin, zearalenone (ZEA), fumonisins (EC, 2006a) threats to human or animal health should be approached and T-2 and HT-2 toxins (EC, 2013). A rather unfortunate with critical reflection. Perspectives