Background last update:13 Nov 2014

Mycotoxin detection techniques: Then and now

Test kits for mycotoxins have become cheaper, faster and more sensitive and specific. Consultant John Richards explains how detection techniques have improved over the years. "It started in the 1960s when aflatoxins were discovered because they were fluorescent under UV light".

By Vera Traar, Romer Labs, Austria

There are some crucial requirements when it comes to mycotoxin testing. When testing for mycotoxins you are working in a challenging environment where not only time is critical, but often also space and sometimes also access to testing equipment is limited. To guarantee the safety of your agricultural products you need a mycotoxin testing method that is easy-to-use and avoids difficult or time-consuming extraction or sample preparations. The optimal testing method should deliver highly accurate results that can be quantified rapidly, while avoiding difficult or time-consuming sample preparation and procedure steps.

Changes over the years
Obviously mycotoxin detection techniques developed over the years. From the beginning of mycotoxin detection with the discovery of aflatoxins in the early 1960s to today's highly reliable and sensitive multi-mycotoxin detection methods like LC-MS/MS systems and rapid, easy-to-use, on-site tests like Lateral Flow Devices, mycotoxin detection techniques went through a highly sophisticated and fascinating process development. Current state-of-the-art technology is split into two major groups, Reference Testing and Rapid Testing. Reference Testing is largely done with HPLC-UV (or -FLD) and LC-MS/MS systems. These are highly accurate but time consuming analysis methods. The most recent group, Rapid Testing, is gaining more and more importance and due to its fast results and the ability for being used as on-site screening tool it is part of daily routine analysis.

Use of spray reagents
Dr John Richard graduated from Iowa State and is the former Research Leader of the Mycotoxin Unit at National Center for Agricultural Utilization Research in IL and former CEO of Romer Labs. He now serves as a consultant for Romer Labs. Speaking about the developments of mycotoxin detection techniques Dr Richard says: "The beginning with detection for mycotoxins first started with the discovery of aflatoxins in the early 1960s. Detection, of course for aflatoxins was based on the fact that they were fluorescent under UV light so that was used initially for detection of them. In some techniques, small columns were developed and by putting extracts through them a fluorescent band indicating aflatoxins was used. Thin Layer Chromatography (TLC) was the method of choice for analysis of aflatoxins, but with the discovery of other mycotoxins that were not fluorescent innately, various spray reagents were used for detection of spots on the TLC plates. The spray reagents were specific for certain chemical entities of the mycotoxin molecule so that helped with identification from other spots on a TLC plate.

LC-MS/MS systems
In the early to mid-1990s, Romer developed the FluoroQuantTM analysis of aflatoxins, one of the first test kit analyses to be sold to customers throughout the US. It uses a very efficient column for purification of the extracts and the eluate is sufficient for analysis for the aflatoxins. At this time also, for laboratory use, High Performance Liquid Chromatography was used and was the "gold standard" for many years until quite recently. It still is used considerably in many labs both in the US and other countries. However, for laboratory analysis, LC-MS or LC-MS/MS systems were developed and today many laboratories use this technology as it is quite specific and multi-mycotoxin detection methods have been developed and are used with internal standards quite successfully. Also, of importance was the ability of being able to produce antibodies specific for many mycotoxins. Monoclonal antibodies were important as well as polyclonal in this endeavour but monoclonals allowed for large quantities of antibody to be produced. Most of these antibodies were quite specific with little cross reaction with other compounds and thus, the development of the Enzyme Linked Immuno-Sorbent Assay (ELISA) was significant not only as a detection and quantitation system for mycotoxins in laboratories but the entire test could be put into a box and was the major development for test kit analysis of mycotoxins. Back in the beginning of testing for aflatoxins and knowing that they occurred in corn, peanuts, cottonseed and other commodities, there was always talk about needing of a dipstick method for detection of mycotoxins that could be used in the field. Well, we now have a variety of "dipstick" methods or lateral flow tests that can be used in the field and allows anyone to set up a "tail gate" laboratory. One of the early tests developed using antibodies was the AflaCupTM, a membrane based detection system for aflatoxins at specific concentration cutoff values."

Sensitivity and specificity
Asking Dr Richard about the "standard" of mycotoxin detection nowadays, he explains: "This is difficult to say, because it depends upon a person's specific application and needs for a given type of analysis. Insofar as laboratory analyses are concerned, I think that LC-MS/MS is making fast inroads in becoming the standard for use today with the double MS being a highly reliable detection system. Basically, the LC-MS/MS is HPLC using a double MS as a detector. So, HPLC is very important as a standard and without the double MS it is lesser expensive means of analysis that is quite reliable, reasonably fast, and sensitive. The detection is what makes the difference in many cases and the columns used are important." Comparing mycotoxin detection techniques from today with the start of mycotoxin testing, he says that sensitivity of the detection systems along with specificity definitely is what changed the most. Further he mentions that there were other kinds of detection/analytical methods for mycotoxins developed such as Surface Plasmon Resonance, Microarray Technology, Molecular Imprinted Polymers and others that were quite sensitive but proved, in most cases, to be expensive and were never fully accepted. In addition he points out that for food manufacturers and other grain and commodity businesses, the ELISA is fairly standard and depending on need, the lateral flow tests are adequate and are quantitative in a sufficient manner to satisfy many of those needs.

New detection techniques make it possible to detect patulin, a mycotoxin produced by a variety of moulds such as the Penicullum clavigerium (pictured here). The toxin is most commonly found in rotting apples, but it has also been found in other foods such as grains, fruits, and vegetables.

Future and outlook
Considering risks or problems that can occur within mycotoxin detection, he thinks that getting rid of matrix effects is important and efforts should be made to avoid matrix effects.

Also, so-called "hidden" mycotoxins are risks that need to be taken into account when doing an analysis. These along with co-occurring or multiple mycotoxin causation of disease is important to consider. In many cases there are multiple mycotoxins in a problem sample and they must all be considered in the intoxication whether additive of synergistic. Looking at future mycotoxin testing techniques, Dr Richard believes that future developments are likely to include "smart" phones or similar devices in mycotoxin detection.

Further he adds: "We are close to measuring some mycotoxins directly as chemical entities in the whole commodity. This effort of research should be continued."

[Source: Managing mycotoxins, 2014]

Vera Traar, Romer Labs

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