Contrary to the Food & Agricultural Organization’s (FAO) suggestion that mycotoxins only affect 20 percent of the world’s grain crops, recent industry studies conclude the number actually hovers closer to 70 to 80 percent.* The realization of this statistic can be attributed to advancements in mycotoxin testing technology.
The existence of mycotoxins has been acknowledged for hundreds of years; however, the bulk of the research and understanding of their chemical structures has evolved only in the last 50 years.
“[Agriculture] has come to the realization that it loses a lot of food and feed to mycotoxins,” explains Dr. Franz Berthiller, head of the Christian Doppler Laboratory for Mycotoxin Metabolism and an assistant professor with the Department of Agrobiotechnology IFA-Tulln of the University of Natural Resources and Life Sciences, Vienna, Austria.
Though regulations are in place to mitigate the risks, despite the feed industry’s best efforts, high levels of mycotoxin contamination remain a major hurdle for feed manufacturers. For example, sampling is the most important phase in mycotoxin determination, but unfortunately it is also the procedure riddled with the most opportunities for error. Such oversights are not necessarily due to the carelessness of the operator, but more attributed to the fact that it’s difficult to draw small amounts of laboratory samples from an inhomogenous bulk of a contaminated commodity.
Once samples are collected, the methods for mycotoxin determination fall into a few different categories: immunochemical, chromatographic and “other methods,” which include optical methods for direct measurement. For the purpose of this review, the focus will be on chromatographic methods coupled to mass spectrometry.
Mass spectrometric methods
Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is hailed as the most accurate of the mycotoxin testing technologies and allows for “the inclusion of a high number of analytes.” The method is able to quantify toxins and other secondary fungal metabolites in feed mixtures rather than limiting the tester to single matrices – streamlining the testing process down to one comprehensive solution.
According to Berthiller, sampling remains a challenge using this model; however, “the variability can be minimized through increasing the size of the sample, number of subsamples, and decreasing the particle size.” After the samples are milled, the next challenge is mycotoxin extraction.
“As mycotoxins are chemically diverse – and range from very polar to very apolar substances – their solubility in liquid solvents is very different,” Berthiller reports. “Then, typically, an acidified mixture of water with organic solvents is used to extract a large amount of different toxins within a single procedure.”
After extraction, liquid chromatography can be employed to separate the toxins from each other and the matrix. Subsequently the compounds are then charged and further separated based on their molecular mass in the mass spectrometer. If tandem mass spectrometry is available, the molecular ions of the mycotoxins are “destroyed” in a collision cell, producing small fragments with characteristic selectivity at very high sensitivity. All of this can be done quickly and with great detail, e.g. within a single second the machine can measure 50 mycotoxins/compounds.
“In 10 years, instruments have gained a factor of 200 in sensitivity,” cites Berthiller.
The LC-MS/MS system only measure ions, meaning that the neutral molecules need to be charged, but the matrix can interfere. However, there are several solutions for dealing with this challenge, specifically: sample dilution; matrix calibration; or the addition of commercially available internal standards (i.e. isotope-labeled mycotoxins).
Why is this methodology important?
While LC-MS/MS is the most accurate method for finding and quantifying mycotoxins, “it is especially important when it comes to legislation and official controls,” Berthiller explains.
It also allows the user to measure multiple compounds at the same time. Today, 380 fungal metabolites can be measured within a single analysis.
Is it necessary to measure so many mycotoxins? Berthiller believes it is, “Masked mycotoxins need to be kept in mind as well. They can be potentially reactivated by cleavage of the conjugate and liberation of the native toxin in the digestive tract of the animals.”
In addition, LC-MS/MS can be used to evaluate a human or animal’s exposure to mycotoxins by identifying biomarkers in urine or blood.
In Berthiller’s opinion, LC-MS/MS instruments most effectively detect “novel toxins and quantify known mycotoxins, but are also key to assess exposure and to determine the efficacy of mycotoxin deactivators.”
*Editor’s note: This article is based off of Dr. Berthiller’s presentation, “Determination of mycotoxins: An overview,” given during the Mycotoxins segment of the BIOMIN’s 2014 World Nutrition Forum.