LC/MS/MS in Detecting and Quantifying Mycotoxins

Mycotoxins are produced primarily by Aspergillus, Penicillium, and Fusarium fungi growing on a variety of agricultural commodities worldwide. They pose a major threat to human and animal health, as they have been implicated as causes of cancer and mutagenicity, as well as estrogenic, gastrointestinal, urogenital, vascular, kidney, and nervous disorders. Some may also impair resistance to infectious disease by compromising the immune system. Their impact on human health, animal productivity, and international trade results in significant economic losses.

The mycotoxins that pose the biggest threat to food safety include the aflatoxins, ochratoxin A, and toxins produced by Fusarium molds, including fumonisins, trichothecenes, and zearalenone. Aflatoxins (B1, B2, G1, G2, and M1), are the most toxic, including damage to DNA that can cause cancer in animals. In fact, AFB1 and mixtures of AFB1, AFG1, and AFM1 are proven human carcinogens, and AFM1 and AFB2 are designated as probable human carcinogens by the International Agency for Research on Cancer (IARC). They contaminate many crops grown in hot and humid regions of the world, including peanuts, corn, cottonseed, and pistachios.

Ochratoxin A is produced by several Penicillium and Aspergillus fungal strains, and it occurs in a large variety of foods. It is classified by the IARC as a probable human carcinogen and is also implicated in kidney damage, birth defects, and immune deficiency.

Fumonisins are the result of fungal infection of maize, tomatoes, asparagus, and garlic, but maize-containing foods are the major food safety concern for fumonisin contamination. There are at least 15 related fumonisin compounds, and fumonisin B1 can cause necrotic lesions in the cerebrum in horses, and pulmonary edema in swine. The fumonisins are weak carcinogens in rodents and probable human carcinogens that have been associated with esophageal cancer in South Africa and China. The level of fumonisin contamination in corn was relatively high in the U.S. between 1988 and 1991, but has been low in recent years.

Only a few of the nearly 200 trichothecenes occur at concentrations high enough to pose significant threats to human health. The most prevalent of these is deoxynivalenol (DON), also known as vomitoxin. DON occurs predominantly in grains such as wheat, barley, oats, rye, and maize, and it is immunotoxic in animal models. It is not a known carcinogen and its major symptom in animals is reduced feed intake. Large amounts of grain with vomitoxin would have to be consumed to pose a health risk to humans. Type A trichothecenes like T-2 toxin or HT-2 toxin are more toxic to mammals than type B trichothecenes such as DON, but fortunately often occur in lower concentrations. Oats are the most prone cereals for contamination by trichothecenes, followed by barley and maize.

Zearalenone is an estrogenic compound found almost entirely in grains that has received recent focus due to concerns that environmental estrogens can disrupt sex steroid hormone functions. In fact, occasional outbreaks of zearalenone mycotoxicosis in livestock have caused infertility. Zearalenone has also been reported to have genotoxic activity.

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Figure 1: Chromatogram of a calibration sample containing all 11 EU regulated mycotoxins illustrating the separation efficiency of the UHPLC method run on an Agilent 1290 Infinity LC system and an Agilent 6490 Triple Quadrupole LC/MS.

Regulating Levels in Food and Feed

Limiting mycotoxin exposure to humans and agricultural animals is paramount, and more than 100 countries regulate levels of mycotoxins in foods and feed because of their public health significance and commercial impact. The U.S. FDA has established advisory levels for DON and fumonisins and action levels for aflatoxin, but regulatory limits have not been established in the U.S. for mycotoxins. China, Brazil, and Mexico have the most comprehensive legislation on aflatoxin. China and Russia have established limits for ochratoxins in cereals and other products. Several countries, including India and Japan, have maximum limits for DON. However, in the international markets, no maximum limits for fuminisins exist in several countries, including Russia, Canada, and many Latin American countries. Several countries do have maximum limits for zearalenone.