If you’re in the dairy or food testing business, you know the threat aflatoxins pose. Aflatoxins are a type of mycotoxin produced by Aspergillus parasiticus, aspergillus flavus , and rarely aspergillus nomius.1 These are likely the most extensively researched group of mycotoxins because of their adverse health effects.2 What’s more, they are widely found in a variety of crops, namely maize, tree nuts, and spices. Believed to be primarily caused by rising temperatures and humidity, these naturally occurring fungi grow on crops in the field, or during storage of feed and raw materials, where they can potentially produce toxins that enter the food chain.

There are four major types of aflatoxins: B1, B2, G1 and G2, that are significant to the food chain.3 Of the four, aflatoxin B1 (AFB1) is the most carcinogenic and most commonly found in food.4

Contamination from aflatoxins also extends beyond those directly produced by the fungi. For example, when a cow ingests AFB1, it can metabolize to become aflatoxin M1 (AFM1). It is through this enzymatic process that AFM1, which also has carcinogenic properties, is indirectly produced and can be detected in bovine milk.

Given these risks, regulators have taken notice. The United States regulations for example, set a maximum residue limit of 500 ng/kg for regular milk and 25 ng/kg for infant milk products.

The complexity of milk

Historically, mycotoxin testing has been a challenge for the food safety industry. Milk contains a complex variety of compounds, including fat, proteins and carbohydrates which can complicate the analysis of contaminants. Also, mycotoxins are a diverse class of compounds with a wide range of polarities and physical properties, which further complicates testing. This can lead to challenges when developing chromatographic methods to separate multiple classes of mycotoxins from each other and their potential interferences.

Fret not, liquid chromatography-tandem mass spectrometry (LC-MS/MS) to the rescue. Luckily, mycotoxins tend to ionize quite readily by electrospray ionization (ESI), which means that LC-MS/MS with ESI can give high sensitivity detection of different mycotoxin profiles, to confirm the presence and quantity of contaminants. The selectivity afforded by tandem mass spectrometry allows for the signals coming from the individual mycotoxins to be independent from each other and from potential background interferences, significantly simplifying the chromatographic separation.

In 2019, a Food Safety and Standards Authority of India (FSSAI) report revealed an AFM1 in milk samples that prompted several states, including Tamil Nadu, to take some measures. In response, our team collaborated with Vaishali Patel of the Food and Drug Testing Laboratory, India, to put together an application note to identify and quantify AFM1 in extracted milk samples using the SCIEX QTRAP 4500 system. Using a relatively simple liquid-liquid extraction protocol, this method was easily able to meet the 500 ng/kg MRL, without the need for laborious solid phase extraction.

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References
1. Creppy, E. E. (2002). Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters, 127(1-3), 19–28. https://doi.org/10.1016/s0378-4274(01)00479-9
2. Trucksess, M. W., & Diaz-Amigo, C. (2011). Mycotoxins in Foods. Encyclopedia of Environmental Health, 888–897. https://doi.org/10.1016/b978-0-444-52272-6.00700-5
3. Joint FAO/WHO Expert Committee on Food Additives (JECFA). (2018, February). Food Safety Digest Ref. No.: Who/Nhm/Fos/Ram/18.1. https://www.who.int/foodsafety/FSDigest_Aflatoxins_EN.pdf. Accessed July 16, 2021.
4. Hamid, A.S, Tesfamariam, I.G ., Zhang, Y.C., & Zhang, Z. G. (2013). Aflatoxin B1-induced hepatocellular carcinoma in developing countries: Geographical distribution, mechanism of action and prevention. Oncology Letters, 5(4), 1087–1092. https://doi.org/10.3892/ol.2013.1169