How to Identify Mycotoxins in Cannabis
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When you think about cannabis contaminants, it’s natural to picture materials or chemicals being physically added to the cannabis material. And this does happen; there are well-publicized cases of potentially dangerous illegal pesticides being spread on cannabis crops, or lead metal being added to street-sold cannabis to increase its weight and subsequent value.
But it’s also crucial for those involved with the quality and safety testing of cannabis to consider naturally occurring sources of contamination.
The cultivation conditions for cannabis plants create an ideal environment for the growth of molds and fungi, which, if allowed to proliferate, can begin to produce chemical mycotoxins. These mycotoxins are a known risk within the food safety industry, and have been linked to kidney and liver damage, reproductive disorders, and immune suppression. Particularly dangerous are the aflatoxins, a type of mycotoxin produced by some aspergillus fungi species, which are potent carcinogens. Even acute exposure can be life-threatening if the dose of aflatoxin is large enough, as the resultant aflatoxicosis can cause severe liver failure.
Cannabis material can also be contaminated with mycotoxins during transportation, storage, and processing. Given the health risks associated with exposure to these mycotoxins, it’s considered essential for the sake of consumer safety that mycotoxin analysis is carried out as part of the drug’s normal testing regimen.
Identifying the mycotoxin risks to cannabis
Due to the continued federal prohibition of cannabis, states in the US with a legal medical and/or recreational cannabis industry often create their own regional limits and requirements for cannabis testing. In a worst-case scenario, this lack of federal direction can lead to a patchwork of state-level regulations across the US, where some states choose to impose strict limits on specific potential contaminants where others might choose to not regulate the substance at all.
When it comes to mycotoxin and aflatoxin testing, the regulations are perhaps surprisingly more uniform across the United States than one may expect. Firstly, almost all state testing requirements tend to focus on five mycotoxins in particular: aflatoxins B1, B2, G1, G2, and ochratoxin A.
While there are more than twenty aflatoxins known to scientists, these four aflatoxins (B1, B2, G1, and G2) have been identified as the most dangerous to human health. Additionally, aflatoxins B1 and B2 can be metabolized by the body to produce aflatoxins M1 and M2 which also pose various health risks, including carcinogenicity and genotoxic activity. Ochratoxin A is a concern as, while its exact health effects on humans are unclear, overexposure to the toxin has been linked to kidney damage and related cancers.
“Exposure to high levels of mycotoxins can be extremely dangerous, especially if the exposure is long-term,” said Amanda Horodyski, the microbiology lab manager at the Maryland-based cannabis and hemp testing laboratory, Atlantic Test Labs, to Analytical Cannabis.
“The mycotoxins relevant to cannabis, aflatoxin and ochratoxin, have been shown to be carcinogenic,” Horodyski continues. “Consumption or inhalation of high levels aflatoxin B1, B2, G1, and, G2 have been shown to cause liver damage. Ochratoxin A has also been demonstrated to be an immune suppressor and can cause kidney damage.”
“Mycotoxins are extremely difficult to break down, making them a dangerous toxin to find in cannabis,” Horodyski warns.
Safety requirements for mycotoxin presence
Once again, in a somewhat unexpected display of uniformity, state testing regulations widely appear to stick to a maximum allowable limit of 20 μg/kg of mycotoxin detected on a sample. However, there is variation in exactly how this limit is applied between states.
For example, the Illinois Department of Agriculture requires each of the five main mycotoxins to separately be under the limit of less than 20 μg/kg, but in Nevada and California, the total sum of detectable aflatoxins B1, B2, G1, and G2 combined must be under 20 μg/kg, with ochratoxin A also separately testing out as less than 20 μg/kg. New Mexico’s Department of Health enforces one of the tightest restrictions on mycotoxin presence, requiring the collective amounts of all five main mycotoxins to sum to less than 20 μg/kg.
According to the World Health Organization, consumption of a food or product containing more than 1 mg/kg (1 part per million) of aflatoxin can be enough to lead to life-threatening aflatoxicosis and liver failure. Alternatively, sustained exposure to lower dosages, on the order of 20-120 μg/kg daily for a period of 1-3 weeks, of aflatoxin B1 specifically can also be acutely toxic and potentially lethal.
The WHO also notes that detecting aflatoxicosis and mycotoxin poisoning in humans can be very difficult, and so the onus is very much on accurate testing methodologies to ensure that consumers of cannabis, and foodstuffs that are also susceptible to mycotoxin contamination, are adequately protected.
How are labs testing for mycotoxins?
There are several different methods that are currently in use within the cannabis industry for mycotoxin testing. These vary from the simplest qualitative strip tests, all the way to highly complex, expensive apparatus, such as the ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS).
Qualitative strip tests, a good preliminary measure
Testing strips are an incredibly straightforward and simple way to test for the presence of mycotoxin on a sample. They generally come in two forms: qualitative or quantitative.
Qualitative strips can tell whether there are mycotoxins present on a sample, but they give very little information on exactly how much aflatoxin or ochratoxin is present. They work by exploiting a natural reaction between antibodies and mycotoxins, which causes the strip change color when exposed to mycotoxins above a certain concentration.
By mixing cannabis flower with a solvent like methanol, it’s possible to create a cannabis solution that can be tested quickly and easily using the strips. The low level of expertise and manpower needed to perform this test makes it a good preliminary screening procedure.
By comparison, quantitative strips take a little longer and often require some additional equipment, but they are able to give a digital readout of the amount of mycotoxin detected – down to a level of just 2 μg/kg or 2 ppb (parts per billion) for some systems. Like the qualitative strips, quantitative strips also rely on the reaction between antibodies and the mycotoxins to accurately detect and measure the presence of a range of mycotoxins.
The first step is carrying out an extraction with a liquid solvent to make a cannabis solution. The solution can then be dropped onto a region of the qualitative strip and left to develop. After five minutes, the qualitative strip can be loaded into a special reader, which interprets the strips and gives a digital readout of the results.
Immunoaffinity columns and fluorometry
While strip tests are a good preliminary reading, cannabis testing laboratories will use more rigorous analysis methods of analysis.
One of the workflows most commonly used involves the usage of immunoaffinity column chromatography to effectively isolate mycotoxins from a sample, and a bench-top fluorometer to accurately quantify each individual aflatoxin and ochratoxin that may be present.
Immunoaffinity columns contain a small plug made up of inert support beads coated with monoclonal antibodies, and a buffer solution. When an extraction of the sample passes through the column, the antibodies will bind to any mycotoxins present and allow the rest of the sample solution to flow through unimpeded. A quick column wash with more buffer solution makes sure that any other impurities are also removed, leaving only the mycotoxins bound to the antibody-coated supports inside the column. These mycotoxins can be removed from the antibodies and isolated by washing the column once more with a solvent like methanol.
The resulting solution can then be analyzed using fluorometry. This works by binding the biological analyte, here the present aflatoxins or ochratoxins, to a fluorescent agent and measuring the intensity of fluorescence. As this intensity will correlate with the number of mycotoxin molecules in the sample, it possible to accurately give a measure of the mycotoxin content in this way.
This method allows for several different mycotoxins to be analyzed at one time, meaning that labs can accurately determine the content of all four of the major aflatoxins and ochratoxin A in a single run. It’s also a highly sensitive technique, able to detect the presence of biological analytes down to concentrations of just 0.5 parts per billion.
More advanced equipment for multi-purpose testing
Mycotoxin testing can also be done directly in conjunction with other forms of analysis, such as pesticide testing, when using advanced chromatography equipment, such as liquid chromatography-mass spectrometry (LC-MS) apparatus, or an ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS).
“Atlantic Test Labs uses an ultra-performance liquid chromatography – tandem mass spectrometer (UPLC-MS/MS) method to identify and quantify mycotoxins,” tells Amanda Horodyski.
Generally, a UPLC-MS/MS workflow first involves a sample preparation step, where a cannabis extract is created through some sort of solid phase extraction or accelerated solvent extraction step. After this, the cannabis solution can be simply injected into the UPLC-MS/MS apparatus for complete analysis.
“This method has many advantages over other methods in the market,” explains Horodyski. “The most important advantage is the time it takes to prepare and run samples. Not only is the sample preparation easier on the UPLC-MS/MS, but it is also able to process pesticide testing in the same injection.”
“Also, the UPLC-MS/MS method is extremely sensitive and can quantify mycotoxin levels as low as parts per trillion (ppt). In order to identify and quantify accurate trace levels of mycotoxins while maintaining a high throughput laboratory a UPLC-MS/MS is fundamental,” she concludes.
However, high-tech chromatography equipment is expensive, and it requires highly trained staff to operate it and interpret its results. This can be a large hurdle to overcome for smaller testing laboratories or new start-ups which may not have the capital to be able to purchase the equipment and employ expert staff. For these facilities, immunoaffinity chromatography and fluorometry analysis remains an effective and accurate method testing methodology, that better suits the needs of that establishment.