From pesticides, heavy metals and residual solvents to microorganisms like mold and bacteria, there are several possible sources of cannabis contamination during the growing, harvesting and post-harvest processes. Cannabis testing labs are tasked with detecting and quantifying these potentially dangerous contaminants to ensure compliance with state and local regulations and keep patients and consumers safe. While cannabis contaminant testing guidelines are relatively new and constantly evolving, the following tips can help you navigate best practices and methods for sample prep, analytical testing and microbiological testing of cannabis products.
1. Know your local requirements
Because cannabis remains illegal at the federal level in the United States, state governments and health agencies have been tasked with developing their own testing guidelines as recreational and/or medical cannabis become legal statewide. As a result, different states may have vastly different requirements, such as different lists of contaminants that require testing and different maximum limits for contaminants. States that have legalized both recreational and medical marijuana may also have different requirements for different consumer types, and some states also differentiate between product types such as edible, inhalable or topical products. Understanding the specific requirements for your state or region is essential, as this can make a difference in which analytes you need to test and even which methods you will need to use, such as total yeast/mold count (TYMC) versus qPCR to identify specific strains. In many cases, state and county health agencies will have up-to-date guidelines available to view on their respective websites.
2. Tailor tried-and-true analytical methods
While cannabis contaminant testing is a relatively new industry, many methods utilized for both analytical and microbial testing are well established and have been successfully translated from methods used to detect contaminants in other industries, such as food, pharmaceutical and environmental sciences. Of course, every method must be individually validated for your cannabis samples, as cannabis represents a unique, complex and often challenging substance to analyze, making effective protocols and instrumentation even more vital.
For testing of pesticides, triple quadrupole liquid chromatography mass spectrometry (LC-MS) is one of the most sensitive methods, and is applicable to most pesticides used in the cannabis industry. However, gas chromatography may be needed for pesticides that are not amenable to ionization, such as pentachloronitrobenzene (PCNB), which is one of the pesticides that require testing in California.
Residual solvents, which can be left behind from the process of extraction used to create cannabis oils and waxes, can be tested with headspace GC-MS to evaporate and analyze common extraction solvents like acetone, butane, ethanol and propane.
For heavy metals, inductively coupled plasma mass spectrometry (ICP-MS) is a popular method due to sensitivity, down to parts per trillion (ppt) levels, and speed. However, optical emission spectroscopy (ICP-OES) is another option with fairly high sensitivity, lower cost and the ability to analyze many elements at once.
The most sensitive method for microbial detection is qPCR, which is required in some states to identify specific strains like E. coli and Aspergillus pathogens. Use free DNA removal methods, such as enzyme-based removal, to prevent false positives from dead DNA. If culture-based methods are used, the method should be thoroughly validated for multiple matrix types and protected from contamination during plating.
For mycotoxins and aflatoxins, dangerous metabolites of mold that could be found in cannabis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides both sensitive detection and throughput benefits due to its speed and ability to also test for pesticides at the same time. However, fluorometry paired with antibody-based immunoaffinity chromatography columns can also offer sensitive and specific mycotoxin and aflatoxin detection.
Tailor your choices of equipment and method to your laboratory’s budget and demand as well as the detection limits necessary to comply with state guidelines, keeping in mind that these guidelines could change and even become more stringent in the future.
3. Beware the matrix effect
One of the unique aspects of testing cannabis is the vast variety of matrices you may encounter in cannabis products, which can include baked goods, chocolates, soaps and lotions, gummy candy, hard candy and more. These different matrices can pose challenges, particularly when dealing with the matrix effect, in which coeluted matrix components interfere with ionization of an analyte such as a pesticide during LC-MS analysis, resulting in either suppression or enhancement of signals and leading to less accurate results. The matrix effect must be taken into account when validating your LC-MS methods for different matrices; one strategy to predict this effect is to compare results from a blank matrix post-spiked with the analyte versus results from the analyte in neat solvent.1 With these results, you can perform matrix-matched calibration to account from the effects you can expect for a given matrix.
4. Don’t skimp on sample prep
Proper sample preparation can make all the difference in cannabis testing as proper homogenization, extraction and clean-up can prevent inaccurate results from the uneven distribution of analytes, the matrix effect and even clogging and contamination of chromatography columns from buildup of matrix components not properly removed. Mechanical and automated grinding methods are essential for providing consistent homogenous samples and small particle sizes necessary for efficient extraction and analysis — freezing the sample with dry ice or refrigeration prior to this step can provide smaller particle sizes from sticky or semi-solid matrices.
Extraction through QuEChERS protocols is one of the easiest and most affordable extraction methods, with various kits for extraction and cleanup available for cannabis and different matrix types, but for certain analytes, like more polar pesticides that can be degraded due to pH changes during QuEChERS methods, solid-phase extraction (SPE) is necessary for accurate results. Cleanup with dispersive SPE (dSPE) reduces matrix effects and risks of column contamination.
5. Don’t let your lab become a contamination source
Cross-contamination is a nightmare in any analytical process and there are several steps that should be taken to mitigate several potential sources of contamination that can befall cannabis samples. Microbial contamination can occur if work and storage areas are not kept clean and climate controlled to avoid excessive humidity and temperature. Equipment including consumable pipette tips and vials should also be properly sterilized to avoid this problem.
A particular concern for cannabis products is cross-contamination due to insufficient cleaning of sticky residues. Caustic cleaners or acid rinsing may be necessary to completely remove these residues. For stubborn residue stuck on glassware, adding an emulsifier during a labware washer cycle, as well as higher wash temperatures and high water turnover rate, can more thoroughly remove these difficult substances. Ultrasonic cleaners are another powerful option for glassware washing. In lieu of equipment and supplies that may be difficult to clean, disposable items that are replaced after each use may be necessary to minimize contamination between samples.
References
- "Tips on the analysis of pesticides and mycotoxins in cannabis products: Matrix matters! (Part I)," Nathaly Reyes, Restek. https://www.restek.com/en/chromablography/chromablography/tips-on-the-analysis-of-pesticides-and-mycotoxins-in-cannabis-products-matrix-matters-part-i/