Although systems for growing, production, and sale of cannabis and cannabis related products are well established, regulation and enforcement of quality and safety testing have lagged behind. However, state governments and private laboratories are focusing on product safety testing with special emphasis on pesticide analysis. This focus is partially the result of various product recalls, media attention, and concern from patient advocacy groups. We evaluated a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation method followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the analysis of multiresidue pesticides. The Association of Analytical Communities International (AOAC) QuEChERS method was used for a reduced 1.5-g amount of plant material and processed with a universal dispersive solid-phase extraction (dSPE) formulation. LC–MS/MS analysis used constant polarity switching electrospray ionization (ESI) and monitored at least two transitions per analyte. Matrix-matched calibration was used for quantitation and both method and instrument internal standards were used. Analyte recovery validation was performed according to the United States Food and Drug Administration (US FDA) guidelines by testing three matrices at three fortification levels in triplicate for more than 200 pesticides. For the large majority of pesticides, in all three matrices and at all three fortification levels, recovery was 70–120%.
The United States legal medical and recreational cannabis market has established cannabis as a high value commodity. Cannabis sales in North America in 2016 were reported to be $6.7 billion, a 30% increase compared to 2015 (1,2). There is also a global movement for legalization adding to the importance of this commodity and the need for testing. Movement toward some form of legalization is occurring in Canada, Israel, Australia, and Spain with many more countries reassessing the legal status of cannabis (1,3,4).
As with any consumer item, product quality and safety are important. Quality and safety are paramount when cannabis is used for medical reasons with immunocompromised patients (5–7). The cannabis analytical testing market is new as well and in the process of building robust testing systems akin to those in industries such as the pharmaceutical and food industries. Potency testing is generally required for product labeling compliance and is often used to value plant material. Material with higher potency cannabinoids content is valued higher than material with lower cannabinoids content. Terpene testing is also a quality test that identifies the terpene content, which is linked to aroma and taste characteristics and thought to participate in medicinal action (8).
Product safety testing falls into several basic categories, including microbiological samples (for example, molds and fungi), heavy metals, aflatoxins, residual solvents, and pesticides. This work focused on pesticide residue testing in cannabis plant material. Concerns about pesticide use are seemingly justified as publicity indicating pervasive use and “high levels” of pesticides are fairly common. Pesticide use has also resulted in product recalls (9–12). However, the use of pesticides is confusing for growers and laboratories because of the lack of regulations or changing regulations, plus they do not have the benefit of the typical Environmental Protection Agency (EPA) oversight (7,13). The EPA registration process requires that extensive data, including toxicity data, be reported to the EPA so that maximum residue levels for a specific commodity can be set (14). This process sets allowable limits intended to protect human health.
The goal of the work described in this article is to evaluate well established techniques used by the food safety industry for use with cannabis plant material. The viability of quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation with liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was investigated using the Association of Analytical Communities International (AOAC) QuEChERS method with a hydration step modification (15). Recovery of about 160 pesticides was determined at multiple fortification levels. Our results allow some indication as to the overall detectability of compounds in this complex matrix.
The QuEChERS sample preparation approach was used and is well established in the food safety industry (16). It is a generic approach that is well suited for multiresidue pesticide analysis in a variety of commodities. QuEChERS provides a “good enough” sample cleanup for analysis, both qualitative and quantitative, when paired with highly sensitive and selective analysis techniques like gas chromatography (GC) and LC–MS/MS. QuEChERS is much less resource intensive than previously used methods and is effective and compatible with many matrices, analytes, and both GC and LC analyses. Although cannabis presents some unique challenges, there is precedent for modifications to typical QuEChERS methods to deal with low water content commodities (17–19).
A multicomponent pesticide mix consisting of compounds varying in physiochemical properties was used. The pesticides represent different pesticide classes including organophosphorus, organonitrogen, and carbamate compounds. In addition, pesticides requiring testing or banned by states with a legal cannabis industry and amenable to LC were included (13).
The pesticide fortification solution at 10 ppm in 0.1% formic acid in acetonitrile was made by combining 10 stock solutions (Restek Corporation) immediately before use. Six deuterated pesticides were used as method internal standards by adding 75 µL of a 10 ppm solution at the beginning of sample preparation. The pesticides included atrazine-d5, diazinon-d10, dichlorvos-d6, dimethoate-d6, diuron-d6, and linuron-d6 and were combined from separate stock solutions at 100 ppm in acetonitrile (Restek Corporation). Linuron-d6 was the internal standard used for quantitative calculations. The other internal standards were analyzed in case the linuron-d6 data was compromised. The addition of 5 µL (10 µg/mL) of carbaryl-d7 (Restek Corporation) in 0.1% formic acid in acetonitrile per 1 mL of sample extract was used as an analytical internal standard to monitor instrument performance.
This article was originally published in "Advancing The Analysis of Medical Cannabis," a supplement to LCGC North America and Spectroscopy magazines in May 2017, pp. 8-22.
- C. Morris, special to CNBC.com, “The Next Big Billion-Dollar Cannabis Markets Investors Are Rushing To,” 2016, available from: http://www.cnbc.com/2016/10/21/the-next-big-billion-dollar-cannabis-markets-investors-are-rushing-to.html.
- Marijuana Business Daily, “Report: North America Marijuana Sales Hit $6.7 Billion in 2016,” 2017, available from: https://mjbizdaily.com/report-north-america-marijuana-sales-hit-6-7-billion-in-2016/.
- D. Giznik, “Israel Just Decriminalized Recreational Cannabis,” Herb, 2017, available from: http://herb.co/2017/03/07/israel-decriminalized-cannabis-2/.
- M. Senthilingam, “Germany Joins Global Experiment on Marijuana Legalization,” CNN.com, 2017, available from: http://www.cnn.com/2016/12/29/health/global-marijuana-cannabis-laws/.
- J. Pizzorno, Integr. Med. (Encinitas) 15(6), 8–12 (2016).
- J.C. Raber, S. Elzinga, and C. Kaplan, J. Toxicol. Sci. 40(6), 797–803 (2015).
- D. Stone, Regul. Toxicol. Pharmacol. 69(3), 284–8 (2014).
- E.B. Russo, Br. J. Pharmacol. 163(7), 1344–64 (2011).
- Denver Department of Environmental Health, “GMC LLC Voluntarily Recalls Medical and Recreational Marijuana and Products Due to Pesticide Residues,” 2017, available from: https://www.denvergov.org/content/denvergov/en/environmental-health/about-us/news-room/2017/GMCRecall.html.
- D. Migoya and R. Baca, “Caregivers For Life Faces Another Recall Over Pesticide-Tainted Pot,” The Denver Post, 2016, available from: http://www.denverpost.com/2016/04/21/caregivers-for-life-faces-another-recall-over-pesticide-tainted-pot/.
- D. Migoya and R. Baca, “Pesticide Recall Hits Life Flower Medical Marijuana Dispensary,” The Denver Post, 2016, available from: http://www.denverpost.com/2016/04/25/pesticide-recall-hits-life-flower-medical-marijuana-dispensary/.
- N. Sullivan, S. Elzinga, and J.C. Raber, J. Toxicol. 2013, 378168 (2013).
- L. Rough, “Leafly’s State-by-State Guide to Cannabis Testing Regulations,” Leafly. com, 2016, available from: https://www.leafly.com/news/industry/leaflys-state-by-state-guide-to-cannabis-testing-regulations.
- US EPA, Pesticide Registration, (U.S. Environmental Protection Agency, Washington, D.C., Pesticide Registration Manual, 2017). Available at: https://www.epa.gov/pesticide-registration/pesticide-registration-manual
- AOAC Official Method 2007.01, “Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate,” (Association of Analytical Communities International, 2007).
- M. Anastassiades et al., J. AOAC Int. 86(2), 412–31 (2003).
- U. Koesukwiwat et al., J. Agric. Food Chem. 58(10), 5950–8 (2010).
- G. Martínez-Domínguez et al., Food Chem. 177, 182–90 (2015).
- K. Mastovska et al., J. Agric. Food Chem. 58(10), 5959–72 (2010).
- P. Rodger Voelker and P. Mowgli Holmes, “Pesticide Use on Cannabis,” Cannabis Safety Institute (2015), available from: http://cannabissafetyinstitute.org/wp-content/uploads/2015/06/CSI-Pesticides-White-Paper.pdf.