In vitro cannabinoid activity-based screening of seized plant materials: a future-proof harm reduction strategy?
Background: In the past few years, adulteration of ‘regular’ cannabis with synthetic cannabinoid receptor agonists (SCRAs) has been brought to attention. Due to the similar appearance with traditional cannabis, people may unknowingly purchase and consume these adulterated products. The consumer’s unwitting SCRA intake, combined with a heightened potency and toxicity of many SCRAs, when compared to THC, increases the risk of severe intoxications. While such ‘spiking’ can be identified via analytical procedures such as (high resolution) mass spectrometry, such analyses are time-consuming, limited in capacity, associated with a significant cost and typically depend on continuously updated mass spectral libraries. Moreover, the varying chemical structure and the dynamic nature of the SCRA market may render the identification of spiked samples a challenging task. Therefore, easy and straightforward detection of counterfeit or SCRA-adulterated cannabis is crucial from a harm reduction perspective. To address this challenge without relying on structural information, we optimized a methodology that employs activity-based screening. In addition, in collaboration with the Belgian Early Warning System on Drugs, we applied this new methodology on a large set of seized plant materials from an international dance festival to evaluate the prevalence of SCRA-laced products/adulterated cannabis.
Methods: The sample preparation, compatible with our bioassay, resulted in a simple procedure, starting from 20 mg plant material, which was extracted with 1 mL methanol, followed by 1:200 dilution before adding the extract onto the bioassay. A live cell-based β-arrestin2 recruitment assay was employed to verify the potential presence of SCRAs via monitoring CB1 receptor activation. Shortly, activation of CB1, fused to one subunit of a nanoluciferase enzyme (NLuc), initializes the recruitment of β-arrestin2, fused to the other subunit. Reassociation of the two subunits allows the restoration of a functional NLuc. After addition of the substrate furimazine, a strong bioluminescent signal is generated and used as a read-out. Positive controls were generated via spiking 20 mg plant material with 100 µg CP55,940. Finally, the method was applied on a large set of plant samples sourced at an international dance festival.
Results: The optimized approach yielded clearly positive results for plant material spiked with 100 µg CP55,940 and for genuine ‘Spice’ samples known to contain SCRAs, thus confirming the validity of the approach. Application on a large set (n=252) of sourced plant samples did not reveal any positives.
Conclusion: The activity-based CB1 bioassay format was, for the first time, successfully used for the screening of a large set of plant materials. The simplicity of the sample preparation, the rapid results, and the universal character of the bioassay render it an effective and future-proof tool to screen herbal materials for a potential fortification with SCRAs.
