In the cannabis industry, it is well known that you can send the same sample to multiple labs (or test the same sample) and get significantly different results. This issue has sparked numerous cannabidiol (CBD) product warning letters from the Food and Drug Administration (FDA) for having cannabinoid test results that do not match the potency labeled on the packaging. The authors of the research paper “Pitfalls in the Analysis of Phytocannabinoids in Cannabis Inflorescence” likewise point out that these products lack the standardization of FDA-approved phytocannabinoid drugs like Sativex and Epidiolex. [1]
Analytical Testing for Cannabinoids
When it comes to cannabis there is no agreement in the scientific community on the validation parameters and criteria for the determination of cannabinoids. There are two widely accepted official methods for testing cannabis flower for cannabinoid potency.
Those are the monograph of cannabis flos of the German Pharmacopoeia and the Union method established by the European Commission for the quantitative determination of the delta-9-tetrahydrocannabinol (Δ9-THC) content in hemp varieties (EU method). The German method uses high-performance liquid chromatography-ultraviolet detection (HPLC-UV) for purity testing and cannabinoid quantification in dried cannabis flower.
The EU method uses gas chromatography-flame ionization detection (GC-FID) to verify the Δ9-THC content in cannabis flower or resin. So far, these are the only two cannabis testing methods that have official acceptance. However, future development of more accurate and reliable cannabis testing methods is needed.
For example, gas chromatography triggers decarboxylation of acidic cannabinoids, but the authors note that this conversion is not fixed and typically incomplete.
Sampling and Sample Preparation
It is important to take the correct part of the flower for testing. Do not use basal flowers that are less developed. The authors recommend collecting and mixing flowers and small leaves from the upper third of a reasonable number of plants located a few meters away in the same geographical area. Cannabis samples need to be properly dried and cured so weight loss on drying does not exceed 10% (w/w). Light and air exposure during storage must be prevented or Δ9-THC can degrade into cannabinol (CBN). Samples need to be ground consistently (1-mm sieve for EU method; 710-μm sieve for German method). Grinding increases surface area and releases resin, improving extraction. Grinding of the sample needs to occur right before testing or samples can degrade.
Extraction Solvent
The German method advises using ethanol as a solvent. Hexane has less affinity for cannabinoids but also carries fewer impurities than ethanol. Hexane is often used to crystalize cannabinoids, so it may also decrease cannabinoid solubility and have less accurate results. However, for chromatograms, its results are cleaner and easier to read. That is why it is used in selective cannabinoid analysis by the European Commission.
Testing Standards
Testing standards can lead to inaccuracies if they are not properly handled. Cannabinoid standards should be aliquoted into smaller units and stored at -18°C for a maximum of 6 months. Cannabinoid standards may come as solutions in methanol, so be aware that the methanol can result in an overestimation of cannabinoid concentration due to reactions that form derivatives with molecular similarity to cannabinoids. That is why it is also important to verify the concentration of cannabinoid standards with in-house testing.
Concluding Notes
Result variability in cannabinoid analysis is a real challenge. Each step of the analysis process has aspects that can alter results. That is why it is extremely important for cannabis companies to have a legitimate quality assurance program based on known standards. Standard operating procedures need to be accompanied by process validations that ensure testing is repeatable and accurate.
References
- Citti C, Russo F, Sgrò S, et al. Pitfalls in the analysis of phytocannabinoids in cannabis inflorescence. Analytical and Bioanalytical Chemistry. 2020;412(17):4009-4022. doi:10.1007/s00216-020-02554-3. [Impact Factor: 4.142; Times Cited: 6 (Semantic Scholar)]
Image: Anna Shvets from Pexels
