New to cannabis potency testing? Click Here!
2 Jun 2022If you’re anything like me, when starting something new I’m always looking to understand why something is done a particular way and also those quick little tips/tricks you learn through experience so I don’t have to waste time or material.
Although there are many types of matrices, such as edibles, beverages, concentrates, lotions, and balms, this blog will focus on flower. If you’re interested in the potency testing of these infused products and concentrates, Restek has technical information on the following analyses on our website:
- Cannabinoids in Gummy Edibles and Multi-Component Analysis of Cannabis Gummies
- CBD Infused Beverage
- Cannabinoids in Chocolate
- CBD Crumble Concentrate and CBD Shatter Concentrate
- Lotions, Balms and Creams
Here’s a few things I’ve learned along the way.
Sample preparation:
Sample prep is key when potency testing. To begin, sample homogeneity is crucial. The quickest and most effective way is to obtain a good grinder. This can be as simple and cost effective as a food processor. You can also purchase fit-for-purpose grinders or mills for this as well. Though a plastic bowl will suffice, I would recommend something with a metal bowl. This is because when processing your sample you will need to use either liquid nitrogen or dry ice. Cryo milling is used to make grinding easier and your sample less sticky. It is also key to ensure you are achieving optimal particles size for extraction as well as preservation of your analytes of interests (Cannabinoids). During the milling/grinding process the sample will heat up which could potentially cause the decarboxylation of your acidic cannabinoids such as ∆9-tetrahydrocannabinolic acid (THCA-A) and cannabidiolic acid (CBDA), returning to their neutral forms of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD) respectively, which is why you’ll want to use cryo milling.1 Regardless of the method you use to pulverize your flower, it is necessary to ensure efficient and effective cleanup of your equipment to prevent cross contamination.
When grinding flower, I prefer to do it all at once, to minimize the loss of sample. This is performed by first adding flower to the grinder, followed by the addition of liquid nitrogen or dry ice. Due to its resistance to cracking from extreme cold, this is where a metal bowl on the grinder is more practical than a plastic bowl. There is not an exact amount of liquid nitrogen/dry ice required. It is more of an art than a science, and will depend on your sample size, how sticky the sample is, as well as the size of your grinder/milling equipment.
Once homogenized, the sample is weighed into a 50 mL centrifuge tube. Most state regulations for potency analysis dictate a minimum sample size of 500 mg to ensure a representative sample is achieved. The extraction solvent is added to the centrifuge tube, which is then vortexed and sonicated. The sample is centrifuged, filtered and diluted prior to injection. It may be necessary to perform two separate dilutions to account for the major and minor cannabinoids. There are many different methods from vendors, AOAC and others. Each of these methods may have different recommendations for sample size, extraction volume and solvent, or preparative protocol. It is important to choose the method that best fits your lab’s needs. In the future, be on the look-out for a follow up blog series comparing recoveries of many of these methods.
Sample Analysis
In order to quantitate your samples, a good calibration curve must be prepared. Be sure to always check your states requirements, for example, in California it is required to have a five point calibration curve. It is recommended to use a calibration curve with at least seven points, analyzed from lowest concentration to highest. If looking at a suite of cannabinoids, using premixed standards, such as the Cannabinoids Acids 7 Standard and the Cannabinoids Neutrals 9 Standard, will greatly reduce the amount of time and error when preparing calibration standards. Another great tip is to implement proper storage of your reference standards to mitigate degradation, as well as preparing fresh calibration standards daily.
Whether you’re a new lab just starting up or an established lab just getting into cannabinoid testing, it is important to have a robust analytical method. If you’re looking for a place to start with method development, a quick and easy isocratic method entitled "16 Cannabinoids on Raptor ARC-18 2.7 µm by LC-UV" can be found on our website. This method is rugged and easily transferrable between instruments and achieves baseline separation for all 16 cannabinoids monitored. So even if initially you are only looking for a handful of specific cannabinoids, it can be advantageous to have a method that can resolve all common cannabinoids to avoid potential peak coelutions.
Detection
Historically, most cannabinoid potency testing has been performed using an HPLC-UV. This methodology is robust and reliable. HPLC-UV is also typically considered to be a cost-effective instrumentation, especially when with an initial startup. However, with more recent discoveries of newer minor cannabinoids, there has been growing interest in LC-MS for its additional spectral deconvolution capabilities.
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Another advantage of using LC-MS, is the need to only resolve isobaric compounds, while being able to achieve lower levels of quantitation. Although, LC-UV is still the preferred technique for meeting regulatory needs due to its cost and ease of use.
Calculations
It is important to be able to accurately quantitate the concentration of ∆9-THC, as these levels are a key differentiator between what is classified as cannabis and hemp. The legal limit for hemp classification is ≤ 0.3% by weight, including both acid and neutral form. It can be easy to get lost in the math, so the concentration of cannabinoids is calculated from the calibration curve here, the following formulas can be used:
Note: Many states are now implementing an expanded Total THC requirements2 Calculation for this includes an additional three cannabinoids:
Do you have any other questions about potency testing? Comment in the section below!
References
- Atkins, P. L. (2019). Sample Processing and Preparation Considerations for Solid Cannabis Products. Journal of AOAC Interanational , 102(2), 427-433. Retrieved from https://watermark.silverchair.com/jaoac0427.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAsswggLHBgkqhkiG9w0BBwagggK4MIICtAIBADCCAq0GCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMwQfTdAFCz2vn2-VDAgEQgIICfjlZrsqBAgWbXUE4cR7aqlAxXL7y7tsdUjOdJWSnnPaxc
- Revenue, C. D. (2022, January 1). Colorado Department of Revenuse SPecialized Business Group MED Rules. Retrieved 05 25, 2022, from Code of Colorado Regulations: https://www.sos.state.co.us/CCR/GenerateRulePdf.do?ruleVersionId=9900