Sample preparation is an essential part of method development and is critical to successful analytical determinations. With cannabis and cannabis-based products, the analyst is faced with a very difficult matrix and targets that can range from trace level to percentage level, placing considerable demands on sample preparation techniques.1 The optimal method of sample preparation, or “extraction,” for cannabis flower potency analysis was determined using methanol extraction coupled with filtration using regenerated cellulose filters.
In the United States (US), Canada and other countries where medicinal and/or recreational cannabis for adults has been legalized, regulatory agencies require a panel of chemical tests to ensure the quality and product safety before retail sale.2. Cannabis testing can be divided into two different categories: Quality and Safety. Quality testing, which includes potency analysis (also called cannabinoid testing or cannabinoid content), is performed to analyze the product in accordance with grower/producer expectations and government regulations. Safety testing is carried out according to regulatory guidelines to ensure that consumers are not exposed to toxic substances such as pesticides, mycotoxins, heavy metals, residual solvents and microbial contaminants.
Potency tests assess the total amount of cannabinoid content, specifically focusing on tetrahydrocannabinol (THC) and cannabidiol (CBD). In the United States, the biggest push for an accurate total THC is to differentiate between hemp (legally grown for industrial or medicinal purposes), which is defined as cannabis sativa with a THC limit ≤ 0.3%, and cannabis (Cannabis spp.), which is any cannabis plant with THC measured above 0.3%3. Potency testing is usually done by liquid chromatography (LC) with UV detection to determine the amount of major cannabinoids.
In addition to signaling THC and CBD, their respective precursors are also important in signaling total potency. Tetrahydrocannabinolic acid (THCA) is the inactive precursor to THC while cannabidiolic acid (CBDA) is the precursor to CBD.4.5
Methods and materials
All samples were homogenized using an immersion blender with a dry matter grinder. The nominal sample quantities were 200 mg of flowers, 500 mg of edibles and 250 mg of candy samples.
Power extraction method (1)
Twenty milliliters (mL) of methanol (MeOH) was added to each sample. Samples were mechanically shaken for 10 minutes and centrifuged for 5 minutes.
Power extraction method (2)
Ten ml of water was added to each sample. The samples were mechanically shaken for 10 minutes. 20 ml of acetonitrile (ACN) was then added to each sample and vortexed. A packet of EN QuEChERS extraction salt was added to the sample. Samples were placed on a mechanical shaker for 2 minutes and then centrifuged for 5 minutes.
Each extract was split and evaluated with two filtration/cleaning steps: (1) regenerated cellulose (RC) syringe filter (Agilent Technologies, 4 mm, 0.45 µm); (2) a PTFE syringe filter (Agilent Technologies, 4 mm, 0.45 µm). The final filtered extracts were injected into the high-performance liquid chromatograph coupled to a photodiode array detector (UPLC-PDA) for analysis.
Standards were obtained for the following cannabinoids at a concentration of 1 mg/mL: cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), tetrahydrocannabinol (9-THC), cannabichromene (CBC), tetrahydrocannabinol acid (THCA). Equal volumes of each standard were mixed with MeOH to make a 10 µg/mL stock standard. Serial dilutions were made from stock to obtain concentrations of 5, 1 and 0.5 µg/mL for the standard curve (Figure 1).
All instrument parameters were followed by Agilent application note 5991-9285EN.8 A UPLC with a PDA detector (Waters Corp, Milford, MA) was used for power analysis. An InfinityLab Poroshell 120 EC-C18, 3.0 x 50 mm, 2.7 µm column (Agilent Technologies, Wilmington, DE) was used for compound separation. The organic mobile phase composition was 0.05% (v/v) formic acid in HPLC-grade MeOH and the aqueous mobile phase composition was 0.1% (v/v) formic acid. formic in HPLC-grade water. The mobile phase gradient is shown in Table 1. Flow rate was 1 mL/min (total program 9.5 minutes), injection volume was 5 µl, and column temperature was 50°C .
Discussion and results
Table 2 summarizes the relative standard deviations (%RSD) found for the THC calibrator (at 1 ug/mL) and an extract from a homogeneous sample (using 7 replicates).
The cannabinoid potency of various samples of cannabis plants and cannabis products was determined for the various extraction techniques. In the chromatograms, THC was observed at around 8.08 minutes and CBD at around 4.61 minutes (Figure 2).
The total potency for THC and CBD was calculated for each sample using the equations below. Equation 1 was used because it takes into account the presence of THCA as well as the difference in specific gravity between THC and THCA (since THCA will eventually convert to THC, this must be taken into account in the calculations ).
Table 3 shows the THC percentage and total THC potency values calculated for the same flower samples that underwent the four different potency sample preparation techniques as previously described. Figure 3 also provides LC chromatograms for flower sample 03281913A-2 and edible sample 03281912-1.
The results indicated that with the “power 2 extraction method” (ACN/QuEChERS extraction) coupled with the RC filter provided a 7.29% higher bias for % total THC compared to the other extraction techniques . Since the other 3 techniques provided total THC values within 2% of each other, the total THC of the sample is more likely around 14%.
Since dilution of the sample for the above data set reduced the CBD content, an undiluted sample was analyzed and analyzed. These data are reported in Table 4.
The CBD results indicated that with “Potency Extraction Method 1” (methanol extraction) coupled with the RC filter, this allowed for greater CBD recovery. This may indicate the loss of CBD with an ACN/QuEChERS extraction.
With an average of approximately 14% total THC and 0.06% total CBD for a homogeneous cannabis flower sample, the optimal extraction for the sample preparation was determined to be methanol extraction coupled with filtration using a regenerated cellulose filter. Since potency remains at the forefront of regulatory cannabis testing, it is important to use the correct sample preparation for your cannabis samples.
- Wang M, Wang YH, Avula B, Radwan MM, Wanas AS, Mehmedic Z, et al. Quantitative Determination of Cannabinoids in Cannabis and Cannabis Products Using Ultra-High Performance Supercritical Fluid Chromatography and Diode Array/Mass Spectrometry Detection. Journal of Forensic Science 2016;62(3):602-11.
- Matthew Curtis, Eric Fausett, Wendi A. Hale, Ron Honnold, Jessica Westland, Peter J. Stone, Jeffery S. Hollis, Anthony Macherone. Cannabis Science and Technology, September/October 2019, Volume 2, Issue 5.
- Sian Ferguson. https://www.healthline.com/health/hemp-vs-marijuana. August 27, 2020.
- Taschwer M, Schmid MG. Determination of the relative percentage distribution of THCA and 9-THC in herbal cannabis seized in Austria – Impact of different storage temperatures on stability. International Forensic Sciences 2015; 254:167-71.
- Beadle A. CBDA Vs CBD: What are the differences? [Internet]. Analytical cannabis. 2019 [cited 2020 Apr 22]; https://www.analyticalcannabis.com/articles/cbda-vs-cbd-what-are-the-differences-312019.
- Storm C, Zumwalt M, Macherone A. Dedicated cannabinoid potency testing using the Agilent 1220 Infinity II LC system. Agilent Technologies, Inc. Application Note 5991-9285EN