The Preparation Process of Cannabis Sampling & Analysis

Preparing Samples of Cannabis for Analysis using Porvair Equipment

With the ever-increasing number of U.S. states that have legalized the use of cannabis (currently 26 states plus the District of Columbia), the need for accurate analysis of the plant material has dramatically increased. The primary focus is the determination of the active ingredients in the plant material. This information is crucial for taxation authorities, medical regulators, and growers alike.

Cannabis plant material contains many different cannabinoids for which the human brain has evolved receptors. The two most important are the psychoactive THC (tetrahydrocannabinol) and the analgesic CBD (Cannabidiol) which has therapeutic uses. In addition, CBD (cannabidiol) and CBN (Cannabinol) may also be of relevance. For revenue and quality control, the ratio of THC to CBD is of importance and is the primary aim of the analysis.

The importance of this ratio is demonstrated by the differing states of medical marijuana and recreational cannabis; Medicinal marijuana typically has a higher CBD and lower THC, typically 21% CBD and 1% THC, whereas recreational cannabis typically has higher THC and lower CBD with levels of around 2% CBD and 24% THC. Thus, the ratio determines the ultimate fate of the product and may influence pricing and taxation.

While most cannabinoids are only found at trace levels, all Sativa species are very efficient at the biosynthesis of eight main cannabinoids. These are:

THC in two optical isomers

8-tetrahydrocannabinol (8THC)
9-tetrahydrocannabinol, primary psychoactive component (9THC)

THC Acid (THCA), the native form of THC found in raw plant material as a carboxylic acid
Cannabidiol (CBD), this is the main therapeutic agent in medical marijuana
Cannabidiolic Acid (CBDA) which is the native form of CBD found in plant material as a carboxylic acid
Cannabinol (CBN) caused by spoilage and aging of the crop
Cannabichromene (CBC)
Cannabigerol (CBG)

Of these, the major cannabinoid in the cannabis plant material is THC Acid (THCA) which is thermally labile and converts to THC by decarboxylation during smoking, cooking, or other heating. Extracts using alcohol or water, from the plant or any preparations which have not been heated, will contain significant levels of both THCA and CBDA according to cultivar.

For the purposes of analysis, it is desirable to determine total THC. This would include the carboxylate free form (THCA), as well as the thermally converted form, THC. The medical value is a function of the CBD content which again must be totaled to include the native carboxylic acid form.

HPLC can identify the acid components of THCA and CBDA before conversion to their corresponding free forms of THC and CBD and is thus often preferred for tinctures and cannabis products to be taken orally. A drawback of analysis by Gas Chromatography (GC) is the destruction of THCA and TBDA in the hot injector which may make the ultimate determination of the THC/TBD ratio inaccurate. For this reason, HPLC/UV or LC/MS should be the preferred methods of analysis. The following method can also be used in both preparations and raw plant material.

For THC/CBD ratio analysis, samples of the cannabis leaf, bud, and flower, if available, should be randomly sampled. We suggest accurately weighing approximately 50mg of dried plant material into each well of a Porvair 219030 Seed Genomics plate.

It is also possible to start with fresh material, using a Porvair Minivap set to 40°C and 50 Liters/minute of dry compressed air, or nitrogen if available, for two hours to dry the material inside the plate. In this case, the plate should be accurately weighed before and after the drying step to determine the moisture content of the fresh material. Once dried, the procedure can be followed as set out below.

Grinding Samples

Add (3) 3mm steel or nickel grinding balls (nickel will be required if grinding seed material) to the Genomics plate. Cap the plate with Cap Mat #219004 and place the capped plate in the GenoGrinder. Grind for two minutes at 1500 r.p.m. Remove the cap mat and transfer the dried ground powder, which should pass through a 1mm sieve, into each well of a Porvair P³ filter Microlute microplates (240100). Accurately add 400μL of methanol or isopropanol (according to preference) to dissolve the oily residues in the plant material. Agitate on a Porvair Microshake shaker fitted with a P³ plate adaptor or other suitable shakers at 330 r.p.m. for 10 minutes.

Draining & Vacuum Extraction

The superhydrophobic frit in the P³ plate will prevent leakage, but an optional drain cap mat is available which, if desired, should be fitted to the underside of the plate before the assay begins (219005). After shaking, remove the Drain cap and place the P³ plate onto a Porvair Microlute manifold (228008) which has a 2mL collection plate in the plenum chamber. This collection plate can be either another seed genomics plate (219030) or a standard 2mL square well plate (219009). Any SLAS/ANSI format deep well plate can be used for the collection step, but 219009 and 219030 use extractable-free polypropylene which will not contribute leachates to the solution to be analyzed. In addition, their plate geometry is optimized for the Porvair Microlute Manifold so that no further adjustment is necessary to prevent cross-contamination between wells. Apply vacuum to draw the purified extract down through the filter and into the wells of the collection plate. Isolate the vacuum supply using the valve on the manifold and release the vacuum in the plenum chamber using the needle valve. The filter plate may now be discarded and the collection plate containing the organic extract can be taken to the HPLC for analysis.

Heat Analysis

If analysis of total THC and CBD is preferred, an extra step will be required. In this case, the THCA and CBDA must be converted to THC and CBD using heat. Take the 2mL collection plate containing the solvent extract and place it on the Porvair Minivap. Set the temperature to maximum (60°C) and evaporate to dryness, continue to heat with hot gas for an additional 45 minutes to allow the dissociation of the thermally labile THCA and CBDA to THC and CBD. After cooling to room temperature, re-suspend in 400μL of solvent, agitate on the Microshake to ensure good mixing, and then proceed to HPLC analysis as before.

Part Number	Description
219030	Porvair 2 mL Deep Well Seed Genomics Microplate
219004	Porvair 96 Well Cap Mat
228008	Porvair Microlute Vacuum Manifold
240100	Porvair Microlute P³ Plate
229206 & 229072	Porvair Minivap Evaporator with 96 Well Spiral Needle Head
219009	Porvair 2.2 mL Square Deep Well Plate
Use Choice	Laboratory Grinder