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Cannabis is among the commonest of street drugs to be subjected to chemical analysis. The medical and legalized use of marijuana also requires laboratory testing for the potency of the product. Potency testing helps consumers decide on which product to use for the purpose they have in mind. Either gas chromatography or high pressure liquid chromatography are employed to test the content of THC (tetrahydrocannabinol) and CBD (cannabidiol), the cannabinoids which chiefly affect how strong the product is.
The total THC in the material is usually measured. Fresh plant material contains both THC and tetrahydrocannabinolic acid (THCA), which is its precursor. Decarboxylation of THCA yields THC and should be performed before the analysis.
Presumptive Tests
Color tests
These give positive results when THC is possibly present, but false positives may occur with henna, nutmeg, mace or agrimony. They must be confirmed using definitive tests such as thin-layer chromatography (TLC) or microscopy. In this combination of tests, at least three compounds must test positive for THC using TLC.
Immunoassays
These have the advantage that they can be performed on the drug itself and not only the plant, but are much more expensive, therefore not preferred.
Definitive Tests
High-Performance Liquid Chromatography (HPLC)
This method has been validated for total THC analysis in the plant form of cannabis after the sample is prepared appropriately. It can also be applied to other forms but requires prior verification.
In HPLC, solvent extraction is carried out to obtain volatile compounds in the specimen, and a sample is passed under pressure through a tube filled with material that has affinity for certain molecules. If these are present in the sample they will move through the tube to a detector at the other end at different speeds depending on the strength of their attraction to the medium in the tube. The detector typically works on the ultraviolet absorbance principle.
Gas Chromatography-Flame Ionization Detection (GC-FID)
This method yields the total or separate amounts of THC and THCA depending on whether the sample is derivatized first or not. The sample solution is passed through a column by the carrier gas or mobile phase (an inert gas) and separates into its components by their interaction with the solid phase coated on capillary tubes or on solid beads packing the column. The stream is finally burnt to detect hydrocarbons and other volatile compounds by the carbon ions produced, which causes a current to flow at the detector.
Gas Chromatography-Mass Spectrometry (GC-MS)
This analysis is analogous to the GC-FID method and depends upon matching to the reference spectra of the commonly found cannabinoids in commercial databases of mass spectra. The chemicals are ionized and sorted by mass/charge ratio.
This method is preferable to mass spectrometry because of its reduced cost and complexity with comparable sensitivity and specificity.
Thin-Layer Chromatography (TLC)
Several TLC methods are also used to analyze a substance for cannabis, each of which must be validated before being accepted for routine laboratory protocol. The mobile phase is drawn up through a thin layer of alumina or silica gel, or cellulose (stationary phase), on a thin glass, paper, aluminum foil or plastic and the test substance moves up according to its affinity to the stationary phase. HPLC is a refined version of TLC.
Ion Mobility Spectrometry (IMS)
This test is not recommended because it cannot distinguish cannabis from heroin and is affected by humidity.
Contamination Testing
Cannabis being a plant product must be screened for molds and bacterial contamination, especially since it may be used by immunocompromised individuals. Moisture testing will help exclude the presence of dampness in the plant. HPLC is chosen for this testing, with a fluorescence detector and a post-column reactor. Real-time PCR is best because of the rapid turnover times and high specificity.
Analysis should also look for the presence of pesticides, herbicides and insecticides because of the potential toxicity of these chemicals to the liver or muscle tissue, and possible carcinogenicity. MS effectively detects and quantitates the presence of even low concentrations of a wide range of pesticides.
Heavy metals must also be excluded, as lead, cadmium and mercury, among others, could affect consumers adversely, especially if the immune system is weakened.
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Additional Techniques
GC-FID Profiling
Using a standard GC column and standardized GC profiles, the substance can be analyzed. Several terpenoids are found in cannabis, these being a class of organic hydrocarbons that have been denatured during curing of the cannabis flowers, and contributing to the structure of the plant resin or oil in most aromatic plants. For this purpose a cluster analysis is performed, with the range of terpenoids being chosen to match the peak pattern sample of cannabis from the same place of origin. Such testing may possibly help determine the region of the world from where the specimen has come. However, this is not accepted as a valid proof of origin yet for several reasons.
A better application may be to determine if different samples come from the same batch, and thus joining up similar samples with regard to the age, phenotypic profile and production plant.
Detecting the presence and level of residual solvent left over from cannabinoid extraction is important as these chemicals can cause allergies, headaches and other health issues. GC-FID with a headspace autosampling technique is used for this purpose.
Solid Phase-Micro Extraction (SPME)
SPME prepares the specimen without the use of solvent, allowing volatile chemicals to be detected either in the headspace or in aqueous solution. Alkaline hydrolysis and derivatization with subsequent GC-MS also allows for headspace analysis using SPME. This helps detect THC, CBN (cannabinol) and CBD (cannabidiol) faster than with liquid-to-liquid extraction techniques.
Stable Isotope Ratio-Mass Spectrometry (IRMS)
The geographical origin of a plant-derived material is best detected by analyzing the ratios of the stable isotopes of carbon and nitrogen, which vary in a stable pattern from region to region. This is especially so with cannabis because it is not chemically processed when prepared as an illicit drug. It can be affected by growing conditions and requires genuine cannabis reference standards whose origins are known definitely.
DNA Profiling
While this determines the presence of identical DNA in separate samples, its use is not clear since this finding of a DNA match does not prove that the samples come from the same plant, owing to the use of cloning techniques as well as the free availability of cuttings.
Sources and Further Reading
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