Chromatography in Cannabis Testing

Key considerations to choosing a method that's best for a lab's specific needs

Michelle Sprawls

Michelle Sprawls is the director of science at CULTA. She graduated from Northern Arizona University with a BS in microbiology and is certified in closed-loop hydrocarbon extraction machinery, adsorbent filtration,...

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The cannabis industry is quickly evolving and growing exponentially throughout most of the US with more states legalizing the medical and recreational markets. One thing that hasn’t changed over the years is the need for these products to be tested before they are released to the market to ensure the health and safety of consumers. 

Many states in the cannabis industry will have certain regulations put in place requiring growers and processors to test/screen for pesticides, mycotoxins, and residual solvent, as well as validate the potency and terpene levels for each batch. Many multidisciplinary labs are looking for ways to diversify their clientele or find new avenues of research for their lab, but don’t know how to apply existing chromatography instruments to cannabis testing or how to select new ones to add to their arsenal. This article covers key considerations for choosing a method and chromatography instruments for your specific needs.

Cannabinoid profile analysis

Cannabinoids are a specific chemical class found in cannabis that are produced in the glandular trichomes of the plant and can be analyzed via high performance liquid chromatography (HPLC). HPLC-based systems with ultraviolet (UV) detectors have been the gold standard in cannabinoid analysis. The amount of each cannabinoid is determined by shinning UV light on the separated compounds. Each cannabinoid absorbs the UV light to a different extent depending on its concentration. By measuring the amount of UV light that is absorbed by each cannabinoid, you can determine the quantity. Potency levels will vary from cultivar to cultivar, making the quantification of tetrahydrocannabinol (THC), cannabidiol, cannabinol, and cannabigerol and other phytocannabinoids, as well as their native acid forms, a necessity for dosage. The analysis of both cannabis flower and extracts is commonly performed using a certified cannabinoid reference standard and 10-minute analysis of the 11 cannabinoids on a C18 column. The main advantage of HPLC versus gas chromatography (GC) is the ability to quantify both acidic and neutral forms of cannabinoids without derivatization because high temperatures are not required for the analysis. HPLC provides a more comprehensive chemical report of cannabis samples compared to GC.3 

Liquid chromatography mass spectrometry (LC/MS) is used for cannabis and hemp potency testing in scenarios when identification is critical. An LC/MS system contains an HPLC as described above but instead of a non-selective UV detector, the detector is a mass spectrometer. Since the molecules are measurable by their mass, LC/MS provides highly selective, quantitative results. LC/MS is the most sensitive and selective testing methodology available on a commercial scale. It delivers accurate results on samples in complex matrices, enabling cannabis and hemp testing labs to identify unique molecular features. 

Terpene and residual solvent analysis

Terpene and terpenoid compounds are naturally occurring aromatic compounds that give cannabis its unique flavor and aroma. Apart from the aromatic properties and their advantageous health benefits, they also have a synergistic relationship with cannabinoids, which further enhance the therapeutic effect of THC. Monoterpenes, diterpenes, and sesquiterpenes can be characterized by investigating the number of repeating units of isoprene, a five-carbon molecule that is the structural hallmark of all terpenoid compounds. The concentrations of individual terpenes vary by strain, harvest time, and drying/curing spaces. A robust analytical method is necessary to chemically profile terpenes in cannabis and cannabinoid products. The most common approach to terpene analysis is headspace GC with flame ionization detection, mass spectrometry (MS), or both. Utilizing headspace via pressure-balanced injection is a rapid, straightforward, accurate, and precise solution. This solution also allows the components of interest (for example, residual solvents and terpenes) to be introduced into the analytical system. 

GC-MS is an efficient and robust technique to investigate cannabis products for terpenes, providing chromatographic resolution, identification, and quantitation. GC columns can enhance identification by overcoming the challenges posed by isomers and differences in the aromatic character of terpenes. The use of high-quality standards, reagents, and chromatography consumables is a prerequisite for accurate and reliable testing and detection of terpenes in cannabis crops and cannabis-derived products. 

Residual solvent analysis is performed through headspace gas chromatography/mass spectrometry analysis. The major benefit of this approach is that headspace is a fast, simple, accurate, and precise technique that allows the components of interest (e.g., residual solvents) to be introduced into the analytical system. A sample is placed in a closed sampling vessel and heated using a known temperature profile. The chromatographic peaks are well separated with a runtime of about seven and a half minutes and a sample-to-sample cycle time of less than 11 minutes.1 Using MS allows for the identification of components without concern for false positives, while still maintaining extremely fast run times. Selecting the proper GC column for residual solvents or terpenes should be based on four significant factors: stationary phase, column internal diameter, film thickness, and column length. The correct choice of CRMs, analytical reference standards, high-purity solvents, and columns form the basis of method development.

Pesticide and mycotoxin analysis

Potentially harmful pesticides and mycotoxins may be present in cannabis crops and extracts despite being legal for sale. Pesticides are classified into seven major groups based on the field of use: insecticides, herbicides, fungicides, rodenticides, acaricides, molluscicides, and nematicides. Most pesticides have a negative health impact on humans and the environment, resulting in their restricted use or a total ban. Mycotoxins are highly toxic secondary metabolites of certain fungi and molds that easily contaminate food crops. 

Accurate methods for identification and quantitation of pesticides and mycotoxins in cannabis are essential for consumer safety; however, there are currently no harmonized guidelines for pesticide and mycotoxin residue tolerances. Consequently, each state has its own list of such contaminants with legal residual tolerance limits that may be quite different in each region. The selectivity and sensitivity required for determining residual pesticides and mycotoxins in these complex matrices can only be achieved through a dual platform approach using both LC/MS/MS and GC/MS/MS. This dual-instrument approach is recommended to get the throughput needed to be successful compared to a LC/MS/MS-only approach. 

LC-MS/MS is the method of choice for pesticide and mycotoxin analysis with superior selectivity and sensitivity, especially for pesticide and mycotoxin residues with different polarities and molecular weights in complex matrices. GC-MS/MS is a selective and sensitive method for volatile and hydrophobic pesticides such as organophosphates and organochlorines.2 The use of analyte protectants can reduce adverse matrix-related effects and derivatization can improve detection and method sensitivity. A combination of both GC-MS/MS and LC-MS/MS is used for multi-residue pesticide and mycotoxin analysis. Tandem quadrupole MS offers high sensitivity and selectivity for simultaneous analysis of hundreds of pesticides at low ng/g (ppb) levels in a single analysis.

Numerous cannabis samples can be analyzed by various GC instruments so long as the compounds are reasonably volatile yet are thermally stable. These factors impact column efficiency, resolution, and sample capacity. There are no easy short cuts to optimizing a good method. Instrument vendors can provide good tools and methods as a starting point, but laboratories should be prepared to customize these further to perfectly suit the workflow and capabilities of their facility.




3. Wang, M., Wang, Y. H., Avula, B., Radwan, M. M., Wanas, A. S., van Antwerp, J., ... & Khan, I. A. (2016). Decarboxylation study of acidic cannabinoids: a novel approach using ultra-high-performance supercritical fluid chromatography/photodiode array-mass spectrometry. Cannabis and cannabinoid research, 1(1), 262-271.