Book:Past, Present, and Future of Cannabis Laboratory Testing and Regulation in the United States/Laboratory testing of cannabis/Methods and guidelines

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3.2 Methods and guidelines

Note: It would be beyond the scope of this guide to include every state's laws and guidelines on cannabis testing; entities such as Leafly Holdings[1] provide such online resources.

Now that we've addressed what's being tested for, we can move on to how they're being tested and what's being done to improve testing methods and procedures, including associated guidelines and recommendations. This section will focus on current and promising techniques using generalizations based on information from multiple sources. If any guidelines and recommendations are known, they'll be included.

3.2.1 Sampling

Random, representative sampling is encouraged. When dealing with solid cannabis, BOTEC Analysis recommends a "quartering" method that divides the sample into four equal parts and takes portions from opposite sections of a square-shaped arrangement of the sample. For liquid cannabis products, remembering to stir before sample collection is advised.[2] Sampling techniques may also vary depending on the constituent being tested, as with terpene testing, which may favor full evaporative technique (FET) headspace-based (HS) sampling for reducing certain sampling biases.[3] Another consideration may be the matrix being tested, as when deriving a sample from a cannabis-laden edible; the QuEChERS approach used by food safety labs for pesticide testing may have practical use.[4] In fact, a variety of parallels have been drawn from the food and herbal medicine industries' sampling guidelines, including from the Codex Alimentarius Commission's CAC/GL 50-2004 General Guidelines on Sampling as well as various chapters of the United States Pharmacopeia and The National Formulary.[2][5] As the Association of Public Health Laboratories (APHL) points out, "[g]ood sampling is key to improving analytical data equivalency among organizations," and it provides a solid base for any future testing and standardization efforts.[2]

Additional sampling insight can be found by examining other states' guidelines, e.g., Massachusetts' Protocol for Sampling and Analysis of Finished Medical Marijuana Products and Marijuana-Infused Products for Massachusetts Registered Medical Marijuana Dispensaries[6], as well as ASTM D8334/D8334M-20 Standard Practice for Sampling of Cannabis/Hemp Post-Harvest Batches for Laboratory Analyses.[7]

3.2.2 Cannabinoid testing

Quantifying cannabinoids for label accuracy is a major goal of testing, though calculation and testing processes may vary slightly from state to state. Despite any differences, laboratorians generally agree that when testing for cannabinoids such as THC and CBD, as well as their respective biosynthetic precursors THCA and CBDA, the methodology used must be scrutinized. The naturally occurring THCA of cannabis isn't psychoactive; it requires decarboxylation (a chemical reaction induced by drying/heating that releases carbon dioxide) to convert itself into the psychoactive cannabinoid THC. Chemical calculations show that the process of decarboxylation results in approximately 87.7 percent of the THCA's mass converting to THC, with the other 12.3 percent bubbling off as CO2 gas.[8] The problem with this in the testing domain is gas chromatography (GC) involves heating the sample solution. If you, the lab technician, require precise numbers of both THCA and THC, then GC analysis poses the risk of under-reporting THC total values.[2] As such, liquid chromatography-diode array detection (LC-DAD) may be required if a concise profile of all cannabinoids must be made, primarily because it provides environmental stability for them all during analysis. If GC is used, the analysis requires extra considerations such as sample derivatization.[2][9][10][11]

The APHL briefly describes analysis methods of cannabinoids using both LC and GC on pages 31–32 of their May 2016 Guidance for State Medical Cannabis Testing Programs. They also point to New York Department of Health - Wadsworth Center's various guidance documents (MML-300, -301, and -303) for methodologies when testing sample types other than solids, particularly using high-performance liquid chromatography photodiode array detection (HPLC-PAD).[2][12] Also worth noting is that ASTM's Subcommittee D37.03 has been working on various standard methods for determining cannabinoid concentrations using different chromatography techniques[13], while the Association of Official Agricultural Chemists (AOAC) has already developed a Standard Method Performance Requirement (SMPR) for analyzing cannabinoids in hemp (i.e., low THC cannabis varieties).[14]

Overall, various methods used in cannabinoid testing include[2][9][12][15][16][17][3]:

Also worthy of note is recent investigation of viably using nuclear magnetic resonance (NMR) spectroscopy as a more affordable and rapid solution to identifying cannabinoid contents and profiles of samples. Conferences[18], research[19][20][21], and articles[22][23][24] over the last four or five years have advanced the use of NMR spectroscopy for cannabinoid analysis.

3.2.3 Terpene testing

Identifying and quantifying terpenes is one of the more difficult tasks facing laboratorians, according to Cassidy[9]:

Terpenes present an analytical challenge because they are nonpolar and structurally similar, and many structural isomers exist. Mass spectrometry (MS) cannot distinguish terpenes that co-elute from a GC column because many have the same molecular weight and share fragment ions.

Goldman et al. share Cassidy's view about MS, though reminding that it has some benefits over flame ionization detection (FID). They note that recent MS methods add another level of confirmation for terpene identification using retention indexing and electron impact mass spectral matching.[3]

Of course, types of gas chromatography work; but like cannabinoids, terpenes can degrade with the high heat of gas chromatography.[17] Combined with the problems mentioned above, highly specialized gas chromatography processes that include additional steps, such as full evaporation technique headspace gas chromatography flame ionization detection (FET-HS-GC-FID), can be used to produce cleaner results, particularly for volatile components.[9] It's less clear if high-performance liquid chromatography (HPLC) is used frequently; some entities such as Eurofins Experchem Laboratories claim HPLC works best for them[17], while others such as Restek Corporation claim the method is problematic at best.[25]

Overall, various published methods for terpene identification and analysis include[9][3][26][16][27][17][28][29]:

  • Full evaporation technique–headspace–gas chromatography–flame ionization detection (FET-HS-GC-FID; tends to be semi-quantitative)
  • Gas chromatography–flame ionization detection (GC-FID)
  • Gas chromatography–mass spectrometry (GC-MS)
  • Gas chromatography–tandem-mass spectrometry (GC-MS/MS)
  • Gas chromatography–vacuum ultraviolet spectroscopy (GC-VUV)
  • Headspace–gas chromatography–mass spectrometry (HS-GC-MS)
  • Headspace–solid-phase microextraction (HS-SPME)
  • High-performance liquid chromatography (HPLC; may have limitations due to coelution of terpenes and cannabinoids at certain ranges[25])

3.2.4 Contaminant testing

LC MS pic.jpg

Pesticides: Gas and liquid chromatography methods are by and large the staple of testing methods for pesticides, which remain "the hardest analyses that are going to be done in the cannabis industry."[9] Goldman et al. echo the sentiment: "pesticide testing is difficult and requires advanced analytical instrumentation and highly skilled staff to meet regulatory demands with a robust, accurate, and precise test method."[3] Notably, high-performance liquid chromatography–tandem-mass spectrometry (HPLC-MS/MS) tends to be one of the most thorough methods says Emerald Scientific's CTO Amanda Rigdon. "Ninety-five percent of the pesticides out there can be analyzed by HPLC-MS/MS, although there are some that you would need a GC-MS/MS for," she says.[9] A popular sample extraction method for detecting multiple pesticide residues in cannabis is the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which shows "acceptable recoveries and relative standard deviations" for almost all known pesticides[3][30][31][32][33], though the release of heat and increase in pH of QuECHERS may degrade particularly sensitive pesticides in the sample.[34] QuECHERS may also not be ideal for some labs due to its organic solvents having a tendency of extracting hydrophobic compounds like cannabinoids.[3] However, other methods such as solvent extraction (such as with acetonitrile) with dispersive solid-phase extraction (dSPE) cleanup[31][33][34] and energized dispersive guided extraction (EDGE) may also been used.[29] Common testing methods that have historically been used, after sample preparation, include[2][28][29][32][33][34]:

  • Gas chromatography–electron capture detection (GC-ECD)
  • Gas chromatography–mass spectrometry (GC-MS)
  • Gas chromatography–tandem-mass spectrometry (GC-MS/MS)
  • Liquid chromatography–mass spectrometry (LC-MS; also high-performance or HPLC-MS)
  • Liquid chromatography–tandem-mass spectrometry (LC-MS/MS; also high-performance or HPLC-MS/MS)

For quantification of pesticides in cannabis, the AOAC's SMPR 2018.011, EPA's Residue Analytical Methods (RAM), and FDA's Pesticide Analytical Manual (PAM) provide guidance to labs.[2][35][36][37]


Solvents: Testing for solvents is largely standardized into a few options, which have parallels to existing pharmaceutical testing standards outlined in Chapter 467 of United States Pharmacopeia and The National Formulary (USP <467>)[38][3][2][9][28][39][40]:

  • Headspace–gas chromatography/mass spectrometry (HS-GC/MS)
  • Headspace–gas chromatography/tandem-mass spectrometry (HS-GC-MS/MS; may be required when high concentrations of terpenes are present)
  • Headspace–gas chromatography–flame ionization detection–mass spectrometry (HS-GC-FID-MS)
  • Full evaporation technique–headspace–gas chromatography–flame ionization detection (FET-HS-GC-FID)

Massachusetts and Oregon—and likely other states—have used a variety of guidance documents such as USP <467>, reports from the Commission of the European Communities' Scientific Committee on Food (now the European Food Safety Authority), and the International Conference on Harmonization's (ICH) Q3C(R5)[2][41][42][38] to set their action level testing values for particular solvents. The AOAC provides another standardized option in the form of their SMPR 2019.002.[43]


Heavy metals: The methods used for quantifying levels of highly toxic metals in plants depend on ease-of-use, level of accuracy, and overall cost. Sample preparation typically includes the use of closed-vessel microwave digestion to get the sample into solution for analysis.[29][44] Once prepared, the following methods are most common for testing cannabis and other plants for heavy metals[3][45][2][9][46][28]:

For quantification of metals in cannabis, the U.S. FDA's ICP-MS methodology document is often used.[2][47]


Mycotoxins and microorganisms: A standard method of testing for the existence of microorganisms is through the process of culturing a sample in a Petri dish, a common diagnostic method in microbiology. Enzyme-linked immunosorbent assay (ELISA) is also used, particularly to identify mycotoxins.[3] However, Petri culture analysis isn't rigorous, and ELISA can at times be time-consuming, as it's limited to one mycotoxin per test.[3][45][9][48] The following are other, more precise techniques that are improving laboratorians' analyses, particularly using DNA snippets of microbiological contaminants[3][45][9][48][49][50]:

The extent of mycotoxin testing required remains in question by several entities. The APHL claims "[t]here is no readily available evidence to support the contention that cannabis harbors significant levels of mycotoxins."[2] The Oregon Health Authority takes a more middle-ground approach, noting that testing for E. coli and Salmonella will "protect public health," though Aspergillus only deserves a warning for people with suppressed immune systems due to its prevalence in the environment.[42] USP <561> recommendations largely limit mycotoxin testing of botanical products to those borne from root or rhizome material[51], "which THC-containing cannabis products presumably do not possess," emphasizes the APHL.[2]

Regardless, U.S. Pharmacopeia's Chapter 561 remains a useful document for testing guidelines and limits regarding microbials[51][2], as does the AOAC's SMPR 2019.001 for the detection of Aspergillus.[52] In the less common case of dealing with powdered cannabis—a relatively new THC extract form—Chapter 2023 provides at least some testing parallels, though Dr. Tony Cundell, a microbiologist consulting for the pharmaceutical industry, suggests USP <2023> doesn't go far enough for immunocompromised patients.[53]

Somewhat related and worth mentioning is moisture content testing. As previously mentioned, warm, moist environments are conducive to microorganism growth, and regularly measuring water activity is useful for the prevention of microbial growth.[42] The APHL references specifications from the Dutch Office of Medical Cannabis that recommend water content be between five to ten percent in cannabis.[2]

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