Difference between revisions of "User:Shawndouglas/sandbox/sublevel3"

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===Analytical aspects of cannabis===
===Analytical aspects of cannabis===
====Cannabinoids====
====Cannabinoids====
As of mid-2015, researchers have identified 104 of the more than 750 constituents of ''Cannabis sativa'' as cannabinoids<ref name="RadwanIso15">{{cite journal |title=Isolation and pharmacological evaluation of minor cannabinoids from high-potency ''Cannabis sativa'' |journal=Journal of Natural Products |author=Radwan, M.M.; ElSohly, M.A.; El-Alfy, A.T. et al. |volume=78 |issue=6 |pages=1271-6 |year=2015 |doi=10.1021/acs.jnatprod.5b00065 |pmid=26000707 |pmc=PMC4880513}}</ref>, active chemical compounds that act in a similar way to compounds our body naturally produces. Many of our body's cells have cannabinoid receptors capable of modulating neurotransmitter release in the brain and other areas.<ref name="WHOTheHealth16">{{cite book |url=http://www.who.int/substance_abuse/publications/cannabis/en/ |title=The health and social effects of nonmedical cannabis use |author=World Health Organization |editor=Hall, W.; Renström, M.; Poznyak, V |publisher=World Health Organization |pages=95 |year=2016 |isbn=978921510240}}</ref> The plant's cannabinoids vary, with each bonding to specific receptors in our body, providing differing effects. From a theoretical and medical standpoint, crafting a strain of cannabis that has specific cannabinoids that can aid with a particular malady, while also carefully reproducing the grow conditions to consistently make that strain in the future, is a desirable but difficult goal to achieve.<ref name="RahnCannab14">{{cite web |url=https://www.leafly.com/news/cannabis-101/cannabinoids-101-what-makes-cannabis-medicine |title=Cannabinoids 101: What Makes Cannabis Medicine? |work=Leafly - Cannabis 101 |author=Rahn, B. |publisher=Leafly Holdings, Inc |date=22 January 2014 |accessdate=03 February 2017}}</ref>
As of mid-2015, researchers have identified 104 of the more than 750 constituents of ''Cannabis sativa'' as cannabinoids<ref name="RadwanIso15">{{cite journal |title=Isolation and pharmacological evaluation of minor cannabinoids from high-potency ''Cannabis sativa'' |journal=Journal of Natural Products |author=Radwan, M.M.; ElSohly, M.A.; El-Alfy, A.T. et al. |volume=78 |issue=6 |pages=1271-6 |year=2015 |doi=10.1021/acs.jnatprod.5b00065 |pmid=26000707 |pmc=PMC4880513}}</ref>, active chemical compounds that act in a similar way to compounds our body naturally produces, and new cannabinoids continue to be identified during cannabis research.<ref name="MudgeChemo18">{{cite journal |title=Chemometric Analysis of Cannabinoids: Chemotaxonomy and Domestication Syndrome |journal=Scientific Reports |author=Mudge, E.M.; Murch, S.J.; Brown, P.N. |volume=8 |page=13090 |year=2018 |doi=10.1038/s41598-018-31120-2}}</ref> Many of our body's cells have cannabinoid receptors capable of modulating neurotransmitter release in the brain and other areas.<ref name="WHOTheHealth16">{{cite book |url=http://www.who.int/substance_abuse/publications/cannabis/en/ |title=The health and social effects of nonmedical cannabis use |author=World Health Organization |editor=Hall, W.; Renström, M.; Poznyak, V |publisher=World Health Organization |pages=95 |year=2016 |isbn=978921510240}}</ref> The plant's cannabinoids vary, with each bonding to specific receptors in our body, providing differing effects. From a theoretical and medical standpoint, crafting a strain of cannabis that has specific cannabinoids that can aid with a particular malady, while also carefully reproducing the grow conditions to consistently make that strain in the future, is a desirable but difficult goal to achieve.<ref name="RahnCannab14">{{cite web |url=https://www.leafly.com/news/cannabis-101/cannabinoids-101-what-makes-cannabis-medicine |title=Cannabinoids 101: What Makes Cannabis Medicine? |work=Leafly - Cannabis 101 |author=Rahn, B. |publisher=Leafly Holdings, Inc |date=22 January 2014 |accessdate=03 February 2017}}</ref> However, even as new strains are developed, identifying an existing strain effectively has its own set of challenges, as Mudge ''et al.'' point out: "the total [tetrahydrocannabinol] and [cannabidiol] content is not sufficient to distinguish strains [though] a combination of targeted and untargeted chemometric approaches can be used to predict cannabinoid composition and to better understand the impact of informal breeding program and selection on the phytochemical diversity of cannabis."<ref name="MudgeChemo18" />


Lab testing of cannabinoids is done primarily as a measure of psychoactive "potency," though cannabinoids have many other potential therapeutic uses. Current laboratory testing looks at only a handful of cannabinoids; more research and development of analytical techniques that can quickly and accurately detect and separate the the rest is required.<ref name="KuzdzalUnrav15">{{cite journal |title=Unraveling the Cannabinome |journal=The Analytical Scientist |author=Kuzdzal, S.; Lipps, W. |issue=0915 |year=2015 |url=https://theanalyticalscientist.com/issues/0915/unraveling-the-cannabinome/ |accessdate=19 January 2017}}</ref> Some of the major cannabinoids tested for include<ref name="KuzdzalUnrav15" /><ref name="APHLGuide16">{{cite web |url=https://www.aphl.org/aboutAPHL/publications/Documents/EH-Guide-State-Med-Cannabis-052016.pdf |format=PDF |title=Guidance for State Medical Cannabis Testing Programs |author=Association of Public Health Laboratories |pages=35 |date=May 2016 |accessdate=01 February 2017}}</ref><ref name="RahnUnder14">{{cite web |url=https://www.leafly.com/news/cannabis-101/understanding-cannabis-testing |title=Understanding Cannabis Testing: A Guide to Cannabinoids and Terpenes |work=Leafly - Cannabis 101 |author=Rahn, B. |publisher=Leafly Holdings, Inc |date=09 October 2014 |accessdate=03 February 2017}}</ref>:
Lab testing of cannabinoids is done primarily as a measure of psychoactive "potency," though cannabinoids have many other potential therapeutic uses. Current laboratory testing looks at only a handful of cannabinoids; more research and development of analytical techniques that can quickly and accurately detect and separate the the rest is required.<ref name="KuzdzalUnrav15">{{cite journal |title=Unraveling the Cannabinome |journal=The Analytical Scientist |author=Kuzdzal, S.; Lipps, W. |issue=0915 |year=2015 |url=https://theanalyticalscientist.com/issues/0915/unraveling-the-cannabinome/ |accessdate=19 January 2017}}</ref> Some of the major cannabinoids tested for include<ref name="KuzdzalUnrav15" /><ref name="APHLGuide16">{{cite web |url=https://www.aphl.org/aboutAPHL/publications/Documents/EH-Guide-State-Med-Cannabis-052016.pdf |format=PDF |title=Guidance for State Medical Cannabis Testing Programs |author=Association of Public Health Laboratories |pages=35 |date=May 2016 |accessdate=01 February 2017}}</ref><ref name="RahnUnder14">{{cite web |url=https://www.leafly.com/news/cannabis-101/understanding-cannabis-testing |title=Understanding Cannabis Testing: A Guide to Cannabinoids and Terpenes |work=Leafly - Cannabis 101 |author=Rahn, B. |publisher=Leafly Holdings, Inc |date=09 October 2014 |accessdate=03 February 2017}}</ref>:

Revision as of 14:46, 15 November 2018

This is sublevel3 of my sandbox, where I play with features and test MediaWiki code. If you wish to leave a comment for me, please see my discussion page instead.

Sandbox begins below

With a sense of history and a better understanding of the regulations and standards affecting cannabis testing and use, this chapter can finally dig into the specifics of the current and future state of laboratory testing of cannabis and related products in the U.S. Here we look at the analytical aspects of cannabis, methods and guidelines used as well as the equipment and software typical to cannabis testing labs.

-----Return to the beginning of this guide-----


3. Laboratory testing of cannabis

Lilly96A.jpg

The overview mentioned how regulators, users, and industry are calling for improved standardization of the production and testing of medical and recreational marijuana. Without proper testing, several issues are bound to arise[1][2][3][4][5][6]:

  • label claims may not match actual contents;
  • contaminants may linger, causing illness or even death;
  • chemical properties and medicinal benefits of specific strains and their unique cannabinoid-turpene profiles can't be isolated; and
  • research on potential therapeutic qualities can't be replicated, hindering scientific progress.

In 2011—a year before any U.S. state had enacted broad legalization of recreational marijuana—California NORML reported that its assessment of analytical cannabis testing laboratories' accuracy found that while California labs broadly reached +/- 20 percent consistency from a replicate sample, three out of 10 provided unfavorable results on at least half of their tests. Similar wide-ranging discrepancies were also found among edibles, extracts, and tinctures, and NORML found that none of the labs could reach two decimal points precision of cannabinoid results despite laboratory claims stating otherwise.[6] Another report out of the state of Washington in January 2015, not long after recreational marijuana sales to the public (requiring accredited lab testing prior) began[7], found blind tests of recreational marijuana at dispensaries could range as much as 7.5 percent in accuracy from its corresponding label.[2] Further issues in 2016 with alleged partiality by some Washington testing laboratories prompted emergency proficiency testing rules to be enacted.[8][9] ("Proficiency testing" essentially requires a laboratory in question to test a sample with known properties, and then those results are compared to those of a neutral third-party lab testing the same sample.) Additional testing problems in Alaska and Washington labs in late 2017 found high disparities between two different testing labs, as well as a laboratory that couldn't "properly perform a coliform test that looks for bacteria."[10]

These discrepancies and deficiencies highlight the growing need for homogenization of testing methods and procedures, if not nationally at least across an entire state. Such homogenization would, in theory, not only positively affect the quality of product but also provide greater consumer confidence that label and product match. As Marketing Director Scott Kuzdzal of Shimadzu pointed out during a January 2017 webinar on analytic testing of cannabis, poor sample preparation, lack of thorough testing, and the manual process itself—which can introduce user error, particularly when good laboratory practices aren’t used—all can contribute to discrepancies between label and product.[11] When dispensaries, edible manufacturers, and supplement companies perform insufficient lab testing or overstate claims on labels, it reduces consumer confidence, and both state and federal authorities—including the FDA—have to interject.[8][9][12]

As was mentioned at the end of the previous section on state regulation, efforts to improve testing methods and procedures, with the goal of seeing the best of them become standards, are ongoing. Where are those efforts now, and where are they going? Before we can examine that, we first need to briefly look at what aspects of cannabis are actually being analyzed.

Analytical aspects of cannabis

Cannabinoids

As of mid-2015, researchers have identified 104 of the more than 750 constituents of Cannabis sativa as cannabinoids[13], active chemical compounds that act in a similar way to compounds our body naturally produces, and new cannabinoids continue to be identified during cannabis research.[14] Many of our body's cells have cannabinoid receptors capable of modulating neurotransmitter release in the brain and other areas.[15] The plant's cannabinoids vary, with each bonding to specific receptors in our body, providing differing effects. From a theoretical and medical standpoint, crafting a strain of cannabis that has specific cannabinoids that can aid with a particular malady, while also carefully reproducing the grow conditions to consistently make that strain in the future, is a desirable but difficult goal to achieve.[16] However, even as new strains are developed, identifying an existing strain effectively has its own set of challenges, as Mudge et al. point out: "the total [tetrahydrocannabinol] and [cannabidiol] content is not sufficient to distinguish strains [though] a combination of targeted and untargeted chemometric approaches can be used to predict cannabinoid composition and to better understand the impact of informal breeding program and selection on the phytochemical diversity of cannabis."[14]

Lab testing of cannabinoids is done primarily as a measure of psychoactive "potency," though cannabinoids have many other potential therapeutic uses. Current laboratory testing looks at only a handful of cannabinoids; more research and development of analytical techniques that can quickly and accurately detect and separate the the rest is required.[17] Some of the major cannabinoids tested for include[17][18][19]:

  • THC (∆9-Tetrahydrocannabinol): This is the most commonly known cannabinoid found in cannabis, notable for its strong psychoactive effects and ability to aid with pain, sleep, and appetite issues. Included is its analogue ∆8-Tetrahydrocannabinol (which shows notably less strong psychoactive effects than ∆9[20]) and its homologue THCV (Tetrahydrocannabivarin), which tends to appear in trace amounts and has a more pronounced psychoactive effect, but for a shorter duration. THCV shows promise in fighting anxiety, tremors from neurological disorders, appetite issues, and special cases of bone loss. Also notable is Δ9-THCA (Δ9-Tethrahydrocannibinolic acid), a non-psychoactive biosynthetic precursor to THC.
  • CBC (Cannabichromene): This non-psychoactive cannabinoid is found in trace amounts; however, it tends to be markedly more effective at treating anxiety and stress than CBD (see next). It's also notable for its anti-inflamatory properties and potential use for bone deficiencies.
  • CBD (Cannabidiol): CBD is a non-psychoactive component of cannabis, typically accounting for up to 35 to 40 percent of cannabis extracts. It acts as a counter-balance to THC, regulating its psychoactivity. It's been researched as a treatment for anxiety, sleep loss, inflammation, stress, pain, and epilepsy, among other afflictions. Included is its homologue CBDV (Cannabidivarin), which is also non-psychoactive and demonstrates promise as a treatment for epileptic seizures. Also notable is CBDA (Cannabidiolic acid), a non-psychoactive biosynthetic precursor to CBD.
  • CBG (Cannabigerol): This cannabinoid is also non-psychoactive but only appears in trace amounts of cannabis. If has potential as a sleep aid, anti-bacterial, and cell growth stimulant. Also notable is CBGA (Cannabigerolic acid ), a non-psychoactive biosynthetic precursor to CBG.
  • CBN (Cannabinol): CBN is mildly psychoactive at best and appears only in trace amounts in Cannabis sativa and Cannabis indica. It occurs largely as a metabolite of THC and tends to have one of the strongest sedative effects among cannabinoids. It shows promise as a treatment for insomnia, glaucoma, and certain types of pain.

Terpenes

Mandated lab testing of terpenes — volatile organic compounds that distinctly affect cannabis aroma and taste — is done primarily as a way to ensure proper labeling of cannabis and related products, including extracts and concentrates, so buyers have confidence in what they are purchasing.[21][22][23] However, additional lab research goes into terpenes as they also show potentially useful pharmacological properties[21][23][24], and they demonstrate synergies (referred to at times as the "entourage effect") with cannabinoids that largely still require further exploration.[5][24][23][25] Testing for specific terpenes (discussed later) is less of a standardized practice, though it's rapidly improving.[21] Commonly tested terpenes by third-party testing labs include[23][22][24][5][19][26]:

  • Bisabolol
  • Caryophyllene
  • Cymene
  • Humulene
  • Limonene
  • Linalool
  • Myrcene
  • Phytol
  • Pinene
  • Terpinolene

Contaminates

Generally speaking, a contaminate is an unwanted substance that may show up in the final product, be it recreational marijuana or a pharmaceutical company's therapeutic tincture. The following are examples of contaminates that laboratories may test for in cannabis products.

Pesticides: Pesticides represent the Wild West of not only growing cannabis but also performing analytical testing on it. One of the core issues, again, is the fact that on the federal level marijuana is illegal. Because it's illegal, government agencies such as the Environmental Protection Agency (EPA) don't test and create standards or guidelines for what's safe when it comes to residual pesticides, let alone how to best test for them.[27][28] Additionally, researchers face their fair share of difficulties obtaining product to test. The end result is we don't know much about how inhalation of pesticide-coated marijuana smoke affects long-term health[27][28], and we don't have standards for pesticide application and testing.[5] With numerous pesticide products and little oversight on what growers apply to their plants, combined with the technical difficulty of testing for pesticides in the lab, pesticides remain one of the most difficult contaminates to test for.[5] That said, several classes of of pesticides are commonly applied during cannabis cultivation and can be tested for by labs[18][4][29]:

  • avermectins: functions as an insecticide that is useful against mites, which are a common problem for cultivators
  • carbamates: functions as an insecticide, similar to organophosphates, but with decreased dermal toxicity and higher degradation
  • organophosphates: functions as the base of many insecticides and herbicides, valued for its easy organic bonding
  • pyrethroids: functions as the base of most household insecticides and exhibits insect repellent properties


Solvents: In 2003, Canadian Rick Simpson published a recipe of sorts for preparing cannabis extract via the use of solvents such as naphtha or petroleum ether. Claiming the resulting oil helped cure his skin cancer, others hoping for a cure tried it, and the solvent method of preparation grew in popularity. Dubious healing claims aside, the solvent extraction method remains viable, though it has evolved over the years to include less harmful solvents such as supercritical carbon dioxide, which has low toxicity, low environmental impact, and beneficial extraction properties.[5][30][31] However, chemical solvents are still used, and if not evaporated out properly, the remaining solvents can be particularly harmful to sick patients using the extract. As for what solvents should be tested for, it gets a bit trickier, though Chapter 467 of United States Pharmacopeia and The National Formulary, the Oregon Health Authority's December 2015 technical report on contaminant testing of cannabis, and the Massachusetts Department of Public Health's response to public comments on cannabis testing provide helpful guidance. Listed solvents include benzene, butane, cumene, dimethoxyethane, hexane, and pentane, among others.[4][18][5][29][32][33]


Heavy metals: 2013 research on contaminant testing on the behalf of Washington State provides insights into heavy metals and why they're looked for in cannabis testing. That research, as well as other sources, tell us[4][18][5][34]:

  • Heavy metals contribute to several health problems, including those of a neurological nature.
  • Cannabis can "hyperaccumulate metals from contaminated soils."
  • Research parallels can be found in tobacco research and how the FDA regulates heavy metal content in foods.
  • The most prominently tested heavy metals include arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), and nickel (Ni).


Mycotoxins and microorganisms: "The ideal conditions for cannabis growth are also ideal for the growth of potentially harmful bacteria and fungi, including yeast and molds," say Shimadzu's Scott Kuzdzal and William Lipps, "therefore microbial contamination poses health risks to consumers and immunocompromised individuals."[17] In truth, these concerns have already borne out; most recently the University of California, Davis reported in February 2017 one of its patients had contracted an incurable fungal infection from inhaling aerosolized marijuana. They later tested 20 marijuana samples from Northern California dispensaries — using specialized techniques — and found a wide variety of potentially hazardous microorganisms.[35]

The degree to which such contaminates commonly appear in grown and stored cannabis material and to which microbiological contaminates should be tested is not clear, however. As mentioned previously, neither the U.S. EPA or neighboring Health Canada provide any significant guidance on cannabis testing, including microbiological contaminates.[36] Like heavy metal testing, parallels are drawn from microbial testing guidelines and standards relating to tobacco and food, where they exist.[36] As warm, moist environments are conducive to microorganism growth, maintaining stable moisture levels during cultivation and storage is essential. Regularly measuring water activity — how moist something is — is particularly useful as a front-line preventative tool to better ensure microbial growth is limited.[29] Regardless, testing of some kind is still required by many U.S. states, including for organisms such as[17][18][5][29][35][36][37][38]:

  • Aflatoxin
  • Alternaria
  • Aspergillus
  • Cryptococcus
  • E. coli
  • Mucor
  • Penicillium
  • Salmonella

Methods and guidelines

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. 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[39] and NORML[40] provide such online resources. Instead, 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.

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.[18] When deriving a sample from a cannabis-laden edible, the QuEChERS approach used by food safety labs for pesticide testing has practical use.[41] 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.[18][42] As the 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.[18]

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.[43]

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.[44] 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.[18] 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.[18][5][45][46]

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-PDA).[18][47] Overall, methods used in cannabinoid testing include[18][5][47][48][49]:

  • Fourier transform infrared spectroscopy (FTIR; has limitations, such as requiring standard samples tested w/ other methods)
  • Gas chromatography flame ionization detection (GC-FID; requires sample derivatization for both acid and neutral compounds; good with standards like 5α-cholestane, docosane, and tetracosane)
  • Gas chromatography mass spectrometry (GC-MS; requires sample derivatization for both acid and neutral compounds; good with standards like deuterated cannabinoids)
  • High-performance liquid chromatography photodiode array detection (HPLC-PDA; stable for all forms of cannabinoids)
  • High-performance liquid chromatography UV detection (HPLC-UV)
  • Supercritical fluid chromatography (SFC; newer technology w/ added benefits)
  • Thin-layer chromatography (TLC; older, less common technology)
  • Ultra-performance chromatography (UPC; newer technology w/ added benefits)

Terpene testing

Identifying and quantifying terpenes is one of the more difficult tasks facing laboratorians[5]:

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.

Of course, types of gas chromatography work; but like cannabinoids, terpenes can degrade with the high heat of gas chromatography.[49] 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.[5] 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[49], while others such as Restek Corporation claim the method is problematic at best.[50]

Overall, methods for terpene identification and analysis include[5][26][48][49][51]:

  • 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)
  • Headspace gas chromatography mass spectrometry (HS-GC-MS)
  • High-performance liquid chromatography (HPLC; may have limitations due to coelution of terpenes and cannabinoids at certain ranges[50])

Contaminate 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."[5] 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.[5] Testing methods that have been used include[18][51]:

  • 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 EPA's Residue Analytical Methods (RAM) or FDA's Pesticide Analytical Manual (PAM) provide guidance to labs.[18][52][53]


Solvents: Testing for solvents is largely standardized into a couple of options, which have parallels to existing pharmaceutical testing standards outlined in Chapter 467 of United States Pharmacopeia and The National Formulary (USP <467>)[32][18][5][51]:

  • Headspace gas chromatography/mass spectrometry (HS-GC/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)[18][33][29] to set their action level testing values for particular solvents.


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. The following methods are most common for testing cannabis and other plants[4][18][5][54][51]:

  • Inductively coupled plasma atomic emission spectroscopy (ICP-AES), sometimes called inductively coupled plasma optical emission spectrometry (ICP-OES) (at times coupled with an ultrasonic nebulizer)
  • Inductively coupled plasma mass spectroscopy (ICP-MS)
  • Inductively coupled plasma tandem-mass spectroscopy (ICP-MS/MS)

For quantification of metals in cannabis, the U.S. FDA's ICP-MS methodology document is often used.[18][52]


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. However, Petri culture analysis isn't rigorous, and ELISA can be time consuming, as it's limited to one mycotoxin per test.[4][5][36] The following are other, more precise techniques that are improving laboratorians' analyses, particularly using DNA snippets of microbiological contaminates[4][5][36][55]:

  • Quantitative polymerase chain reaction (qPCR)
  • Whole metagenome shotgun (WMGS) sequencing
  • Matrix-assisted laser desorption/ionization (MALDI)
  • High-performance liquid chromatography (HPLC)
  • Liquid chromatography tandem-mass spectrometry (LC-MS/MS)

The extent of mycotoxin testing required remains in question by several entities. The Association of Public Health Laboratories (APHL) claims "[t]here is no readily available evidence to support the contention that cannabis harbors significant levels of mycotoxins."[18] 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.[29] USP <561> recommendations largely limit mycotoxin testing of botanical products to those borne from root or rhizome material[56], "which THC-containing cannabis products presumably do not possess," emphasizes the APHL.[18] Regardless, U.S. Pharmacopeia's Chapter 561 remains a useful document for testing guidelines and limits regarding microbials.[56][18] 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.[57]

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.[29] The APHL references specifications from the Dutch Office of Medical Cannabis that recommend water content be between five to ten percent in cannabis.[18]

Reports

There's little in the way of standardization for lab reporting of cannabis test results, though some U.S. states have outlined requirements for what must be included in such reports. The Oregon Health Authority's Oregon Administrative Rules, Chapter 333, Division 64, Section 0100: Marijuana Item Sampling Procedures and Testing stipulates that any report must include total THC and total CBD (by dry weight) and, if discovered, "up to five tentatively identified compounds (TICS) that have the greatest apparent concentration." It also lays out requirements for pesticides, failed tests, limits of quantification, and specimen identifiers such as test batch number.[58]

In late January 2017, Pennsylvania released its temporary regulations in support of its new medical marijuana program (28 Pa. Code Chapter 1171), which includes a section on test results and reporting (1171.31). The regulations stipulate reporting by electronic tracking system, with stipulations on using certificates of analysis which include lot/batch number and the specific compounds and contaminates tested.[59] Regulations aside, it's largely up to the laboratory — and often by extension, the software they're using — to decide how a report is formatted. Some labs like Seattle-based Analytical 360 offer clean, color-based certificates of analysis, with high-magnification photographs, the chromatogram, potency, cannabinoid content, contaminate content, and explanation of limits, with the name of the approving analyst.[60][61] Others may simply generate a computer printout with the basic data and a legend.[62] Reports may originate from the measuring device itself (e.g., an integrator in a chromatography device), a middleware or data station attached to the instrument, or a laboratory information management system that accepted data from the instrument.[63]

Though not directly related to laboratory testing, it's worth noting states also have their own reporting requirements for growers, processors, and dispensaries. Both Oregon and Washington, for example, require monthly reports related to medical marijuana transfers.[64][65]

Lab equipment

As indicated in previous sections, spectrometry and chromatography have played and will continue to play an important role in cannabis laboratory testing. This should not be surprising: "mass spectrometry is superior to other spectral techniques in such features as sensitivity, selectivity, generation possibility of molecular mass/formula, and combinability with chromatography."[66] Analyzing complex chemical compounds that have many features and which are at times difficult to differentiate from each other proves challenging, but these technologies excel in meeting that task.[66] Refer to the previous "Methods and guidelines" section to note the specific technology associated with each molecule and contaminate. Aside from spectrometry and chromatography equipment, the analysis of microorganisms in cannabis may turn to DNA analysis methods that require additional equipment such as a thermal cycler (qPCR) or sequencer (WMGS), or ELISA, which utilizes a photometer or spectrophotometer. Of course, preparing and storing samples requires equipment as well, such as microplates, centrifuges, comparison standards, capillaries, chemicals, columns, Petri dishes, scales, and disposable gloves. Software-based data management systems may also constitute equipment and are discussed in the next section.

When it comes to purchasing lab equipment specifically for cannabis testing, a 2015 interview with Emerald Scientific's CTO Amanda Rigdon (then with Restek Corporation) provides good advice[67]:

  • Industry-specific instrumentation isn't needed in most cases as most of the techniques and equipment used in food and herbal medicine testing have strong parallels to cannabis testing.
  • That said, some sample preparation tools, standards, and consumables specifically marketed to the industry may very well make the job quicker and more reliable.
  • Appropriate sample preparation techniques are just as vital as the equipment you use.
  • Do your research; many instrument companies are examining methodologies usable on conventional equipment, lessening the need for more expensive devices.
  • If buying used equipment, make sure the original manufacturer is still in business and producing consumables and replacement parts. Make sure your planned methods match the equipment, and make sure it's not so old that it can't be serviced by a qualified technician.

Software

Laboratories increasingly depend on software to analyze, store, and share critical data from instruments and experiments.[68] This has led to the development of laboratory-specific software like the laboratory information management system (LIMS), electronic laboratory notebook (ELN), and chromatography data management system (CDMS; sometimes simply CDS). These and other software systems such as "seed-to-sale" programs can also play an important role in the cannabis testing laboratory.

LIMS

Laboratories of all types use LIMS software to manage the wide variety of data, testing and analysis workflows, and other enterprise activities typical of them. This generally includes — but is not limited to — sample receipt, workflow management, sample tracking and analysis, quality control, instrument data management, data storage, reporting and document management.[69] The cannabis testing laboratory is no exception, though its activities differ slightly from, for example, a clinical pathology laboratory. As such, a few additional features outside of what's typically found in a generic LIMS are required.

Features that may be incorporated into a cannabis testing LIMS that you might not necessarily find in an all-purpose LIMS include[70][71][72][73]:

  • sample loading screens optimized for the industry, including differentiation between medical and recreational marijuana
  • pre-loaded compliant test protocols, labels, and reports optimized and readily adjustable for a rapidly changing industry
  • tools for creating new, compliant test protocols, labels, and reports
  • a web API to integrate with state-required compliance reporting systems
  • chain-of-custody (CoC) tracking, when necessary
  • support for inventory reconciliation

As previously discussed, industry-specific test protocols largely focus on cannabinoids, terpenes, and a wide variety of contaminates, including excess water. However, as regulations continue to be in a state of flux and not particularly standardized, most LIMS developers are including the ability for users to adjust their protocols and even add new ones. And while CoC functionality is not entirely foreign to generic LIMS, it's particularly important in an industry where currently transporting even a cannabis test sample across state lines can create huge problems.

In cases where daily sample processing is infrequent and only a couple of chromatography machines are used, laboratories may weigh a decision between a LIMS and a chromatography-specific CDMS, although the ability to produce an acceptable certificate of authenticity (CoA) and document the CoC are still factors, along with any state reporting requirements.

CDMS

Scientists on the research side of cannabis are certainly using CDMSs from Agilent, Thermo Scientific, Waters, and other to manage the data coming out of their chromatography equipment[74][75][76], and slowly but surely some of those CDMSs are beginning to also support spectrometer data management in a similar way.[77] Additionally, some chromatography system developers will collaborate with CDMS vendors to develop software drivers — code that essentially acts as a translator between a device and a program — so chromatography devices can interact fully with the CDMS.[78]

The CDMS likely has a place in the cannabis testing lab as well, though it may depend on the lab's data management needs and goals. In more complex labs with multiple instruments and significant daily processing workflows, a LIMS may make more practical sense.

Seed-to-sale

The use of seed-to-sale software is an emerging trend that is only tangentially related to laboratory testing of cannabis. Rather than at testing laboratories, seed-to-sale software is found at cultivation sites, production facilities, and dispensaries, and that software is typically designed to be able to integrate with testing laboratory or other software. The goal: create a complete record of transaction, from the grown plant to the lab, producer, and seller. This sort of tracking is mandated in various ways by many U.S. states with legalization laws. "It’s there to prevent the diversion of marijuana, which the federal government still lists as a Schedule I substance, the most dangerous class of drugs," wrote Daniel Rothberg of the Las Vegas Sun in December 2015. "Tracking also ensures product safety, assists with audits and helps facilitate recalls."[79] This type of software is able to track plant yields, attempted theft or diversion, patient preferences, extraction methods, batch weights, and various financial statistics for analysis.[80][79]

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Citation information for this chapter

Chapter: 3. Laboratory testing of cannabis

Title: Past, Present, and Future of Cannabis Laboratory Testing and Regulation in the United States

Author for citation: Shawn E. Douglas

License for content: Creative Commons Attribution-ShareAlike 4.0 International

Publication date: November 2018


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