The last column dealt with measuring peak widths and using them in separation quality calculations, but what causes chromatographic peaks to have different widths? One of the culprits is band spreading, which is caused by diffusion. In this column, we discuss diffusion and see how a quantity called plate number is used to measure the amount of band spreading.

Peak Widths and Chromatographic Resolution: A Review

In my last column (1) we discovered that in addition to peak positions chromatographic features have peak widths. The technique we chose to measure peak widths was the triangulation method (1) seen in 

In this method a baseline is drawn under a peak, and then lines are drawn along the sides of the peak that intersect with the baseline to form two baseline endpoints. The width of the peak is measured between the two baseline endpoints to give the triangulated peak width. Using this method in Figure 1 the peak width w

 for peak a is 2 min, whereas the peak width w

For the chromatogram seen in Figure 1, the peak widths do not pose a problem because the peaks are baseline separated, that is, there is flat baseline between them. However, as we saw in the last installment of this series (1), excessively wide peaks affect separation quality as seen in 

The retention times for peaks a and b in Figures 1 and 2 are the same, namely 4 min and 8 min, respectively, but in Figure 2 the separation is of poor quality because the peaks are overlapped due to excessive peak width. We can see this by comparing the widths of these peaks in the two figures. Peak a is 2 min wide in Figure 1 and 5 min wide in Figure 2, while peak b is 1 min wide in Figure 1 and 6 min wide in Figure 2.

The challenge we faced in the last column (1) was deriving a measure of chromatographic separation quality that took peak width into account. We found that this quantity is called the 

The units in the numerator and denominator are both time, which means R is unitless. Note in the numerator of Equation 1 that the elution times for two chromatographic peaks are subtracted from each other. The stipulation that t

 exists so that the numerator, and hence R, have a positive value. The denominator in Equation 5 is the average of the two peak widths. Because of this, as average peak width goes up R goes down. That is, the broader the chromatographic peaks the worse the quality of a separation and the lower the R value. Using Equation 1, the separation in Figure 1 has a resolution of 2.67, whereas the separation in Figure 2 has a resolution of 0.73. Baseline separated peaks have an R value of 1.5 or greater, which is always our goal when developing separation methods.

I refer you to the literature (2-4) for more details and background on the following discussion.

 illustrates in red a slug or band of analyte after it has been injected at the head of a chromatographic column. The analyte band traverses the column from left to right. Note that upon injection at time T = 0 the analyte has adsorbed and the band has a width w

. You can think of the red bars in Figure 3 as locations where the analyte is adsorbed on the stationary phase, and the white spaces as locations where the analyte is in the mobile phase.

Note that there are five spots along the column where the analyte adsorbs, and as the analyte band traverses the column it gets wider and wider. What’s going on here? A phenomenon called 

 caused by the process of diffusion (5). 

 occurs when chemical species spontaneously move from areas of higher concentration to areas of lower concentration. You can observe diffusion when you put cream in your morning coffee. The slug of cream doesn’t just sit there, you can see it move throughout your coffee cup. Note that the longer you watch the cream the more diffusion takes place. Stirring the coffee and cream mixture with your spoon speeds the process of diffusion, but left to its own devices for long enough the cream will infiltrate all parts of your coffee cup until the concentration of cream is the same everywhere.

Diffusion happens to analytes in chromatographic columns. When an analyte is adsorbed on the stationary phase it does not diffuse because it is held fast. However, when the analyte is in the mobile phase, such as in the white areas in Figure 3, the analyte molecules will diffuse along the longitudinal axis of the column broadening out the band and giving it increased peak width. That is why each red band in Figure 3 is wider and wider as it moves from left to right down the column until it elutes at retention time ta. Like the cream in your coffee, the more time your analyte spends in solution, the more it will diffuse and the more widespread the distribution of the analyte. The upshot here is that the more time your analyte spends in the mobile phase, the more diffusion will take place and the broader your peak will become.

To get the narrowest peaks then and to optimize separations you want to choose a method that minimizes the time your analyte spends in the mobile phase. Recall (6) that the elution time of an analyte depends upon the fraction of time an analyte spends adsorbed on the stationary phase as given by 

 is the fraction of time an analyte spends adsorbed on the stationary phase.

Also recall (7) that the total time a molecule spends in a column is simply the sum of the time it is adsorbed on the stationary phase and the time it is in the mobile phase as given by 

 is the fraction of time an analyte spends in mobile phase and F

 is the fraction of time an analyte spends in the stationary phase.

While the analyte is in the column it is either in the mobile or stationary phases, so the fractions in Equation 3 add up to 1.

We said above that the more time the analyte spends in the mobile phase the more diffusion can take place and the broader our peak is when it elutes from the chromatographic column. In other words, the larger F

Measuring Band Spreading: The Plate Number

Given the above then our measure of band spreading must be related to F

In this figure the retention time of our analyte, t

, is 7 min and the triangulated peak width w is 4 min. A measure of band broadening for a peak like this is called the 

 is the retention time for a chromatographic peak, and w is the chromatographic peak width measured with the triangulation method.

 and w are measured in units of time, and that when we divide them the plate number N becomes a unitless quantity. For the peak seen in Figure 4, the plate number is found through 

Now, Equation 4 contains the quantity tr/w, but this is all easier to understand if we think about its inverse, w/t

. This quantity, the peak width divided by the retention time, measures the percentage of the elution time taken up by the peak width. The more band spreading, the broader the band hence the bigger w/t

. In Equation 4 then we see the inverse of w/t

/w, so as band spreading, and hence peak width, goes down N goes up. This means we want N to be large for a separation because it means the peak is narrow compared to the retention time of the peak. So, N is yet another measure of chromatographic separation quality.

A grossly oversimplified way of thinking about the plate number N in a chromatographic separation is that it measures the average number of times an individual molecule adsorbs on the stationary phase before it elutes. Thus, for the peak seen in Figure 4 the average molecule adsorbed 49 times. Now, remember that band spreading goes with F

. The more times an analyte molecule adsorbs on the column, the less time it spends mobile, the less diffusion and hence band spreading and the narrower the peak will be. This is why N is a measure of band spreading and separation quality.

This is the sixth in a series of articles I have written on chromatographic theory (1,6-9). Across these articles we have derived a number of quantities that I have claimed are measures of chromatographic separation quality including the capacity factor k (6), the separation factor α (9), and now here the plate number N. Given all these quantities how do we get the truest measure of separation quality? Well, there has been a method to my madness. What we ultimately need to do is discover what I will call the master resolution equation for chromatography because it includes k, α, and N. Because the master resolution contains these three variables, we had to define them and discuss them thoroughly before introducing this equation. The master resolution equation is the best summary of how to measure separation quality and to determine what variables to adjust in what way to optimize a separation. More on this next time.

Chromatographic peaks have both peak heights and widths. Narrower peaks are preferred because it is easier to obtain baseline separation. Wide peaks are a result of band spreading, which is caused by the physical phenomenon of diffusion. More diffusion—hence band spreading—takes place the longer an analyte is in the mobile phase. The plate number, N, is a measure of band spreading because it is related to the number of times an analyte molecule adsorbs on the stationary phase, which means it spends less time mobile and hence less time diffusing.

Smith, B., Chromatography Theory, Part III: Calculating Elution Times and Capacity Factors, 

Skoog, D.A., West, D.M., and Holler, E.J., Analytical Chemistry: An Introduction, 

Robinson, J.W., Undergraduate Instrumental Analysis, 

Smith, B., Chromatography Theory, Part II: How Separations Occur Illustrated and Quantitated, 

Smith, B., Principles of Chromatography, Part I: Theory, 

Smith, B., Chromatographic Theory, Part IV: Measuring Separation Quality with Capacity and Separation Factors, 

, PhD, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific. He is the inventor of the BSS series of patented mid-infrared based cannabis analyzers. Dr. Smith has done pioneering research and published numerous peer-reviewed papers on the application of mid-infrared spectroscopy to cannabis analysis, and sits on the editorial board of 

. He has worked as a laboratory director for a cannabis extractor, as an analytical chemist for Waters Associates and PerkinElmer, and as an analytical instrument salesperson. He has more than 30 years of experience in chemical analysis and has written three books on the subject. Dr. Smith earned his PhD on physical chemistry from Dartmouth College.
Direct correspondence to: 

Smith, B., Chromatographic Theory, Part VI: Peak Widths, Band Spreading, and Plate Number

Articles

Cannabis Analysis 

Here we discuss diffusion and see how a quantity called plate number is used to measure the amount of band spreading.

Chromatographic Theory, Part VI:  Peak Widths, Band Spreading, and Plate Number

A cannabis extraction facility is often less expensive and more versatile than a cannabis cultivation facility. These are some of the reasons there are more cannabis processors and extractors than cultivation facilities in states that are not vertically integrated. Mature cannabis markets have consistent, if not an excess, supply of cannabis biomass. Prices for biomass have extreme swings depending on the season or supply and demand of cannabis biomass. For cannabis processors that do not cultivate, sourcing biomass is critical to determining the cost and quality of the cannabis extracts that are produced. Here, we review the variables in sourcing cannabis biomass and the impact they have on extraction outcomes.

Commercial cannabis can be grown in many different environments. Cultivators choose between outdoor, indoor, and greenhouse depending on the facility’s location, budget, and production goals. Cultivation of cannabis involves various factors, such as genetics, cultivation methods, and processing techniques, that affect the quality of the final product. The cultivation method and scale of the facility can significantly impact the cost, range of quality, and supply of biomass. These variables dramatically impact processors who source extraction material from their own cultivation facility or other cultivators.

Indoor cultivation is the practice of growing cannabis in an indoor facility with a controlled environment. The indoor facility is equipped with artificial lighting, temperature and humidity control, and ventilation systems. The controlled environment of indoor cultivation allows cultivators to manipulate various growth factors, such as lighting, temperature, and humidity, to optimize plant growth and increase yields. Greenhouse cultivation is a hybrid of outdoor and indoor cultivation; however, the cultivation methods and end product are more similar to indoor cultivation.

On the other hand, outdoor cultivation involves growing cannabis in an open environment, such as in a field or a greenhouse, without the use of artificial lighting or environmental control systems. The natural environment of outdoor cultivation can provide various benefits to the plant, such as exposure to natural sunlight and fresh air, which can enhance the terpene and cannabinoid profiles.

The aforementioned differences in cultivation methods significantly impact the outcomes of the extracted end products.

The terpene and cannabinoid profiles of cannabis can be significantly affected by the cultivation method used. Cannabinoids, like tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), are naturally produced by the cannabis plant. However, there are cannabinoids found in cannabis that are oxidation or degradation products, such as cannabinol (CBN) and cannabitriol (CBT) isomers. Outdoor cultivation methods can produce cannabis with a more diverse and complex terpene profile due to exposure to sunlight, Earth’s elements, and the genetics of plants successfully grown outdoors (1). Cannabis with a more diverse and complex terpene profile can result in a more flavorful and aromatic final product. Outdoor cannabis often has a lower concentration of cannabinoids and a higher concentration of terpenes than indoor cannabis (

). For processors seeking diverse chemical profiles, which will produce diverse effects of cannabis, outdoor cannabis may be the best source. Exposure to the outdoors also increases the amount of wax the plant produces to protect itself, which ends up in the raw extract. This wax is a byproduct that can add time to the refinement process and can interfere with the quality of the extract. This is something to consider when evaluating source material and processing time.

Indoor cultivation methods can produce cannabis with a more consistent and predictable terpene profile due to the controlled environment. Because of this, the cannabis flower and final extract often have a lighter color than outdoor cannabis. Interestingly, the cannabinoid profiles of indoor cannabis have higher concentrations of THCA and extrinsic cannabinoids (please see graphics in reference 1). Higher concentrations of THCA produce a more potent THC product as opposed to the balanced and diverse chemical profile of outdoor cannabis. All these factors have a significant impact on the end product quality and chemical profile.

The use of pesticides and fertilizers can also impact the quality of the final product. Indoor cultivators may use more pesticides and fertilizers to combat pests and optimize plant growth. Outdoor cultivators, on the other hand, may use fewer pesticides and fertilizers due to the natural environment and natural defense mechanisms of the plant.

The yield and cost of cannabis may be the most important metrics to monitor when sourcing cannabis for extraction. They are both significantly affected by the cultivation method and grade of the biomass. 

 is an example of how the final cost of the extract can be estimated by using biomass cost and THC concentration.

Indoor cultivation methods can consistently produce a harvest multiple times a year and the product will have higher concentrations of cannabinoids. This means processors sourcing from indoor cultivators will have access to consistent biomass year-round and extraction yields of cannabinoids will be higher. However, the cost of production is significantly higher due to buildout cost, operating cost, and heavy utility use for lighting and environmental control systems. The high cost of indoor biomass may be worth it depending on the extraction yield and quality.

Outdoor cultivation methods produce one harvest per year and the product will have lower concentrations of cannabinoids. Depending on the scale of the outdoor cultivation site, one outdoor harvest can outweigh the annual production of the annual harvest weight of indoor cannabis facilities. However, it depends on the scale of the outdoor cultivator. Starting with material that has a lower concentration of cannabinoids produces lower yields, but the cost of production is lower due to the use of natural resources.

The differences between indoor versus outdoor cannabis has been evaluated here, but the grade of the material will also impact the quality and cost of the final extract. Cannabis flower is the highest grade of material, however, there is high- and low-quality cannabis flower. Indoor and outdoor flower will also have different cannabinoid and terpene content. Cannabis trim is a byproduct and is often lower in cannabinoid and terpene content. It is also usually exposed to lower quality care and handling, which can impact the quality of the end extract. All these variables can be evaluated by analyzing the cannabinoid content and terpene content as well as visually inspecting the biomass prior to purchase.

Ultimately, all these factors can be used to make the best choice when sourcing cannabis for extraction. Supply and demand will fluctuate throughout the year and should be anticipated when budgeting for purchasing throughout the year. To make the best decision for extraction sourcing, consider yield, cost, quality, and what end product the extract will produce.

Zandkarimi, F., Decatur, J., Casali, J., Gordon, T., Skibola, C., Nuckolls, C., Comparison of the Cannabinoid and Terpene Profiles in Commercial Cannabis from Natural and Artificial Cultivation, 

. 2023; 28(2):833. doi: 10.3390/molecules28020833.

 is the founder, CEO, and Chief Extractor of Heylo. With a background in chemistry and clinical research, Lo was inspired to explore cannabis as a medicine and to enter the emerging industry. She joined Eden Labs, a leading CO

extraction equipment manufacturer to support and expand a Research and Development department. There she managed the development of their latest and greatest CO

 extraction system. In 2017, after working with Eden Labs and another cannabis processor, Lo launched Heylo with a mission to help people get more out of life with cannabis.

Friesen, L., Cannabis Sourcing and Extraction Outcomes, 

Extraction Science

For cannabis processors that do not cultivate, sourcing biomass is critical to determining the cost and quality of the cannabis extracts that are produced. Here, we review the variables in sourcing cannabis biomass and the impact they have on extraction outcomes.

Cannabis Sourcing  and Extraction Outcomes

Often when scientists or analysts speak of analysis and contamination in the cannabis market it is regarding chemical components such as heavy metals, pesticides, and other potentially hazardous chemical substances. In previous columns, we have taken a deep dive into the chemical aspects of cannabis analysis and now we change direction into another important study involving cannabis—microbiology. Chemical compounds are far from the only potential contamination found in cannabis products, in fact, in the world of consumer products, chemical contamination makes up for only a small amount of product recalls in comparison with microbiological contamination. In this column, we start the discussion on the important microbiological targets for the cannabis market and understand their significance as a threat to health and safety.

Biological contaminants in the form of microbes are by far one of the greatest concerns for illness. According to the World Health Organization (WHO), there are more than 300 million cases of foodborne illnesses in the world each year resulting in almost half a million deaths (1). In the US, the Centers for Disease Control and Prevention (CDC) estimates that 48 million people get sick from foodborne illnesses and up to 3000 people die from foodborne diseases (2).

As cannabis becomes legal in more states and countries, there has been a corresponding increase in the amount of testing, and the cannabis testing market to try to ensure accuracy of labelling and claims in addition to product safety from physical, chemical, and biological pathogens.

Microorganisms, or microbes, are organisms that are microscopic in size and are composed of either single cells, colonies of cells, or small multicellular structures. The ways in which human beings have classified life since the early beginnings of scientific study has changed dramatically over the centuries (and even over the last few years!). The most current consensus based on the three-domain system is that living organisms fall into three superkingdoms or domains: archaea, bacteria, and eukarya.

Archaea and bacteria are prokaryotes lacking a nucleus and membrane-bound organelles. These two domains consist of only single-celled organisms. There are few to no widespread pathogens in the Archaea domain, but they do share similarities in their genetics and metabolic pathways to some bacteria, especially 

. Most pathogens fall into the domains of bacteria and eukarya.

In this three-domain model, viruses are not included in one of the domains but instead are submicroscopic infectious agents that can only survive and replicate inside other cells. From the two dominant domains and viruses there are at least five types of biological agents (microbes) possible of causing disease including: bacteria, viruses, and the eukaryotes (parasites, protozoa, and fungi).

Parasites (multicellular) and protozoans (unicellular) are single celled organisms that can be free-living or parasitic living off other organisms or their detritus. There are dozens of human parasitic protozoans that cause a variety of disease from waterborne amoebas to mosquito driven Plasmodium malarias. In general, cannabis products are minimal risk targets for protozoans or human parasite contamination, instead the primary source of human pathogens are bacteria and fungi. However, protozoa, parasites and viruses can still be agricultural targets for the cannabis industry in need of analytical testing.

In a previous column (3) we looked at fungal targets and their biological by-products, mycotoxins. The mobility of fungal spores and their minute size makes fungus and molds a significant threat to agriculture and human health. Of particular concern are the toxic fungi from the phylum Ascoymcota (molds, yeasts, mildew, and so forth), which can produce mycotoxins. The same species of fungi can produce multiple mycotoxins and many mycotoxins can be produced by more than one type of fungi (see 

Agricultural crops, such as botanicals and cannabis, are prone to fungal growth because of agricultural conditions and moisture content. Plant materials with more than 14% moisture can encourage mold growth. Mycotoxins, especially aflatoxins and ochratoxins, need oxygen to grow, so the reduction of the oxygen in the storage areas can retard growth.

For the cannabis industry, the focus has been placed on measuring and controlling the 

 species of fungi, especially for inhalable products since these fungi and their mycotoxin product can affect the respiratory system. The fungi were named after an aspergillum (holy water sprinkler) when it was first observed under a microscope. The similarity of the shape of the fungus has also been compared to a dandelion seedhead (see 

grows on rotting vegetable matter and can be easily inhaled. While there are numerous species of 

 the species of most concern for the cannabis industry are 

Aspergillus niger, terreus, flavus, and fumigatus. 

In most areas where cannabis products are legal there are some requirements for testing for 

species in select products. In many cases there must be no 

species detected for a product to pass because of the highly toxic nature of the mycotoxins produced by the fungus.

Toxic and lethal dosages for mycotoxins can be quite small for acute poisonings. Ochratoxin A has a tolerable daily intake designated by the WHO of 5 ng/kg of body weight per day. Ochratoxin A is very toxic with an LD50 of 20-25 mg/kg of body weight. WHO recognizes products containing more than 1 mg/kg of aflatoxins as potentially dangerous or life-threatening. The Food and Drug Administration (FDA) has limits for mycotoxins in human and animal feed up to 20 µg/kg for direct human exposure.

While fungi and mycotoxins are of concern in products that can be inhaled, the larger danger to human health from contamination of cannabis products come from bacteria. On the US CDC list of the top 30 human food and waterborne pathogens, 22 pathogens were bacteria, and five each were viruses and parasites. The majority of bacteria targets of concern for the cannabis industry are from three families of bacteria:

These bacteria of concern can be classified in multiple ways depending on their family, morphology, status as an anaerobic or aerobic organism, and ability to stain under different reagents—just to name a few categories. Morphology is one of the primary methods of differentiating families of bacteria. There are five basic morphologies of bacteria: spherical (cocci), rod (bacilli), comma or bent rod (vibrios), spiral (spirilla), or corkscrew (spirochaetes) (see 

The bacteria that are important targets for cannabis are almost all bacilli or rod-shaped bacteria, which can be detected by negative gram stain. Gram staining is a method of labelling bacteria into gram-negative and gram-positive groups by taking advantage of the chemical and physical properties of the bacterial cell walls. Positive cells have a thicker cell wall that retains the crystal violet stain while negative cells can wash off the stain. Different counterstains and additives can strengthen or bind the stain to the cell membranes allowing for preliminary identification of certain bacterial groups.

In a variety of regulations, the target is called bile-tolerant gram-negative bacteria (BTGN) which includes gram negative bacteria of the 

. Another type of test measure that can be required is total coliform, which is a different measurement of 

All these bacteria potentially have cause for concern in cannabis products, however, the most regulations are created around the quantitation and detection of 

. Salmonella causes almost 80 million illnesses worldwide with hundreds of deaths each year. Salmonella are rod-shaped, gram-negative bacteria from the 

 with six subspecies and more than 2600 serotypes or strains; some (mostly typhoidal strains) can cause severe illness or death (see 

Salmonella infections are caused by the ingestion of food that has been contaminated by feces (animal or human) either in the agricultural or handling phase of preparation. The serotypes can be divided into two main groupings: typhoidal or nontyphoidal. Typhoidal serotypes are transferred from human to human and invade the bloodstream. Nontyphoidal serotypes are zoonotic (transfer animal to human) and invade the gastrointestinal tract.

is a second important target for microbiological analysis in cannabis.This bacterium is a gram-negative, rod-shaped bacteria found commonly in the intestine of warm-blooded organisms (see 

). Most strains are either harmless or can be beneficial to the hosts while other strains are pathogenic and cause food poisoning.

Worldwide there are more than 300 million infections and 200,000 deaths due to some forms of 

that produce diarrhea and gastrointestinal illness. There are six pathogen types of the bacteria associated with diarrhea:enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), and Shiga toxin-producing E. coli (STEC). There are further serotypes of some of these pathogen types determined by the presence of O and H surface antigens. There are about 200 known serotypes. 

is one of the most diverse groups of bacteria sharing only about 20% of its genetic code amongst all the strains.

One of the most important targets because of its toxicity is the Shiga toxin 

 strain (STEC), especially the serotype O157:H7. The Shiga toxin produced by this strain of the bacteria causes severe abdominal pain, diarrhea, and life-threatening conditions such as dysentery, hemolytic uremia, hemorrhagic colitis, kidney failure, and death.

Due to the severe nature of the consequences of these microbiological infections most regulatory bodies for cannabis have proposed testing for microbiological contamination in cannabis products.

Agricultural products (including cannabis) around the world are routinely evaluated for contaminants and impurities such heavy metal ions, pesticides, mycotoxins, and residual solvents. This is true for all agricultural products that are consumed by human beings. Heavy metals are tested by inductively coupled plasma-mass spectrometry (ICP-MS) or inductively coupled plasma-optical emission spectroscopy (ICP-OES). Residual solvents, which are small, volatile impurities, are tested by gas chromatography (GC). Pesticides and mycotoxins, which are products of pathogens like 

, are routinely tested by liquid chromatography (LC). Many countries have specific requirements for biological contamination such as microorganisms. The presence of live microorganisms is assessed in several ways. The best quantitative results are obtained by quantitative polymerase chain reaction (qPCR), but other methods exist, such as cell culture techniques, and enzyme-linked immunosorbent assays (ELISA).

There are numerous ways to detect microorganisms in agriculture products, but the most common methods include cell culture, chromogenic media, ELISA assays, and polymerase chain reaction (PCR). Let’s discuss each of these in more detail.

The methodology for the detection and quantitation of microorganisms has traditionally been performed by utilizing various forms of cell culture. Many different culturing media have been used over the years that are enriched with specific concentrations of nutrient broths mixed with agar for support and grown in individual dishes or plates. The microorganisms are allowed to grow for specific durations of time (hours to days) and the direct number of colonies are counted per milliliter of fluid (CPM) or through viable cell counting to determine cell proliferation using colony forming units/mL (CFU).

In more recent years, much attention has been given to the use of chromogenic media for use in plating microorganisms. This application combines the use of synthetic substrates for the detection of enzymatic microbial activities with colorimetric reactions, allowing for more specific detection of species or sub-species, often with only visual analysis. Cell culturing requires a good working knowledge of scientific technique and requires designated laboratory space for laminar flow hoods, orbital shakers, incubators, and other necessary equipment. Moreover, microbiologists that have become experts in these techniques are reticent to accept some of the newer methodologies, so some debate exists in cannabis and throughout pathogen detection as to whether cell culture is indeed the “gold standard” of microbiology. Moreover, since culturing is a manual and time-consuming process, the need for more rapid results has pushed the development of other assays, namely ELISA and qPCR.

ELISA kits are enzyme-linked immunosorbent assays designed for both the rapid detection and quantitation of target proteins, antibodies, and other soluble targets of interest including cytokines, growth factors, and signaling molecules, as well as transcription factors and post-translational gene regulators (RNAi). ELISAs are highly sensitive, with a low limit of detection typically in the range of 0.1-0.01 ng. Their use for detecting microorganisms involves using a heat-inactivated bacterial strain or a target protein at known concentration for positive control and allows users to automate their sampling and produce far more replicates for statistical derivation in a typically 96-well format using a multiplate reader and minimal additional equipment needs. Additionally, the reactions do not require a high degree of technical expertise, making them more suitable for use in the cannabis space.

PCR, and now the use of qPCR, has allowed for the detection and quantitation of genes found with less than 10 copies in each sample. The combined use of fluorophores also allows a researcher to quantify five genes simultaneously, with one as a reference or “housekeeping” gene. This ensures that the deoxyribonucleic acid (DNA) amplified occurs in known relative concentrations to a DNA baseline, thereby minimizing human error. qPCR is slowly becoming the “go to” technology for operators in the cannabis space for microorganism detection and quantitation by virtue of such rapid generation of results and diversity of application. Despite having a reputation of being error prone, numerous studies have shown that qPCR-based microorganism detection protocols are faster and more sensitive than classical culturing or plating techniques for many organisms and the process can be automated and routinely performed with a modest amount of equipment and technical expertise.

Cannabis, like every other agricultural product, is tasked with providing for the safety of the consumer by limiting or eliminating pathogens. The agricultural practices and handling processes in the cannabis industry can lead to increased exposure to microbes. There are significant health concerns regarding microbiological contamination of products made into edible foods, inhalation products, and medicines for critically ill patients with potentially compromised immune systems. It is critical to understand the type, nature, and detection of the microbiological species to separate the potentially harmless serotypes and species from the toxic variants. In the next column, we will continue into a deeper discussion of the methods for detection and quantitation of the diverse types of microbiological targets along with their strengths and weaknesses as analytical techniques.

Estimating the Burden of Foodborne Diseases 

https://www.who.int/activities/estimating-the-burden-of-foodborne-diseases

https://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html

Atkins. P., Thomas R., Measuring Microbiology: A Look at the Microbiological Contaminants in Cannabis, Cannabis Science and Technology, 2019, 2(6), 20-23.

 is a Senior Applications Scientist with Spex, an Antylia Scientific company and has been a member of many cannabis advisory committees and working groups for cannabis including NACRW, AOAC and ASTM.

Atkins, P., MeasuringMicrobiology, Part I: A Look at the Microbiological Contaminants in Cannabis, Cannabis Science and Technology, 

Navigating the Lab

In part I of this series, we start the discussion on the important microbiological targets for the cannabis market and understand their significance as a threat to health and safety.

Measuring Microbiology, Part I: A Look at the Microbiological Contaminants in Cannabis

Cannabis testing labs in the US are facing the pressing concern of tetrahydrocannabinol (THC) inflation. Producers are indulging in “lab shopping,” which involves engaging with laboratories that artificially inflate THC numbers. Laboratories join in to keep customers returning to them. The laboratories that uphold their scientific integrity and report true results are shutting down. The practice is undermining consumer confidence, damaging the industry’s integrity. This tutorial article delineates the rising threat to the US cannabis industry and proposes solutions to overcome them.

The legalization and decriminalization of cannabis in several states in the US has led to the meteoric rise of the cannabis industry, with industry analysts projecting that the value of the regulated cannabis market will ascend to an astounding $82.3 billion by the year 2027 (1). The impetus for this exponential expansion of the cannabis industry can be attributed to the growing societal acceptance and legalization of cannabis for both therapeutic and recreational purposes. As of April 24, 2023, 38 states, three territories, and the District of Columbia have granted legal recognition to cannabis for medical use, while 22 states have passed laws allowing its recreational use (2). This trend is poised to continue as more states come to acknowledge the manifold advantages of legalizing cannabis, including the generation of increased tax revenue, the creation of new employment opportunities, and the delivery of medicinal benefits to those in need. Moreover, the surging demand for cannabis products, such as edibles, oils, and tinctures, is serving as a powerful engine driving the growth of this industry.

The legalization of cannabis has created new possibilities and spurred a rush of exciting innovations, paving the way for an extraordinary boom in the cannabis industry. The multifaceted nature of this industry has given rise to a multitude of ventures. Whether it be the cultivation, harvesting, processing, testing, or distribution of cannabis products, there is an unending stream of business opportunities for enterprising individuals and investors who wish to capitalize on this burgeoning market. The high growth potential of this sector coupled with the significant demand for cannabis-based products ensures that the market is a hotbed of activity. The rapid influx of new players and businesses has fueled unprecedented growth, and the industry shows no signs of slowing down. The vast opportunities presented by the cannabis industry are not only limited to the aforementioned aspects of the value chain but extend to ancillary services as well. Legal and financial services, marketing, branding, and consulting are all crucial components of the industry, and a multitude of cannabis-related businesses specialize in providing these services. This multifarious industry requires the services of experts and professionals from diverse fields, thus opening up opportunities for a wide array of individuals to contribute to the growth and development of this nascent sector.

However, despite the exciting developments in the cannabis industry, it has encountered some setbacks in recent times. One such notable challenge that came to light in 2022 is the phenomenon of tetrahydrocannabinol (THC) inflation, which has garnered significant attention (3). As cultivators strive to achieve the maximum possible THC concentrations in their produce, this predicament has become a major cause for worry, jeopardizing the integrity of the entire industry.

A Widely Held Misconception and the Malpractices of THC Inflation and Lab Shopping

There is a widely held belief among cannabis enthusiasts that products with higher THC levels are inherently more potent and lead to stronger effects. However, this belief is a misconception, as THC levels alone do not solely determine the overall potency of a cannabis product. To accurately assess the potency and effects of cannabis products, it is crucial to consider a variety of factors. These factors include the presence of other cannabinoids and terpenes, the method of consumption, and an individual’s metabolism and tolerance. For instance, a variety of cannabis that has reduced THC content, but elevated levels of other cannabinoids and terpenes may result in more powerful impacts compared to a variety with greater THC levels but lower levels of other compounds.

The mistaken notion that higher THC levels equate to greater potency has contributed to an increase in demand for high-THC products. This demand has resulted in producers offering incentives to laboratories that report inflated THC numbers for their products. As a result, some laboratories have engaged in a practice known as “lab shopping,” wherein they provide reports that meet the producers’ desired THC levels, rather than accurately reflecting the actual levels present in the product.

The term “THC inflation” refers to the practice of falsely elevating the reported levels of THC in a sample to indicate a higher THC concentration than the actual concentration. Strains with less than 10% of THC are classified as low THC, while those with more than 20% THC are considered high THC strains. The act of “lab shopping” arises from THC inflation, wherein dishonest manufacturers search for laboratories that boost THC levels artificially. The extent of this practice is such that certain laboratories openly advertise their services by emphasizing the elevated THC figures in their certificates of analysis (CoAs).

THC inflation and lab shopping undermine the integrity of the cannabis industry and reduce consumer trust. By working only with laboratories that give them the desired results, growers and producers can create the illusion of a higher quality product than what they actually have. This, in turn, can lead to consumers receiving products that do not meet the quality standards they are promised, which can erode trust in the industry as a whole. Moreover, it creates a situation where laboratories that strive to maintain their integrity and provide accurate testing results are unfairly disadvantaged. As a result, customers may become skeptical of all cannabis products and the entire industry may suffer.

Mitigating THC Inflation and Lab Shopping in the Cannabis Industry

Growing THC inflation is largely fueled by the absence of standardized testing protocols within the cannabis industry. As different laboratories use varying methods and equipment, the results are prone to differences in outcomes. This discrepancy is often exploited by corrupt laboratories that capitalize on the lack of standardization to report exaggerated THC values. Moreover, the possibility of manual intervention creates opportunities for such dishonest laboratories to manipulate testing outcomes and deceive regulators. To effectively combat the problem of THC inflation, it is imperative to establish a set of industry-wide standards that would eliminate these gaps in the testing process. The cannabis industry must establish a uniform standard for testing that ensures consistency and accuracy across all laboratories.

In response to the rampant issue of lab shopping, some states have attempted to adopt a uniform standard for testing. For instance, California’s cannabis regulator has made it mandatory for all laboratories in the state to conduct a standardized cannabinoid test. This test will help alleviate the crisis and guarantee that consumers are provided with precise and uniform information about the cannabinoid content of the cannabis and cannabis products they purchase or consume, as stated by the Department of Cannabis Control (4). Likewise, the cannabis regulatory body in Oregon has recently introduced a policy known as the “second lab” policy. This requires cannabis companies in the state to acknowledge that their product samples may be sent to a second laboratory, designated by the regulator, for the purpose of verifying the contents of the product. By doing so, the state regulatory authority can effectively cross-check the cannabinoid content of products and identify any discrepancies that may arise. This measure is aimed at ensuring transparency and accuracy in the cannabis industry, thus promoting consumer safety.

Another feasible solution to thwart THC inflation is to have different laboratories conduct tests on identical samples, as the variations in results among different laboratories can be instrumental in identifying and flagging inflated THC numbers. Once the potential outliers have been identified, regulators must take stringent action against laboratories that report inflated THC levels to the detriment of the industry and the consumers. This measure will aid in maintaining the integrity of the cannabis industry and ensuring that products are marketed truthfully and transparently. However, to implement such an approach, standardized guidelines for testing must be adopted and implemented to ensure the accuracy, consistency, and reliability of testing protocols. Only then can we guarantee fair play and avoid exploitative practices that may damage the cannabis industry’s reputation
and credibility.

It is also imperative to educate and inform the public about the fallacy that equates higher THC levels with increased potency. By disseminating accurate information and promoting awareness, we can combat the issue of THC potency inflation, and thereby, tackle the issue of lab shopping and curb its proliferation.

In addition, laboratories must take the initiative to pursue accreditation to the ISO/IEC 17025 standard to validate their expertise in generating reliable and trustworthy results (5). This move will not only strengthen the credibility of these laboratories but also restore faith in the legitimacy of the regulated cannabis market among consumers.

The Importance of Laboratory Software for CBD and THC Laboratories

Laboratory software for cannabidiol (CBD) and THC laboratories, also known as laboratory information management system (LIMS), is crucial for meeting the requirements of ISO/IEC 17025. This system is paramount in ensuring an added level of assurance and confidence in the accuracy of laboratory results. Laboratory software for CBD and THC laboratories automates processes through analytical instrument integration and adherence to strict quality standards, thereby reducing the potential for manual manipulation of test results (6).

Moreover, laboratory software for CBD and THC laboratories generates reports automatically, complete with scannable QR codes that can be shared with customers in real-time. By configuring the QR code, it is possible to include a link to the original CoA produced by the laboratory, which permits buyers to authenticate the reported composition on the product label by referring to the authentic test outcomes on the CoA. This fosters transparency, trust, and accountability in the cannabis industry.

Laboratory software for CBD and THC laboratories meticulously tracks and records all activities, including staff login activities, changes to documents, sample records, and test results. This comprehensive record-keeping eliminates the possibility of manual manipulation of laboratory data, further bolstering the reliability and defensibility of test results. Additionally, the system manages the results of various quality control (QC) samples to ensure accurate test results (7). It identifies analytical errors by comparing the test results of QC samples with the test samples, and this helps cannabis testing laboratories maintain high-quality standards.

The decriminalization of cannabis in multiple states across the US has propelled the cannabis industry into a meteoric rise, with the industry expected to reach a value of $82.3 billion by 2027. The surge in demand for cannabis products and the creation of new opportunities for employment and tax revenue are some of the advantages of legalizing cannabis. However, the industry has faced setbacks, including the problem of THC inflation and lab shopping. This issue is fueled by the absence of standardized testing protocols within the cannabis industry. Unscrupulous producers and laboratories exploit this discrepancy to deceive regulators and consumers. To mitigate the problem, industry-wide standards for testing must be established to ensure consistency and accuracy across all laboratories. California and Oregon have already implemented measures to tackle the issue by mandating laboratories to perform standardized tests and implementing the “second lab” policy.

The importance of regulators to enhance the accuracy and dependability of cannabis testing, along with fortifying consumer confidence, is indicative of an ambitious objective—to secure maximal profitability for the cannabis industry. As substantial amounts of capital have already been injected into the development of cannabis products, as well as the establishment of laboratories for testing, it is in the interest of growers, producers, and testing facilities alike to ensure that the actual worth of cannabis products really does align with the projected worth of the cannabis industry. Laboratories that are complicit in suspect practices must contemplate how such association harms their own repute, whilst also augmenting the risk of decertification and legal action, both of which could severely damage their economic viability during this pivotal moment for the industry. Given the number of competitors who are willing to lower their standards, the time is ripe for investment in and showcasing the quality and trustworthiness of one’s laboratory, so as to garner the trust, loyalty, and attention of reputable growers and distributors within the industry.

The cannabis industry is an exciting and innovative sector with vast opportunities for entrepreneurs and investors, but industry-wide standards must be put in place to promote consumer safety and guarantee transparency and testing accuracy.

Cannabis Market, Latest Global Industry Size Forecast Report

https://www.marketsandmarkets.com/Market-Reports/cannabis-market-201768301.html

https://www.ncsl.org/health/state-medical-cannabis-laws

Leveraging a Laboratory Information Management System for Cannabis Testing to Address THC Inflation & Lab Shopping Concerns, 

https://cloudlims.com/leveraging-a-laboratory-information-management-system-for-cannabis-testing-to-address-thc-inflation-lab-shopping-concerns

How US Cannabis Testing Laboratories Can Thrive, Despite Lab Shopping 

https://www.lgcstandards.com/FR/fr/Resources/Articles/DRE-cannabis-lab-shopping

On-Demand Webinar: Prepare Your Lab for ISO 17025 Accreditation Using a Cannabis LIMS 

https://cloudlims.com/on-demand-webinar-prepare-your-lab-for-iso-17025-accreditation-using-a-cannabis-lims/ 

What Is A LIMS: Benefits of Using A LIMS 

Achieving Quality Analytical Testing of Cannabis Using LIMS 

https://cloudlims.com/lims-posters/achieving-quality-analytical-testing-of-cannabis-using-lims/

 is the CEO of CloudLIMS in Wilmington, Delaware. Direct correspondence to: 

Apte, A., The Rising Threat to the US Cannabis Industry: How THC Inflation and Laboratory Shopping are Undermining Trust, 

Feature

 A tutorial article that delineates the rising threats of “lab shopping” and THC inflation to the US cannabis industry and proposes solutions to overcome them.

The Rising Threat to the US Cannabis Industry:  How THC Inflation and Laboratory Shopping are Undermining Trust

Led by expert program chairs and an educational steering committee, the first Cannabis Science Conference of 2023 returned to its roots in Portland, Oregon! To kick off the event, we held the pre-conference cannabis education workshop—the Canna Boot Camp—where attendees gained hands-on knowledge and experience in several aspects of the cannabis industry. The two-day conference featured two keynote speakers, several panel discussions, and plenty of insightful research from the four tracks: analytical science, medical cannabis, cultivation, and psychedelics. The exhibit hall provided attendees more opportunities to connect and collaborate with industry experts at various booths and poster presentations. Below we share some additional highlights from the event.

The full day, pre-conference Canna Boot Camp workshop was held at ChemHistory, a premier quality control testing laboratory in Milwaukie, Oregon. Attendees visited four interactive zones to learn about topics ranging from sample prep methods to in depth cultivation work and medical cannabis education as well as hands-on analytical testing. Each zone offered the chance to get a firsthand perspective, ask questions, and learn something new.

Thank you to ChemHistory, Fritsch, Spex CertiPrep, Milestone, Medical Marijuana 411, Christian West and Zac Hildenbrand for providing an in-depth learning experience for us!

Watch our onsite interview with ChemHistory Business Director Alex Hoggan 

Cannabis Science Conference Spring: Day One

Dustin Sulak, DO, launched the first day of the conference with an engaging presentation titled “Low and Ultra-Low Doses of Cannabinoids: Are We Missing the Sweet Spot?” Dr. Sulak examined several preclinical studies with ultra-low doses of cannabinoids—such as tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), and cannabidiolic acid (CBDA)— explaining how the cannabinoids could be working with or against each other. He also compared methods and results from human studies with diabetes, pediatric epilepsy, fibromyalgia, as well as experience from his own practice. He concluded with his recommendations for researchers, cannabis medicine producers, clinicians, and patients. See a 

Following Dr. Sulak’s presentation, the analytical track held a fireside chat discussion with a group of esteemed panelists led by moderator Patrick Bird, MS, Sr. Manager, Scientific Affairs, bioMerieux, on ways to reduce microbial product failure rates. The panel featured Heather Krug, Regulatory Programs Branch Chief, Colorado State Public Health Lab; Antionette Duncan, Sr. Director Quality Assurance, Curaleaf; Chris Hudalla, President and CEO, ProVerde Labs; Steve Jetter, Program Analyst for Cannabis and Psilocybin Policy, Oregon Health Authority’s Oregon Environmental Laboratory Accreditation Program (ORELAP); and Shane Flynn, Senior Director Laboratory Proficiency Testing, AOAC. The analytical science track covered a wide variety of topics throughout the day, such as analyses of aerosol generation and collection and delivery systems with Dr. Mark Crosswhite, laboratory testing, cannabinoid extraction, cannabis compliance, and evaluation and examination of terpenes. Watch a 

quick clip of analytical track program chair Dr. Julie Kowalski

 on what she looked forward to at the conference.

Moderated by program chair Cherissa Jackson, RN, the medical track included studies on cannabis and obesity, breast cancer, chronic pain, and precision cannabinoid medicine, plus a panel discussion on results from the first NIH funded population-based study of medical cannabis. To end the track on day one, Eloise Theisen, RN, Chief Nursing Officer and Board President of Leaf 411, presented case reviews of cannabis to reduce opioid intake in palliative care. For more on her presentation, watch our 

The cultivation track, led by program chair Dr. Zacaraiah Hildenbrand, covered cannabis-specific plant nutrition, solutions to lower CapEx and OpEx costs, 3D printed construction, and finding a source and solution to microbe contamination presented by Carly Bader. Get an in-depth look into her presentation and insights in our recent 

For the first presentation of the day in the psychedelics track, program chair Dr. Denise Vidot presented her research from “Cardiovascular Disease Risk Among Cannabis + Psilocybin Co-Consumers.” The track also featured topics such as content variety in psilocybe cubensis strains, and using HPLC to analyze psychoactive alkaloids.

At the end of day one, a networking mixer held in the exhibit hall provided the opportunity for everyone to exchange knowledge in-person with speakers, attendees, and exhibitors and poster presentations from all facets of the cannabis industry.

Cannabis Science Conference Spring: Day Two

Dr. Sue Sisley began the second day of the conference with her informative presentation, “MDMA/Psychedelic Assisted Therapy: The Path to Potential FDA Approval.” She presented her recently published Phase 3 study on 3,4-Methyl​enedioxy​methamphetamine (MDMA)-assisted therapy for treating post-traumatic stress disorder (PTSD) and explained the results, safety data, addiction potential, dose, and treatment time. She also addressed issues of the cost of psychedelic therapy, the limited efficacy of current treatment options, and the economic and social impact of untreated PTSD in the US. She described the neurological effects of MDMA, the neurobiology of PTSD, and what a typical MDMA session looks like and how it seeks to get to the root of trauma. MDMA, Dr. Sisley said, acts as “a catalyst, not the real treatment.” She ended her presentation with the changes she would like to see made for the future of psychedelic therapy, plus a real-life example of treating PTSD in the police force. Questions from the audience included the possibility of training psychiatric nurses or social workers to bolster the number of current therapists, why the FDA is appearing to embrace MDMA faster than cannabis, and the uniqueness of a therapy protocol around a medication.

One the second day of the analytical track, three different panel discussions covered the topics of the value of proficiency testing, which was moderated by program chair, Dr. Julie Kowalski; the standardizing and data sharing for the future of testing and quality control; and quality assurance and safety in cannabis psilocybin, and hemp, which was moderated by Kim Stuck. Watch our 

, CEO of Allay Consulting and author of the 

Across the medical track, patients, researchers, nurses, pharmacists, and doctors presented on topics such as impending cannabis regulation, cannabis in palliative care, the role of machine learning in treating autism spectrum disorder with cannabis, an integrative understanding of smoking cannabis, and the use of cannabinoids in outpatient primary care. Closing out the track, Jeremy Robbins, MS, gave a patient’s perspective on cannabis and spinal cord injury.

The psychedelics track was closed out on day two with an hour-long panel discussion that explored ketamine assisted psychotherapy from an integrative medical clinic perspective and included three experts from the AIMS Institute: Dr. Alison Draisin, PsyD, LMHC, Dr. Emily Whinkin, and Madeleine Turner, LMHC-A, Certified JAMP

 Transformational Coach. This panel will be covered in more detail in our upcoming psychedelics digital issue coming out in June 2023—stay tuned!

In the cultivation track, James Schwartz elaborated on the danger of the 2018 Farm Bill, Kevin McKernan presented information on the genetics of hop latent viroid, and presentations also covered the expanded soil food web and hemp production in North Dakota.

Finally, the conference concluded with a screening of the film 

, with commentary from filmmaker Steve Ellmore. The film advocates for veteran access to medical cannabis, highlighting the research of Dr. Sisley and testimony from veterans, veteran family members, advocates, and more. For more on this important film, please read our 

Deep Dive interview with Cherissa Jackson, RN,

 medical track program chair, as she provides insights into veterans, medical cannabis, and why 

Join us for Cannabis Science Conference Fall in Providence, Rhode Island September 20-22, 2023 as the industry continues to move forward!

https://www.cannabisscienceconference.com/

McEvoy, E., Cannabis Science Conference Spring 2023: Keeping Science at the Forefront of the Industry, 

Cannabis Science Conference Spring was held in Portland, Oregon from April 19-21, 2023. Here we provide a few highlights from the event.

Cannabis Science Conference Spring 2023: Keeping Science at the Forefront of the Industry

In this Q&A, Lake Superior State University’s Ben Southwell and Agilent Technologies’ Jean-Francois Roy suggest that laboratories should network and share best practices to optimize analytical procedures and overcome the lack of standardization in the cannabis market. As a step toward method standardization, they offer the relative merits and practical considerations of performing potency evaluation using mass spectrometry (MS) versus ultraviolet (UV).

The cannabis market has seen incredible growth in recent years as it transitions into various industrial applications. In 2022, the cannabis market in the United States alone was $13.2B and is projected to surpass $40B by 2030 (1). Cannabis is finding expanding applications in the cosmetics and food and beverage industries. Still, the pharmaceutical sector is the most significant market contributor. In terms of products, the market is led by cannabidiol (CBD) and tetrahydrocannabinol (THC), found in Food and Drug Administration (FDA)-approved drugs such as Epidiolex to treat seizures associated with Lennox-Gastaut Syndrome and dronabinol to treat chemotherapy-induced nausea and vomiting.

Such efforts are increasing the demand for regulatory oversight and product quality assurance. However, the industry that monitors compounds like CBD and THC is challenged by a need for more standardization and uniformity in its testing protocols. Testing laboratories often use different sample preparation, chromatographic, and detection techniques, making inter-laboratory result comparisons challenging. Some producers participate in “lab shopping,” which entails sending samples to multiple laboratories and selectively choosing data from the laboratory offering the most beneficial results.

To overcome this lack of standardization, Lake Superior State University’s Ben Southwell and Agilent Technologies’ Jean-Francois Roy suggest that labs should network and share best practices to optimize analytical procedures in this Q&A style article. As a step toward method standardization, they offer the relative merits and practical considerations of performing potency evaluation using mass spectrometry (MS) versus ultraviolet (UV).

In this Q&A, Lake Superior State University’s Ben Southwell and Agilent Technologies’ Jean-Francois Roy suggest that laboratories should network and share best practices to optimize analytical procedures and overcome the lack of standardization in the cannabis market.

June 2023

Chromatographic Theory, Part VI: Peak Widths, Band Spreading, and Plate Number

Cannabis Sourcing and Extraction Outcomes

Measuring Microbiology, Part I: A Look at the Microbiological Contaminants in Cannabis

The Rising Threat to the US Cannabis Industry: How THC Inflation and Laboratory Shopping are Undermining Trust

Cannabis Science Conference Spring 2023: Keeping Science at the Forefront of the Industry

The Future of Cannabis Potency Testing