This column continues the discussion of the practicalities and pitfalls of quantitative spectroscopy started in the last two columns (1,2). The reason that a discussion of quantitative spectroscopy matters for cannabis analysis is that many cannabis potency methods use high performance liquid chromatography (HPLC) along with ultraviolet-visible (UV-vis) spectroscopic detection (3). Also, there exist cannabis potency analyzers based on mid-infrared spectroscopy (4).

The fundamental equation of quantitative spectroscopy is Beer’s Law (5), whose form is seen in 

where A is the absorbance, the amount of light absorbed by a sample; ε is the absorptivity, a fundamental physical constant of a molecule; L is the pathlength or sample thickness; and C is the concentration.

I will dispense with any further discussion of Beer’s Law or the basics of quantitative spectroscopy and refer you to my previous columns and book on these topics (1,2,5-8). In this, the third in a series of pitfalls to avoid and practicalities to know about, we will discuss the importance of validating and checking your calibration to ensure quality.

The Fundamental Assumption of Quantitative Spectroscopy

Our tendency once we have a calibration in our hands is to immediately begin using it because they are a lot of work, and we may be anxious to start getting results. This is, however, the wrong approach. Recall (5-8) that when we generate a spectroscopic calibration, we have to make up samples of known concentration called 

, measure the absorbance of these standards, and plot their absorbance versus concentration to obtain a calibration line. We can then use our calibration to predict concentrations in unknown samples using 

 is the concentration of analyte in the unknown sample, A

 is the absorbance of the analyte in the unknown sample, and εL is the product of the absorptivity and pathlength obtained from the slope of the calibration line.

Note in Equation 2 that the concentration and absorbance refer to the unknown sample, but that εL is derived from a plot based on the standard samples. The assumption we are making here is that εL is the same for the standards as it is for the unknown samples. This is what I call the 

fundamental assumption of quantitative spectroscopy 

(FAQS) because if it is violated it means εL is different for the standards and unknowns. Applying an incorrect value of εL to unknown samples will give incorrect values of C

 because we are using a value of εL that applies to the standards but not to the unknowns. Therefore, violating the fundamental assumption of quantitative spectroscopy can be very damaging. So then, how do we know if we are violating this assumption? This is where calibration validation comes in.

To check if we are violating the FAQS we must test whether εL is the same for the standard and unknown samples. Why would εL ever be different for the standards and unknowns? The pathlength, L, can be different if the sampling cells or devices you are using for the standards and unknowns are not identical. The best way to ensure this is to use the same sample cell for all standards and unknowns. Ensuring the absorptivity, ε, is the same for samples and unknowns is trickier. Recall that the absorptivity is matrix sensitive and changes with variables such as temperature, pressure, concentration, and composition (5,6). This is why it is so important to ensure that all the experimental variables are the same when you measure the absorbance of the standard and unknown samples.

Given the vicissitudes of experiments and our inability to always perfectly control all variables, we must perform calibration validations before using them to analyze unknown samples. To test the FAQS, we make up a standard sample of known concentration but do not use it in the calibration. We will call this sample a 

We treat the validation sample as an unknown, measure its absorbance, apply Equation 2, and predict its concentration. We then compare the predicted concentration of the validation sample to its known concentration to see how well they agree. If they agree within the accuracy you are looking for this means you can go forward implementing your calibration. If not, back to the drawing board, and more on that in future columns.

Recall that accuracy is a measure of how far away a measurement is from its true value (9). The difference between the known and predicted concentration for the validation sample is the measure of the accuracy of your calibration. It is important to use at least one validation sample to test that your calibration does not violate FAQS. It is even better to create and analyze several validation samples. In this case for each sample, like above, measure its concentration, apply the calibration, and predict its concentration value. The beauty of analyzing multiple validation samples is that it allows you to calculate what is, in my opinion, the best measure of calibration accuracy called the standard error of prediction (SEP), which is seen in 

where SEP is the standard error of prediction, ∑

 is the validation sample number index, C

 is the actual concentration, and n is the total number of validation samples.

Let’s say there are three validation samples. To calculate the SEP the first thing to do is subtract the actual concentration from the predicted concentration for the three samples, square each of the differences, and then add these together. This gives the numerator in Equation 3. You then divide this sum by the number of validation samples minus 1, this is the denominator in Equation 3. Then, take the square root, and you have yourself a standard error of prediction. The SEP is the best measure of calibration quality because it shows you how well your calibration does on samples that are not included in the calibration, which is exactly how a calibration is used in real life. The units of the SEP will be in the units of your concentration measurement. For example, if C

 are in weight percent (Wt. %) then the SEP will be in Wt. %.

What value of the SEP tells me if my calibration does or doesn’t violate the FAQS? Strangely enough after all this math this ultimate answer is a judgement call. It depends on how accurate you need the calibration to be, that is, the needed accuracy is application specific. For example, if you want to know the total tetrahydrocannabinol (THC) in a cannabis bud that is normally around 20 Wt. %, an accuracy of ±1 Wt. % might be fine. However, if you are a hemp grower and have to ensure your crop is less than 0.3% weight total THC to comply with federal law, an accuracy of ±1 Wt. % total THC is worthless because the error bar is bigger than 0.3 Wt. % and overlaps with zero. In this case, an acceptable total THC accuracy would need to be significantly less than 0.3% total THC. For example, mid-infrared spectroscopy is capable of an accuracy of ±0.04 Wt. % for total THC in dried, ground hemp (10).

A mistake I find many people make is demanding more accuracy out of a calibration than is necessary. For example, for cannabis growers in states such as California where cannabis is legal, an accuracy of ±0.04 Wt. % for total THC is not needed since the state allows a ±10% relative error between your label and the actual product (11). For example, a bud labeled at 20% total THC can contain anywhere from 18% to 22% total THC and still be within the law. Because increasing accuracy is always time consuming and expensive, determine before you start calibrating what accuracy your calibration requires, and then stop work once you achieve that accuracy level.

Now that we have a validated calibration, we can begin using it to legitimately predict concentrations in unknown samples. Does this mean our job is done? By no means. Remember that because of the FAQS we must always ensure that εL is the same for the standards and the unknowns. Things change over time, and the only way to make sure you are not violating the FAQS over time is to run frequent calibration checks. A calibration check is not a full calibration and is very much like a validation. To perform a 

, make up some validation samples, measure their absorbance, predict their concentration, and compare the predicted and known values like we did above. Calculate an SEP if you used more than one calibration check sample. If things still agree within your required accuracy you can continue to use your calibration. If not, you need to immediately stop using the calibration and investigate what went wrong and how to fix it (more on this in later columns).

How often should you run a calibration check? The answer is as often as possible. Some laboratories will run a calibration check every time they use a quantitative spectroscopic calibration. This is great, but it can be time consuming and expensive. If you are using your calibration every day certainly a calibration check every week at least should be performed. If you use your calibration less often, you really should think about running a calibration check every time you use the calibration.

The fundamental assumption of quantitative spectroscopy (FAQS) was introduced, wherein we assume that the product of the pathlength and concentration, εL, is the same for standard and unknown samples. We test this assumption by running a calibration validation where a standard sample of known concentration, a validation sample, is analyzed with the calibration and the known and predicted values are compared. The difference between these two is the accuracy of your calibration. Ideally several validation samples should be run so that a standard error of prediction, the best measure of calibration accuracy, can be calculated. Whether or not your calibration is accurate enough to be used depends upon your application. To insure the FAQS is not violated over time, calibration checks should be run as often as is practicable.

M.W. Giese, M.A. Lewis, L. Giese, and K.M. Smith, 

Quantitative Spectroscopy: Theory and Practice

 (Elsevier, Boston, Massachusetts, 2002).

https://cannabis.ca.gov/wp-content/uploads/sites/2/2021/10/DCC-Cannabis-Regulations-Sept.-2021.pdf.

, 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: 

Articles

Cannabis Analysis 

The third part in this series of pitfalls to avoid and practicalities to know about discusses the importance of validating and checking your calibration to ensure quality.

Quantitative Spectroscopy Pitfalls and Practicalities, Part III:  Calibration Validation and Calibration Checks

In last month’s article, the wide variety of cannabis concentrates were defined and clearly differentiated. Every concentrate type, from crude ethanol extract to distillate, can be used to produce a cannabis edible. Cannabis extract is an ingredient in cannabis edibles production and, as with your food, the quality and makeup of the ingredients affect the quality of the end product. Many have claimed that differences in extract type can affect cost, ease of formulation, and the experience of the finished product. In this article, a review is done of each of these claims to better understand if it truly matters which type of cannabis extract is used in the production of a cannabis edible.

The cost of production for cannabis extracts has a wide range because of the number of variables. Cost of cannabis plant material, solvents, labor, and equipment are the main factors in calculating cost. If the cost and quality of cannabis plant material were fixed, cost could be assessed purely based on the extraction method. To keep things simple, tetrahydrocannabinol (THC)-dominant extracts are the focus of this cost evaluation. 

 shows a list of concentrates used in the production of edibles ranked by price and by THC content, with 1 being the highest and 4 being the lowest.

The amount of extract used to produce a THC infused edible is dependent on the concentration of THC in the extract. If the target dose of an edible is 10 mg, a higher quantity of extract is required to be used if the THC concentration is lower. Based on this, more extract is required to infuse THC edibles when using a live resin extract versus a distillate. The cost of production is directly correlated with the wholesale price of extract. Many edibles manufacturers select their extract type based on the cost per mg of THC. This is one of the reasons most edibles are produced with distillate.

Cannabis extracts are a complex matrix of many different compounds. Just ask your local analytical chemist! This complexity translates to how the extract interacts with ingredients in an edible. An edible needs all of its ingredients to be stable and maintain its target structure, flavor, and makeup until expiration. The more complex the extract, the more challenging it can be to integrate the extract with an edible. Flavor is another factor in formulation as the extract type will affect the flavor of the final product. The higher the concentration of cannabinoids, like THC, the lower the concentration of other compounds such as terpenes, aldehydes, ketones, and esters. These “other compounds” have unique flavors, which can pass through the edible when the consumer eats or drinks the infused product. Many consumers don’t like the flavor of cannabis, which has influenced how manufacturers produce edibles. Manufacturers can select high-THC distillate, which has little to no flavor, or formulate an edible that can mask the flavor of the cannabis extract. The market contains edible options that use distillate, full spectrum, live resin, or full extract canna oil (FECO) with a wide range of flavor profiles.

THC in cannabis is one of hundreds of compounds that influence the effect of cannabis. The jury is still out about whether ingesting terpenes and flavor compounds can influence the cannabis experience. However, there is anecdotal evidence to demonstrate that extract type does impact the effect of an edible. THC, whether in a full spectrum or distillate extract, is still THC. However, the hundreds of other compounds that can be found in full spectrum, live resin, or FECO are not found in distillate.

Science has yet to prove any theories about the effects of extract type in an edible, but it is hard to ignore the fact that different extraction methods produce different chemical profiles. The chemical makeup of distillate is vastly different from the chemical makeup of a live resin, full spectrum, or FECO. So, it is easy to theorize that a different chemical profile will result in a different experience. These differences seem to present themselves in the duration of the high and the complexity of the high. While it is hard to articulate how someone feels when they are “high,” anecdotal evidence suggests that distillate-based edibles are more cerebral and affect the mind. Whereas full spectrum, live resin, or FECO-infused edibles can be felt in the body and some have a distinct energizing or sedative effect depending on the product. These richer, more complex experiences can leave consumers feeling more satisfied with the product. This is why markets are beginning to see consumers shift their purchasing from distillate-based edibles to edibles infused with other extract types (1). Consumers are associating distillate-based edibles as potentially lower quality products than other infusions and it is causing a shift.

Consumers drive the market. Many consumers are still searching for the most cost-effective products on the market. However, market trends are shifting and demonstrating that consumers are willing to pay more for “higher quality” products. Manufacturers are left with making decisions on how to formulate edibles to meet their business model. Manufacturers must balance cost, ease of formulation, and consumer experience to understand which path to take. Time will tell whether consumers have a preference, but for now full spectrum and live resin edibles are taking the market by storm.

https://www.headset.io/the-best-selling-cannabis-products/oregon-edibles

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 CO2 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.

Extraction Science

A review of the claims that differences in extract type can affect cost, ease of formulation, and the experience of the finished product.

Does Extract Type Make  a Difference in Cannabis Edibles?

In this column, we attempt to explore in depth the theory and methodology of our common laboratory techniques and show how an understanding of interworking of those techniques allow for more flexibility in analysis. The knowledge of the theories behind the scenes can be used to manipulate the method variables for a more accurate and precise result. Up until this point, we have been mainly focused on the techniques commonly used in molecular spectroscopy and chromatography, which all have organic molecules as their target analytes. In the next few columns, we will look at another critical aspect of analytical chemistry—atomic spectroscopy and elemental analysis.

In the previous columns we have examined spectroscopy as a tool of organic chemical analysis. 

is the study or measurement of the interaction of matter and electromagnet radiation resulting in spectra (wavelength or frequency of the radiation). In organic analysis, spectroscopy examines the interaction of energy and molecules, but in inorganic chemistry we focus on atomic spectroscopy or the interaction of atoms and energy. The designation of the type of spectroscopy is categorized by the area of the electromagnetic spectrum targeted (for example, infrared, ultraviolet [UV], or X-ray, and so forth), the atomization source and interaction (such as emission, adsorption, transmittance, and so on), or the type of spectroscopy being employed (optical or mass spectrometry).

Electromagnetic (EM) radiation spectrum encompasses a wide band of energy including radio waves, microwaves, infrared, visible light, ultraviolet, X-rays, and gamma rays (see 

Many laboratory analytical techniques are focused on the range of waves associated with light from infrared to the X-ray range of wavelengths.

There are numerous interactions between EM and matter. These include some of the most commonly used interactions in instrumentation including absorption, transmission or refraction, reflection, and emission. All of these processes become the basis for some form of detection or spectroscopy measurement technique (see 

Atomic Spectroscopy Properties and Methods

Spectroscopy techniques are divided into atomic and molecular spectroscopy depending on the target to be measured and the material being evaluated.Spectrometry instruments are often further divided and defined by the type of interactions they produce and the type of energy that is measured like 

 (XRF), which measures X-rays and the amount of fluorescent energy a material emits during exposure. Some techniques focus on a narrow band of interactions (such as emission only) or a small part of the EM spectrum (like X-rays) while other techniques monitor an array of interactions and wavelengths on the spectrum (see 

In atomic spectroscopy, changes in energy are most commonly measured by the movement of electrons in atomic orbitals when exposed to an energy source such as light. The energy movement of electrons in orbitals use or produce measurable changes in energy usually as photons (

The characteristic movement of electrons when exposed to particular types or wavelengths of energy produces signature energy fingerprints called 

. Each element of the periodic table has a unique spectrum based on the number of electrons where changes in wavelength and intensity can be calculated as the number of atoms of a particular element in a sample (

 (AAS), light of a set wavelength is passed through a sample of atoms and if that wavelength corresponds to the difference in energy as their electrons move to higher energy states, the light (or a portion of the light) is absorbed, and that difference is calculated as an absorbance value.

The basic components of an atomic spectroscopy system include an energy source, an atomizer or nebulizer to distribute and reduce the sample to its corresponding atoms, a focus or monochromator, and finally a detector (

The energy for most of the atomic absorption instrumentation comes from a variety of lamps that produce metal atoms similar to the sources found in other molecular spectroscopy techniques. Sources for absorption spectroscopy are divided into two groups: continuum and line sources.

Continuous sources provide a wide range of wavelengths of energy and do not require very high sensitivity monochromators to differentiate the wavelengths. These sources include some of the common lamps we discussed when examining molecular spectrometers in previous columns (for example, tungsten, mercury, xenon, and deuterium lamps). The most common use for these types of sources are molecular absorption spectrometers or fluorometers. In atomic absorption, these lamps can be used for background correction.

Line sources produce specific portions or bands of a spectrum and can have large gaps between spectral lines because of the fact that they only contain a few wavelengths. Line sources do require high resolution monochromators and separate lamps for each range of wavelengths required for analysis. Most atomic absorption spectroscopy instruments use some type of line source either as a hollow cathode lamp, electrodeless discharge lamp, sodium, or mercury vapor lamps.

One of the most commonly used lamps is the hollow cathode lamp, which contains a tungsten anode and a hollow cylindrical cathode made with an element selected for its spectrum. The cathode tube is a sealed glass vessel filled with an inert gas (argon or neon). A high voltage is applied to ionize the gas and as a current flows between the anode and cathode the metal atoms are liberated from the cathode and collide with the gas and are excited producing characteristic radiation. The cathode of the lamp is created using the metals targeted for analysis. Some multielement lamps are created by the fusion of several target metals together producing the ability to measure more than one element at the expense of overall sensitivity for any one element. These multielement lamps can also be a source of spectral interferences in the analysis. Modern atomic absorption spectrometers can contain anywhere from 2 to more than 12 lamps in a system.

In order for the sample to be analyzed, the material must be broken down into atoms before exposure to the energy sources. The first step of many liquid matrix samples is nebulization or the process of creating fine droplet spray from a liquid sample. Nebulizers and the supporting pumps pull samples into the system at a controlled rate and create a fine spray that is then injected into the atomizer along with some type of fuel and oxidizer.

The atomizers that are most commonly found in use are either flame or electrothermal atomizers. The oldest technology still in common use is the flame atomizer. Flames are produced by a mix of air or nitrous oxide with acetylene. The mix of air-acetylene has a temperature of about 2300 °C while the nitrous oxide mix can be up to 2700 °C. Once liquid sample (via a nebulizer) enters the flame it undergoes drying then vaporization to transfer the material to the gas phase. These systems are straightforward and economical, but can be subject to increased levels of interference during the introduction and atomization phases and possible ionization of the sample leading to reduced sensitivity because of ground state depletion. This technique generally has sensitivity in the parts per million range with some elements having lower detection.

In electrothermal atomizers a graphite tube about 20 mm long and 5 mm diameter becomes a heated chamber via an electrical current allowing for precise control of the temperature of the furnace or chamber. The samples (either solid, liquid, or gas) enter the tube and are subjected to a temperature program that dries then pyrolizes the sample before atomization transitions samples to the gas phase. This technique is most commonly referred to as 

graphite furnace atomic absorption spectroscopy

 (GFAAS). It has the most flexibility of the atomic absorption techniques and can analyze many different forms of sample and provides part per billion (ppb) and sub-ppb analytical ranges. GFAAS is also known to have less interferences than traditional flame AA, which has made it a popular technique for atomic absorption.

Atomic absorption techniques are limited to a small number of elements in the periodic table (usually about 70). Both techniques require separate lamps for each targeted element. Flame absorption systems require flammable gas mixtures and rely upon the skill of the operator and cleanliness of the samples and system to avoid interferences and ionization. GFAAS has a higher degree of sensitivity but is still limited by the number of elements that can be analyzed. The GFAAS systems do have a wider range of functionality as to sample type and matrix being able to analyze liquids, gases, and solids.

 (AES), a high intensity source of light energy such as a spark, arc, flame, or plasma is produced to excite atoms. The sample pathway is similar to atomic absorption where the sample as a gas or a solution is aerosolized in a nebulizer before being vaporized and atomized. The atoms are then excited to a higher energy state by the source before moving back to lower energy and ground states. The movement of the electrons from excited to ground states emits photon energy (such as, light) and is a reason AES is also known as 

 (OES). The emissions from the element are focused by mirrors and slits to a detector (

The nebulizer or atomizer is the basis of the sample introduction system. Nebulizers by definition create a mist of particles from liquids. There are three basic groups of nebulizers: ultrasonic, electrothermal, and pneumatic nebulizers based on their primary mode of nebulization. Ultrasonic nebulizers, as those found in many medical settings, force liquids onto a surface of vibrating surface. Electrothermal nebulizers use a closed chamber evaporator with a method of heating and cooling to produce nebulized particles. There are spectroscopy systems that do use electrothermal nebulizers. But the most common type of analytical nebulizers are pneumatic nebulizers that use induction to atomize liquids. When a gas of a higher-pressure flows into a lower-pressure gas it creates a jet and pulls in the lower-pressure aerosol. The liquid is pumped into the system and the high-pressure gas draws in and disperses the particles into the jet.

There are many types of modern nebulizer designs that fit into several categories including: concentric, cross flow, Babington or V-Groove, porous disc, parallel path, flow blurring, and vibrating mesh. The first two (concentric and cross flow) are the most used configuration of nebulizer for AES and OES.

In the concentric nebulizer the gas surrounds the sample introduction or vice versa so that concentric rings of sample and gas are formed producing a plume at the end of nebulized material (

). Cross flow nebulizers have a configuration where the gas and the liquid flow are at right angles to each other usually meeting at the tip of the nebulizer to come together to form the plume in a spray chamber (

). Cross flow nebulizers allow for more sample to be introduced to the system, but can be prone to clogging.

The next stop for the nebulized sample is the light source which excites the atoms and causes the emission of photons. Again, there are many different types of sources for AES including arc, spark, flame, and plasma.

The earliest types of AES systems used arcs, sparks, and flames, as the source of excitation. Sparks are short bursts of excitation energy generated by an electrode usually composed of graphite. Arcs are sustained AC or DC currents of energy between two electrodes with frequency similar to sparks. DC arcs are considered low-current discharges of about up to 15 A, while AC arcs are higher between 15 to 30 A. The current and the temperature of the arc discharges are controlled by adjusting the applied potential.

These techniques are used in the analysis of metallic solid samples. Materials that are nonconductive are mixed with a conductive matrix such as graphite to facilitate analysis. The sample is heated to a high temperature by the passage of the spark or arc through the sample. The excited atoms emit characteristic wavelength light that is filtered by the monochromator for the detector. Older arc and spark technologies were almost always qualitative or at best semi-quantitative, but newer advances have increased the sensitivity and accuracy of both types of systems. Despite the advances, these systems are not for precise and fine analytical work with low levels of detection.

 (FES). FES uses the energy from the flame to excite species of certain metals like Group I alkali metals and Group II alkaline metals. The high temperatures of the flame can cause some instability in some elements.

This type of instrumentation uses a burner and a flammable gas or liquid to produce a flame. A sample is mixed with an oxidant and nebulized in a mixing chamber with the fuel gas. The flame then vaporizes and atomizes the sample. The energy of the change of states as the high temperature changes the energy levels is focused by lenses and filtered by a monochromator before reaching the detector (

There are different types of burners and mixtures of gas that are used in these applications. Gases are chosen regarding temperature and the nature of the target elements; more energy and higher temperatures are needed to excite larger numbers of atoms to higher states. Some metals also require higher temperatures to vaporize. The most common mix of gases for the burner contain a mix of air, oxygen, hydrogen, nitrous oxide, or acetylene depending on the target elements and temperatures that need to be achieved to vaporize those elements without oxidation or instability (

The most widely used types of atomic emission spectrometers use a plasma source to vaporize and atomize the samples. A plasma is a homogenous mixture a fine mist or fog of ionized gases composed of electrons, ions, and neutral species. The most common gas for spectroscopy plasma is argon. The method in which the plasma is depends on the source of the electric field that produces it. The mostly widely used types of plasma are either by 

(ICP). Other types of plasma with less applications in the analytical world are laser-induced plasma, laser breakdown, and microwave-induced plasma.

In DCP, ionization occurs in a discharge tube with between two and three electrodes (often graphite anode blocks with a tungsten cathode block). Argon gas flows around the anode blocks and the plasma is produced by the contact of the cathode with the anodes. The temperature at the core can be more than 8000 K.

For ICP, there are three concentric silica quartz tubes (called the torch) in which argon gas moves the sample in its aerosol through an applied Rf power supply. The plasma is ignited by a spark from a Tesla coil. The hottest area of the plasma torch is at the base around 10,000 K while the further away from the tip of the torch the temperature drops to 6000 K (

Plasma sources for atomic emission spectroscopy offer a much broader range of applications than other types of both atomic emission and atomic absorption spectrometry because of their sensitivity and their ability to produce spectra for a wide range of the periodic table of elements. Detection limits can be dropped even further with the coupling of mass spectrometers to ICP systems thereby allowing for sub-ppb levels of routine detection and expanding the analytical reach to ultra-trace level detection. In upcoming columns, we will look deeper into applications and the configurations of ICP-OES and ICP-MS systems and how they have become indispensable tools for the cannabis laboratory.

 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.

Navigating the Lab

In this column, we attempt to explore in depth the theory and methodology of our common laboratory techniques and show how an understanding of interworking of those techniques allow for more flexibility in analysis.


Energy and Elements, Part I: Understanding Atomic Spectroscopy

In pursuit of a more detailed understanding of the naturally rare occurring cannabinoid analogue, hexahydrocannabinol (HHC), safety becomes a question when possible human consumption is introduced. We explore the safety profile of HHC, using a third-party lab to produce preclinical in vitro safety profile data on the cyto-toxicity of cardiomyocytes through the human ether-à-go-go-related gene (or the human ether related gene) (hERG), hepatocytes, and lung fibroblasts. hERG (1) is a gene that codes for a protein known as Kv11.1, the alpha subunit of a potassium ion channel. This ion channel (simply denoted as “hERG”) is best known for its contribution to the electrical activity of the heart: the hERG channel mediates the repolarizing current in the cardiac action potential, which helps coordinate the heart’s beating. Preceding studies of HHC, neglect the base scaffold structure of HHC (2), and test on synthetic derivatives, and various analogs. The purpose of these studies is to demonstrate HHC as a safe cannabinoid for potential human consumption. The preclinical assessments of HHC did not indicate any cardiac safety issues via data from a hERG (3) fast patch assay. HHC also demonstrated no cytotoxicity in the plated human liver hepatocyte cell viability assay. HHC, according to in vitro data produced potential cytotoxic effects in human lung fibroblasts when exceeding 10 mm concentrations, when reviewing the data, safe possible human consumption is feasible without complications.

The use of cannabinoids dates to ancient China around 2500 BC (4). These substances, which are found in nature, have been used for recreational and medicinal purposes: to alleviate pain (5), gastrointestinal (GI) issues (6), multiple sclerosis (7), anxiety (8), cancer (9), and more. The explosion of cannabis usage around the world caused an increasing number of studies to be conducted to determine the therapeutic benefits of these compounds, in the fight against some of the most debilitating and chronic diseases. 

. produces more than 120 minor cannabinoids (10).

Current and relevant studies of hexahydrocannabinol (HHC) (11) involve substituted groups such as alcohols and carbonyls, which affect the target and binding affinities and can pose risks that are not widely studied, while we focus purely on HHC which has a methyl at the C9 position rather than other functional groups. Since this cannabinoid (HHC) is found naturally in trace amounts (12,13), it can be extracted from hemp and cannabis via the conventional isolation methods, but this is an inefficient and costly process. Our group has developed an efficient and robust way to synthesize and isolate (R/S)-HHC starting from D8 and D9-tetrahydrocannabinol

(THC) while also focusing on the safety study of this rare cannabinoid, including but not limited to cell viability and cytotoxicity studies.

 studies were performed for (R/S)- HHC. The studies were conducted at Charles River Laboratories (cell viability); Cleveland, OH (hERG); and Skokie, IL (Ames). All studies were exploratory and carried out under a non-good laboratory practice (GLP) environment. All procedures carried out in the laboratory were reviewed and approved by the institutional authorities.

The Ames test is one of the most frequently applied tests in toxicology. Almost all new pharmaceutical substances and chemicals used in industry are tested by this assay. The 

 reverse mutation assay is a bacterial short-term test for identification of carcinogens using mutagenicity in bacteria as an endpoint. The Ames test detects mutations in a gene of a histidine-requiring a bacterial strain that produces a histidine-independent strain. Both direct and indirect (for example, chemicals that require metabolic activation) mutagens can be identified using the Ames test.

Evaluation of hERG Current Using Patch Clamp Analysis

One of the most frequent adverse side effects, leading to the failure of drugs, is cardiac arrhythmias. Such failure is mostly related to the capacity of the drug to inhibit the hERG cardiac potassium channel. Inhibition of the hERG current causes QT interval prolongation resulting in potentially fatal ventricular tachyarrhythmia called Torsade de Pointes (note: the QT interval is the section on the electrocardiogram [ECG] that represents the time it takes for the electrical system to fire an impulse through the ventricles and then recharge). To evaluate anticipated cardiovascular effects, early evaluation of hERG toxicity has been strongly recommended, for instance by the regulatory agencies such as the US Food and Drug Administration (FDA) and European Medicines Agency (EMA).

Cell Viability Assay Using Human Lung Fibroblasts

The cell viability assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. The CellTiter-Glo Luminescent Cell Viability Assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the adenosine triphosphate (ATP) present, an indicator of metabolically active cells.

Cell Viability Assay Using Plated Human Hepatocytes

Similar to the lung fibroblasts, the highly sensitive ATP CellTiter-Glo Luminescent Cell Viability Assay was used to determine the potential of 

 liver toxicity of (R/S)-HHC. The test compound ([R/S]-HHC) and the control (Terfenadine) were tested at a concentration range of 0.05 to 50.0 µM for cell viability (cytotoxicity) in human hepatocytes.

A 5 L reactor equipped with a reflux condenser and an addition funnel was purged with argon for 15 min. Pd/C (10% by mass) was added to the reaction slowly using a powder funnel under argon. The reactor was then purged with argon for 15 min. Ethanol (200 mL) was added slowly to avoid sparking the solvent. A mixture of D8- and D9-THC (100 g, 318 mmol) was dissolved in ethanol (100 mL). The solution was added to the reactor under argon and purged for 15 min. Afterwards, the atmosphere of argon was stopped, and an atmosphere of hydrogen (1 bar) was introduced. The reaction was then stirred at 25 °C until completion by high performance liquid chromatography (HPLC). Upon completion, the reaction was purged with argon for 20 min. The reaction mixture was filtered over 1–3-µm filter paper on a Buchner funnel and then the solution was concentrated 

. The crude oil is then dissolved in hexane and purified over silica (0–5% EtOAc). The fractions of interest were concentrated 

 and then distilled to afford a colorless to light yellow oil with three compounds of similar m/z ratios. HPLC (C18): 7.940 min, 8.141 min, 8.395 min LRMS: [M+1]

CN) δ 0.89, 0.93, 1.18, 1.30, 1.43, 1.53, 1.62, 2.06, 2.38, 2.42, 2.60, 3.06, 6.08, 6.68.

The final product was isolated and 99.9 % (R/S)-HHC is shown in 

. In addition, 1H nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC–MS) analysis were preformed to determine the structure of (R/S)-HHC, as illustrated in 

Potential of (R/S)-HHC to induce reversion mutations in 

 strains TA98, TA100, TA1535, and TA1537 and 

 strain WP2 uvrA both with and without metabolic activation was evaluated. The plate incorporation screen for (R/S)-HHC was tested in duplicate with strains TA98, TA100, TA1535, TA1537, and WP2 uvrA in the presence and absence of a metabolic activation system (phenobarbital/5,6-benzoflavone-induced rat liver S9 microsomal fraction) at 1, 5, 10, 50, 100, 500, 1000, and 5000 µg/plate. The positive controls without metabolic activation were 2-nitrofluorene (TA98, 2.5 µg/plate), sodium azide (TA100 and TA1535, 1 µg/plate), ICR-191 Acridine (TA1537, 0.5 µg/plate), and 4 nitroquinoline N oxide (WP2 uvrA, 2.0 µg/plate). The positive control with metabolic activation for all strains was 2-aminoanthracene (2.5 µg/plate for 

strain). Dimethyl sulfoxide (DMSO) was used as the vehicle control.

(R/S)-Hexahydrocannabinol was not mutagenic, up to concentrations at ≥ 100 µg/plate in strain TA1535 without metabolic activation, and at ≥ 500 µg/plate in strains TA98, TA100, TA1537, and WP2 uvrA both with and without metabolic activation and in strain TA1535 with metabolic activation. The data from the Ames test suggests that (R/S)-HHC is a non-mutagenic compound.

The effect of (R/S)-HHC on cloned hERG potassium channels (encoded by the KCNH2 gene and expressed in HEK293 cells) was examined using the QPatch II (Sophion Bioscience A/S), an automatic parallel patch clamp system. (R/S)-HHC was exposed to hERG at 6.25, 12.5, 25 and 50 µM (n ≥ 3). The duration of exposure to each test article concentration was a minimum of 3 min. The positive control data confirmed the sensitivity of the test systems to ion channel inhibition.

A summary of the results for (R/S)-HHC and the positive controls are shown in 

. The data suggests that (R/S)-HHC does not block HERG-encoded channels expressed in HEK293 cells.

(R/S)-HHC and the control (chlorpromazine) were tested at a nominal concentration range of 0.156 to 50.0 µM for cell viability (cytotoxicity) in human lung fibroblasts. In lung fibroblasts, the IC50 for (R/S)-HHC was 14.4 µM, and the percent cytotoxicity at 50 µM was 74.8%. The control, chlorpromazine, showed an IC50 value of 14.3 µM and 86.4% cytotoxicity at 50 µM. The data is summarized in 

In human hepatocytes, (R/S)-HHC was non-cytotoxic and the IC

 for (R/S)-HHC was not measured, and the percent cytotoxicity at 50 µM was also very low (8.9%) because of its weak response. Terfenadine showed an IC

 value of 15.8 µM and 99.9% cytotoxicity at 50 µM. The data is summarized in Table III. The results indicated that there is no potential for hepatotoxicity, with very weak results in this plated human hepatocyte cell viability assay.

(R/S)-HHC derived from Colorado Chromatography Labs, using their patent pending technology demonstrated the following results: (R/S)-HHC was not mutagenic in 

 genotoxic assay (Ames test). Preclinical assessments in the hERG patch clamp study did not indicate that (R/S)-HHC had any cardiac safety issues, which suggests that there does not appear to be an effect of (R/S)-HHC and QTc prolongation. The finding in the cell viability studies demonstrated potential cytotoxic effects of (R/S)-HHC in human lung fibroblasts at > 10 µM concentrations. (R/S)-HHC was not cytotoxic or damaging to human liver cells in 

The future of this compound can be shifted since there was some slight toxicity to the lung fibroblasts, with directed remediation of molar concentrations as well as possible derivatizing of the future HHC compounds either the virgin HHC compound or derivatized compound could be used as a possible anti-tumor drug and treat certain lung cancers. With this initial cell viability and safety study we are providing evidence that although this compound was made in a laboratory setting, to increase the production of this rare cannabinoid that is difficult to extract, this compound is safe to consume, and can be opened in the world of research to see what effects and ailments that HHC can treat like other cannabinoids.

K.P.R. and W.C. gratefully acknowledge NMR spectroscopy support from Dr. Jin Hong at Custom NMR Services, Inc. from Woburn, MA and mass spectrometry support from Dr. Jasper van Heemst at KCA Laboratories in Nicholasville, KY.

A. Garrido, A. Lepailleur, S.M. Mignani, P. Dallemagne, and C. Rochais, 

https://doi.org/10.1016/j.ejmech.2020.112290

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https://doi.org/10.1152/physrev.00036.2019

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R. Abalo, G. Vera, A.E. López-Pérez, M. Martínez-Villaluenga, and M.I. Martín-Fontelles, 

The Gastrointestinal Pharmacology of Cannabinoids: Focus on Motility Pharmacology

M. Iskedjian, B. Bereza, A. Gordon, C. Piwko, and T.R. Einarson, 

F.S. Guimaraes, T.M. Chiaretti, F.G. Graeff, and A.W. Zuardi, 

L. De Petrocellis, D. Melck, A. Palmisano, T. Bisogno, C. Laezza, M. Bifulco, M., et al. “The Endogenous Cannabinoid Anandamide Inhibits Human Breast Cancer Cell Proliferation.” 

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K.B. Walsh, A.E. McKinney, and A.E. Holmes, 

https://doi.org/10.3389/fphar.2021.777804

S.P. Nikas, S.O. Alapafuja, I. Papanastasiou, C.A. Paronis, V.G. Shukla, D.P. Papahatjis, A.L. Bowman, A. Halikhedkar, X. Han, and A. Makriyannis, 

J. Basas-Jaumandreu, and F.X.C. de las Heras, 

R. Adams, Marihuana Active Compounds, US Patent No 2,419,937, May 6, 1947.

 are with Colorado Chromatography Labs in Parker, Colorado. Direct correspondence to: 

A. Collins, T. Tesfastion, G. Ramirez, K. Ray, and W. Cruces, 

Peer-Reviewed Article

In the work presented here, the authors pursue a more detailed understanding of the naturally rare occurring cannabinoid analogue, hexahydrocannabinol (HHC), by exploring the safety profile using a third-party lab to produce preclinical in vitro safety profile data. The purpose of these studies is to demonstrate HHC as a safe cannabinoid for potential human consumption.

Nonclinical In Vitro Safety Assessment Summary of Hemp Derived (R/S)-Hexahydrocannabinol ((R/S)-HHC)

In legal cannabis markets, two molecules dominate the conversation for both adult use and medical use cannabis: delta-9-tetrahydrocannabinol (Δ

-THC is the dominant cannabinoid found in most adult-use cannabis. Many 

 cultivars have been selectively bred for it, and CBD is the most commonly found cannabinoid in the supplemental market. Both molecules have had successful drug formulations developed for specific medical conditions such as epilepsy (Epidolex; CBD) and reduce nausea from medical conditions or drugs (Dronabinol; Δ

-THC). While these drugs are an important step toward legitimizing cannabinoid-based medicines, they reduce the significance of the other 400 chemical entities within the plant, such as terpenes and other phytocannabinoids. More importantly, the synergistic properties of the molecules working together don’t exist in our current adult-use and medical products. These molecules working together may have led to many of the anecdotal medical successes attributed to 

. It is a major shift away from the traditional single-molecule compounds in the pharmaceutical industry. While we do not know the effects of every molecule found within the plant, we have research that shows many phytocannabinoids beyond Δ

-THC and CBD have medical benefits. These molecules include the precursor to Δ

-THC, tetrahydrocannabinolic acid (THCA), and a long list of lesser-known cannabinoids such as cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), and more.

-THC and CBD will allow us to leverage the compounds that 

 contains fully. This is important for furthering the pharmaceutical and herbaceutical medicines brought to market or commoditized. When using most cannabinoid-based drugs, there is a significant side effect: inebriation or being “high.” While individuals who use cannabis frequently don’t mind or enjoy the psychoactive effects of cannabis, it is a side effect that many people don’t enjoy. As with all modern medicines, some side effects are to be expected, but the psychoactive effects of cannabis specifically come from Δ

-THC. Suppose we can develop cannabinoid medicines that reduce or potentially eliminate the psychoactive effects. In that case, we can engage medical professionals, legislatures, and individuals skeptical of the drugs because of this particular side effect. We may do this without losing the therapeutic efficacy of current formulations by exploring other phytocannabinoids present in cannabis.

THCA is the most abundant phytocannabinoid found in the resin glands of 

. THCA is typically extracted from the plant and converted to Δ

-THC for its psychoactive properties by breaking the carboxyl group off of the molecule through decarboxylation. THCA has potential for medical value, and the research suggests that we should consider it for product development. An article published in the National Library of Medicine showed that THCA dosed at 0.05/0.5 mg/kg may be more effective at reducing lithium chloride (LiCl) induced vomiting compared to Δ

-THC, which did not suppress nausea at the same dosage 

. THCA’s potential doesn’t stop with nausea. According to a scientific journal published in 2012, THCA, Δ

-THC, and CBD were given to mice to study the molecules’ protective properties against MPP+, a neurotoxin that leads to cell death and Parkinson’s disease 

. The study concludes that while no neurite outgrowth (potential nerve restoration) was observed from the cannabinoids, a high concentration increased overall cell counts by 123% from THCA, and THCA protects against MPP+ induced cell death. THCA also has other neuroprotective properties, as discovered by an article published in the 

. This work showed that THCA in mice treated with 3-NPA, a mycotoxin that is a mitochondrial inhibitor, increased mitochondrial mass in neuroblastoma cells, improving motor deficits and preventing striatal degeneration (

The cannabinoid called CBN has gained much traction in the commoditized market of both adult-use cannabis and nutraceuticals. In fact, 5-10 mg of CBN can be found in many products marketed as “sleep” or “rest” aides, and unsurprisingly a study published in November 2021 (

), seems to support this. While CBN isn’t the next Ambien, the study performed by the University of Western Australia’s Center for Sleep Science shows that a formulation containing THC, CBN, and CBD at a ratio of 20:2:1 showed an improvement in the 24 participants with insomnia in a sleep journal. The participants specifically noted that it took less time to fall asleep, they slept longer, reduced nighttime wakefulness, and ultimately felt more rested. In addition to their sleep journals, the participants partook in polysomnography measurements (an analysis of brain waves, heart rate, and REM sleep) that did not conclude any difference between the placebo group and the group that took the cannabinoid drug. The potential for CBN doesn’t stop at sleep; there is a potential for CBN to be used in topical applications because of its inhibiting keratinocyte proliferation properties and agnostic effects on the transient receptor potential vanilloid type 2 (TRPV2) cation channels, which are high threshold thermosensors. As published in 

), these properties of CBN show, there is a potential for the treatment of psoriasis and burn wounds.

CBC, a nonpsychotropic cannabinoid, has different observed effects that need further research. Like CBN, “CBC interacts with transient receptor potential cation channels that inhibit endocannabinoid inactivation, and stimulate the CB

). The specific channels that CBC interacts with have a role in inflammation and pain within mammals, specifically nociceptor firing. This significant interaction with the different transient receptor potential (TRP) channels can cause strong anti-inflammatory effects, which are supported by a study published in 2011 (

) where CBC stimulated pathways of antinociception and caused analgesia in mice. What is even more fascinating is that much of the research also shows an additive result on the nociception effects when THC is co-administered with CBC. To further demonstrate the synergistic effects of the cannabinoids, we could look at an article published in 2011 which provided a pharmacological evaluation of CBC’s impact on pain and inflammation. The research showed that CBC significantly reduced lipopolysaccharide (LPS)-induced paw edema in rat models and produced a subset of effects in the mouse tetrad array (series of behavioral paradigms in rodents treated with cannabinoids). What is fascinating is that the tetrad effects were not CB

-THC was given alongside CBC, it showed anti-inflammatory effects in the LPS-induced paw edema model because of CB

 receptor activation. In addition to this additive relationship surrounding the anti-inflammatory properties of CBC and Δ

-THC, there was also an observation that higher doses of CBC led to increased psychoactive effects of Δ

-THC, suggesting a potential pharmacokinetic interaction when the two molecules are combined (

The list of cannabinoids with potential medical value doesn’t stop here, as relevant research has been performed on other cannabinoids. CBG, which is the decarboxylated form of cannabigerolic acid (CBGA), the “parent molecule” for all other cannabinoids, has promising new research regarding the therapeutic potential in treating neurological disorders. In 2022, diseases such as Huntington’s disease, Parkinson’s disease, multiple sclerosis (MS), and inflammatory bowel disease (IBD), could be reduced in severity based on the in vitro and animal model studies when intaking CBG (

). Cannabitriol (CBT), a cannabinoid that shares a very similar chemical structure to Δ

-THC, has the potential to reduce the psychoactive effects of Δ

-Tetrahydrocannabivarin (THCV) is another interesting cannabinoid because it does not have the psychoactive properties of Δ

-THC but does have a measurable effect on appetite stimulation and weight gain—further research must be done on this molecule, however, because it can exhibit either agnostic or antagonistic effects on the CB

). Cannabinoids such as cannabielsoin (CBE), cannabinodivarin (CBV), and cannabiripsol (CBR) have been identified, but no research has yet to be published. Today more cannabinoids are being identified and quantified than ever before. We know of 120+ different cannabinoids, most of which we have done zero dedicated research on.

 is among a remarkable variety of plants that may be able to provide us with a broad range of medical developments ranging from a sleep aid to pain management. While we are currently using phytocannabinoids in a way that is very similar to traditional pharmaceuticals—one molecule targeting one ailment or symptom—the future of cannabinoid drugs must explore all of what the plant has to offer. Δ

-THC and CBD have paved a pathway for research—truly valuable research—to become more mainstream in the scientific community. These two molecules have also brought mainstream media attention to the cannabinoid medicines with products specifically designed to treat adolescent epilepsy. But to move cannabinoid-based medicines forward, we must look beyond the obvious most dominant cannabinoids and into the relatively uncommon ones. To provide efficacious medicines with minimal side effects, we need to be exploring the synergistic effects of primary and minor cannabinoids. The cannabis research should not stop here; we must also learn how the different terpene ratios interact with the various cannabinoids.

E.M. Rock, R.L. Kopstick, C.L. Limebeer, and L.A. Parker,

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792001/

R. Moldzio, T. Pacher, C. Krewenka, B. Kranner, J. Novak, J.C. Duvigneau, and W.D. Rausch, 

https://doi.org/10.1016/j.phymed.2012.04.002

https://pubmed.ncbi.nlm.nih.gov/22571976/

X. Nadal, C. Del Río, S. Casano, B. Palomares, C. Ferreiro-Vera, C. Navarrete, C. Sánchez-Carnerero, I. Cantarero, M.L. Bellido, S. Meyer, G. Morello, G. Appendino, and E. Muñoz, 

https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.14019

J.H. Walsh, K.J. Maddison, T. Rankin, K. Murray, N. McArdle, M.J. Ree, D.R. Hillman, and P.R. Eastwood, 

https://pubmed.ncbi.nlm.nih.gov/34115851/

This article examines why we should be studying the cannabinoids beyond THC and CBD in order to explore their potential medical value.

September 2022

Quantitative Spectroscopy Pitfalls and Practicalities, Part III: Calibration Validation and Calibration Checks

Does Extract Type Make a Difference in Cannabis Edibles?

Energy and Elements, Part I: Understanding Atomic Spectroscopy

Nonclinical In Vitro Safety Assessment Summary of Hemp Derived (R/S)-Hexahydrocannabinol ((R/S)-HHC)

The Case for THCA and Other Minor Cannabinoids

Reading the Leaves: Adventures in the Leaf-Level Physiology of Cannabis