The simple and rapid determination of cannabinoid concentrations in cannabis extract

North America has seen a notable increase in the production of cannabis extracts and oils for recreational and medicinal products. This increased production has been driven by patient demand for greater diversity in cannabis products and market demand as states legalize cannabis use.1,2,3

Regardless of the instrument or solvent used, most cannabis extraction processes undergo a decarboxylation step.

This process sees the carboxylic acid functional group removed from the cannabinoids, converting naturally occurring acid forms of the cannabinoids - for example, tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) - to their neutral, more potent forms - tetrahydrocannabinol (THC) and cannabidiol (CBD).

The carboxylic acid group is thermally labile, prompting the application of a heat source to decarboxylate the cannabinoids. A catalyst is also employed in some circumstances.

There has been a great deal of discussion in the industry around this heat-promoted decarboxylation reaction, but an extensive literature search reveals relatively few papers on the process.4,5,6

Available data incorporates a wide range of reaction conditions, for example, diverse reaction temperatures, reaction times and instrumental setups. This has led to a notable lack of universal agreement around the ideal reaction conditions for the decarboxylation process in cannabis extract.

The decarboxylation reaction’s sensitivity to water adds additional challenges, with additional water content promoting the reaction.7 Studies have also highlighted that competing oxidation, isomerization and decomposition reactions may occur at higher temperatures.7

This combination of factors and challenges frequently result in inconsistent cannabis extract products and wide-ranging quality control issues in the laboratory.

It has historically been difficult for cannabis extraction manufacturing staff and quality control laboratory technicians to predict the reaction time and temperature required to achieve maximum decarboxylation.

Contemporary manufacturing practices routinely require the cannabis extract to be placed in a glass beaker on a stirred hot plate or on aluminum sheet trays in a vacuum oven.

The necessary conditions for these techniques to result in a complete reaction are generally undefined and these heating processes rarely monitor decarboxylation.

In cases where decarboxylation is monitored, extractors must rely on physical observations, for example, reduced carbon dioxide off-gassing.

This lack of chemical information during a key processing step results in extremely subjective determination of the completeness of the reaction, in turn resulting in less efficient extraction processes that are not scientifically robust.

A combination of extended reaction times and ambiguous optimum temperature can lead to a lack of overall process control and inefficient use of vital laboratory resources.

This article explores the use of Fourier transform infrared spectroscopy (FT-IR) with Attenuated Total Reflectance (ATR) in providing an appropriately accurate quantitative estimation of the decarboxylation reaction progress in cannabis extract.

Experimental

A selection of cannabis plant matter was milled to a 2 mm particle size (Fritsch P19, Germany), with carbon dioxide supercritical extraction used to produce cannabis extracts in a 20 L, 2000 psi system (Apeks Supercritical®, USA).

Different cannabis oil amounts were tested, and the cannabis extracts contained various THC and THCA concentrations.

Table 1. Details for the different cannabis extracts tested by FT-IR-ATR. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Reaction Reaction Temperature (°C) Extract (g) Initial THC (%) Initial THCA (%)
A 150 100 19.1 29.1
B 140 100 59.0 20.9
C 140 370 18.7 21.8

 

Decarboxylation reaction

Decarboxylation was attained by using a hot oil bath with a programmable hot plate to heat the cannabis extract. Even heat distribution was promoted using an overhead stirrer.

Recordings were taken of oil bath and extract temperatures at 5-minute intervals throughout the 80-minute heating process.

Mid-infrared spectra were also acquired at 5-minute intervals. This was done by pipetting a small amount of heated extract onto the crystal of a Universal Attenuated Total Reflectance (UATR) accessory from PerkinElmer.

Aliquots at varying time points throughout the experiment were obtained, with high performance liquid chromatography (HPLC) used to determine cannabinoid concentration.

Spectral data collection

The PerkinElmer Spectrum Two™ equipped with a UATR accessory was used to acquire infrared spectra. These were collected over the 4000 – 450 cm-1 spectral range at 4 cm-1 resolution with each spectrum being the result of four averaged spectra.

The PerkinElmer Spectrum Quant™ software was used to develop quantitative chemometric models of sample spectra and corresponding reference HPLC cannabinoid concentrations.

Spectral pre-processing was performed to include the 3665 – 2775 cm-1 and 1755 – 450 cm-1 regions with absorbance threshold blanking at values greater than 1.5 absorbance units.

Regions with very little chemical significance were excluded from the model development.

Quantitative models for THCA and THC were generated using Principal Component Regression (PCR). During the calibration step, leave-one-out cross-validation was performed and a total of 29 calibration spectra were utilized in building models to predict concentrations of THC and THCA in the cannabis extract.

Example spectra of cannabis extract throughout the course of decarboxylation by the  application of heat.

Figure 1. Example spectra of cannabis extract throughout the course of decarboxylation by the  application of heat. Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions

Correlation plots showing the relationship between reference HPLC cannabinoid content and those predicted using the PCR models for THC (left) and THCA (right).

Figure 2. Correlation plots showing the relationship between reference HPLC cannabinoid content and those predicted using the PCR models for THC (left) and THCA (right). Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions

Results and discussion

Figure 1 displays infrared spectra of the in-process extracts. Changes observed in the infrared spectra illustrate the conversion of THCA to THC as the reaction progresses, as well as a loss of water.

PCR models were individually optimized for each specific cannabinoid – whether this was THCA or THC.

Table 2 shows regression details for the final regression models. Coefficient of determination (R2) values of 0.990 and 0.998 for THCA and THC, respectively, exhibit an exceptional correlation between reference HPLC cannabinoid concentrations and concentrations predicted using FT-IR.

Figure 2 shows correlation plots for calibration data points of both the THCA and THC models. Calibration data points are evenly distributed throughout the full calibration range for each individual cannabinoid.

Table 2. Regression summary for THC and THCA PCR models (where SEP is the standard error of prediction and CVSEP is the cross validation standard error of prediction). Source: PerkinElmer Cannabis & Hemp Testing Solutions

Property Value Average Property Value Number of PCs SEP CV-SEP R2
THC (%) 42.61
(75.69-15.9)
4 1.24 1.62 0.998
THCA (%) 16.08
(34.42-0.6)
5 1.48 1.73 0.990

 

Decarboxylation reaction monitoring

A total of three distinct decarboxylation reactions were performed using oil bath temperatures ranging from 140 – 150 ˚C.

Initially, THCA and THC concentrations fell within 20.9 – 29.0% and 18.7 – 59.0% ranges, respectively.

THCA is converted to THC as the decarboxylation reaction progresses.

Cannabinoid concentration plots over the course of decarboxylation for three separate decarboxylation reactions.

Cannabinoid concentration plots over the course of decarboxylation for three separate decarboxylation reactions.

Cannabinoid concentration plots over the course of decarboxylation for three separate decarboxylation reactions.

Figure 3. Cannabinoid concentration plots over the course of decarboxylation for three separate decarboxylation reactions. Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions

Figure 3 plots the cannabinoid concentrations throughout the reaction, highlighting this trend. Figure 3 also includes cannabinoid concentrations predicted by FT-IR-ATR overlaid with the HPLC reference values.

This data shows exceptional agreement between the two techniques, showcasing FT-IR-ATR’s potential for real-time, accurate monitoring of decarboxylation reactions.

Conclusion

The use of the PerkinElmer Spectrum Two with the UATR accessory facilitates the rapid, straightforward determination of cannabinoid concentrations in cannabis extract, with the FT-IR-ATR technique offering a robust real-time solution to the challenges associated with decarboxylation reaction monitoring.

The ability to monitor this reaction over time allows cannabis extract manufacturers to enhance and optimize extraction conditions, identifying and addressing process deviations as required.

The FT-IR-ATR technique benefits from small sample quantity requirements, no sample preparation requirements and minimal operator training requirements.

References

  1. Weed, Julie. “Cannabis Industry Delivers 100,000 + Jobs and Billions in Tax Revenue.” Forbes, 22 May 2018, https://www.forbes.com/sites/julieweed/ 2018/05/22/cannabis-industry-delivers-100000-jobs-and-billions-in-tax- revenue/#3d3068b72879.
  2. “State Marijuana Laws in 2018 Map.” Governing, http://www.governing.com/ gov-data/state-marijuana-laws-map-medical-recreational.html.
  3. Lindsey, Nick. “Data Shows The Demand for Legal Cannabis Is Increasing.” HighTimes. 13 April 2018, https://hightimes.com/news/data-shows-demand- legal-cannabis-increasing/.
  4. Ardent Cannabis. “What Is Decarboxylation, and Why Does Your Cannabis Need It?.” Leafly, 30 April 2018, https://www.leafly.com/news/cannabis-101/ what-is-decarboxylation.
  5. Cannabis Extracts. “Catalyst for THCa decarboxylation?” reddit. https://www.reddit.com/r/CannabisExtracts/comments/6ven87/catalyst_for_ thca_decarboxylation/.
  6. DOI: 10.1089/can.2016.0020 B) Journal of Chromatography 520 (1990) 339-347
  7. Roggen, Markus. “Latest Advances in Cannabis Production Processes.” https://www.slideshare.net/MarkusRoggen/presentations.

Acknowledgments

Produced from materials originally authored by Dr. Markus Roggen and Antonio M. Marelli from Outco; and Doug Townsend and Ariel Bohman from PerkinElmer.

About PerkinElmer Cannabis & Hemp Testing Solutions

With the cannabis and hemp markets continuing to grow rapidly and regulations strengthening, labs increasingly need streamlined access to best-in-class testing solutions geared toward the unique requirements of the industry. Whether your lab is well established or just starting up, PerkinElmer is a single-source vendor for instruments, methods, reagents, and consumables on hand to help enhance your testing capacity and get ahead of the competition.

They help drive analytical best practices and operating procedures and commit to ensuring your laboratory has maximum uptime. Learn about their various instruments, testing methods, and applications for cannabis analyses. Let them work with you to build an efficient workflow, so you can focus on growing your business.


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Last updated: Sep 24, 2021 at 10:52 AM

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