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Scientists Develop New Method to Simultaneously Measure Cannabinoids and Terpenes

Feb 05, 2021

Scientists Develop New Method to Simultaneously Measure Cannabinoids and Terpenes

Alexander Beadle
Science Writer

Researchers from the University of Ljubljana, Slovenia, have developed of a new method for analyzing cannabinoids and terpenes simultaneously.

Both compound classes are important to study when analyzing cannabis, and yet the two have very different physical properties that often require two distinct sample preparation and analysis procedures. While there has been at least one previously proposed methodology for the simultaneous analysis of the two, it required complex techniques that make it suitable for advanced studies and chemotaxonomy, but broadly unsuitable for routine use.

By experimenting with different sample preparation procedures and analytical instrument setups, the University of Ljubljana researchers have seemingly demonstrated a much simpler methodology for simultaneous analysis, using a gas chromatography-based technique.


Why do analysts need a new method?

From an analytical point of view, both the cannabinoids and the terpenes are major points of interest in cannabis analysis.

Cannabinoids have to be determined by law in many areas with legal cannabis ordinances, and generally provide a good picture of the potential physiological effects that the drug material might have on the body if ingested. Terpenes are responsible for cannabis’ unique smell and are often characteristic of a particular variety or strain of cannabis.

To improve efficiency, analysts would ideally study both compound classes simultaneously in a single analysis run.

Normally, cannabinoids are analyzed using gas chromatography (GC) or high-performance liquid chromatography (HPLC) techniques. The former is more common technique on account of its simplicity, but the latter provides the most direct measurement of the cannabinoids present in a given sample, as it avoids the high temperatures that cause GC analyzers to decarboxylate some cannabinoids found naturally in raw cannabis.

In terpene analysis, however, GC still a powerful technique when combined with a headspace sampling technique that leverages the natural volatility of the terpene compounds. Terpenes can also be studied using a solvent extraction preparation step followed by traditional GC analysis, but the polarity and concentration of the terpenes in cannabis material differ significantly from that of the cannabinoids, making it a challenging feat to combine the study of both compound classes in a single method.


Using gas chromatography for simultaneous analysis

The University of Ljubljana researchers set out to conquer the challenge of finding a suitable sample preparation step that would make simultaneous analysis possible.

After settling on solvent extraction as the most promising extraction method, the team excluded possible solvents known to have a poor impact on the environment, eventually narrowing the selection down to acetone or ethyl acetate. After some trial runs, the researchers settled on acetone as the most appropriate solvent, as it was found to return better extraction recoveries. The researchers also experimented with different sample-to-volume ratios during extraction, finding that a ratio of 1:17, or 300 milligrams per 5 milliliters of solvent, struck the best balance between extraction efficiency and preserving sufficient concentrations of the terpenes in the working sample solutions.

Given the expense associated with HPLC analysis, the team opted for a GC machine with a flame ionization detector (GC-FID). A coupled mass spectrometer was also used to confirm the identity of the terpenes. As terpenes and cannabinoids naturally have a large difference in polarity, separating the two compound classes was not be the biggest issue. The major concern was making sure that all terpenes present in the sample could be properly analyzed before their natural volatility caused them to evaporate off the column, while still providing good separation between cannabinoids with similar retention times, such as cannabichromene (CBC) and cannabidiol (CBD).

The researchers found that this was possible using a more polar stationary phase (50 percent phenyl, 50 percent dimethylpolysiloxane, as opposed to the more common 5 percent phenyl, 95 percent dimethylpolysiloxane) and an oven temperature program that started relatively cool before ramping up to complete the analysis.

This methodology was validated and evaluated for its performance by comparing with results obtained using a previously published and validated HPLC analysis method. The simultaneous GC analysis was found to correlate well with the results obtained using HPLC analysis, even at higher concentrations.

“Despite many of the method parameters being near the practical limits in terms of instrumentation capacity like temperature, detector response, etc., it provides a robust tool for simultaneous quantitative analysis of these two chemically different groups of analytes,” the researchers wrote in their study, which was published in the journal Molecules.

While GC is inherently less sensitive than HPLC, the researchers say that sensitivity limits established for this GC method are still sufficiently low to be appropriate for the routine analysis of cannabis and hemp samples.

 

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