New Mass Spec Method Can Identify 17 Cannabinoids Simultaneously, Say Researchers

Potency testing for THC and CBD alone just isn’t enough these days. With the progression of the cannabis science sector, many more cannabinoids have been found to hold interesting and unique pharmacological properties.

As these cannabinoids have become increasingly more sought-after, there has been a rising demand for accurate and reliable testing methods that are able to detect and quantify these compounds, in addition to the two traditional major cannabinoids.

While such simultaneous quantification methods do exist, they have tended to rely on very sophisticated and expensive ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) systems.

Now, researchers from Southern Cross University, Australia, have developed a more accessible form of simultaneous cannabinoid quantification. Using high performance liquid chromatography (HPLC) for separation and a simple time-of-flight mass spectrometer (TOF-MS) for detection, this new method is able to detect and quantify a total of 17 individual cannabinoids simultaneously, using equipment that is already commonly owned by most natural products research laboratories.

LC-TOF-MS for cannabinoid quantification

In the newly published research paper, the scientists explain the decision-making processes behind their equipment and method selections.

While gas chromatography (GC) was previously a popular method for cannabis potency testing due to its speed and simplicity, many labs now recognize liquid chromatography (LC) as the preferred method for comprehensive cannabis analysis. This is because the high heat needed for GC methods also converts many cannabinoids from their acidic forms to their neutral forms. These cannabinoid acids are becoming increasingly more sought-after for their potential therapeutic benefits and so care should be taken to preserve them.

UV spectrophotometry was also previously a popular detection technique. However, its low specificity placed an increasing importance on the cannabinoid separation technique being used.

“Due to the lack of specificity of UV detection, any unknown compound/s co-eluting at the same retention time as the target cannabinoid can cause overestimation of its concentration,” the researchers wrote. “To overcome this specificity problem, the detection method of choice has been changing to mass spectrometry (MS).”

Specifically, tandem mass spectrometry (MS-MS) is the method most often used in testing laboratories. In this technique, two or more MS analyzers are used to examine the sample — one performing the traditional MS analysis after ionization and the others conducting further analysis after additional rounds of fragmentation. The extra fragmentation step creates even smaller fragments, which can often be used to distinguish between different compounds with very similar mass-to-charge ratios.

“Most of the methods using MS detection of cannabinoids are based on tandem mass spectrometry (MS-MS). However, there is no real advantage of using this approach over basic mass spectrometry (MS), as cannabinoids of the same molar masses produce similar fragmentation patterns in the second MS event thus not providing additional selectivity,” the researchers wrote.

Since there is no loss of benefits associated with not using MS-MS analyzers, the researchers elected to use a simple time-of-flight mass spectrometer (TOF-MS) for detection as this equipment is already widely owned by many product research laboratories.

Accessible method can simultaneously quantify 17 cannabinoids

Standard solutions for 17 cannabinoids — including THC, CBD, cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidivarin (CBDV), and cannabigerol (CBG) — and medicinal cannabis inflorescences supplied by the New South Wales Department of Primary Industries were used to develop and validate this LC-TOF-MS method, as well as optimize the sample preparation and extraction steps.

The final validated method was able to detect all 17 cannabinoids in a mixture of standard solutions, and 16 (all but delta-8 THC) when testing on real and pooled cannabis samples.

By using a low flow rate, the method was able to achieve very good chromatographic separation even at low pressure HPLC conditions, though this naturally extended the analysis run time. No significant loss of selectivity was seen despite using MS instead of MS-MS analysis. However, the relatively high limit of detection and quantification (LOD and LOQ) values would indicate some loss of sensitivity.

Despite these limitations, the researchers believe that the method is still more than capable of carrying out routine cannabinoid analyses and profiling measurements within the analytical laboratory environment.

“Due to the simplicity of instrumentation, and the robustness resulting from a high resolution in the chromatography of isobaric cannabinoids, the method is well suited for routine phytocannabinoid analysis for a range of applications,” the researchers concluded.

The future of cannabinoid analysis

Innovation in the cannabis testing sector has perhaps never been so important. As the research effort into the therapeutic potential of cannabinoids continues, these compounds will attract more attention. For the cannabis testing space, this means more demand from producers and consumers to develop methods that can adequately detect and quantify these compounds in finished cannabis products.

“I think definitely the cannabinoid list in general is going to grow,” Dr Tania Sasaki, chief science officer at Confidence Analytics, told Analytical Cannabis. Sasaki previously presented a talk on how to identify and analyze novel cannabinoids at the Analytical Cannabis Science of Cannabis Testing 2021 Online Symposium.

“In most states, you’re only required to test for anywhere between about four and maybe six cannabinoids. The consumer and the producer tend to like to see a bigger list. But it is still an emerging industry.”

Comprehensive analysis is still a tricky endeavor for this emerging industry to grapple with. Cannabis is a complex matrix made up of hundreds of potentially biologically active chemical entities. And when two such compounds of interest happen to be structurally very similar, this can make things extremely difficult for analysts.

“I started getting phone calls about delta-8 [THC] probably a year to a year-and-a-half ago, and it was clear to me that this was going to be an interesting issue for chromatography,” Sean Orlowicz, business development manager for food and cannabis at Phenomenex, told Analytical Cannabis earlier this year.

Delta-8 THC is an isomer of the more commonly known (and legislated against) delta-9 THC. Delta-8 THC recently made headlines when questions were raised over the legality of delta-8 THC, leading some to view it as a kind of legal high. The two compounds share very similar chemical structures, and so this has necessitated the development of new high-resolution analysis methodologies to tell the two compounds apart.

“There come times in which you must adapt your methodologies in order to get better results for the current marketplace, and I think this [delta-8 THC] is just another example of that,” Orlowicz said.