Overcoming the Unique Challenges of Novel Cannabinoids With Chromatography

Comprehensive cannabinoid profiling is one of the cornerstones of the cannabis sector. As the market has matured in recent years, attitudes in cannabis testing have shifted away from the narrow scope of quick potency tests and towards the detailed insights afforded by full cannabinoid profiling. This type of testing relies in a large part on chromatography techniques.

To discuss the chromatographic challenges posed by novel cannabinoids, Analytical Cannabis spoke with Sean Orlowicz, business development manager for food and cannabis at Phenomenex. As a manufacturer of chromatography columns and other related consumables, Phenomenex has been working closely with cannabis laboratories in North America to provide chromatography solutions suitable for cannabis testing.

Orlowicz explored the challenges of cannabinoid testing further in his Analytical Cannabis webinar The Analytical Landscape of Cannabis Testing Session 1: Potency Testing, which took place on January 20, 2022. Register here to watch the webinar on-demand now.

Dealing with THC isomers in chromatography

One of the cannabis compounds dominating the industry's thoughts over the past few years has been the rise of delta-8 THC. While discussions between regulators, lawyers, and cannabis industry firms over the compound’s legality under the 2018 Farm Bill are still ongoing, the compound also presents a complex analytical challenge to cannabis testing laboratories.

In a chromatogram, the delta-8 peak elutes shortly before the peak correlating to delta-9 THC, on account of their very similar chemical structures. The amounts of delta-8 THC present in most cannabis strains are naturally quite low, and since delta-9 THC is the intoxicating cannabinoid that is usually focused on by cannabis cultivators and consumers, this rarely caused any issue in analysis. But with the advent of delta-8 THC products, this is changing.

“I started getting phone calls about delta-8 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,” Orlowicz recalls. “When you get a delta-8 concentrate or something at 80 plus percent [potency], the game changes chromatographically.”

Testing laboratories had encountered a problem. Products with significant amounts of delta-8 THC were returning chromatograph readouts where the peak shape for delta-8 THC was non-Gaussian — making it more difficult for testing labs to accurately quantify the analytes detected. In some cases, delta-8 THC peaks were so large that they would absorb the delta-9 peak entirely.

“I’m not going to get into what compliance on a delta-8 sample is; it’s not for me to judge, I’m not a lawyer, I’m not a registered regulator, I’m a chromatographer. But I know this: that if you are trying to measure down to 0.3 percent delta-9 THC and you have a huge delta-8 peak, chromatography matters,” said Orlowicz. “You must resolve those peaks well to get adequate quantitation.”

Adopting new methods will be necessary as the market matures

Orlowicz compares the current situation to that of the discovery of vitamin E acetate in certain vape products linked to the EVALI outbreak of 2020, another occasion in which laboratories were calling up to discuss how best to address this new analyte of interest.

“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.

Due to the continued federal prohibition of cannabis, the regulation of cannabis testing is primarily a state issue. Each state will have different required standards for its labs, but fundamentally each lab will be looking to better resolve the delta-8 and delta-9 THC isomers in order to provide more comprehensive testing results.

“What I’ve seen so far is that the best way to improve the resolution between delta-8 and delta-9 is actually through the use of methanol-based mobile phases,” said Orlowicz.

“Some of the more legacy-type chromatographic methods for potency are acetonitrile-based as their organic modifier, and there’s reasons for this. Acetonitrile-based methods resolve a comprehensive list of cannabinoids very well; they’re very fast, and they’re very low back pressure so you can run them on just about any instrument.”

“This is certainly up for debate, and I look to the industry for better ways, but I have found that methanol as an organic modifier, that resolves the isomers much better.”

There are several drawbacks to methanol-based techniques, which Orlowicz readily points out. The largest hurdle is the high back pressure demands for methanol-based methods, which often means that laboratories need the finances to invest in high-end machinery or else have the luxury of time so that they can run this method for longer at lower back pressures. Alternatively, labs may start to investigate non-traditional or non-typical analysis methods for the cannabis sector, such as normal phase chromatography, to see if this may be a viable option that suits their needs.

Delta-10 THC: the next big hurdle?

Adapting methodology to better suit trends in cannabinoid popularity, product compositions, and novel cannabinoids is a crucial forefront for the cannabis testing community. Trying to predict the next challenge on the horizon is difficult and at times totally impossible. But already analytical scientists have a few ideas about what lies ahead.

“Delta-10 [THC] is a really important one from a chemistry perspective, specifically a chromatography perspective, because it's the first THC isomer in which we’re going to start talking about chirality,” Orlowicz explains.

“Delta-10 has two chiral centers. Many cannabinoids have one, and sometimes two, but we typically ignore that chiral center because in the plant the enzymatic process that creates a delta-8 THC – the most important isomer, I would argue – is enzymatically and chirally specific, so it only produces one enantiomer. When people start pursuing semi-synthetic and synthetic pathways, that enantiomeric-specific chemistry goes away.”

Will testers need to chromatographically separate these enantiomers in the future? Will they need to be quantitated separately? These are the questions that the marketplace is already beginning to ask. If so, new methods will need to be developed by scientists that can become widely adopted by labs.

“​​We are working on delta-10 separations today because of the market need,” said Orlowicz, referring to Phenomenex. “But it’s going to bring another level of analytical requirements; it’s going to educate the market into other forms of chromatography, and it’s going to really bring the relationship between the chemistry of the market and the analytical capabilities of the testing laboratories even closer together. That’s what I’m really excited about with delta-10 THC. Not about the challenges it brings, but excited about the topics that it’s going to return to the prime.”