Analyzing an Extended Terpenes List in a Commercial Cannabis Laboratory
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Terpenes are nearly ubiquitous across nature. These aromatic compounds are most commonly found in plants, but some terpenes have also been identified in marine organisms, insects, and, to a lesser extent, higher-order animals.
In cannabis, the terpenes that are present in a specific batch of flower will determine its unique aroma and flavor profile. Oftentimes, the strains that end up placing well at cannabis competitions are those with a particularly intense or interesting combination of terpenes. As a result, interest in terpene profiling has risen in recent years as producers look to create unique experiences for the consumer.
At the Analytical Cannabis Expo West Online 2021, Think20 Laboratories’ commercial operations manager Adam Floyd explained to attendees how this type of extended terpene analysis is done in a commercial cannabis laboratory and detailed the considerations needed when building a suitable analytical method for terpene analysis.
Terpene analysis in cannabis
To date, over 30,00 unique terpene compounds have been discovered in nature. More than 150 terpenes have been identified in different strains of cannabis, and this number is likely to grow further as cannabis research continues. As a result, testing laboratories offering terpene analysis need to make a choice about which terpenes they want to test for, balancing the ease of data analysis and method development versus the specific terpenes that clients and consumers might be interested in.
“The general starting place for terpene analysis is a standard 22 terpene mix,” Floyd said. “It's available from a number of different vendors and as far as I know those 22 terpenes are essentially the same within those mixes.”
“And so you have two choices when deciding to expand on this: there is extended terpene mixtures [containing] 40 to 44 terpenes, and then there’s numerous individual standards that are available. You can make the decision to either just extend your list from a commercial mix stand standard or add individual terpenes that you may be interested in.”
Deciding which terpenes to analyze is just the first of many major decisions that needs to be made to begin the method development process. The core of these methods will be gas chromatography, but there are a number of different variables that need to be considered when deciding on the chromatograph set up, sample injection, and detector to be used.
“The next question I always ask people is if you are going to run residual solvents on the same instrument, because that definitely influences your [chromatography] column selection,” Floyd said. “There’s columns that are better for residual solvent analysis, and ones that are better specifically for terpene analysis. But it’s not practical to continually swap columns out.”
The length of a column is another factor. Longer columns will result in better separation of the sample compounds in the final spectra, but this will also make the analysis runs last longer. Some commercial labs might decide that time is their biggest constraint and elect for a slightly shorter column instead.
Laboratories also need to consider the desired detection limits that they might want to achieve with their analysis; many of the less common terpenes are only present in relatively low concentrations and this can present additional challenges if they are being studied.
Developing an ideal analysis method
In terpene analysis, labs generally choose either liquid injection or headspace sampling as the sample injection method. Liquid injection is a very fast technique and most modern gas chromatographs (GCs) have autosamplers that allow for simple sample injection. However, because of the nature of the liquid injection method, all of the volatile and non-volatile compounds in the sample are being injected into the column. The build-up of these non-volatile compounds means that technicians need to change out the GC’s liners relatively frequently and maintenance may be required more often.
“Headspace is, for the most part, my preferred technique,” said Floyd. “One of the biggest advantages to headspace is that flower samples can be analyzed directly for terpenes.”
In headspace sampling, the cannabis flower is placed in a sealed vial and the volatile gases – which include terpenes – in the headspace above the flower are what is sampled and analyzed. Since only the volatile compounds enter the detector, this sample injection method is very clean, reducing the amount of potential downtime needed for maintenance.
“The biggest disadvantage of headspace is the time involved in method development,” Floyd explained. “You have to pay the price of 15 minutes incubation per sample, and then if you have a 15-minute run you are looking at 30 minutes from injection to injection, whereas it’s roughly half that for liquid injection.”
With an injection technique decided, the next step is to focus on chromatographic separation and the detector type. For laboratories that choose to use a flame ionization detector (FID), achieving baseline separation is very important to achieve clear and useable results. This separation can be improved using slow temperature ramps. But in complex terpenes samples, this can be difficult to achieve.
“The biggest issue with using an FID is the lack of mass selective detection, which for an extended terpenes list is, in my mind, extremely critical,” said Floyd. “So I would sway against choosing an FID if at all possible when choosing to go down the route of developing an extended method. At the end of the day, you’re going to end up with extremely long run times.”
“If a lab can afford mass spectrometry, I really always strongly encourage people to choose that. Mass spectrometry really allows for a more dynamic style of analysis. Having this spectral data allows for us also to be able to identify untargeted terpenes. This is very useful, especially with method development and selecting terpenes that you want to use in your method.”
One of the biggest challenges with terpene analysis is separating out the peaks from co-eluting compounds. Using specific masses and mass ratios of known terpenes, it is possible to deconvolute these co-eluting segments in the spectra using mass spectroscopy, which effectively shortens runtimes by avoiding the need to significantly stretch out the temperature ramping or use very long columns.
The method validation process
Once all these decisions have been made and the laboratory has a complete analysis methodology, it is time to consider how the method will be validated. Depending on the type of sample (flower, oil, infused edibles, etc.), matrix spike studies will need to be conducted during validation to unravel any possible matrix effects affecting the analysis results. But more broadly, there is a range of other validation studies that are normally required by regulators.
“There are a lot of different areas that can be covered with validation, so you have to refer to your state regulations for what’s required,” said Floyd. “But in general, accuracy, precision robustness, LOD/LOQ [limit of detection/quantitation], matrix recovery studies, and so on and so forth, are all required to really validate your method. And this is extremely important because it allows you to understand the limitations of your method.”
Accuracy is arguably the most important aspect of this – it is the measure of how close in agreement a test result is to an accepted reference value. This accuracy is normally measured using an initial calibration verification (ICV) process, where standards from a secondary source are run and the results are compared against the known values.
Confirming good sensitivity, selectivity, and linearity of the method is also central to the validation process. Sensitivity is generally understood as the change in instrument response that corresponds to a change in the quantity being measured – so here, that would be how well a method can determine changes in the analyte concentration. A method with good sensitivity should have good limits of detection and quantification (LOD/LOQs), which can be determined experimentally by looking at the height of the spectral peaks compared to the standard deviation of the baseline noise.
Selectivity describes the extent to which a method can determine a particular analyte in a matrix without interference from other similar analytes, and is demonstrated by showing consistent analyte recovery in both solvent and matrix samples. Linearity is the ability of a method to provide an instrument response that is proportional to the quantity of the analyte.
With success in all of these measurements, a lab will have fully designed, developed, and validated a terpene analysis method that is capable of handling an expanded terpene list. This can give research labs a better insight into this very intricate part of the cannabis experience and give commercial cannabis testing labs an exciting new offering to clients with a special interest in terpene profiling.
“Award-winning cannabis oftentimes has very complex and diverse terpene profiles, and so these are really important to understand,” Floyd said. “And I think that allowing your lab to have this additional offering of an extended terpenes list can be very beneficial – over the years, I’ve just seen interest in terpenes grow further and further.”
This article originally appeared in Analytical Cannabis' Advances in Cannabis Testing eBook in September 2021.