LABTips: Troubleshooting Tricky Terpenes

Terpene profiling is an important area of cannabis testing that goes beyond just determining the aromatic qualities of a product. Terpene contents and concentrations can be used as a “fingerprint” to identify certain cannabis strains, and terpenes have also been shown to enhance the medicinal properties of cannabinoids, such as the muscle relaxant effects of THC.¹ Some regions also require terpene testing for quality assurance in addition to cannabinoid and contaminant analysis. Overall, more than 140 different terpene components can be found in cannabis, and you may be tasked with testing anywhere from a handful, to tens, to dozens of these components, some of which may be more difficult to recover and separate than others. Below are a few handy tips to keep in mind while performing your terpene profiling analyses using gas chromatography (GC) methods.

1. Keep it cool to prevent volatile analyte loss

Terpenes are volatile or semi-volatile compounds with low boiling points which make them a well-suited analyte for separation via gas chromatography. This volatility also means that a certain amount of terpene loss can be expected between the harvest of the cannabis plant to the time of analysis; the amount of loss depends on factors such as time, storage conditions and processes such as drying. Monoterpenes such as myrcene, pinene, camphene and limonene, which consist of two isoprene units, are more volatile and thus more susceptible to loss through evaporation than sesquiterpenes like caryophyllene, which have three isoprene units and are less volatile. To prevent further loss of these analytes during sample preparation, samples such as plant material should either be frozen before grinding or ground under liquid nitrogen,² as heat from the grinding process can lead to excessive, premature volatilization.  Other measures for preserving terpenes include keeping samples and solvents chilled, keeping samples frozen while in storage, and minimizing exposure to moisture and light prior to analysis.³

2. Try these methods to improve sesquiterpene recovery

As mentioned, sesquiterpenes are less volatile terpenes with higher boiling points and higher molecular weights than monoterpenes, and can pose their own challenges during analysis. For example, you may see poorer recovery of less volatile terpenes and terpenoids (terpenes with additional functional groups) when conducting headspace sampling,⁴ which is among the favored methods for terpene analysis via GC. Liquid injection may show better recovery for these particular terpenes, but comes with its own drawbacks such as increased sample preparation time and more components other than analytes being injected into the system. One method for coaxing the less volatile sesquiterpenes and sesquiterpenoids into the headspace is adding a carrier solvent such as water and salt (NaCl),⁵ or glycerol,⁶ to the headspace vial. Additives such as salt help to increase the vapor pressure and lower the partitioning coefficient of volatile and semi-volatile analytes, increasing their concentration in the headspace.⁷

Another problem you may run into is condensation of higher boiling point and higher molecular weight analytes in the headspace syringe.⁵ This could be resolved by trying an alternate method such as solid phase microextraction (SPME), in which the analytes are absorbed or adsorbed onto a fiber coated with an extracting phase. The affinity of the terpenes to an appropriate extracting phase, such as a combination of divinylbenzene (DVB) and polydimethylsiloxane (PDMS), will keep the analytes on the fiber until they are desorbed into the instrument inlet. Already using SPME? Then you may want to try a direct immersion (DI) SPME method instead of headspace to ensure capture of those stubborn sesquiterpenes.⁸

3. Effective sample handling/extraction strategies can help bust matrix effects

Cannabis products come in a strikingly diverse range of matrices beyond just plant material; this includes edible products, waxes, oils, lotions and more. This can present a challenge for analysis due to interference from non-analyte matrix components. Fortunately, there are numerous techniques that can minimize the amount of unwanted components entering the GC system, starting with sample handling and extraction. As previously discussed, headspace sampling is incredibly popular for cannabis terpene testing as it allows for partitioning of the volatile components of interest into the headspace while leaving non-volatile matrix components behind in the vial.⁹ This is advantageous compared to liquid injection, which can introduce more matrix contaminants into the system and lead to clogging and increased maintenance time,¹⁰ especially as some plant components such as chlorophyll are often co-extracted in common solvents like ethyl acetate and methanol (MeOH).¹¹

Headspace sampling can even be used to directly analyze some samples, such as dried plant material, without prior solvent extraction, further cutting back sample prep time and reagent use, which is where methods like SPME come in handy to selectively capture your volatiles and semivolatiles.¹² Other matrices, however, such as viscous oils, have a tendency to hold onto analytes and make recovery more difficult, in which case solvents such as dimethylacetamide (DMA) and/or MeOH can be used prior to headspace analysis.¹⁰ The full evaporation technique (FET) is another headspace sampling approach that minimizes both sample prep and matrix effects by completely evaporating a small amount of sample to create a single gas phase, ensuring that all analytes volatilized and freed from the matrix.^13,14 This can be used for complex, varied solid and semi-solid cannabis extracts without the need for matrix matching.

4. Choose more selective techniques to resolve coeluting peaks

One of the incredible things about terpenes is that they can each confer different sensory experiences and medicinal properties while at the same time having extremely similar structures. This is especially true of terpene isomers, which contain the exact same atoms but in a different arrangement, such as α-pinene and β-pinene. These similarities can cause issues with coelution, making accurate quantitation more difficult. For GC-flame ionization detection (FID) analysis, this can mean a much longer runtime (up to an hour or more) may be needed to achieve sufficient separation, depending on the number and types of terpenes you are analyzing. If you are developing a method to analyze a panel that includes terpenes with similar retention times, you may want to consider mass spectrometry (MS) over FID to ensure you will be able to resolve those coeluting peaks. The benefit of MS is that certain co-eluting peaks, such as those of limonene, p-cymene and ocimene, can be deconvoluted based on masses and mass ratios for accurate quantification.¹³ This takes away any uncertainty in your analysis while keeping runtimes short, which is especially practical for large terpene panels.

Another approach that provides greater separation efficiency for similarly-structured analytes is two-dimensional gas chromatography (GCxGC). Running the sample through two columns with different stationary phases can provide the extra dimension needed to differentiate between coeluting compounds without drastically increasing runtime. GCxGC can be used with an FID detector to provide better separations and greater data quality¹⁴ or, paired with an advanced detector such as a time-of-flight mass spectrometer (TOF-MS), can create clear distinctions between terpenes and their terpenoid derivatives, and even help resolve nearly-identical isomers such as cis- and trans-nerolidol when parameters are optimized.¹⁵ Considering the specific terpenes you will encounter and the challenges they may pose, choosing the right methods and technology for your needs can help nip these common problems in the bud.

References

1. "Simplified Cannabis Terpene Profiling," Application Note No. GCMS-1604, 2016, Shimadzu. https://www.shimadzu.de/sites/shimadzu.seg/files/GC_Shimadzu-GCMS-Terpene-Application.pdf 
2. "Analysis of Terpenes in Cannabis Using the Agilent 7697A/7890B/5977B Headspace GC-MSD System," Application Note, Agilent Technologies. https://www.agilent.com/cs/library/applications/5991-8499EN_cannabis_terpenes_application.pdf 
3. "The Importance of Terpene Preservation," Blog, Luna Technologies. https://blog.lunatechequipment.com/importance-of-terpene-preservation 
4. "Terpenes Analysis in Cannabis Products by Liquid Injection using the Agilent Intuvo 9000/5977B GC/MS System," Application Note, Agilent Technologies. https://www.agilent.com/cs/library/applications/cannabis-terpenes-Intuvo-5994-2032en-agilent.pdf 
5. "Terpene Analysis Approaches - Part II," Blog, 2019, Restek. https://www.restek.com/en/chromablography/chromablography/terpene-analysis-approaches---part-ii/ 
6. Nguyen, T. D.; Riordan-Short, S.; Dang, T. T. T.; O’Brien, R.; Noestheden, M. Quantitation of Select Terpenes/Terpenoids and Nicotine Using Gas Chromatography-Mass Spectrometry with High-Temperature Headspace Sampling. ACS Omega 2020, 5, 5565–5573. DOI: https://doi.org/10.1021/acsomega.0c00384. 
7. "SPME Fundamentals: Don't forget the salt for HS VOCs!," Blog, Restek. https://www.restek.com/en/chromablography/chromablography/spme-fundamentals-dont-forget-the-salt-for-hs-vocs/ 
8. "Terpene Analysis Approaches - Part III," Blog, Restek. https://www.restek.com/en/chromablography/chromablography/terpene-analysis-approaches---part-iii/ 
9. "PerkinElmer Cannabis & Hemp Analytical Solutions," Application Note Compendium, PerkinElmer. https://resources.perkinelmer.com/lab-solutions/resources/docs/APP_Cannabis_Hemp_Science_Compendium_2020_014714_01.pdf 
10. "Headspace Gas Chromatography Mass Spectrometry Analysis of Terpenes," Presentation by Adam Floyd, CannMed 2019. https://www.youtube.com/watch?v=GgRpuaUA9tE 
11. "Medical Marijuana Solvent Extraction Efficiency - Potency Determinations with GC-FID," Blog, Restek. https://www.restek.com/en/chromablography/chromablography/medical-marijuana-solvent-extraction-efficiency--potency-determinations-with-gc-fid/ 
12. Stenerson, K.; Halpenny, M.R. Analysis of Terpenes in Cannabis Using Headspace Solid-Phase Microextraction and GC-MS. LCGC Supplements. 2017, 35(5), 27–32. https://www.chromatographyonline.com/view/analysis-terpenes-cannabis-using-headspace-solid-phase-microextraction-and-gc-ms 
13. "Developing a Comprehensive Terpenes Analysis Method Using Gas Chromatography Mass Spectrometry," Webinar, Presented by Adam Floyd, Hosted by Labroots. https://www.youtube.com/watch?v=nfo6Bo4IETg 
14. "Improving confidence in the quantitative analysis of cannabis terpenes using flow-modulated GCxGC-FID," White Paper, SepSolve Analytical. https://www.sepsolve.com/analysis-of-cannabis-terpenes/ 
15. McGregor, L.; Hughes, E. Improved Profiling of Cannabis Terpenes for Accurate Products Labelling Using GCxGC. The Column. 2020, 16(8), 11–16. https://www.chromatographyonline.com/view/improved-profiling-of-cannabis-terpenes-for-accurate-product-labelling-using-gc-gc