Quantifying Terpenes in Hydrodistilled Cannabis sativa Essential Oil with GC-MS

A recent study conducted at the University of Georgia, (Athens, Georgia) presented a validated method for quantifying 18 terpenes in Cannabis sativa essential oil, extracted via hydrodistillation. The method, utilizing gas chromatography–mass spectrometry (GC–MS) with selected ion monitoring (SIM), includes using internal standards (n-tridecane and octadecane) for accurate analysis, with key validation parameters—such as specificity, accuracy, precision, and detection limits—thoroughly assessed. This approach offers a simple, solvent-free extraction process combined with a precise analytical technique, supporting both therapeutic and industrial applications of C. sativa terpenes. LCGC International spoke to Noelle Joy of the University of Georgia, corresponding author of this paper discussing the method, about its creation and benefits it offers the analytical community.

Your recently published paper (1) reports on a method developed for quantifying 18 terpenes in C. sativa essential oil obtained through hydrodistillation. What are terpenes, and what makes them worth studying?

Terpenes are a naturally produced class of chemical compounds that contribute to the flavor and aroma in plants, and essentials oils are comprised mainly of terpenes. I think Cannabis terpenes are worth studying because they are becoming more popular! The terpenes give cannabis its unique taste and smell, and are an indicator of quality, as certain post-harvest handling methods can decrease the terpenes. They are also believed to provide some potential enhancements in the health benefits of cannabis.

Briefly describe hydrodistillation and why this technique was used.
Hydrodistillation is a process that involves submerging plant material in water and heating until boiling. Terpene compounds have a high boiling point (150 to 300 °C), but during distillation they are released from the plant matrix and form a heterogeneous mixture with water, which allows for compounds to be separated and volatilized at relatively low temperatures (97-98 °C). The vapor turns to liquid in a condenser and falls into a Clevenger trap where the essential oil forms a layer on top and can be removed. This technique was used because we wanted to measure the total mass of essential oil in the plant material, in addition to the terpene composition.

Terpene analysis is primarily performed using gas chromatography with a mass spectrometer detector (GC–MS). What benefits did using this technique offer?

The terpene composition in essential oil can be very complex; sometimes up to 100 compounds can be found in a single essential oil sample. Cannabis essential oil can have issues with compounds co-eluting, so GC–MS offers the advantage of being able to look at the mass spectrum to identify co-eluting compounds and quantify them based on their unique ions.

What were your main findings? Was there anything surprising or interesting?

I think our main finding was that we were able to do what we set out to do, which was utilize distillation to obtain our essential oil and reliably quantify major terpenes in a complex essential oil containing co-eluting and isomeric compounds.

What were the main analytical challenges you encountered, and how did you overcome them?

A big analytical challenge was working with volatile samples that contained a large range of different molecular weight compounds. We didn’t want to lose any terpenes to volatization during sample preparation, so we kept the samples cold by placing them in a frozen metal autosampler tray sitting on top of an ice pack. However, we found that some of the terpene compounds were solid when frozen. We overcame this challenge by allowing the terpenes to come to room temperature and vortexing the sample to make sure it was homogenized and then placing the vials in a cold tray before opening the cap to reduce volatilization. We did this for our external standards as well.

How did you process the data to obtain the results you were looking for?

Because we were using SIM for the quantification, we built a quantitation database with our standard curves using the Chemstation software. We transferred the quantitation results from Chemstation to an Excel spreadsheet, set up the hold the results of 30 samples. On the spreadsheet we had formulas to calculate all the corrections to be able to statistically compare the results. For each sample we first corrected the results by the mass of essential oil added to sample solution, volume of sample solution, and dilution factor of the injection solution. Then, we corrected for essential oil yield, mass of plant material added to distillation and moisture content of plant material. For each batch of 30 samples, we also had internal and continuous calibration verification samples, matrix spikes, and sample duplicates that we included in the spreadsheet to make sure we met our % recovery target ranges for method validation.

You mention in your paper that, while the primary standard solution contained 21 compounds, but it was determined during method development that fully resolving all peaks in the solution would not be possible. Why do you believe this to be the case?

This is a great question. To be honest, it might be possible, and I just didn’t know how at the time. I was teaching myself how to use the GC–MS to be able to analyze C. sativa samples dried under different treatments for my Ph.D. research. I found a method development training program that suggested a stepwise approach for building an oven program for separation. I tried a large range of starting temperatures, and oven ramp programs, as well as two different columns, and found that I couldn’t separate them all because of the large complexity of the sample. Therefore, I prioritized separating the compounds that were most commonly present in the C. sativa samples we planned to analyze, as well as what we found reported in the literature. I am proud of this method but know there is always more to learn. With more experience, I might be able to improve this method to separate more compounds.

Were there any other limitations to this research that are important to note?

A big limitation is the distillation time. At over 3 h per sample, it is a much longer and more labor-intensive separation compared to a solvent extraction.

How could the findings of this method be applied to detect different essential oils in C. sativa, or in other plants?

What I think is valuable (and unique) about this method is the ability to quantify complex samples containing co-eluting compounds and determine the total yield of essential oil from a plant sample. I believe it has a wide range of possible applications, especially research on the production and post-harvest handling of aromatic and medicinal crops.

What are the next steps in this research?

When using this method to analyze samples on the GC–MS, I ran each sample twice. First in SCAN mode and then in SIM mode. My understanding was that SIM mode would lead to more accurate and precise quantification because I was only monitoring 3-4 ions per compound versus all the ions in a specified mass range. I think an interesting next step would be to compare quantitation results between the two. I also collected NIR data on my C. sativa samples and I am curious to see if we could create a model to predict the concentration of selected terpenes C. sativa flower from an NIR scan.

References

1. Joy, N.; Jackson, D.; Coolong T. A Validated GC-MS Method for Major Terpenes Quantification in Hydrodistilled Cannabis sativa Essential Oil. Phytochem Anal. 2025. DOI: 10.1002/pca.3526

Noelle Joy is currently finishing up a PhD in horticulture at the University of Georgia in Athens, Georgia. Before her PhD, she spent five years as the program manager of UGArden Herbs, a medicinal herb teaching farm and herbal products business at the University of Georgia. She has a Certificate of Herbal Medicine from the Botanologos School of Herbal Studies and an MS in Horticulture where she focused on holy basil (tulsi). She is passionate about the idea of farming secondary metabolites and doing research to understand the complicated relationships of how growing and post-harvest handling affect the quality of medicinal plants. Through her research, she hopes to improve the quality, efficiency, and profitability of growing herbs.