Profiling Terpenoids in Cannabis with GC×GC-MS

A joint study conducted by the University of Ferrara (Italy) and the Council for Agricultural Research and Economics (Rome, Italy) focused on the analysis of terpenes and terpenoids—key bioactive compounds responsible for the distinctive flavor and potential therapeutic effects of cannabis. For this study, the research team used comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC-MS) coupled with dynamic headspace extraction (DHS) to profile these compounds in cannabis inflorescences. The chromatographic process was further optimized using cryogenic trapping to improve resolution and peak quality. Ultimately, the study successfully profiled terpenes and terpenoids across different cannabis chemovars, revealing notable differences in their chemical compositions. LCGC International spoke to Flavio A. Franchina of the University of Ferrara, corresponding author of the paper which resulted from this study, about their research.

You recently published a paper using comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC–MS) to profile terpenes and terpenoids in cannabis inflorescences (1). Why are you investigating these analytes?

We are interested in deciphering the chemical composition of complex samples through high-resolution hyphenated techniques and tailored workflows. Natural products like cannabis have a rich distribution of specialized metabolites which play fundamental roles during their living activities. They might also leave a peculiar fingerprint related to the original composition or to the transformation process of the raw material. On the other hand, these complex samples represent the perfect test samples to challenge and improve our chemical analysis tools and workflows, including extraction, separation and detection, and data analysis.

In this specific research, we investigated the terpenoids because they are also the main responsible for the distinctive flavor of natural products, and cannabis is very rich indeed.

Traditionally, the determination of terpenoids in cannabis, as in other plants' raw materials, is commonly performed with one-dimensional gas chromatography (GC) with a flame ionization detector (FID). What limitations does GC–FID have that led you to explore alternative techniques?

The separation capacity of one-dimensional (1D) GC system might not be sufficient for profiling studies, in which a specific chemical class is characterized at the analyte level. In addition, the level of identification exploiting GC-FID would only rely on retention time matching from certified standards, making the profiling very limited and challenging. These were the main reasons to explore the samples using a GC×GC–MS system.

Does GC×GC–MS offer additional benefits to other existing methods other than those you just covered for this application?

When we use GC×GC–MS (and we exploit it a lot in our research!), we always highlight the benefits we experience in that given application. In this work, we pinpointed the ordered clustering in the 2D space of terpenoids, specifically related to monoterpenes and sesquiterpenes, and the superior separation power that resulted in higher 2D chromatographic resolution and cleaner mass spectra for database matching. The sensitivity was not an issue in this specific application since the samples were naturally rich. On top of this, we used an enrichment technique like dynamic headspace extraction into sorbent traps.

However, in other applications where trace and ultra-trace analytes are sought, the sensitivity gain provided by the band focusing in space of the GC×GC system is without any doubt another handy advantage.

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

In the paper, we discuss several layers of findings, both from the technical and the sample point of view. Initially, we proved the advantages of a secondary trapping/release stage, avoiding the use of an external unit which makes the system more compact with fewer hardware components, and which provides a direct and more efficient injection into the GC column head. Moreover, the overall chromatography benefitted from the additional trapping/release stage from the use of a cryotrap.

For the extraction, we obtained different analytical performances based on the trapping materials (for example, because of the water content or due to the strength and type of interaction with the analytes). From such an evaluation, we selected a porous polymeric material which obtained the best reproducibility, recovery, and analyte coverage.

When analyzing the samples, we uniquely detected two analytes eluting between the monoterpenes and sesquiterpenes. These two analytes were only detected with the selected adsorbent material and of course were not present in the various blanks and controls. Once again, there, we had a demonstration of the significance of the sample preparation step.

It was curious that for these analytes we could not find a reliable match with spectral libraries and retention indices: we labelled them as “unknowns,” and we are investigating their identity further.

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

We had to carefully optimize inlet desorption and select the most appropriate adsorbent material for our objective. Having the thermal desorption straight at the column head needs careful optimization to ensure efficient analyte transfer. In such a configuration, the use of a cryotrap, and thus a secondary trapping/release stage helped with the injection band broadening.

Regarding the selection of the adsorbent materials instead, since there are many available (also in combination), we decided to evaluate experimentally those which were, from theory, the most suitable on our samples.

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

It was fun to perform data analysis during this research. Initially, we had to calculate fundamental chromatographic metrics (peak width, height, resolution, and peak quality) to select the proper injection conditions. We also worked on both the total response of the entire monoterpene/sesquiterpene classes, and the individual analytes. The first approach was helpful to evaluate efficiently and comprehensively the extraction methods; the latter was useful to assess in detail the differences and the similarities between samples.

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

We could not properly quantify the analytes of interest. Thus, we only compared the cross-sectional response of terpenoids and relate them to the different types of cannabis samples. Having a double FID/MS detection would have made possible a detailed profiling with quantitative data.

How could the findings of this method be applied practically in analytical laboratories for other hydrocarbons, or for other sources containing them?

The method we discussed in the research paper can be adapted for any aroma profiling, of course taking into consideration the specific challenges associated with the given sample.

Are you planning to develop this research further?

Definitively! We are working on the detailed profiling of cannabinoids from various chemovars, exploiting both GC×GC–MS and LC–MS…Stay tuned!

Reference

1. De Poli, M.; Chenet, T.; Felletti, S.; Spadafora, D.; Cavazzini, A.; Franchina, F. A. Sorbent-Based Sampling with Two-Stage Trapping/Desorption Coupled to Comprehensive Two-Dimensional Gas Chromatography and Mass Spectrometry for Terpenoids Profiling in Cannabis. Anal. Sci. Adv. 2024, 6 (1), e202400044. DOI: 10.1002/ansa.202400044

Flavio A. Franchina is an associate professor of analytical chemistry at the University of Ferrara (Italy), bringing a wealth of international research experience from the University of Messina (Italy), Dartmouth College (Hanover, New Hampshire), and the University of Liège (Belgium). He is committed to harmonizing analytical strategies for targeted and non-targeted bioanalysis. This includes a comprehensive approach to sample preparation, separation techniques, quantification methods, and data processing, all within application-driven workflows. Franchina leverages chromatography and mass spectrometry to investigate specialized metabolites in biological systems and emerging environmental contaminants. His collaborative research across academia and industry has yielded numerous publications covering metabolomics, natural products, food authenticity and safety, and environmental contaminants.