Journal:Development of a gas-chromatographic method for simultaneous determination of cannabinoids and terpenes in hemp

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Full article title Development of a gas-chromatographic method for simultaneous determination of cannabinoids and terpenes in hemp
Journal Molecules
Author(s) Zekič, Jure; Križman, Mitja
Author affiliation(s) National Institute of Chemistry - Ljubljana, University of Ljubljana
Primary contact Email: mitja dot krizman at ki dot si
Year published 2020
Volume and issue 25(24)
Article # 5872
DOI 10.3390/molecules25245872
ISSN 1420-3049
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/1420-3049/25/24/5872/htm
Download https://www.mdpi.com/1420-3049/25/24/5872/pdf (PDF)

Abstract

An original gas-chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were not only the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimized by avoiding any analyte derivatization. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium-polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established high-performance liquid chromatography (HPLC) methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.

Keywords: cannabinoids, terpenes, cannabis, hemp, gas chromatography, capillary column

Introduction

The hemp plant (Cannabis sativa and Cannabis indica), or simply Cannabis, is a plant that has elicited much interest throughout history because of its characteristics and various possibilities of use. Over the last few years, the popularity of the Cannabis plant and its constituents has particularly increased, and a widespread recognition of its usefulness, including for medical purposes, is becoming increasingly noticeable.[1][2][3][4][5] Hemp is known to contain various groups of compounds, probably the most characteristic among them being cannabinoids. Furthermore, cannabis also contains a diverse array of terpenes and flavonoids, as well as other groups of compounds.[6][7][8][9]

Cannabinoids are probably the most studied metabolites of cannabis. Many of their beneficial effects on human health are already known, and there is also a lot of ongoing research, discovering new ones.[10] As a result, the use of cannabinoids in a wide variety of preparations is growing, which is also reflected in increased cannabis production. At the same time, a need for an efficient, routine analytical method for monitoring the cannabinoid content in plant material has arisen. A number of methods for the analysis of cannabinoids in cannabis have indeed already been developed; among various approaches, the predominant is chromatographic analysis, in particular using gas chromatography (GC)[11][12][13][14][15][16][17][18] or high-performance liquid chromatography (HPLC).[16][19][20][21][22][23][24][25][26][27]

Even though gas chromatography used to be the most common technique for analysis of cannabinoids in cannabis extracts, HPLC is currently increasingly gaining popularity in this field of application. HPLC determination of cannabinoids, in comparison to the analysis with GC, has some significant advantages: above all, it avoids the potential aggravating circumstances caused by the high temperature of analysis associated with GC, which affects the results mainly during the phase of sample injection and also indirectly during the analysis itself. Cannabinoids are found mainly in acidic forms in the plant, which eventually decarboxylate if they are exposed to raised temperature.[28] The temperature in the gas chromatograph also causes the process of decarboxylation, which is reflected in the results in two ways: we cannot separately determine acidic and decarboxylated forms of a particular cannabinoid, but only their total content. On the other hand, there is a significant probability that decarboxylation in the injector will not proceed completely.[29] Especially at higher cannabinoid concentrations, this may be reflected in apparently lower values measured and consequently irregular results of analysis. Both problems can be successfully solved by the derivatization of cannabinoids (including their acid forms) in the sample.[17][30][31][32] However, this represents an additional step that is often not desirable, because it increases probability for experimental error and prolongs analysis time, which may be a considerable drawback in terms of method suitability for routine use. With HPLC, all of these problems have been successfully avoided, as some relatively rapid, simple, and effective methods for the determination of both acidic and decarboxylated cannabinoids in cannabis samples have already been developed.[16][19][20][21][22][23][24][25][26][27][33]

Thus, two major approaches to chromatographic analysis of cannabinoids in hemp most often appear in the literature; direct analysis of a suitably diluted sample extract by liquid chromatography[16][19][20][21][22][23][24][25][26][27][33], or preliminary derivatization of the extract and subsequent analysis by gas chromatography.[17][30][31][32] Despite its mentioned drawbacks, the latter approach is still quite in use, somewhat for traditional reasons, but also for entirely practical reasons, since GC instrumentation is simpler and less expensive than HPLC, more economical for use, or maybe even the only option available.

For direct gas chromatographic analysis of cannabinoids, traditionally, the most commonly used stationary phase is 5% phenyl 95% dimethylpolysiloxane[7][16][18], followed by 100% dimethylpolysiloxane phase.[11][13] Recently, more polar stationary phases like 35% phenyl 65% dimethylpolysiloxane have also been used, with a potential gain in selectivity.[34][35]

Another relatively important group of compounds in cannabis are terpenes.[6][7][8][9] Different varieties of cannabis contain mainly different monoterpenes and sesquiterpenes, which also give a distinctive scent to hemp plants. From an analytical point of view, they are interesting because their profile is often characteristic of a particular variety or population of cannabis, which may enable identification of different plant specimens.[36][37] Terpenes in cannabis are also often credited for the so-called “entourage” effect.[37] The analysis of terpenes is most often performed using gas chromatography as a separate type of analysis; successful separation and determination on different types of columns is usually quite fast, effective, and simple.

An unavoidable step in chromatographic analysis of plant material is analyte extraction from the sample. Both groups of compounds, terpenes and cannabinoids, can be extracted from the plant material by different approaches. For cannabinoids, the most common is classical extraction with a relatively apolar solvent (usually ethanol) either by mechanical shaking or by ultrasonic extraction. On the other hand, GC analysis of terpenes can also be done by the headspace sampling technique.[38] The alternative to headspace sampling is of course solvent extraction, in such a case a solvent of appropriate polarity must be chosen in relation to the analytes of interest. Terpenes and cannabinoids differ both in terms of volatility and polarity, as well as in the concentrations found in hemp samples. Terpene levels are usually significantly lower compared to cannabinoids. As demonstrated by Namdar et al.[7], an optimum solvent for terpene extraction was found to be a mixture of ethanol:hexane (3:7, v/v), while for cannabinoids they corroborated the use of ethanol as the optimum solvent.

Given the above, a simultaneous analysis of terpenes and cannabinoids in hemp samples is not a trivial task. A notable example of such a type of methodology has been published by Franchina et al.[39] In that case, the methodology involved the use of sorptive extraction and thermal desorption sampling, two-dimensional gas chromatography, and mass detection. Such a methodology is certainly very detailed and useful when advanced studies have to be done, like in chemotaxonomy. For routine analyses, however, such a setup is probably too complicated and expensive.

The aim of this work was to find appropriate conditions, mainly in terms of sample preparation, for a simultaneous analysis of both groups of compounds, while keeping the overall experimental and instrumental setups simple.

Results and discussion

Sample preparation

As already stated in the introduction, the main challenge in combined analysis of terpenes and cannabinoids is the sample preparation step, and more precisely the extraction conditions. As Namdar et al. have noted[7], optimum solvent composition for terpenes and cannabinoids differs. During their work, they found the mixture of ethanol:hexane (3:7, v/v) to be the best compromise for extracting both groups of compounds. In this present work, the quest for a single solvent similar in properties to the mentioned work was undertaken. The solvent selection was then narrowed to acetone and ethyl acetate, with the added benefit of them being solvents with low environmental impact.[40] Finally, acetone was selected as the most appropriate solvent, based on the extraction recoveries obtained.

Another important parameter highly affecting the results is the sample-to-volume ratio during extraction. Besides having a direct effect on extraction efficiency as well, this ratio also has implications on the final analyte concentrations. Good overall analyte recoveries were obtained with sample-to-volume ratios between 1:10 and 1:25. During method development, it was found that a ratio of about 1:17 (i.e., 300 mg per 5 mL of solvent) was a good compromise between extraction efficiency while at the same time still providing sufficient concentrations of terpenes in working sample solutions in order to be quantified without concentrating the solution. Terpenes are unfortunately very volatile, and significant analyte losses can be expected with any of the solvent evaporation techniques.[7] Therefore, this sample preparation step was deliberately avoided. At the same time, cannabinoid concentrations in sample solutions proved to be below the upper practical limit in terms of detector linearity. Compared to terpenes, cannabinoids are more problematic to analyze. Besides their lower volatility, the possibility of incomplete decarboxylation of cannabinoid acidic forms during sample vaporization and injection must be prevented. This phenomenon is more pronounced at higher concentrations.[29]

In practice, the concentrations of major cannabinoids in the extracts were kept at up to 1.5 mg/mL or below. By using such high cannabinoid concentrations, it was less challenging to quantify minor cannabinoids as well.

Gas chromatographic separation

Terpenes and cannabinoids differ widely, both in terms of polarity and volatility. These two groups of compounds are therefore easily separated between each other using GC, although a wide temperature gradient program is needed due to a large difference in volatility. Successful separation of individual terpenes is not particularly challenging, as many works demonstrate.[7][8][36][38][41][42][43] The main challenge was to provide good separation of some cannabinoids, the most critical being the resolution between cannabichromene (CBC) and cannabidiol (CBD). Using the ubiquitous stationary phase based on 5% phenyl 95% dimethylpolysiloxane, the resolution between those two peaks proved to be unsuitable, since these two peaks overlapped, as proven by preliminary tests (data not published). Much better results are obtained using more polar stationary phases like 35% phenyl 65% dimethylpolysiloxane, as recent applications also demonstrate.[34][35] As a consequence, the choice for an even more polar stationary phase was made, namely 50% phenyl 50% dimethylpolysiloxane. According to initial expectations, a good resolution was obtained, and no issues related to overlapping of cannabinoid peaks were observed anymore. At the same time, using a relatively polar stationary phase did not impair the separation of terpenes. In fact, even more polar stationary phases were employed for terpenes.[41][42][43] Chromatograms of standard solutions and sample extracts are depicted on Figure 1, Figure 2. and Figure 3.


Fig1 Zekič Molecules2020 25-24.png

Fig. 1 Chromatograms (displayed tR = 13.5–17.0 min) of cannabinoid standard solution (top) and hemp plant (cannabigerol (CBG) chemotype) extract (bottom). Peak labelling: IS—internal standard, CBC—cannabichromene, CBD—cannabidiol, Δ8-THC—Δ8-tetrahydrocannabinol, Δ9-THC—Δ9-tetrahydrocannabinol, CBG—cannabigerol, CBN—cannabinol.

Fig2 Zekič Molecules2020 25-24.png

Fig. 2 Chromatograms (displayed tR = 3.0–9.5 min) of terpene standard solution (top) and hemp plant extract (bottom). *β-caryophyllene was identified by mass-spectrometric data.

Fig3 Zekič Molecules2020 25-24.png

Fig. 3 Chromatogram of hemp sample extract (full scale).

Method performance and validation

The developed method exhibited good overall analytical performance within a relatively short analysis time, since it provided separation of two major groups of compounds in the cannabis plant. The most difficult to separate, CBD and CBC, were fully baseline resolved. Other analytes were also separated with excellent resolution. Validation and stability data (Table 1) confirmed the suitability of the method also for quantitative use. Sufficiently low sensitivity limits, which are important (especially from the terpenes standpoint), while also maintaining accurate results for higher cannabinoid concentrations, allow for the use of non-concentrated or non-diluted working sample solutions. This is a great advantage, especially in view of the process' simplicity.

Table 1. Method validation parameters. LOD = limit of detection, LOQ = limit of quantification. a Determination of LOD and LOQ was based on extrapolation of signal-to-noise responses. Data in parentheses are validation parameters obtained with our HPLC method[33] for comparison purposes (where applicable). b Second consecutive extraction of samples gave no detectable peaks for the analyte.
Analyte Injection Precision (% RSD, n = 5) Accuracy (%) Extraction Efficiency (%) Repeatability (% RSD, n = 3) Intermediate Precision

(% RSD, n = 9)

LOD a (µg/mL) LOQ a (µg/mL) Regression Coefficient (R) Stability 48 h (%)
cannabidiol
cannabigerol
Δ8-tetrahydrocannabinol
Δ9-tetrahydrocannabinol
cannabichromene
cannabinol
α-pinene
β-pinene
myrcene
limonene
α-terpinene
α-humulene


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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.