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Comparison of Three Gas Chromatographic Methods – Identification of Terpenes and Terpenoids in Cannabis sativa L.

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05 June 2024

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07 June 2024

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Abstract
Terpenes and terpenoids content in cannabis plant was already studied in the past with three used methods. Since these works did not compare the content of these substances under the same conditions, we tried to make this comparison exactly. Three different gas chromatography/mass spectrometry (GS/MS) methods - hexane based liquid extraction (Liq), static head-space extraction (HS), and head-space solid phase microextraction (SPME) – were compared to identify volatile compounds in four different cannabis chemotypes - Green fields, Titan chemotype, Black Domina chemotype, and Neptune chemotype. The main compounds focused on were monoterpenes/monoterpenoids and sesquiterpenes/sesquiterpenoids. Extraction with hexane gave comparable results, however the other two methods allowed for the identification of more substances. For the final evaluation of the comparison of the three methods of analysis, extraction with hexane gives comparable results (which is advantageous for quantitative analysis), although the other two methods allowed for the identification of more substances. This means that the same method should be used everywhere for the quantitative evaluation of constituents in cannabis.
Keywords: 
Gas chromatography
;  
liquid sample
;  
head-space
;  
SPME
;  
terpenes
;  
terpenoids
Subject: 
Chemistry and Materials Science  -   Analytical Chemistry

1. Introduction

Today we know, without any doubt, that the presence of terpenes and terpenoids in the Cannabis sativa L. plant is important, not only from the biogenetical point of view but also from the medical perspective. These bioactive compounds play an important role concerning use of cannabis as a medicament. Unfortunately, up-to-date there is not enough knowledge concerning the importance of these compounds and their ratio with others bioactive compounds, mainly cannabinoids. At present, it is very important to clarify the importance of the compounds quantity, the content compound types and their ratios, and to understand the medicinal power of this plant for the treatment of various diseases.
The biosynthesis of terpenes in cannabis is comprised of two different pathways [1,2] . The first pathway is the plastidial methylerythritol phosphate pathway [3], which starts with the substrates pyruvate and glyceraldehyde-3-phosphate [4,5]. Through several steps geranyl diphosphate is produced, which then interacts with olivetolic acid for cannabigerolic acid origination. Additionally, this pathway is also a precursor for monoterpene origination [6,7]. The second pathway is the cytosolic mevalonate pathway [8] which starts with acetoacetyl-CoA and after several steps forms farnesyl diphosphate followed by sesquiterpenes formation.
Terpenes found in hemp are not unique to this plant, as they are also found in other plants. Many terpenes have medical potential but their bioactivity obviously depends on the cannabis chemotype, due to terpenes and cannabinoids having different quantitative contents and ratios [9,10,11,12,13,14,15]. Of course, both the cannabis chemotype used and the patient’s genetics play a major role in the treatment. In addition, the amount used in one patient may be too high and in another one insufficient. At present, we need to know much more about the biodynamic effect of terpenes in cannabis in order to be able to breed cannabis plants that are suitable for therapeutic use. We must understand that the therapeutic effectiveness of terpenes contained in cannabis is different when we use plant material (by smoking, vaporization or in capsules), or as extracts (in oil, capsules, suppositories, creams and the other preparations). We must also understand that many terpenes are not stable compounds. It is possible that some of these phytochemicals are artifacts that formed in the resin on the plant due to various reasons (as described below). For instance, UV sunlight exposure, harvesting, drying, storage and processing of the plant, and even during the analysis. Smoking and vaporization gives rise to other substances, not fully studied, that can also affect patients (or other users) either positively or negatively. So far, there is a discussion on whether terpenes act as such or have a synergistic or entourage effect [16,17,18,19,20,21].
LaVigne [22] found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice [23,24,25,26], suggesting cannabimimetic activity. Further, some mice behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action.
We must take into consideration that upon aging essential oils can undergo oxidation and polymerization which may result in a loss of pharmacological properties. Heat, light and air can lead to its oxidation, polymerization, isomerization, thermal rearrangement, or dehydrogenation [27]. Inflorescence stored using the novel packaging approach is a significant step towards providing patients with cannabis inflorescence of reproducible and reliable terpene content, an important component of inflorescence efficacy [28].
The aim of this study was to compare three different gas chromatography/mass spectrometry methods - hexane based liquid extraction (Liq), static head-space extraction (HS), and head-space solid phase microextraction (SPME), - in order to determine which method best identified volatile compounds in cannabis samples, mainly monoterpenes/monoterpenoids and sesquiterpenes/sesquiterpenoids. We used four chemotypes to also compare possibilities of content compounds identification in different samples.

2. Experimental

2.1. Methods

Standards: Commercially available standards for α-pinene, camphene, β-pinene, myrcene, Δ3-carene, α-terpinene, p-cymene, limonene, 1,8-cineole, α-ocimene, trans-β-ocimene, γ-terpinene, terpinolene, linalool, isopulegol, geraniol, β-caryophyllene, α-humulene, cis-nerolidol, trans-nerolidol, caryophyllene oxide, guaiol, and α-bisabolol were obtained from Restek (Bellefonte, PA, USA).
Plant material: Dry female flowering tops of four different chemotypes (LOH LL1 - Green fields chemotype, LOH LL2 – Titan chemotype, LOH LL3 – Black Domina chemotype, LOH LL4 – Neptune chemotype) used for medical treatment, cultivated in Israel, were used for analysis. These four varieties were selected randomly to compare different chemotypes.
Sample preparation: The ground plant material was extracted with n-hexane (final concentration was 1 mg/ml) with occasional shaking for half an hour. One microgram of the sample thus prepared was injected for Liq analysis. 25 mg of plant material was used for HS and 0.3 mg for SPME analysis.
Instrument: GC/MS [Agilent 7890B GC, Agilent 5977B MSD, PAL 3 (RSI 85)].
Column: Agilent Technologies, Inc., HP-5MS UI, 30 m x 250 μm, film 0.25 μm.

2.2. Experimental Conditions for HS

Incubation time: 6 min; Incubation temperature: 80ºC.

2.3. Experimental Conditions for SPME:

Incubation time: 10 min, incubation temperature: 60ºC, GC cycle time: 5 min, fiber conditioning station temperature: 250 ºC, pre desorption conditioning time: 2 min, sample extraction time: 10 min, sample desorption time: 1 min

2.4. Experimental Condition for All Three Analyses

The column temperature was initially 35 °C for 5 min, followed by temperature ramping from 35-150 °C at 5 °C/min, then to 250 °C at 15 °C/min (inlet: 250 °C; detector: 280 °C; split ratio 5:1;); gas: helium (flow rate: 1 mL/min).
Analytical method validation - selectivity, specificity, accuracy, precision, linearity, range, limit of detection, limit of quantification, ruggedness, and robustness were performed [29]. They are beyond the scope of this manuscript and will be published in another publication.

2.5. Identification

The content compounds were identified by comparison to standards, retention times, retention indices, mass spectra of particular compounds, and the spectral matching of libraries NIST/EPA/NIH Mass Spectral Library 2017, Wiley Registry of Mass Spectral Data 11th Edition, FFNSC3, ©2015, and Adams Essential Oils Library.

3. Results

As we did not have all the main compounds as standards, it was impossible to quantify all these terpenes/terpenoids exactly. We therefore “quantified” volatile compounds by a normalization method. We chose the biggest peak (by space) as 100% and calculated the relative ratios of the other ones (in Tables as. % norm). The ten main peaks in each method are marked in Tables below in bold.
Sample LOH LL1 (Table 1) revealed β-myrcene as the biggest peak in HS and β-caryophyllene in Liq and SPME methods. There are 7 different compounds above 50%; in norm Liq – 7 ones above 50%, in HS – 1 above 50%, and in SPME – 6 above 50%. Between the ten main compounds above 50% were identified β-myrcene - 3 x, β-caryophyllene – 2 x, γ-elemene – 1 x, α-humulene – 2 x, α-bulnesene 2 x, γ-selinene – 2 x, selina-3,7(11)-diene - 2 x. Altogether within the ten main compounds 14 different terpenes/terpenoids were identified. Altogether 51 compounds were identified by HS (97.53% of total volatiles), 46 compounds by SPME (88.36%) and 37 compounds by liquid (72.73%) GC/MS, what was together 67 different volatile compounds. The same 28 compounds were found in all three analyses. Comparison of all three methods of analysis is presented in Chromatogram 1 - Liq (Chromatogram 1a), HS (Chromatogram 1b), and SPME (Chromatogram 1c).
Chromatogram 1a. Hexane based liquid extraction method (Liq) - sample LOH LL1 chemotype. Counts vs. Acquisition Time (min)
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Chromatogram 1b. Static head-space extraction method (HS) - sample LOH LL1 chemotype. Counts vs. Acquisition Time (min)
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Chromatogram 1c. Head-space solid phase microextraction method (SPME) - sample LOH LL1 chemotype. Counts vs. Acquisition Time (min)
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Chromatogram 1. Comparison of all three methods of analysis - sample LOH LL1 chemotype. Counts vs. Acquisition Time (min).
Chromatogram 1. Comparison of all three methods of analysis - sample LOH LL1 chemotype. Counts vs. Acquisition Time (min).
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Results of analysis of the sample LOH LL2 are presented in Table 2. Limonene was the biggest peak in HS and β-caryophyllene in Liq and SPME methods. Three different compounds were above 50%; in norm Liq – 3 above 50%, in HS – 1 above 50%, and in SPME – 2 above 50%. Of the ten main compounds identified above 50% were limonene 1 x, β-caryophyllene – 2 x, and α-humulene – 1 x. Altogether 17 different terpenes/terpenoids were found between the ten main compounds. The same 24 compounds were found in all three analyses. Altogether 74 compounds were identified by HS (97.87% of total volatiles), 57 compounds by SPME (90.80%) and 38 compounds by liquid (80.02%) GC/MS, what was together 94 different volatile compounds. Comparison of all three methods of analysis is presented in Chromatogram 2.
In Table 3 limonene was the biggest peak in HS and SPME and selina-3,7(11)-diene in Liq. Five different compounds were above 50%; in norm Liq – 4 above 50%, in HS – 2 above 50%, and in SPME – 5 above 50%. Between the ten main compounds identified using all three methods was above 50% β-myrcene – 2 x, limonene – 3 x, β-caryophyllene – 2 x, γ-selinene – 2 x, selina-3,7(11)-diene – 2 x. Altogether there were 20 different terpenes/terpenoids between the ten main compounds. The same 21 compounds were found in all three types of analysis. Altogether 55 compounds were identified by HS (96.45%), 49 compounds by SPME (85.43%) and 45 compounds by Liq (86.85%) GC/MS, what was together 84 different volatile compounds. Comparison of all three methods of analysis is presented in Chromatogram 3.
Table 4 pointed out limonene as the biggest peak in HS and β-caryophyllene in Liq and SPME. Four different compounds were above 50%; in norm Liq – 1 above 50%, in HS – 3 above 50%, and in SPME – 2 above 50%. Between the ten main compounds gathered from all three methods were above 50% β-myrcene – 1 x, limonene – 1 x, β-caryophyllene – 3 x, and α-humulene – 1 x. Altogether 17 different terpenes/terpenoids were identified between the ten main compounds were. The same 21 compounds were found in all three types analysis. Altogether 57 compounds were identified by HS (97.17%), 47 compounds by SPME (88.85%), and 34 compounds by Liq (88.10%) GC/MS, what was together 77 different volatile compounds. Comparison of all three methods of analysis is presented in Chromatogram 4.
A comparison of the number of identified substances for the four different chemotypes by the three different methods can be found in the Table 5 and results of quantitative determination of compounds for which we had commercially available standards are in Table 6.

4. Discussion

Gas chromatography analysis of the essential oil from Cannabis sativa was published already in 1957 [30]. In oil from fresh large leaves of female Cannabis sativa the compounds identified by gas chromatography were myrcene, limonene, α-humulene, and β-caryophyllene [31]. By steam distillation of fresh Indian Cannabis sativa L. from Kashmir 21 terpenes/terpenoids were identified in the essential oil [32].
Static headspace gas chromatography has already been used for marijuana and hashish analysis by Hood et al. [33,34]. They identified 16 terpenes and 1 terpenoid in the samples. For simultaneous quantification of 93 terpenoids present in air-dried Cannabis inflorescences and extracts static headspace – GC/MS/MS was used [35]. We also used GC/MS for identification of volatiles in different chemotypes of cannabis (medical and industrial) and published content volatiles, mostly terpenes/terpenoids, and their ratios in cannabis inflorescences and essential oils [36]. Thirteen chemotypes with different main terpenes/terpenoids were presented.
Solid-phase microextraction GC/MS was used to identify cannabidiol, Δ8-tetrahydrocannabinol, Δ9-tetrahydrocannabinol, and cannabinol in pure water and human saliva [37]. For cannabinoids determination in cannabis samples GC/MS method was developed [38]. Yang et al. [39] identified 13 monoterpenes, 4 monoterpenoids, and 14 sesquiterpenes in cannabis essential oil. The three mentioned gas chromatography techniques (HS, SPME, and liquid injection) were compared [40]. All three were excellent for the lower boiling monoterpenes. In HS sesquiterpenes were underrepresented. SPME gave a stronger signal for early eluting sesquiterpenes. Higher boiling sesquiterpenes were only adequately represented in liquid injection (hexane extract). Myers et al. [41] compared headspace-syringe and liquid injection-syringe techniques to the more modern headspace-solid phase microextraction arrow and direct immersion-SPME arrow. They used 23 terpene/terpenoids standards and determined from the results that the liquid injection-syringe method is the most straightforward and robust method.
We compared three different gas chromatography/mass spectrometry methods - static head-space extraction, head-space solid phase microextraction, and hexane based liquid extraction - to identify volatile compounds in cannabis samples, mainly monoterpenes/monoterpenoids and sesquiterpenes/sesquiterpenoids. We found hexane to be the best solvent for analysis of a liquid samples. Liquid samples give the most complex spectrum of the main mono- and sesquiterpenes/terpenoids as sesquiterpenes/sesquiterpenoids can be seen with higher retention times. Such liquid samples also have the advantage for absolute quantification of terpenes. The static headspace chromatogram gives the best representation of monoterpenes and monoterpenoids but a weaker signal for sesquiterpenes and sesquiterpenoids. Solid-phase microextraction gives significant spectrum of sesquiterpenes and sesquiterpenoids with shorter retention times and weaker signal for monoterpenes and monoterpenoids.
It seems that the results of Liq and SPME are the most similar (but not in all cases), so the analysis of the extract prepared with an organic solvent (hexane) will be the most suitable for the quantitative determination of these substances.
Altogether 26 terpenes/terpenoids were between the ten main present in four different chemotypes. They were divided with Liq containing 16 terpenes/terpenoids (2 terpenes, 1 terpenoid, 9 sesquiterpenes, and 4 sesquiterpenoids), 15 in HS (5 terpens, 2 terpenoids, 8 sesquiterpenes) and 17 in SPME (4 terpenes, 3 terpenoids, 10 sesquiterpenoids). The main terpene in chemotype LOH LL3 from the HS and SPME methods was limonene, but as we can see from Table 3 the other terpenes/terpenoids do not follow the same relative ratio. Chemotype LOH LL4 has a similar situation. The main terpene in Liq and SPME is β-caryophyllene, but as we can see from Table 4 the other terpenes/terpenoids do not follow the same relative ratio. As we can see from Tables 1 to 4, the terpenes found most often from amongst the ten main were β-myrcene, limonene, β-caryophyllene, α-humulene, γ-selinene, and selina-3,7(11)-diene. The most reproducible analysis for quantitative analysis appears to be from Liq samples.
It is now well known that different cannabis chemotypes (also mentioned as variety, strain, chemovar, cultivar, phenotype, or genotype) with the same content of major cannabinoids act differently in the treatment of the same patient. Not every chemotype is suitable for a given patient. Cannabis constituents (whether cannabinoids, terpenes/terpenoids or the other bioactive substances) interact with each other and can thus increase (or decrease) the effectiveness of a given chemotype [16,42]. If these effects are independent, synergistic or entourage, they still need to be thoroughly studied. In addition, each of us is genetically different and therefore one chemotype that is suitable for one patient might not be suitable for another one. It should also be emphasized that a given chemotype of cannabis that is suitable for a particular patient in a given disease may not (but sometimes may) cure a different disease.

5. Conclusions

In conclusion we can say that for the final evaluation of the comparison of the three methods of analysis, extraction with hexane gives balanced results (which is advantageous for quantitative analysis), although the other two methods allowed for the identification of more substances. This means that the same method should be used everywhere for the quantitative evaluation of constituents in cannabis. Only in this way it will be possible to objectively compare the results from different laboratories. The differences for the same sample analyzed in different laboratories will then be within the allowable error range.

Acknowledgments

My thanks to Margalit Lillie Beck for manuscript reading and language corrections.

Statement of Ethics

The author has no ethical conflicts to disclose.

Conflicts of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

There were no external funding sources to the study in the preparation of data or the manuscript.

Abbreviations

Liq: hexane based liquid extraction; HS: static head-space extraction; SPME: head-space solid phase microextraction; Key: RT = retention time, % norm = % of the given peak relative to the main peak (main peak = 100 %), RI = retention index

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Chromatogram 2. Comparison of all three methods of analysis - sample LOH LL2 chemotype. Counts vs. Acquisition Time (min).
Chromatogram 2. Comparison of all three methods of analysis - sample LOH LL2 chemotype. Counts vs. Acquisition Time (min).
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Chromatogram 3. Comparison of all three methods of analysis - sample LOH LL3 chemotype. Counts vs. Acquisition Time (min).
Chromatogram 3. Comparison of all three methods of analysis - sample LOH LL3 chemotype. Counts vs. Acquisition Time (min).
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Chromatogram 4. Comparison of all three methods of analysis - sample LOH LL4 chemotype. Counts vs. Acquisition Time (min).
Chromatogram 4. Comparison of all three methods of analysis - sample LOH LL4 chemotype. Counts vs. Acquisition Time (min).
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Table 1. GC/MS identification of the dry flowering tops - sample LOH LL1 chemotype.
Table 1. GC/MS identification of the dry flowering tops - sample LOH LL1 chemotype.
Peak RT % norm (Liq) % norm (HS) %
norm (SPME) 		Compounds Type RI
1 6.429 0.02 2,3-butanediol glycol 788
2 7.789 0.72 2,4-dimethylheptane hydrocarbon 821
3 9.212 0.20 ethylbenzene aromatic hydrocarbon 855
4 9.379 0.48 4-methyloctane hydrocarbon 863
5 9.509 0.75 p-xylene aromatic hydrocarbon 865
6 9.524 0.08 1-hexanol organic alcohol 868
7 10.718 0.37 0.05 heptanal alkyl aldehyde 901
8 11.223 0.92 1.29
 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane bicyclic monoterpene 921
9 11.600 0.04 α-thujene bicyclic monoterpene 929
10 11.808 4.56 5.55 0.54 α-pinene bicyclic monoterpene 937
11 12.329 1.35 1.52 0.26 camphene bicyclic monoterpene 952
12 12.810 0.02 benzaldehyde aromatic aldehyde 962
13 13.372 8.38 10.07 1.50 β-pinene bicyclic monoterpene 979
14 13.893 0.03
 6-methyl-5-hepten-2-one
 unsaturated methylated ketone 986
15 14.053 95.78 100.00 86.64 β-myrcene acyclic monoterpene 991
16 14.887 0.23 α-terpinene monocyclic monoterpene 1017
17 15.111 0.02 0.08 p-cymene monocyclic monoterpene 1025
18 15.247 32.07 30.90 30.66 limonene monocyclic monoterpene 1030
19 15.328 0.13 1,8-cineole bicyclic monoterpenoid 1032
20 15.624 0.06 cis-β-ocimene acyclic monoterpene 1038
21 15.969 0.01 0.19 trans-β-ocimene acyclic monoterpene 1049
22 16.273 0.06 0.15 γ-terpinene monocyclic monoterpene 1060
23 16.554 0.04 sabinene hydrate bicyclic monoterpenoid 1068
24 17.243 0.34 1.13 terpinolene monocyclic monoterpene 1088
25 17.636 6.38 2.90 2.86 linalool acyclic monoterpenoid 1099
26 18.037 4.64 1.29 3.46 fenchyl alcohol bicyclic monoterpenoid 1113
27 18.286 3.07 0.71 cis-pinene hydrate bicyclic monoterpenoid 1121
28 18.590 0.25 neo-allo-ocimene acyclic monoterpene 1131
29 19.079 0.03 ipsdienol acyclic monoterpenoid 1147
30 19.672 0.96 0.15 0.52 borneol bicyclic monoterpenoid 1166
31 20.025 1.12 0.20 0.70 terpinen-4-ol monocyclic monoterpenoid 1177
32 20.426 3.40 0.39 0.91 α-terpineol monocyclic monoterpenoid 1189
33 20.706 0.10 dodecane alkane hydrocarbon 1200
34 24.931 0.06 α-cubebene tricyclic sesquiterpene 1351
35 25.516 0.56 0.09 1.18 ylangene tricyclic sesquiterpene 1372
36 25.637 0.23 0.07 0.63 α-copaene tricyclic sesquiterpene 1376
37 25.813 0.18 β-patchoulene tricyclic sesquiterpene 1381
38 25.997 0.03 0.31 7-epi-sesquithujene bicyclic sesquiterpene 1391
39 26.206 0.31 tetradecane alkane hydrocarbon 1400
40 26.462 0.03 1.56 cis-β-caryophyllene bicyclic sesquiterpene 1406
41 26.639 1.11 0.19 3.33 cis-α-bergamotene bicyclic sesquiterpene 1415
42 26.791 100.00 12.73 100.00 β-caryophyllene bicyclic sesquiterpene 1419
43 26.879 0.02 γ-maaliene tricyclic sesquiterpene 1430
44 27.024 0.02 β-copaene tricyclic sesquiterpene 1433
45 27.144 71.31 14.69 γ-elemene monocyclic sesquiterpene 1434
46 27.160 5.60 1.35 15.36 α-bergamotene bicyclic sesquiterpene 1436
47 27.248 19.06 2.44 25.89 α-guaiene bicyclic sesquiterpene 1439
48 27.376 0.43 0.06 1.39 guaia-6,9-diene bicyclic sesquiterpene 1443
49 27.585 2.32 humulen-(v1) bicyclic sesquiterpene 1455
50 27.649 55.13 5.56 61.27 α-humulene monocyclic sesquiterpene 1454
51 28.042 0.27 0.02 0.86 4,5-di-epi-aristolochene bicyclic sesquiterpene 1467
52 28.194 6.30 0.39 6.68 γ-muurolene bicyclic sesquiterpene 1477
53 28.282 0.07 α-amorphene bicyclic sesquiterpene 1485
54 28.370 4.35 0.36

 5.68

 4a,8-Dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene bicyclic sesquiterpene 1492
55 28.434 13.06 0.92 16.48 β-selinene bicyclic sesquiterpene 1486
56 28.555 3.73 δ-selinene bicyclic sesquiterpene 1488
57 28.627 16.16 1.21 23.47 α-selinene bicyclic sesquiterpene 1494
58 28.835 56.69 4.39 56.51 α-bulnesene bicyclic sesquiterpene 1505
59 28.988 0.08 1.86 γ-cadinene bicyclic sesquiterpene 1513
60 29.124 13.65 0.87 16.32 δ-amorphene bicyclic sesquiterpene 1519
61 29.380 69.01 3.66 67.76 γ-selinene bicyclic sesquiterpene 1544
62 29.493 73.42 4.35 89.15 selina-3,7(11)-diene bicyclic sesquiterpene 1542
63 29.757 31.07 4.40 0.37 germacrene B monocyclic sesquiterpene 1557
64 30.158 3.53 0.08 caryophyllene oxide bicyclic sesquiterpenoid 1581
65 31.312 0.57
 cadalene
 bicyclic aromatic hydrocarbon 1674
66 31.575 4.56 juniper camphor bicyclic sesquiterpenoid 1691
67 32.378 0.17 guaiazulene bicyclic sesquiterpene 1775
Table 2. GC/MS identification of the dry flowering tops - sample LOH LL2 chemotype.
Table 2. GC/MS identification of the dry flowering tops - sample LOH LL2 chemotype.
Peak RT % norm (Liq) % norm (HS) % norm (SPME) Compound Type RI
1 2.966 0.12 2-methylbutanal saturated fatty aldehyde 662
2 4.578 0.21 2-methyl-1-butanol alcohol 739
3 6.373 0.67 DL-2,3-butanediol glycol 773
4 6.510 0.11 2,3-butanediol glycol 788
5 7.799 0.12 2,4-dimethyl-heptane hydrocarbon 821
6 9.508 0.53 1-hexanol alcohol 868
7 10.718 0.16 heptanal saturated fatty aldehyde 901
8 11.231 0.32
 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane bicyclic monoterpene 921
9 11.608 0.09 α-thujene bicyclic monoterpene 929
10 11.808 4.55 15.82 0.93 α-pinene bicyclic monoterpene 937
11 12.33 1.39 4.88 0.98 camphene bicyclic monoterpene 953
12 12.811 0.05 benzaldehyde aromatic aldehyde 962
13 13.372 7.74 25.48 1.24 β-pinene bicyclic monoterpene 979
14 13.901 0.75 2,2,4,6,6-pentamethylheptane hydrocarbon 991
15 14.013 10.24 37.32 8.73 β-myrcene acyclic monoterpene 991
16 14.358 0.12 ethyl hexanoate fatty acid ester 1000
17 14.839 0.16 0.52 α-terpinene monocyclic monoterpene 1017
18 14.983 0.05 p-menth-1-ene monocyclic monoterpene 1025
19 15.111 0.07 0.74 p-cymene monocyclic monoterpene 1025
20 15.272 36.55 100.00 23.48 limonene monocyclic monoterpene 1030
21 15.336 0.31 1,8-cineole bicyclic monoterpenoid 1032
22 15.624 0.20 0.13 cis-β-ocimene acyclic monoterpene 1038
23 16.017 0.65 trans-β-ocimene acyclic monoterpene 1049
24 16.282 0.11 0.29 γ-terpinene monocyclic monoterpene 1060
25 16.546 0.21 cis-sabinene hydrate bicyclic monoterpenoid 1070
26 16.739 0.13 linalool oxide monocyclic monoterpenoid 1086
27 17.283 1.69 terpinolene monocyclic monoterpene 1088
28 17.644 7.96 11.36 11.42 linalool acyclic monoterpenoid 1099
29 17.730 1.53 undecane hydrocarbon 1100
30 17.789 0.06 nonanal saturated fatty aldehyde 1104
31 18.037 7.21 7.27 6.74 fenchol bicyclic monoterpenoid 1113
32 18.286 5.65 4.61 1.64 cis-pinene hydrate bicyclic monoterpenoid 1121
33 18.438 0.19 0.25 methyl octanoate fatty acid ester 1126
34 18.590 0.49 neo-allo-ocimene acyclic monoterpene 1131
35 18.919 0.67 0.53 trans-pinene hydrate bicyclic monoterpenoid 1140
36 19.015 0.07 camphor bicyclic monoterpenoid 1145
37 19.119 0.22 0.20 camphene hydrate bicyclic monoterpenoid 1148
38 19.408 0.03 isoborneol bicyclic monoterpenoid 1157
39 19.665 1.42 0.94 1.69 borneol bicyclic monoterpenoid 1166
40 20.025 0.11 0.32 terpinen-4-ol monocyclic monoterpenoid 1177
41 20.418 5.93 3.01 7.99 α-terpineol monocyclic monoterpenoid 1189
42 20.635 0.89 0.79 1.63 ethyl octanoate fatty acid ester 1196
43 23.192 0.12 0.32 bornyl acetate bicyclic monoterpenoid 1286
44 23.801 0.07 0.33 (E)-4-decenoic acid methyl ester fatty acid ester 1299
45 24.202 0.04 0.32 methyl decanoate fatty acid ester 1325
46 24.939 0.10 α-cubebene tricyclic sesquiterpene 1351
47 25.525 0.15 0.79 ylangene tricyclic sesquiterpene 1372
48 25.685 0.33 1.60 ethyl trans-4-decenoate fatty acid ester 1375
49 25.837 0.28 1.20 hexyl hexanoate fatty acid ester 1384
50 25.990 0.17 0.43 7-epi-sesquithujene bicyclic sesquiterpene 1391
51 26.086 0.30 1.27 ethyl decanoate fatty acid ester 1396
52 26.246 0.07 tetradecane hydrocarbon 1400
53 26.302 0.15 cyperene tricyclic sesquiterpene 1399
54 26.390 0.79 0.26 sesquithujene bicyclic sesquiterpene 1402
55 26.463 0.17 2.37 cis-caryophyllene bicyclic sesquiterpene 1406
56 26.647 1.33 0.97 cis-α-bergamotene bicyclic sesquiterpene 1415
57 26.807 100.00 48.85 100.00 β-caryophyllene bicyclic sesquiterpene 1419
58 27.047 2.19 10,10-dimethyl-2,6-dimethylenebicyclo[7.2.0]undecane bicyclic sesquiterpene 1440
59 27.16 10.57 5.28 14.84 α-bergamotene bicyclic sesquiterpene 1435
60 27.248 0.62 0.26 1.15 α-guaiene bicyclic sesquiterpene 1439
61 27.376 0.18 guaia-6,9-diene bicyclic sesquiterpene 1444
62 27.465 0.06 epi-β-santalene bicyclic sesquiterpene 1448
63 27.480 0.30 α-himachalene bicyclic sesquiterpene
64 27.649 42.09 17.38 55.63 α-humulene monocyclic sesquiterpene 1454
65 27.809 0.24 β-santalene bicyclic sesquiterpene 1462
66 28.338 2.08 α-curcumene aromatic sesquiterpene 1483
67 28.370 2.17 0.85 selina-4,11-diene bicyclic sesquiterpene 1474
68 28.402 4.05 4a,8-dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene bicyclic sesquiterpene 1492
69 28.443 2.29 0.67 4.04 β-selinene bicyclic sesquiterpene 1486
70 28.587 0.84 4.19 valencene bicyclic sesquiterpene 1492
71 28.627 1.93 0.67 3.90 α-selinene bicyclic sesquiterpene 1494
72 28.747 0.09 0.36 β-dihydroagarofuran tricyclic sesquiterpenoid 1496
73 28.819 7.14 2.28 9.18 α-farnesene acyclic sesquiterpene 1508
74 28.931 0.62 β-curcumene monocyclic sesquiterpene 1514
75 28.972 0.11 sesquicineole bicyclic sesquiterpenoid 1516
76 29.140 3.00 0.88 4.59 β-sesquiphellandrene monocyclic sesquiterpene 1524
77 29.389 4.11 0.36 3.48 γ-selinene bicyclic sesquiterpene 1538
78 29.493 3.88 0.83 5.12 selina-3,7(11)-diene bicyclic sesquiterpene 1542
79 29.757 0.29 germacrene B monocyclic sesquiterpene 1557
80 29.781 0.94 nerolidol acyclic sesquiterpenoid 1544
81 30.158 4.86 0.51 caryophyllene oxide bicyclic sesquiterpenoid 1581
82 30.334 16.49 0.46 1.11 guaiol bicyclic sesquiterpenoid 1596
83 30.455 0.84 0.02 5-epi-7-epi-α-eudesmol bicyclic sesquiterpenoid 1616
84 30.527 0.06 humulene epoxide II bicyclic sesquiterpenoid 1606
85 30.663 22.81 0.55 1.91 10-epi-γ-eudesmol bicyclic sesquiterpenoid 1619
86 30.799 5.38 0.04 γ-eudesmol bicyclic sesquiterpenoid 1631
87 30.859 1.07 agarospirol bicyclic sesquiterpenoid 1645
88 31.040 7.59 0.07 β-eudesmol bicyclic sesquiterpenoid 1649
89 31.072 13.73 0.11 α-eudesmol bicyclic sesquiterpenoid 1653
90 31.224 19.51 0.09 0.35 bulnesol bicyclic sesquiterpenoid 1667
91 31.320 0.34 cadalene bicyclic aromatic hydrocarbon 1674
92 31.575 1.62 juniper camphor bicyclic sesquiterpenoid 1691
93 32.523 0.31 α-phellandrene dimer tricyclic terpene 1801
94 33.733 0.13 hexadecanoic acid saturated fatty acid 1968
Table 3. GC/MS identification of the dry flowering tops - sample LOH LL3 chemotype.
Table 3. GC/MS identification of the dry flowering tops - sample LOH LL3 chemotype.
Peak RT % norm (Liq) % norm HS %
norm (SPME) 		Compound Type RI
1 8.987 0.10 3-hexen-1-ol 856
2 9.398 0.51 4-methyl-octane hydrocarbon 863
3 9.528 0.74 p-xylene aromatic hydrocarbon 865
4 9.508 1.02 1.70 1.58 1-hexanol organic alcohol 868
5 11.239 0.21
 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane bicyclic monoterpene 921
6 11.608 0.50 0.45 α-thujene bicyclic monoterpene 929
7 11.824 11.86 24.64 4.11 α-pinene bicyclic monoterpene 937
8 12.345 3.43 7.13 0.93 camphene bicyclic monoterpene 952
9 13.388 17.21 33.51 4.92 β-pinene bicyclic monoterpene 979
10 14.045 41.18 76.91 70.38 β-myrcene acyclic monoterpene 991
11 14.406 0.11 α-phellandrene monocyclic monoterpene 1005
12 14.646 29.77 Δ3-carene bicyclic monoterpene 1011
13 14.879 2.48 α-terpinene monocyclic monoterpene 1017
14 14.999 0.10 p-menth-1-ene monocyclic monoterpene 1025
15 15.119 0.13 38.91 p-cymene monocyclic monoterpene 1025
16 15.288 63.52 100.00 100.00 limonene monocyclic monoterpene 1030
17 15.344 0.75 1,8-cineole bicyclic monoterpenoid 1032
18 15.632 0.20 cis-β-ocimene acyclic monoterpene 1038
19 15.977 0.10 0.79 trans-β-ocimene acyclic monoterpene 1049
20 16.290 0.25 1.92 γ-terpinene monocyclic monoterpene 1060
21 16.554 0.21 cis-sabinene hydrate bicyclic monoterpenoid 1070
22 16.322 0.42 p-cresol phenol derivative 1077
23 16.875 5.67 m-cymenene aromatic compound 1082
24 17.252 1.68 terpinolene monocyclic monoterpene 1088
25 17.308 8.92 p-cymenene aromatic compound 1090
26 17.652 18.06 14.53 11.77 linalool acyclic monoterpenoid 1099
27 17.730 1.46 undecane hydrocarbon 1100
28 18.045 11.22 7.01 8.98 fenchol bicyclic monoterpenoid 1113
29 18.286 6.74 3.40 trans-pinene hydrate bicyclic monoterpenoid 1132
30 19.127 0.40 0.22 camphene hydrate bicyclic monoterpenoid 1148
31 18.871 0.34 5-methyl-undecane hydrocarbon 1156
32 19.296 0.17 2,3-dimethyldecane hydrocarbon 1157
33 19.673 3.14 1.34 2.52 borneol bicyclic monoterpenoid 1166
34 19.817 0.25 0.70 3-methyl-undecane hydrocarbon 1170
35 20.025 0.15 2.48 terpinen-4-ol monocyclic monoterpenoid 1177
36 20.178 1.34 p-cymene-8-ol monocyclic monoterpenoid 1183
37 20.426 8.93 2.37 4.41 α-terpineol monocyclic monoterpenoid 1189
38 20.627 0.34 myrtenal bicyclic monoterpenoid 1193
39 20.705 0.56 dodecane hydrocarbon 1200
40 23.192 0.10 bornyl acetate bicyclic monoterpenoid 1286
41 23.585 1.08 carvacrol monoterpenoid phenol 1299
42 24.931 0.10 α-cubebene tricyclic sesquiterpene 1351
43 25.525 0.57 0.36 2.34 ylangene tricyclic sesquiterpene 1372
44 25.637 0.15 0.71 copaene tricyclic sesquiterpene 1376
45 25.845 0.08 hexyl hexanoate fatty acid ester 1384
46 25.893 0.22 α-bourbonene tricyclic sesquiterpene 1384
47 26.182 0.42 tetradecane hydrocarbon 1400
48 26.471 0.69 cis-β-caryophyllene bicyclic sesquiterpene 1406
49 26.791 50.61 19.34 54.32 β-caryophyllene bicyclic sesquiterpene 1419
50 27.121 14.06 1.55 γ-elemene monocyclic sesquiterpene 1434
51 27.160 3.24 1.49 6.27 α-bergamotene bicyclic sesquiterpene 1435
52 27.248 0.10 0.49 α-guaiene bicyclic sesquiterpene 1439
53 27.376 0.13 0.88 guaia-6,9-diene bicyclic sesquiterpene 1444
54 27.545 1.70 humulen-(v1) bicyclic sesquiterpene 1455
55 27.649 17.43 5.37 22.06 α-humulene monocyclic sesquiterpene 1454
56 28.202 2.60 0.79 4.01 γ-muurolene bicyclic sesquiterpene 1477
57 28.290 0.17 1.77 α-amorphene bicyclic sesquiterpene 1482
58 28.386 2.92

 4a,8-dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene bicyclic sesquiterpene 1492
59 28.378 1.49 0.52 selina-4,11-diene bicyclic sesquiterpene 1474
60 28.443 5.84 1.34 9.13 β-selinene bicyclic sesquiterpene 1486
61 28.531 0.22 δ-selinene bicyclic sesquiterpene 1495
62 28.627 8.87 2.26 13.55 α-selinene bicyclic sesquiterpene 1494
63 28.749 0.87 β-dihydroagarofuran tricyclic sesquiterpenoid 1496
64 28.819 7.49 1.74 6.98 α-farnesene acyclic sesquiterpene 1508
65 28.996 0.73 0.19 1.31 γ-cadinene bicyclic sesquiterpene 1513
66 29.132 18.11 δ-amorphene bicyclic sesquiterpene 1497
67 29.388 73.83 11.61 63.98 γ-selinene bicyclic sesquiterpene 1544
68 29.469 9.56 α-bisabolene monocyclic sesquiterpene 1540
69 29.501 100.00 15.20 84.02 selina-3,7(11)-diene bicyclic sesquiterpene 1542
70 29.757 12.81 3.04 germacrene B monocyclic sesquiterpene 1557
71 30.158 2.54 0.12 caryophyllene oxide bicyclic sesquiterpenoid 1581
72 30.334 22.79 0.36 guaiol bicyclic sesquiterpenoid 1596
73 30.450 1.30 5-epi-7-epi-α-eudesmol bicyclic sesquiterpenoid 1616
74 30.663 27.80 0.38 10-epi-γ-eudesmol bicyclic sesquiterpenoid 1619
75 30.799 9.26 0.06 γ-eudesmol bicyclic sesquiterpenoid 1631
76 30.859 1.46 agarospirol bicyclic sesquiterpenoid 1645
77 31.045 10.68 0.10 β-eudesmol bicyclic sesquiterpenoid 1649
78 31.072 23.23 0.14 α-eudesmol bicyclic sesquiterpenoid 1653
79 31.224 26.57 0.17 bulnesol bicyclic sesquiterpenoid 1667
80 31.312 1.17
 cadalene
 bicyclic aromatic hydrocarbon 1674
81 31.377 14.80 0.08 α-bisabolol monocyclic sesquiterpenoid 1684
82 31.575 7.52 juniper camphor bicyclic sesquiterpenoid 1692
83 32.523 0.36 α-phellandrene dimer tricyclic terpene 1801
84 32.719 1.67 selina-4,7-diol bicyclic sesquiterpenoid 1826
Table 4. GC/MS identification of the dry flowering tops - sample LOH LL4 chemotype.
Table 4. GC/MS identification of the dry flowering tops - sample LOH LL4 chemotype.
Peak RT % norm (Liq) % norm (HS) %
norm (SPME) 		Compound Type RI
1 9.398 0.36 4-methyl-octane hydrocarbon 863
2 9.515 0.98 1-hexanol organic alcohol 868
3 0.51 o-xylene aromatic hydrocarbon 887
4 10.341 0.10 2-heptanone ketone 891
5 10.734 0.20 heptanal alkyl aldehyde 901
6 11.239 1.29
 5,5-Dimethyl-1-vinylbicyclo[2.1.1]hexane bicyclic monoterpene 921
7 11.600 1.58
 0.52 3-methyl-2-butenoic acid ethyl ester fatty acid ester 924
8 11.824 3.89 19.50 0.81 α-pinene bicyclic monoterpene 937
9 12.345 1.19 5.88 0.99 camphene bicyclic monoterpene 952
10 12.818 0.10 benzaldehyde aromatic aldehyde 962
11 13.379 5.53 26.49 β-pinene bicyclic monoterpene 979
12 13.941 0.23 6-methyl-5-heptene-2-one unsaturated methylated ketone 986
13 13.917 0.73 2,2,4,6,6-pentamethylheptane hydrocarbon 991
14 14.037 20.17 87.07 32.79 β-myrcene acyclic monoterpene 991
15 14.398 0.10 α-phellandrene monocyclic monoterpene 1005
16 14.846 0.22 0.29 α-terpinene monocyclic monoterpene 1017
17 14.999 0.07 p-menth-1-ene monocyclic monoterpene 1025
18 15.039 0.39 isomyrcenol acyclic monoterpenoid 1022
19 15.119 0.16 0.32 p-cymene monocyclic monoterpene 1025
20 15.271 26.80 100.00 32.54 limonene monocyclic monoterpene 1030
21 15.343 0.53 1,8-cineole bicyclic monoterpenoid 1032
22 15.632 0.22 cis-β-ocimene acyclic monoterpene 1038
23 16.025 0.58 trans-β-ocimene acyclic monoterpene 1049
24 16.289 0.22 0.26 γ-terpinene monocyclic monoterpene 1060
25 16.562 0.16 cis-sabinene hydrate bicyclic monoterpenoid 1070
26 17.291 1.41 terpinolene monocyclic monoterpene 1088
27 17.644 7.85 14.47 10.37 linalool acyclic monoterpenoid 1099
28 17.813 0.19 nonanal aldehyde 1104
29 18.045 3.93 5.52 3.88 fenchol bicyclic monoterpenoid 1113
30 18.294 2.38 2.79 0.82 trans-pinene hydrate bicyclic monoterpenoid 1140
31 18.598 0.53 allo-ocimene acyclic monoterpene 1144
32 18.927 0.26 cis-pinene hydrate bicyclic monoterpenoid 1121
33 19.135 0.22 camphene hydrate bicyclic monoterpenoid 1148
34 19.672 1.34 1.07 1.08 borneol bicyclic monoterpenoid 1166
35 20.033 0.10 0.56 terpinen-4-ol monocyclic monoterpenoid 1177
36 20.426 3.63 2.13 4.27 α-terpineol monocyclic monoterpenoid 1189
37 21.147 0.21 2,4-dimethyl-benzaldehyde aromatic aldehyde 1181
38 24.931 0.10 α-cubebene tricyclic sesquiterpene 1351
39 15.196 0.54 clovene tricyclic sesquiterpene 1440
40 25.524 0.17 0.81 ylangene tricyclic sesquiterpene 1372
41 25.645 0.18 0.45 copaene tricyclic sesquiterpene 1376
42 25.989 0.22 0.36 7-epi-sesquithujene bicyclic sesquiterpene 1391
43 26.390 0.35 sesquithujene bicyclic sesquiterpene 1402
44 26.462 0.25 2.04 cis-caryophyllene bicyclic sesquiterpene 1406
45 26.647 1.01 1.05 cis-α-bergamotene bicyclic sesquiterpene 1415
46 26.807 100.00 68.80 100.00 β-caryophyllene bicyclic sesquiterpene 1419
47 27.023 0.08 β-copaene tricyclic sesquiterpene 1432
48 27.168 13.59 7.26 20.70 trans-α-bergamotene bicyclic sesquiterpene 1435
49 27.256 17.05 12.19 25.39 α-guaiene bicyclic sesquiterpene 1439
50 27.464 0.08 epi-β-santalene bicyclic sesquiterpene 1448
51 27.585 2.17 humulene-(v1) bicyclic sesquiterpene 1455
52 27.657 45.78 24.62 63.07 α-humulene monocyclic sesquiterpene 1454
53 28.042 0.11 aristolochene bicyclic sesquiterpene 1476
54 28.050 0.62 drima-7,9-diene bicyclic sesquiterpene 1461
55 28.330 3.10 α-curcumene monocyclic sesquiterpene 1483
56 28.378 1.15 4.70 4a,8-dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene bicyclic sesquiterpene 1492
57 28.442 7.71 2.95 16.02 β-selinene bicyclic sesquiterpene 1486
58 28.627 9.44 3.48 20.62 α-selinene bicyclic sesquiterpene 1494
59 28.835 42.10 14.65 45.64 α-bulnesene bicyclic sesquiterpene 1505
60 29.012 1.09 γ-cadinene bicyclic sesquiterpene 1513
61 29.380 25.36 6.27 27.14 γ-selinene bicyclic sesquiterpene 1538
62 29.468 5.77 α-bisabolene monocyclic sesquiterpene 1540
63 29.493 33.77 8.89 37.76 selina-3,7(11)-diene bicyclic sesquiterpene 1542
64 29.757 5.04 1.32 germacrene B monocyclic sesquiterpene 1557
65 30.158 4.70 0.31 caryophyllene oxide bicyclic sesquiterpenoid 1581
66 30.342 28.03 0.82 0.41 guaiol bicyclic sesquiterpenoid 1596
67 30.438 0.09 β-atlantol
 monocyclic sesquiterpenoid 1607
68 30.454 2.11 0.04 5-epi-7-epi-α-eudesmol bicyclic sesquiterpenoid 1616
69 30.663 30.65 0.83 1.04 10-epi-γ-eudesmol bicyclic sesquiterpenoid 1619
70 30.799 11.05 0.22 γ-eudesmol bicyclic sesquiterpenoid 1631
71 30.859 2.03 agarospirol bicyclic sesquiterpenoid 1645
72 31.040 15.65 0.24 β-eudesmol bicyclic sesquiterpenoid 1649
73 31.072 29.06 0.45 α-eudesmol bicyclic sesquiterpenoid 1653
74 31.232 29.44 0.42 0.14 bulnesol bicyclic sesquiterpenoid 1667
75 31.312 0.30 cadalene bicyclic aromatic hydrocarbon 1674
76 31.380 3.67 α-bisabolol monocyclic sesquiterpenoid 1684
77 31.575 3.29 juniper camphor bicyclic sesquiterpenoid 1691
88.10% 97.17% 88.85%
34 cpd 57 cpd 47 cpd
Table 5. Number of identified compounds in different samples by three different methods and all identified different compounds in each chemotype from all three methods.
Table 5. Number of identified compounds in different samples by three different methods and all identified different compounds in each chemotype from all three methods.
GC/MS LOH LL1 LOH LL2 LOH LL3 LOH LL4
Liquid 37 38 45 34
HS 51 74 55 57
SPME 46 57 49 47
all identified different compounds 67 94 84 77
Table 6. Quantitative determination of terpenes/terpenoids for which were commercially available standards.
Table 6. Quantitative determination of terpenes/terpenoids for which were commercially available standards.
Compound LOH LL1
µg/g 		LOH LL2
µg/g 		LOH LL3
µg/g 		LOH LL4
µg/g
α-pinene 45.6 60.0 101.6 47.0
camphene 13.5 18.33 29.38 14.4
β-pinene 83.7 102.0 147.5 62.0
β-myrcene 2136.2 294.3 744.1 544.2
limonene 473.3 705.9 803.7 457.1
linalool 151.5 236.1 367.6 231.3
β-caryophyllene 1322.1 1704.0 542.1 1599.4
α-humulene 535.0 538.4 152.1 605.2
caryophyllene oxide 0.1 0.2 0.2 0.2
guaiol - 313.7 288.8 522.3
α-bisabolol - - 170.0 64.7
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