Headspace SPME-GC/MS Analysis of Terpenes in Hops and Cannabis

Katherine K. Stenerson Principal Scientist
 

In this application, headspace-SPME combined with GC/MS was used to analyze some of the terpenes present in both common hops and cannabis.

Terpenes are small molecules synthesized by some plants. The name terpene is derived from turpentine, which contains high concentrations of these compounds. Terpene molecules are constructed from the joining of isoprene units in a head-to-tail configuration (Figure 1). Classification is then done according to the number of these isoprene units in the structure (Table 1). The configurations of terpenes can be cyclic or open, and can include double bonds, and hydroxyl, carbonyl or other functional groups. If the terpene contains elements other than C and H, it is referred to as a terpenoid.1

 

Figure 1. Isoprene Unit

Isoprene Unit

 

Table 1. Classification of Terpenes

Classification Number of Isoprene Units
Monoterpene 2
Sesquiterpene 3
Diterpene 4
Triterpene 6
Tetraterpene 8

Terpenes are present in essential oils derived from plants and often impart characteristic aromas to the plant or its oil. For example, d-Limonene, which is found in lemon, orange, caraway and other plant oils, has a lemon-like odor. Essential oils, with their component terpenes and terpenoids, have been applied in therapeutic use known as aromatherapy to aid in the relief of conditions such as anxiety, depression, and insomnia.2 This has led to the use of plants which contain these compounds in preparations such as oils, teas, and tonics.

Using Terpene Profile for Plant Identification

The cannabis sativa (cannabis or marijuana) plant contains over 100 different terpenes and terpenoids, including mono, sesqui, di, and tri, as well as other miscellaneous compounds of terpenoid orgin.3 Although the terpene profile does not necessarily indicate geographic origin of a cannabis sample, it can be used in forensic applications to determine the common source of different samples.4 In addition, different cannabis strains have been developed which have distinct aromas and flavors; a result of the differing amounts of specific terpenes present.5 Humulus lupulus (common hops) and cannabis are both members of the family Cannabaceae.6 Consequently, there are similarities in the terpenes each contains. Terpenes give both plant commodities characteristic organoleptic properties and, in the case of cannabis, produce characteristic aromas when the buds are heated or vaporized.7

Experimental

Dried cannabis sample was obtained courtesy of Dr. Hari H. Singh, Program Director at the Chemistry  & Physiological Systems Research Branch of the United States National Institute on Drug Abuse at the National Institute of Health. The extract strain of the sample was not known. Hop flowers of an unknown variety were purchased from an on-line source. Pelletized of Cascade and US Golding hop varieties were purchased at a local home-brew supply shop. Chromatographic separation was performed on an Equity®-1 capillary GC column, and identification was done using retention indices and spectral library match. Final analytical conditions appear in the figures.

SPME Method Optimization

The SPME method was developed using a sample of dried hops flowers (0.2 g in 10 mL vial). The initial SPME parameters were based on previously published work.8 The GC/MS results of this analysis are shown in Figure 2. This initial set of parameters used the 100 µm PDMS fiber, a 1 g sample size, and 60 minute equilibration at room temperature prior to extraction. The sample size was then scaled down to 0.2 g, and the equilibration temperature increased to 40 °C. This increased temperature allowed the equilibration time to be decreased from 60 to 30 minutes without a loss in sensitivity (Figures 3 and 4). The initial extraction time used was 20 min, and a shorter extraction time of 10 minutes was evaluated. However a loss in sensitivity was noted, thus extraction time was maintained at 20 minutes. The DVB/CAR/PDMS fiber was then evaluated (Figure 5). As expected, this fiber extracted more of the lighter compounds, which by MS spectral match, were identified as short chain alcohols and acids.

 

Figure 2. Headspace SPME-GC/MS Analysis of Dried Hops Flowers (100 µm PDMS Fiber, 1 g Sample)

Sample/matrix: 1 g ground hop flowers
SPME fiber: 100 µm PDMS (57341-U)
Sample equilibration: 60 min, room temperature
Extraction: 20 min, headspace, 40 °C
Desorption process: 3 min, 270 °C
Fiber post bake: 3 min, 270 °C
Column: Equity®-1, 60 m x 0.25 mm I.D., 0.25 µm (28047-U)
Oven: 60 °C (2 min), 5 °C/min to 275 °C (5 min)
Inj. temp.: 270 °C
Detector: MSD
MSD interface: 300 °C
Scan range: full scan, m/z 50-500
Carrier gas: helium, 1 mL/min constant flow
Liner: 0.75 mm ID SPME

Headspace SPME-GC/MS Analysis of Dried Hops Flowers

 

Figure 3. Headspace SPME-GC/MS Analysis of Dried Hops Flowers (100 µm PDMS Fiber, 0.2 g Sample)

Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers

Headspace SPME-GC/MS Analysis of Dried Hops Flowers

 

Figure 4. Headspace SPME-GC/MS Analysis of Dried Hops Flowers, Increased Sample Equilibration Temperature (100 µm PDMS Fiber, 0.2 g Sample)

Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers
sample equilibration: 30 min, 40 °C

Headspace SPME-GC/MS Analysis of Dried Hops Flowers

 

Figure 5. Headspace SPME-GC/MS Analysis of Dried Hops Flowers, Increased Sample Equilibration Temperature (DVB/CAR/PDMS Fiber, 0.2 g Sample)

Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers
SPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C
sample/matrix: 0.2 g ground hop flowers
sample equilibration: 30 min, 40 °C

Headspace SPME-GC/MS Analysis of Dried Hops Flowers

Identification of Terpenes Using GC/MS

Using the DVB/CAR/PDMS fiber, samples of hops and cannabis were analyzed using the optimized SPME method. Peak identifications were assigned using MS spectral matching against reference spectra in the Wiley and NIST libraries. Confirmatory identification was done based on retention index. Retention indices were calculated for the compounds identified in each sample using an n-alkane standard analyzed under the same GC conditions. This data was compared with published values (Tables 2 and 3), and final identifications were assigned, as shown in Figures 6 and 7.

Terpenes in Hops Samples

For the dried hop flower sample (Figure 5), the terpene profile should have shown a predominance of β-myrcene, humulene, and caryophyllene, which are typical aroma compounds in hops and hop oil.9 While caryophyllene was identified, both β-myrcene and humulene were not present at levels high enough to be detected by a library search. This may be due to the condition of the sample or the actual variety of hops analyzed since terpene profiles are known to vary between different hop varieties.10 The variety of the hop flowers analyzed is unknown, as the identity was not indicated on the packaging. For comparison, samples of two different varieties of pelletized hops were analyzed after grinding.

These samples appeared green in color, and had a much more characteristic hops-like odor than the dried flowers. Analysis of these samples showed a characteristic terpene profile, with high levels of β-myrcene, caryophyllene, and humulene present in both (Figure 6). The SPME method was able to detect differences in the terpene profiles between the two hops varieties. For example, farnesene (peak 18) was identified in the Cascade hops, but was too low to be confirmed in the US Goldings sample. The level of farnesene in Cascade hops is expected to be 3-7% of total oils, while in US Goldings the level should be <1%.13

Terpenes in Cannabis Sample

The terpenes identified in the cannabis sample (Figure 7) are indicated in Table 3. The profile was similar to those found previously in the analysis of dried cannabis.4,8 Peaks 1-27 in Figure 7 (with the exception of peak 7) were monoterpenes and monoterpenoids. The later eluting peaks consisted of sequiterpenes and caryophyllene oxide, which is a sequiterpenoid. The most abundant terpene was caryophyllene. The predominance of this compound could be due to the specific strain of cannabis tested, and/or the nature of the sample tested, which was dried. Previous studies have shown the level of this compound to increase significantly relative to other terepenes and terpenoids with drying.4 Consequently, the levels of the more volatile monoterpenes and terpenoids would be expected to be less, and this was observed to some degree. Among the monoterpenes and terpenoids the most abundant were α-pinene and d-Limonene.

 

Table 2. Terpenes in Hops Pellets Identified by MS Spectral Library Match and Retention Index.

Peak No. RT (min) Name RI (calculated) RI (literature) Reference
1 8.58 Hexanal   —   780 11
2 12.84 α-Pinene   939   942 11
3 13.28 Camphene   953   954 11
4 13.71 6-Methyl-5- hepten-2-one   966   968 11
5 14.1 β-Pinene   979   981 11
6 14.41 β-Myrcene   988   986 11
7 15.32 Cymene 1018 1020 11
8 15.65 d-Limonene 1030 1030 11
9 15.98 β-Ocimene 1041 1038 11
10 16.72 cis-Linalool oxide 1066 1068 11
11 17.49 Linalool 1089 1092 11
12 21.86 Geraniol 1239 1243 11
13 25.28 Geranyl acetate 1363 1364 11
14 25.85 α-Ylangene 1384 1373 8
15 25.97 α-Copaene 1388 1398 11
16 27.22 Caryophyllene 1437 1428 11
17 27.4 trans-α- Bergamotene + unknown 1445 1443 12
18 17.63 trans-β-Farnesene 1454 1450 8
19 28.11 Humulene 1473 1465 11
20 28.41 γ-Muurolene 1484 1475 11
21 28.45 γ-Selinene 1486 1472 12
22 28.68 Geranyl isobutyrate 1495 1493 11
23 28.79 β-Selinene 1499 1487 8
24 28.94 α-Muurolene 1505 1500 11
25 28.97 α-Selinene 1507 1501 12
26 29.31 γ-Cadinene 1521 1518 11
27 29.37 Calamenene 1524 1518 11
28 29.45 Δ-Cadinene 1527 1524 11
29 30.93 Caryophyllene oxide 1590 1584 8
30 31.5 Humulene oxide 1614 1599 12

 

Figure 6. Headspace SPME-GC/MS Analysis of Hops Pellets Using Final Optimized Method

The peak elution order is listed in Table 2.
Conditions same as Figure 2 except:
sample/matrix: 0.5 g ground hop flowers (hops pellets)
SPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C

Headspace SPME-GC/MS Analysis of Hops Pellets

 

Figure 7. Headspace SPME-GC/MS Analysis of Dried Cannabis Using Final Optimized Method

The peak elution order is listed in Table 3.
Same as Figure 2 except:
sample/matrix: 0.5 g dried, ground cannabis
SPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C

Headspace SPME-GC/MS Analysis of Dried Cannabis

 

Table 3. Terpenes in Dried Cannabis Identified by MS Spectral Library Match and Retention Index

Peak No. RT (min) Name RI (calculated) RI (literature) Reference
1   8.57 Hexanal   —   —
2 10.05 Hexene-1-ol   —   —
3 10.89 2-Heptanone   —   —
4 12.56 α-Thujene   928   938 11
5 12.86 α-Pinene + unknown   939   942 11
6 13.27 Camphene   953   954 11
7 13.69 6-Methyl-5- hepten-2-one   966   968 11
8 14.09 β-Pinene   979   981 11
9 14.27 β-Myrcene   984   986 11
10 15.09 δ-3-Carene 1010 1015 12
11 15.2 α-Terpinene 1014 1012 12
12 15.29 Cymene 1018 1020 11
13 15.6 d-Limonene 1028 1030 11
14 16.42 γ-Terpinene 1056 1057 11
15 16.6 trans-Sabinene hydrate 1062 1078 11
16 16.72 cis-Linalool oxide 1066 1068 11
17 17.43 Linalool 1087 1092 11
18 18.04 d-Fenchyl alcohol 1107 1110 11
19 18.82 trans-Pinocarveol 1135 1134 12
20 19.59 Borneol L 1161 1164 11
21 19.81 1,8-Methandien- 4-ol 1168 1173 8
22 19.81 p-Cymen-8-ol 1168 1172 12
23 19.92 Terpinene-4-ol 1172 1185 11
24 20.22 α-Terpineol 1181 1185 11
25 24.2 Piperitenone 1322 1320 12
26 24.76 Piperitenone oxide 1344 1352 12
27 25.85 α-Ylangene 1384 1373 8
28 25.97 α-Copaene 1388 1398 11
29 26.76 γ-Caryophyllene 1419 1403 12
30 27.01 α-Santalene 1429 1428 12
31 27.16 Caryophyllene 1435 1428 11
32 27.36 trans-α- Bergamotene + unknown 1443 1443 12
33 27.49 α-Guaiene 1448 1441 8
34 27.56 trans-β-Farnesene 1451 1446 12
35 27.98 Humulene 1467 1465 11
36 28.17 Alloaromadendrene 1475 1478 11
37 28.25 α-Curcumene 1478 1479 12
38 28.75 β-Selinene 1497 1487 8
39 28.97 α-Selinene 1507 1497 8
40 28.97 β-Bisobolene 1507 1506 8
41 29.13 α-Bulnesene 1514 1513 12
42 30.12 Selina-3,7(11)- diene 1556 1542 12
43 30.94 Caryophyllene oxide 1590 1595 12
44 31.5 Humulene oxide 1614 1599 12
45 32.48 Caryophylla-3, 8(13)-dien-5-ol A 1658 1656 12

Conclusion

A simple headspace SPME-GC/MS method was used in the analysis of the terpene/terpenoid profiles of both hops and cannabis. The method was able to detect the characteristic terpenes and terpenoids of both, and to distinguish between different hops varieties.

 

References

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Materials