Application Note

Food

AuthorJessica Westland Agilent Technologies, Inc.

IntroductionThe increased legalization of cannabis in the U.S. States and in countries throughout the world have pushed for qualifying and quantifying terpene concentrations to the forefront of the analytical industry. The development of a faster, more efficient method to produce rapid and accurate results at a low cost is needed. The high vapor pressures and volatility of terpenes make them excellent candidates for static headspace (HS) gas chromatography/mass spectrometry (GC/MS) analysis. This approach offers several advantages compared to solvent extraction and GC/FID. It:

• Does not require the use of organic solvents

• Does not coextract matrix interferences

• Provides additional means of peak identification and purity using spectral data

Please note that this application of cannabis flower terpene analysis by SPME is meant to be a proof of principal study illustrating the capabilities of SPME extraction for a variety of complex matrices. This methodology is not recommended for routine terpene testing in cannabis and hemp matrices without further method development and verification.

SPME-GC/MS of Selected Terpenes Using Agilent DVB/CAR-WR/PDMS SPME Fiber

2

Experimental

DVB/CAR-WR/PDMS Fiber reproducibilityA 10 μL amount of a 5 ppm terpene mixture (part numbers TPM-100-1 and TPM-105-1) was added to 8 mL of Milli-Q 18.2 Ω water in a 20 mL headspace vial.

SamplesCannabis flower samples were obtained from the University of Mississippi Marijuana Project with permission from the U.S. Drug Enforcement Administration (DEA).

Terpene profiling of various cannabis samplesAn 8 mL volume of Milli-Q 18.2 Ω water was added to approximately 0.1 g of homogenized cannabis plant material in a 20 mL headspace vial.

Solid phase microextraction (SPME)In SPME, analytes establish equilibria within the sample matrix, the headspace above the sample, and a polymer coating on a fused silica fiber. Extracted analytes are thermally desorbed from the fiber to a capillary GC column. Table 1 shows the SPME headspace parameters.

GC/MS analysisSelected terpenes were analyzed using SPME headspace with a PAL RTC rail system. This was combined with an Agilent 7890B GC system coupled with an Agilent 5977B high efficiency source GC/MSD (Figure 2).

Table 1. SPME headspace parameters.

Parameter Value

Script Name ARROW-STD-V2.0

Tool SPME 1

SPME Fiber Phase 80/10 µm DVB/CAR-WR/PDMS (p/n 5191-5874)

Incubation Time 5 minutes

Stirrer Heatex Stirrer 1

Heatex Stirrer Speed (Agitation) 1,000 rpm

Heatex Stirrer Temperature (Extraction Temp) 40 °C

Agitator None

Sample Extract Time 20 minutes

Extraction Temperature 40 °C

Sample Vial Penetration Depth 40 mm

Sample Vial Penetration Speed 20 mm/s

Inlet Penetration Depth 40 mm

Inlet Penetration Speed 100 mm/s

Injection Signal Mode Before fiber expose

Sample Desorption Time 3 minutes

Conditioning Port SPMEArrowCond 1

Pre-Desorption Conditioning Time 5 minutes (analytical run)/60 minutes (precondition)

Fiber Conditioning Station Temperature 270 °C

Post Desorption Conditioning Time 0 minutes

GC Cycle Time 5 minutes (set for sequence overlap)

Figure 1. DVB/CAR-WR/PDMS SPME Fiber (part number 5191-5874).

Figure 2. The PAL RTC rail system combined with an Agilent 7890B GC and an Agilent 5977B GC/MSD.

3

Results and discussion

DVB/CAR-WR/PDMS Fiber reproducibilityTwelve replicate injections of a 5 ppm standard were performed on three different DVB/CAR-WR/PDMS 100 µm Fibers within a single batch. Percent RSDs were calculated for each Fiber, then averaged together. Each set of replications maintained a percentage of RSDs lower than 20%. Table 3 shows the average results.

Table 2. Agilent 7890B GC settings.

Parameter Value

Inlet Liner Inlet liner, Ultra Inert, splitless, straight, 0.75 mm id (p/n 5190-4048)

Injection Mode/Temperature Splitless/270 °C

Oven Program60 °C (hold 2 minutes), 5 °C/min to 140 °C (hold 1 minute), 15 °C/min to 250 °C (hold 4 minutes)

Equilibration Time 0.5 minutes

Control Mode Constant flow (1 mL/min)

Column Agilent J&W DB-1ms, 60 m, 0.25 mm, 0.25 µm GC column (p/n 122-0162)

Septum Purge Flow Mode Standard at 3 mL/min

Purge Flow to Split Vent 15 mL/min at 0.35 minutes

Agilent 5977B GC/MS Conditions

Transfer Line 280 °C

Acquisition Mode SCAN

Solvent Delay 8 minutes

Tune File atune.u

Gain 1

MS Source Temperature 280 °C

MS Quad Temperature 150 °C

Table 3. Compound %RSD results per DVB/CAR-WR/PDMS Fiber.

Compound Fiber 01 Fiber 02 Fiber 03 Average

Camphene 4.77 3.54 1.79 3.37

Sabinene 7.99 5.04 3.41 5.48

b-Pinene 5.94 3.77 2.93 4.21

3-Carene 7.09 2.41 3.17 4.22

(R)-(+)-Limonene 4.64 1.81 3.54 3.33

Ocimene 16.60 5.57 4.84 9.00

γ-Terpinene 9.54 8.00 5.49 7.68

Fenchone 20.07 20.11 5.27 15.15

(–)-Camphor 9.73 4.83 5.18 6.58

(–)-Isopulegol 8.96 7.18 4.68 6.94

Isoborneol 16.46 10.55 6.93 11.31

Terpineol 9.34 18.86 12.17 13.46

Pulegone 15.44 8.29 10.18 11.30

b-Caryophyllene 19.65 5.26 5.36 10.09

(–)-Guaiol 24.43 18.32 14.55 19.10

(+)-Cedrol 17.85 12.90 10.69 13.81

(–)-alpha-Bisabolol 22.31 18.24 13.84 18.13

4

Terpene profiling of various cannabis samplesTerpenes account for the vast array of smells and flavors in cannabis flowers and extracts, and provide the notable aromas of cannabis varieties.

Several cannabis flower samples were profiled with the use of the DVB/CAR-WR/PDMS SPME Fiber (part number 5191-5874). Selected identified terpenes are reported in Table 4, and representative chromatograms are shown in Figures 3 to 13.

Limonene2

Limonene is the second most abundant terpene in all cannabis strains, but not all strains necessarily have it. Limonene gives strains a citrusy smell that resembles lemons.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.014.814

D-Limonene

14.410 14.507

15.062

14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5

×102

Acquisition time (min)

Co

un

ts (

%)

Figure 3. (R)-(+)-Limonene identified in cannabis flower sample 1103.

Table 4. Qualitative determination of terpenes in samples (concentration >5 ppm).

Name 1367 1330G THC000A 3486 3653 1103 2524 3401 3535 3648 3658 3812

Camphene X X

Sabinene X

b-Pinene X X X X X X X

3-Carene

(R)-(+)-Limonene X X X X X X X X

Ocimene X

g-Terpinene X X X

Fenchone X X X X

(–)-Camphor X X X X X X X X

(–)-Isopulegol X X X X X X X X X X X

Isoborneol X X

Terpineol X X X X X X X X X X X X

Pulegone

b-Caryophyllene X X X X X X X X X X X X

(–)-Guaiol X X X

(+)-Cedrol X X X X X X X X X X

(–)-alpha-Bisabolol X X X X X X X X X X X

5

gamma-Terpinene (g-Terpinene or γ-Terpinene)2

g-Terpinene has an herbaceous, citrusy sweet aroma. It is commercially extracted from tea tree oil.

Isopulegol3,4

Isopulegol, the chemical precursor to menthol, is a terpene known for its minty smell. It is commonly used as a flavoring agent in foods and fragrance in cosmetics.

×101

Acquisition time (min)

Co

un

ts (

%)

-0.4

-0.20

0.20.40.6

0.81.01.21.4

1.61.82.02.2

2.42.62.83.0

3.23.43.63.8

4.0

15.641gamma-Terpinene

15.3 15.35 15.40 15.45 15.50 15.55 15.60 15.65 15.70 15.75 15.80 15.85 15.90 15.95 16.00 16.05

Figure 4. g-Terpinene identified in cannabis flower sample 3653.

Figure 5. (–)-Isopulegol identified in cannabis flower sample 3535.

×101

Acquisition time (min)

Co

un

ts (

%)

-0.2

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

18.061

(–)-Isopulegol

17.6 17.7 17.8 17.9 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.7

6

Terpineol3

Terpineol occurs naturally in lilacs, pine trees, lime blossoms, and eucalyptus sap. It is frequently used to create pleasant aromatic profiles in products such as soap, lotion, and perfume. Terpineol is characterized by its ability to relax the consumer.

Caryophyllene2

Caryophyllene is the only terpene that binds to cannabinoid receptors. It is best known for its spicy and peppery notes. Caryophyllene is found in black pepper, cinnamon, cloves, and spices such as oregano, basil, and rosemary.

×101

Acquisition time (min)

Co

un

ts (

%)

-0.2

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

19.434

L-alpha-Terpineol

19.0 19.05 19.1 19.15 19.2 19.25 19.3 19.35 19.4 19.45 19.5 19.55 19.6 19.65 19.7 19.75 19.8 19.919.85

Figure 6. Terpineol identified in cannabis flower sample 3658.

×101

Acquisition time (min)

Co

un

ts (

%)

-0.4-0.2

00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8 23.165

Caryophyllene

22.90 22.94 22.98 23.02 23.06 23.10 23.14 23.18 23.22 23.26 23.30 23.34 23.38 23.42 23.46

Figure 7. b-Caryophyllene identified in cannabis flower sample 2524.

7

Cedrol5

Cedrol is a sesquiterpene alcohol found in the essential oil of conifers (cedar oil), especially in cypress and juniper. Studies have shown cedrol to have deeply sedative effects when inhaled.

alpha-Bisabolol2

alpha-Bisabolol has been primarily used in the cosmetics industry, but lately has been seen to be effective in treating bacterial infections and wounds. Its pleasant floral aroma can also be found in the chamomile flower, and is

an antioxidant with anti-irritation and analgesic properties.

Different cannabis strains have been developed containing distinct aromas and flavors that make up its specified terpene profile.

×101

Acquisition time (min)

Co

un

ts (

%)

-0.20

0.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8

27.262

alpha-Bisabolol

27.06 27.10 27.14 27.18 27.22 27.26 27.30 27.34 27.38 27.42 27.46 27.50

Figure 9. (–)-alpha-Bisabolol identified in cannabis flower sample 3812.

Figure 8. (+)-Cedrol identified in cannabis flower sample 3401.

×101

Acquisition time (min)

Co

un

ts (

%)

0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

26.661

(+)-Cedrol

26.5026.46 26.54 26.58 26.62 26.66 26.70 26.74 26.78 26.82 26.86 26.90 26.94

8

×107

Acquisition time (min)

Co

un

ts

0

0.1

0.2

0.3

0.4

0.5

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0.7

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0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

14.822

D-Limonene

23.165

Caryophyllene19.434

L-alpha-Terpineol

27.262

alpha-Bisabolol

18.055

Isopulegol

15.646

g-Terpinene

15 16 17 18 19 20 21 22 23 24 25 26 27

Figure 10. Selected terpenes in cannabis flower sample 1367: (R)-(+)-limonene, g-terpinene, (–)-isopulegol, terpineol, b-caryophyllene, and (–)-alpha-bisabolol.

×107

Acquisition time (min)

Co

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ts

0

0.1

0.2

0.3

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1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9 14.914

D-Limonene

23.182

Caryophyllene

19.440

alpha-Terpineol

27.262

alpha-Bisabolol

15 16 17 18 19 20 21 22 23 24 25 26 27

Figure 11. Selected terpenes in cannabis flower sample 1330G: (R)-(+)-limonene, terpineol, b-caryophyllene, and (–)-alpha-bisabolol.

9

×107

Acquisition time (min)

Co

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ts

0.1

0.2

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0.7

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1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8 19.463

L-alpha-Terpineol

27.262

(–)-alpha-Bisabolo

18.067

(–)-Isopulegol

15.664

gamma-Terpinene

16 17 18 19 20 21 22 23 24 25 26 27

Figure 12. Selected terpenes in cannabis flower sample THC001A: g-terpinene, (–)-isopulegol, terpineol, and (–)-alpha-bisabolol.

×102

Acquisition time (min)

Co

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ts (

%)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.023.182

Caryophyllene

19.440

L-alpha-Terpineol

26.661

(+)-Cedrol

18.061

Isopulegol

18.0 18.4 18.8 19.2 19.6 20.0 20.4 20.8 21.2 21.6 22.0 22.4 22.8 23.2 23.6 24.0 24.4 24.8 25.2 25.6 26.0 26.4 26.8

Figure 13. Selected terpenes in cannabis flower sample 3648: (–)-isopulegol, terpineol, b-caryophyllene, and (+)-cedrol.

www.agilent.com/chem

This information is subject to change without notice.

© Agilent Technologies, Inc. 2019 Printed in the USA, October 8, 2019 5994-1411EN

Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories where such use is permitted under state/country law.

ConclusionSince terpenes have high vapor pressures, and are extremely volatile, they are excellent candidates for static headspace (HS) gas chromatography/mass spectrometry (GC/MS) analysis. This application of cannabis flower terpene analysis by SPME is meant to be a proof of principal study illustrating the capabilities of SPME extraction for a variety of complex matrices. Similar SPME extraction methodology may be useful in the measurement of volatile phytochemicals found in fruit, vegetables and other plant-based matrices.

References1. SPME Arrow Sampling of Terpenes

in Cannabis Plant Material. Agilent Technologies Application Note, publication number 5994-1046EN, July 2019.

2. Greencamp. https://cannacon.org/15-terpenes-cannabis-explained/

3. Potbotics. https://www.potbotics.com/learn/terpenes/isopulegol/

4. The Apothecarium. https://apothecarium.com/blog/nevada/2018/6/7/terpenes-the-essentials-isopulegol/

5. Kagawa, D. et al. The Sedative Effects and Mechanism of Action of Cedrol Inhalation with Behavioral Pharmacological Evaluation. Planta medica, 69,7, 637–641. doi: 10.1055/s-2003-41114