No.

SSI-GCMS-1604

Gas Chromatograph Mass Spectrometer

Simplified Cannabis Terpene Profiling

by GCMS
No. GCMS-1604
■ Introduction
Terpene and terpenoid compounds are naturally
occurring aromatic compounds that give cannabis its
unique flavor and fragrance. Aside from their
aromatic properties, terpenes have advantageous
health benefits. They also have a synergistic
relationship with cannabinoids, which further
enhance the therapeutic effect of THC.
Cannabis has over 140 terpene components, many
of which are of medicinal interest.1 Predominant
terpenes in cannabis include β-myrcene, which has
antibiotic properties and enhances the THC muscle
relaxant effect; α-pinene, which has anti-
inflammatory properties and enhances the THC
bronchodilator effect; and β-caryophyllene, which
also has anti-inflammatory properties and enhances
the THC gastric cytoprotective effect amongst other
health benefits. 2, 3 The concentration of individual
terpenes varies by strain, can be anywhere from 0.1
to 1.5% of its total dry weight, and can vary
depending on harvest time, drying and storage
conditions.1, 4 Terpene levels can decrease over time,
and after three months of storage, can reduce
terpene levels by more than half.4 The decrease in
terpene amount over time varies for different
terpenes.
accordance with NRS 453A.368 in May of 2015.5
Due to the uniqueness of terpene profiles, they can
be used by cultivators as a “fingerprint” to partially
ID the specific strain in question. This application
note describes the analysis of several strains of
cannabis for 41 terpenes on Gas Chromatography
Mass Spectrometry (GCMS) with headspace
injection.
■ Experimental
Terpene analyses were conducted using a Shimadzu
GCMS-QP2010SE single quadrupole mass
spectrometer with the HS-20 headspace autosampler
for sample introduction. As terpene concentration
can be in the percent level, effluent from the HS-20
was split 50-to-1 to allow for analyzing high
concentration standards. The MS was run using the
FAAST (Fast Automated SCAN/SIM Type) where in
the SCAN mode was used for identification and the
SIM mode was used for greater quantitation. The
instrument and operating conditions are shown in
Table 1.

Recent proliferation of new terpene profiling

methods can be attributed to the ever-increasing
state legalization of cannabis use. The state of
Nevada added the testing of ten terpenes in
Shimadzu GCMS-QP2010SE
No. SSI-GCMS-1604

Table 1: Instrument Operating Conditions and Method Parameters

Head Space HS-20 Loop Model
Operation Mode Static headspace with loop
10uL sample volume
Sample
10-mL headspace vial
Equilibration 30 minutes at 150oC
1-mL loop
Vial pressurization 1.00 min, equilibration 0.20 min
Sample Loop
Loop load time 1.00 min, equilibration 0.20 min
Injection time 1.0 min
Sample Pathway Temperature 150oC
Transfer Line Temperature 150oC

Gas Chromatogram GC-2010 Plus

Injection Split injection from HS-20, with 50:1 split ratio
Rxi-624 Sil MS 30.0m x 0.25 mm x 1.40 µm
Helium carrier gas
Column Constant linear velocity, 47.2 cm/sec
Column Flow 1.64 mL/min
Purge Flow 3.0 ml/min
80 oC, hold 1.0 min
12 oC /min to 150 oC, hold 1.0 min
Oven Program 9 oC /min to 250 oC, hold 1 min
Total GC run time 19.94 min
Total cycle time 24 min

Detector GCMS-QP2010 SE
Operating mode Selected Ion Monitoring (SIM) and SCAN
Ion Source 200 oC, EI mode, 70eV
Solvent Cut Time 2 min
MS Interface 300 oC

Sample Preparation
In order to obtain a more complete terpene profile, Table 2: Calculated amount of terpene standard on column
two sets of standards were used. Standard one, Standard Concentration Injection Amount On-
(µg/mL) Volume Column (µg)
purchased from SPEX (Metuchen, NJ), included 38
(mL)
terpenes in a 100µg/mL stock solution. Standard SPEX 12.5 0.01 0.125
two, purchased from Restek (Bellefonte, PA), SPEX 25 0.01 0.25
included 23 terpenes in a 2500 µg/mL stock solution. SPEX 50 0.01 0.50
SPEX 75 0.01 0.75
As plant material does not dissolve in solvent, the full SPEX 100 0.01 1.0
evaporation headspace technique (FET) is used for Restek 78.125 0.01 0.78
quantitation. Using FET, a small amount of standard Restek 156.25 0.01 1.25
Restek 312.5 0.01 3.25
and sample is used to create a single phase gas
Restek 625 0.01 6.25
system, as compared to a two phase liquid-gas Restek 1250 0.01 12.5
system as in traditional headspace techniques.6 A Restek 2000 0.01 20.0
five-point calibration curve was created from the Restek 2500 0.01 25.0
SPEX terpene standard with concentrations ranging
from 12.5-100 µg/mL. An aliquot of 10µL of the A part of the flower weighing 1.0 gram was frozen,
standard was placed in a 10mL headspace vial and followed by grinding to ensure a representative
capped. A seven-point calibration curve was created sample. Ten to 30mg of the flower were then
from the Restek standard in a similar fashion as weighed into a headspace vial and capped. The final
described above, with the concentration range from result was calculated to give wt %.
78.125 µg/mL to 2500 µg/mL. The calculated
amount of terpene injected on column is shown in
Table 2.
No. SSI-GCMS-1604

■ Results and Discussion

Chromatography
Figures 1a and 1b show the TIC chromatographic
separation using the SPEX and Restek standards.
(x100,000)
7.0

6.0

5.0

4.0

3.0

2.0

1.0

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0

Figure 1a: TIC from SPEX standard

(x1,000,000)

2.5

2.0

1.5

1.0

0.5

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0

Figure 1b: TIC chromatogram from Restek standard

Calibration
Calibration standards were prepared and analyzed the state of Nevada with corresponding correlation
using the optimized HS-20 and GC-MS parameters coefficients illustrating linearity. The SIM
with concentrations ranging from 0.125µg to 0.1µg chromatograms represent the lowest calibration
using the SPEX terpene standard, and 0.78µg to point (0.78µg) for each terpene. All of the
25µg using the Restek standard. All points on the chromatographic peaks were easily integrated at
calibration curve were run in replicates of six. Figure each level. Full statistics on all calibration points are
2 shows the terpene calibration curves required by listed in Table 3a and 3b.

Alpha-Pinene
(x100,000) Area(x1,000,000)
93.00
6.051

1.5 91.00
92.00
7.5
1.0
5.0

0.5
2.5

0.0
5.9 6.0 6.1 6.2 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
Beta-Mycene
(x100,000) Area(x1,000,000)
93.00
6.726

1.5 41.00 7.5

69.00

1.0 5.0

0.5 2.5

0.0
6.6 6.7 6.8 6.9 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
No. SSI-GCMS-1604

Beta-Pinene
(x100,000) Area(x1,000,000)
93.00

6.779
1.5 69.00
7.5
1.0
5.0

0.5
2.5

0.0
6.6 6.7 6.8 6.9 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
alpha.-Terpinene
(x100,000) Area(x1,000,000)
121.00
7.290

1.25 93.00
136.00
1.00 5.0
0.75

0.50 2.5
0.25

0.0
7.2 7.3 7.4 7.5 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
(R)-(+)-Limonene
(x100,000) Area(x1,000,000)
68.00
7.460

1.00 67.00
93.00
5.0
0.75

0.50
2.5
0.25

0.0
7.3 7.4 7.5 7.6 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
Linolool
(x10,000) Area(x1,000,000)
71.00
9.002

93.00
55.00
5.0

2.5
2.5

0.0
8.9 9.0 9.1 9.2 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
No. SSI-GCMS-1604

Beta-Caryophyllene
(x10,000) Area(x1,000,000)
93.00

14.422
4.0 91.00 3.0

3.0
2.0
2.0

1.0
1.0

0.0
14.3 14.4 14.5 14.6 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
alpha.-Humulene
(x100,000) Area(x1,000,000)
93.00
14.972

80.00
1.00 121.00 7.5
0.75
5.0
0.50
2.5
0.25

0.0
14.8 14.9 15.0 15.1 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.999
Caryophyllene oxide
Area(x1,000,000)
(x10,000)
79.00
17.372

41.00 2.0
43.00
2.0

1.0
1.0

0.0
0.0 5.0 10.0 15.0 20.0 Conc.
17.2 17.3 17.4 17.5 R2 = 0.997
Alpha-Bisabolol
(x10,000) Area(x1,000,000)
43.00
18.218

119.00
5.0 69.00 3.0

2.0
2.5
1.0

0.0
18.1 18.2 18.3 18.4 0.0 5.0 10.0 15.0 20.0 Conc.
R2 = 0.994
Figure 2: Calibration curves for Restek terpene testing required by the state of Nevada; ranges shown are 1.25µg to 25µg on column
No. SSI-GCMS-1604

Table 3a: 5-point calibration and precision results for analysis of terpenes (SPEX standards)
Calibration Results Precision at 12.5ppm Precision at 100 ppm
Terpene (n=6) (n=6)
RF RSD (%) R2 Mean Conc RSD (%) Mean Conc RSD (%)
Alpha-Pinene 5.549 0.998 0.123 0.796 0.948 2.980
Camphene 3.642 0.999 0.124 1.164 0.964 3.734
Sabinene 3.440 0.999 0.121 1.447 0.972 2.005
beta.-Myrcene 3.732 0.997 0.126 1.317 0.994 3.038
beta.-Pinene 3.998 0.998 0.124 1.180 0.963 2.294
.alpha.-Phellandrene 4.367 0.999 0.124 3.533 0.959 2.602
(1S)-3-Carene 5.247 0.999 0.124 3.386 0.978 2.841
alpha.-Terpinene 5.916 0.996 0.107 1.269 0.935 1.982
trans-.beta.-Ocimene 3.757 0.998 0.125 1.041 0.957 2.857
(R)-(+)-Limonene 5.817 0.997 0.123 0.887 0.950 1.914
Ocimene (mixture of
4.109 0.997 0.124 1.334 0.950 2.776
isomers)
Eucalyptol 3.675 0.998 0.124 0.766 0.952 2.720
gamma.-Terpinene 3.695 0.998 0.124 0.908 0.947 1.954
Terpinolene 4.095 0.998 0.124 1.024 0.948 1.426
Sabedine Hydrate 9.252 0.995 0.122 2.926 0.950 5.300
Linolool 2.863 0.999 0.126 1.763 0.989 2.055
Fenchone (mix of 0.607 0.999 0.125 1.011 - -
isomers)*
(R)-Endo-(+)-Fenchyl 5.338 0.999 0.125 4.560 0.980 0.979
Alcohol
Isopulegol 2.433 0.999 0.126 2.210 0.981 2.109
Camphor (mix of isomers)* 0.785 0.999 0.125 0.952 - -
Isoborneol 8.659 0.992 0.130 6.949 1.050 2.745
Hexahydrothymol 4.979 0.998 0.128 3.287 1.005 1.939
Borneol (isomers) 4.774 0.997 0.125 5.422 0.983 2.094
Terpineol (mix of isomers) 1.376 0.999 0.125 1.909 0.981 1.242
gamma.-Terpineol 2.161 0.999 0.126 1.657 0.985 1.359
Nerol 11.23 0.998 0.127 6.418 0.997 3.828
Geraniol 14.27 0.999 0.125 8.386 0.985 2.858
Pulegone 1.859 0.999 0.124 2.093 0.973 1.841
Geraniol acetate 3.574 0.998 0.126 0.922 0.979 2.142
alpha.-Cedrene 3.058 0.999 0.124 1.087 0.958 1.630
Trans-Caryophyllene 2.899 0.999 0.126 1.899 1.021 4.524
alpha.-Humulene 2.146 0.999 0.125 1.180 0.969 1.652
Valencene 2.382 0.999 0.126 1.427 0.979 2.107
cis-Nerolidol 3.688 0.999 0.126 1.141 0.993 2.531
trans-Nerolidol 1.577 0.999 0.125 5.449 0.988 1.360
Guaiol 1.565 0.999 0.125 1.246 0.976 1.237
(+)-Cedrol 3.077 0.999 0.126 1.211 0.984 1.750
.alpha.-Bisabolol 2.911 0.999 0.126 1.862 0.986 1.536
* Data at the high level is not applicable
 No. SSI-GCMS-1604

Table 3b: 7-point calibration and precision results for analysis of terpenes (SPEX standards)
Calibration Results Precision at 78.125 ppm Precision at 2500 ppm
Terpene (n=6) (n=6)
RF RSD (%) R2 Mean Conc RSD (%) Mean Conc RSD (%)
Alpha-Pinene 2.545 0.999 0.764 1.478 24.614 2.12
Camphene 2.095 0.999 0.766 1.674 24.501 2.195
beta.-Myrcene 1.833 0.999 0.768 1.599 24.604 3.013
beta.-Pinene 1.973 0.999 0.767 1.386 24.725 1.948
(1S)-3-Carene 2.029 0.999 0.767 1.412 24.690 2.390
alpha.-Terpinene 2.150 0.999 0.766 1.611 24.706 1.940
trans-.beta.-Ocimene 1.746 0.999 0.777 1.349 25.381 2.525
(R)-(+)-Limonene 1.611 0.999 0.777 1.630 25.395 1.914
p-cymene 2.777 0.999 0.767 1.780 24.712 2.390
Ocimene (mixture of
1.452 0.999 0.772 1.395 25.011 2.494
isomers)
Eucalyptol 3.597 0.998 0.768 1.498 25.664 4.007
gamma.-Terpinene 2.231 0.999 0.766 1.562 24.578 2.489
Terpinolene 1.831 0.999 0.768 1.553 24.726 2.380
Linolool 3.545 0.999 0.784 1.156 25.783 1.876
Isopulegol 2.816 0.999 0.780 1.773 25.432 1.992
Geraniol 10.282 0.999 0.796 1.273 25.944 2.812
Trans-Caryophyllene 2.077 0.999 0.768 1.493 24.565 2.487
alpha.-Humulene 2.213 0.999 0.768 1.430 24.436 2.553
cis-Nerolidol 2.532 0.999 0.772 1.640 24.095 3.597
trans-Nerolidol 3.043 0.999 0.769 1.699 23.772 3.692
Guaiol 5.176 0.996 0.757 1.535 22.852 3.800
Caryophyllene oxide 9.397 0.997 0.804 7.127 26.803 4.869
.alpha.-Bisabolol 9.873 0.993 0.046 28.412 24.053 3.741

Cannabis Results
Table 4 shows the wt% of terpenes in three different Figure 5 shows the chromatograms of the required
strains of cannabis: CB Diesel, Blue Dream, and Haze terpenes by the state of Nevada.
Wreck.

Table 4: wt% of terpenes in three different strains of cannabis

Wt% of Terpenes in Cannabis

0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Terpineol (mix of…
Fenchone (mis of…

Camphor (mix of…

Ocimene (mixture of…

(R)-Endo-(+)-Fenchyl…
alpha.-Terpinene

Borneol (isomers)

Caryophyllene oxide
(1S)-3-Carene

gamma.-Terpinene
Terpinolene

Beta-Caryophyllene
trans-.beta.-Ocimene

p-cymene

Sabedine Hydrate

trans-Nerolidol

.alpha.-Bisabolol
alpha.-Pinene

beta.-Pinene

gamma.-Terpineol

Trans-Caryophyllene

Valencene
Eucalyptol

Isopulegol

Hexahydrothymol

Geraniol
Pulegone
Linolool

cis-Nerolidol
Sabinene

(R)-(+)-Limonene

Geraniol acetate

alpha.-Humulene
.beta.-Myrcene

.alpha.-Phellandrene

Isoborneol

(+)-Cedrol
Camphene

alpha.-Cedrene
Nerol

Guaiol

CB Diesel Blue Dream Haze Wreck

No. SSI-GCMS-1604

Figure 5: Chromatograms for terpenes required by the state of Nevada for three strains of cannabis

Terpene CB Diesel Blue Dream Haze Wreck

Alpha-Pinene (x1,000,000) (x1,000,000)
1.00 93.00
(x1,000,000)
3.0 93.00 3.0 93.00

6.052

6.046

6.052
91.00 91.00 91.00
92.00 0.75 92.00 92.00
2.0 2.0
0.50
1.0 1.0
0.25

6.0 6.1 6.2 6.0 6.1 6.2 6.0 6.1 6.2

Beta-Mycene (x1,000,000) (x1,000,000) (x1,000,000)

93.00 1.5 93.00 2.0 93.00

6.727

6.721

6.730
5.0 41.00 41.00 41.00
69.00 69.00 69.00
1.0
2.5 1.0
0.5

6.6 6.7 6.8 6.6 6.7 6.8 6.6 6.7 6.8

Beta-Pinene (x1,000,000) (x1,000,000) (x1,000,000)

93.00 1.5 93.00 2.0 93.00
5.0 69.00 69.00 69.00

1.0

6.774
6.779

2.5 1.0

6.782
0.5

6.7 6.8 6.9 6.7 6.8 6.9 6.7 6.8 6.9

alpha.- (x10,000) (x10,000) (x10,000)

7.5 121.00 121.00 121.00
Terpinene 93.00 93.00 7.5 93.00
136.00 1.0 136.00 7.286
136.00
5.0
5.0
0.5
7.291

2.5 2.5

7.2 7.3 7.4 7.2 7.3 7.4 7.2 7.3 7.4

(R)-(+)- (x1,000,000) (x1,000,000) (x1,000,000)

68.00 1.0 68.00 68.00
Limonene 1.5 67.00 67.00 67.00
93.00 93.00 1.0 93.00
7.455
7.461

7.465
1.0
0.5
0.5
0.5

0.0
7.4 7.5 7.6 7.4 7.5 7.6 7.4 7.5 7.6

Linolool (x100,000) (x100,000) (x100,000)

71.00 71.00 71.00
9.004

8.998

93.00 5.0 93.00 93.00 9.007

55.00 55.00 2.0 55.00
2.5
2.5
1.0

0.0 0.0
8.9 9.0 9.1 8.9 9.0 9.1 8.9 9.0 9.1

Beta- (x100,000) (x100,000)

5.0 93.00
(x100,000)
93.00 93.00
14.418

Caryophyllene
14.427

14.429

91.00 91.00 91.00

5.0

2.5 2.5
2.5

0.0
14.3 14.4 14.5 14.3 14.4 14.5 14.3 14.4 14.5
 No. SSI-GCMS-1604

alpha.- (x100,000) (x100,000) (x100,000)

5.0 93.00 93.00 93.00
Humulene

14.974

14.968

14.978
80.00 80.00 80.00
121.00 121.00 3.0 121.00

2.5 2.0
2.5
1.0

0.0
14.9 15.0 15.1 14.9 15.0 15.1 14.9 15.0 15.1

Caryophyllene (x1,000) (x1,000) (x1,000)

79.00 79.00 7.5 79.00
oxide 41.00 7.5 41.00 41.00
5.0 43.00 43.00 43.00
5.0
5.0

2.5 2.5 2.5

0.0
17.3 17.4 17.5 17.3 17.4 17.5 17.3 17.4 17.5

Alpha- (x10,000) (x10,000) (x10,000)

43.00 3.0 43.00 1.5 43.00
Bisabolol
18.223

18.217
119.00 119.00 119.00
69.00 69.00 69.00
2.0 1.0
2.5

1.0 0.5

0.1 0.0
18.1 18.2 18.3 18.1 18.2 18.3 18.1 18.2 18.3

■ Conclusion
The first sample of cannabis analyzed, CB Diesel, was change terpene results over time,4 and this should be
analyzed shortly after harvest. The resulting wt% of taken into consideration when analyzing cannabis
terpenes is similar to that in current literature.1 The samples as the results show less than expected
other two samples, Blue Dream and Haze Wreck, results. Varying storage conditions and degradation
were stored at ambient temperature and exposed to experiments should be the next study in the ever-
light for one month prior to analyzing. It has been changing world of regulatory cannabis testing.
demonstrated that different storage conditions can

■ References
1. Giese, M.W., Lewis, M.A., Giese, L., and Smith, K.M. Development and Validation of a Reliable and
Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Napro Research, California,
2015.
2. Parland, J.M., and Russo, E.B. Cannabis and Cannabis Extracts: Greater than the Sum of Their Parts? The
Haworth Press, Pennsylvania, 2001
3. [1] E. Russo, Taming THC: Potential Cannabis Synergy and Phytocannabinoid-Terpenoid Entourage Effects,
British Journal of Pharmacology 163 (2011) 1344.
4. Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents. Humana Press, New
Jersey
5. Information on all Policies related to Nevada’s Medical Marijuana Establishments can be found at
http://dpbh.nv.gov/Reg/MME/MME_-_Home/
6. http://www.restek.com/Technical-Resources/Technical-Library/Foods-Flavors-Fragrances/fff_FFAN2045-UNV

For more information on Shimadzu’s solutions for cannabis testing, please visit www.GrowYourLab.com

First Edition: September 2016

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