Throughout history, cannabis has been used as a panacea, an herbal remedy for nearly all medical concerns from simple headaches to severe pain. Now that many states have legalized medical cannabis, it is important to have analytical methodologies to study the compounds that the patients will be ingesting or inhaling. Terpenes are a major class of compounds found in cannabis. They are volatile hydrocarbons responsible for the plant’s aroma. These compounds are found in other plants as well. Through various clinical trials they were found to be medically relevant. In terms of cannabis, these compounds reportedly assist the cannabinoids in their effects. The cannabinoids bind to the cannabinoid receptor in the brain, and thus have medical relevance. Cannabichromene, cannabidiol, cannabigerol, and cannabinol are the main four cannabinoids that are implicated in relieving symptoms of pain, nausea, and directly reducing seizures. Delta-9-tetrahydrocannabinol is responsible for the euphoria experienced when smoked or ingested. With the increase in usage of cannabis due to its medical legalization in many states, it is important to have analytical methods for testing potency and variance of the cannabinoids and terpenes within the plant material. To do this, terpenes and cannabinoids were analyzed using a GC-FID. As the terpenes have higher volatility, several injection techniques were tested, including liquid injection, SPME, and headspace. The cannabinoid method was then applied to test the variance in subsequent doses of the same size, mimicking that of doses distributed to patients.

1. Chemical Assessment of Cannabis by
GC-FID
By:
Rebecca Plessel
Dorman Lab
Penn State University

2. Estimated over 8% of the US population uses
cannabis
 2014 Census puts the US population at 318.9 million
Hall et al, The Lancet 2014, 374 (9698), 1383-1391

3. Over 25.5 million
cannabis users

4. 1.5 million
Registered State-legal Medical Cannabis Patients
https://www.mpp.org/issues/medical-marijuana/state-by-state-medical-marijuana-laws/medical-marijuana-patient-numbers/

5. What is Medical Cannabis?
 As defined by the NIH:
“The term medical marijuana
refers to using the whole,
unprocessed marijuana plant or its
basic extracts to treat a disease or
symptom.”
https://www.drugabuse.gov/publications/drugfacts/marijuana-medicine

6. Medical Cannabis Helps
 Reduce pain
 Reduce anxiety
 Reduce severity and frequency of seizures
 Prevent nausea and vomiting
 Increase appetite
Strouse, Cannabis and Cannabinoid Research 2016, 1(1), 38-43

7. The Cannabinoids
Cannabidiol (CBD) Cannabigerol (CBG)
Cannabichromene (CBC)
Delta-9-tetrahydrocannabinol (THC)
Cannabinol (CBN)

8. CBD
 “There is a growing interest in the marijuana chemical
cannabidiol (CBD) to treat certain conditions such as
childhood epilepsy, a disorder that causes a child to
have violent seizures.”
 Scientists breed marijuana plants high for CBD, which
coincides with a decrease in the plant’s production of
THC.
 These drugs are less intoxicating, if at all
"Is Marijuana Medicine?" NIH, July 2015.

9. Why isn’t it FDA
approved?
http://www.dea.gov/druginfo/ds.shtml
https://www.drugabuse.gov/publications/drugfacts/marijuana-medicine

10. Schedule I
https://www.dea.gov/divisions/hq/2016/hq081116.shtml

11. Other Schedule 1 Drugs
DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml

12. Schedule 2 Drugs
DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml

13. THC has various symptoms
http://headsup.scholastic.com/students/the-science-of-marijuana
Strouse, Cannabis and Cannabinoid Research 2016, 1(1), 38-43

14. How much do you take?
 Depends on the disease/symptom:
 Amyotropic lateral sclerosis (ALS) 
10mg/day THC for 1mo
 Nausea from chemotherapy  5mg THC, 4-6
times/day
 Chronic pain  2.5-20mg/day THC for roughly
25 days
 All currently self-determined
http://www.mayoclinic.org/drugs-supplements/marijuana/dosing/hrb-20059701

15. How much your body
receives from smoking
 10% - 20% THC in the plant
 20% - 70% enters the lungs
 5% - 24% reaches the brain
 Incredible amount of variance
 1g plant  1mg – 33.6mg THC entering your brain
 If dose is 10mg  1/10 or 3X amount
Hall et al, The Lancet 2009, 374(9698), 1383-1391,

17. 4 Studies

18. Method Development
 Rtx – 5 column
 Couldn’t resolve the CBC and CBD compounds
 Resolved peaks with Rtx-200
 Previous Surrogates
 JWH 007
 Expensive
 Undesirable basic nitrogen
 4-hydroxycoumarin
 Sky liner, IP liner, Cyclosplitter
 Degradation in the liner, high pKa
 New surrogate is 1-Naphthol

19. Marijuana Samples
 Seized by University Park
Police
 Marked for destruction
 Provided by Lt. Stephanie
Brookes and Srgt. Monica
Himes for analysis

20. Sample Prep and Extraction
 100mg plant material
 Spike in 1-Naphthol
 Solvate in 2ml Methanol
 Sonicate for 15 minutes
 Syringe Filter (0.45um,
PTFE)

21. Instrument
Agilent GC-dual FID
Equipped with a
Hydrogen generators,
and Nitrogen and
Helium tanks
Fitted with Gerstel
Rail MPS 2

22. GC Parameters
 Oven Temp: 80°C  300°C
 Ramp: 12°C/min
 Split mode
 Total Run Time: 23 min
 Inlet:
 250°C
 19.2 psi
 Split ratio: 10:1
 Carrier Gas: helium
 Front Detector:
 350°C
 H2 Flow: 40ml/min
 Air Flow: 450ml/min
 Makeup Flow: 48.4
ml/min
 Column:
 RTX-200, 30m, 0.25mm
ID, 0.5 um df

23. Cannabinoid Peaks
CBC
(16.680)
CBD
(16.815)
THC
(17.334)
CBG
(17.658)
CBN
(18.041)

24. Mass Spectrum – THC
60 80 100 120 140 160 180 200 220 240 260 280 300 320
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
m/z-->
Abundance
Scan4153(26.826min): 02181603.D
299
314
231
271
243
258
193174 2179177 107 137121 28555 161149 205

25. Mass Spectrum – CBD
60 80 100 120 140 160 180 200 220 240 260 280 300 320
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
m/z-->
Abundance
Scan 4043 (26.197 min): 02181603.D
231
246
314
193174121
207107 2999177 137 159 27155 258219 286

26. Reagent (Methanol)
1-
Naphthol
CBC CBD THC CBG CBN
Mean:
(ng/ul)
20.46 18.33 18.29 17.96 17.06 18.02
%RSD: 2.12% 4.34% 4.07% 10.04% 6.75% 9.78%
1-
Naphthol
CBC CBD THC CBG CBN
Mean:
(ng/ul)
23.26 21.99 21.76 21.58 21.39 22.09
%RSD: 10.63% 11.34% 11.81% 13.18% 11.73% 11.56%
Samples 1-10
Sample 10 – 10x

27. Parsley
Samples 1-10
Sample 10 – 10x
1-
Naphthol
CBC CBD THC CBG CBN
Mean:
(ng/ul)
13.61 13.08 12.90 12.42 13.42 13.41
%RSD: 7.88% 7.92% 8.51% 8.19% 7.78% 7.25%
1-
Naphthol
CBC CBD THC CBG CBN
Mean:
(ng/ul)
13.10 12.40 12.42 11.84 12.61 12.81
%RSD: 1.11% 1.36% 1.12% 1.34% 1.36% 0.96%

28. Non-Homogenized Cannabis
(% by Weight)
Samples 1-5
Sample 5– 5x
CBC CBD THC CBG CBN
Mean(%): 0.21 0.04 8.77 0.78 4.18
%RSD: 9.33 5.92 8.56 9.89 7.44
CBC CBD THC CBG CBN
Mean(%): 0.19 0.04 9.75 0.42 3.60
%RSD: 2.56 1.93 0.50 0.75 0.40

29. Homogenized Cannabis
(% by Weight)
Samples 1-5
Sample 5– 5x
CBC CBD THC CBG CBN
Mean(%): 0.18 0.04 6.66 0.43 4.70
%RSD: 5.99 5.05 6.16 4.29 4.98
CBC CBD THC CBG CBN
Mean(%): 0.18 0.04 6.67 0.44 4.48
%RSD: 3.02 3.25 3.55 3.67 3.59

30. Conclusions
 If smoked, homogenized cannabis is the better
method of distribution
 Provides a more consistent dosage that makes taking
this drug safer for patients

31. Future Studies
 Use QuEChERS as alternative extraction
method
 Exploring terpene and pesticide analysis in
cannabis

32. Terpenes
 Terpenes are volatile unsaturated hydrocarbons that often
produce a scent.
 Plants (and some animals) use to ward off predators or
attract pollinators.
Elsohly, Mahmoud A., Slade, Desmond. “Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids”, Life Sciences, 78 (2005), 539-548.

33. Compounds
 There are 120 terpene in cannabis and only about 70
cannabinoids.

34. Terpenes Elsewhere
 Plants:
 β-Myrcene  Mangoes, Bay Leaves, and Eucalyptus
 Pinene  Conifers, Pine woods, Citrus Fruits
 Limonene  Lemons, Oranges, and Rosemary
 Personal Care Products
 Limonene  aftershave lotions, bath products,
cleansing products, hair products, makeup,
moisturizers, perfumes and colognes, skin care
products, and suntan products

35. Why are we interested?
 In general aroma is associated with quality
 Forensic perspective:
 Link between Terpene and cannabinoid production between
strains
 Medical perspective:
 Link between Terpenes and THC diffusion through the
blood brain barrier
 Increase effective distribution of medication
 Problem with lung irritation if smoked
Oier Aizpurua-Olaizola et. al, Jour. Nat Prod. 2016, 79, 324-331
Sidney Cohen, JAMA, 1978, 240(16), 1761-1763
Casano et al, Fundación Canna 2011, 925, 115-121

36. Medicinal Properties of
Terpenes
 β-myrcene is known to prevent peptic ulcer disease
 Caryophyllene and limonene combined shows anti-
inflammatory properties
 CBD, linalool, limonene, and pinene are used to treat acne
 Linalool reduces lung irritation caused by smoking
Jeena et al, Jour. Essential Oil Bearing Plants 2014, 17(1), 1-12
Aizpurua-Olaizola et al, Jour. Nat. Prod. 2016, 79, 324-331
Bonamin et al, Chem-Bio Interactions, 2014, 212, 11-19
Ma et al, International Immunopharmacology, 29, 708-713

37. Terpene Standards
Restek Medical Cannabis Terpenes Standard #1
(-)-α-Bisabolol
Camphene
Δ-3-Carene
β-Caryophyllene
Geraniol
(-)-Guaiol
α-Humulene
p-Isopropyltoluene
(p-cymene)
(-)-Isopulegol
d-Limonene
Linalool
β-Myrcene
Nerolidol
Ocimene
α-Pinene
(-)-β-Pinene
α-Terpinene
γ-Terpinene
Terpinolene
Restek Medical Cannabis Terpenes Standard #2
(-)-Caryophyllene oxide 1,8-Cineole (Eucalyptol)

38. Goals of Terpene Analysis
 To develop an analytical method for the qualitative
and quantitative analysis of Terpenes in Cannabis
 Headspace (HS)
 Liquid injection
 Solid Phase Microextraction (SPME)
 Thermal Desorption Unit of solid material

39. HS GC-FID Method

40. Full Evaporation
Technique
 Samples were prepared by spiking 10ul of analytical
standards into a headspace vial
 Plant matrix spikes contained 10mg of parsley in
headspace vial + 10ul of analytical standards

41. Elution of
Terpenes on
Rxi-624SilMS
Compound Retention Time
alpha pinene 8.309
camphene 8.638
beta myrcene 8.926
beta pinene 9.041
delta 3 carene 9.372
alpha tepinene 9.494
ocimene 9.644
limonene 9.685
p cymene 9.727
eucalyptol 9.852
gamma terpinene 9.994
terpinolene 10.401
linalool 10.796
isopulegol 11.579
geraniol 12.769
beta caryophyllene 14.813
alpha humulene 15.196
nerolidol 1 15.726
nerolidol 2 16.03
(-)-guaiol 16.756
caryophyllene oxide 16.927
alpha bisabolol 17.402

42. Headspace Results
 Resolved terpenes on Rxi624SilMS
 Unable to analyze for cannabinoids on Rxi624SilMS
 Observed area differences between reagent spikes and
matrix spikes
 Not always higher or lower in area counts
 Adsorption, volatility, matrix interferences
 Area count bias throughout chromatogram
 More retained compounds displayed lower area counts
 Calibration showed less linearity for later eluting
compounds

43. Liquid Injection GC-MS

44. Liquid Injection GC-MS

45. Elution on
Rtx200MS
Retention Time Identification Confirmation
8.03 alpha pinene
retention match to
standard
8.58 camphene
retention match to
standard
9.15 beta pinene
retention match to
standard
9.33 beta myrcene NIST match, no standard
9.42 delta 3 carene
retention match to
standard
9.83 alpha terpinene
retention match to
standard
9.87 d-limonene NIST match, no standard
10.06 ocimene
isomer, NIST match, no
standard
10.34 gamma terpinene
retention match to
standard
10.42 p-cymene
retention match to
standard
10.50 ocimene
isomer, NIST match, no
standard
10.72 tepinolene NIST match, no standard
10.66 eucalyptol
retention match to
standard
12.15 linalool
retention match to
standard
13.62 isopulegol
retention match to
standard
14.73 geraniol
retention match to
standard
16.25 beta-caryophyllene
retention match to
standard
16.71 alpha humulene
retention match to
standard
17.64 nerolidol (1 or 2)
retention match to
standard
17.99 nerolidol (1 or 2)
retention match to
standard
18.84 guaoil NIST match, no standard
19.47 alpha-bisbolol
retention match to
standard
20.21 cayophyllene oxide
retention match to
standard
20.91
phthalate- lab
contaminant NIST match
Restek Medical Cannabis Terpenes Standard #1
Restek Medical Cannabis Terpenes Standard #2

46. Cannabinoids on
Rtx200MS
CBC CBD THC CBG CBN
26- 28 minute elution period

47. Liquid Injection Results
 Resolved all terpene standards on Rtx200MS column
 No area count bias based on retention in column
 Analyzed for Terpenes and Cannabinoids in a single
analysis

48. Headspace SPME
 50/30 µm
divinylbenzene/Carboxen
on polydimethylsiloxane
on a StableFlex fiber
 Rxi-624SilMS (30m x
0.25mm x 1.4um)
 1mm SPME liner

49. Parameter Optimization
 Desorption/ Inlet Temperature
 230, 250, 270 C
 Extraction/ Fiber Exposure Time
 5, 10, 20, 40, 60 min
 Extraction/ Fiber Exposure Temperature
 30, 40, 60, 90, 115, 140, 180 C
 Vial Incubation Time
 5, 15, 30, 45 min

50. Optimized Parameters
 Desorption/ Inlet Temperature: 230 C
 Extraction/ Fiber Expose Time: 10 minutes
 Extraction/Fiber Extraction Time: 60 C
 Vial Incubation Time: 30 minutes
 Used same GC parameters as headspace method

51. SPME Results

52. SPME Results
 Matrix interference observed in parsley spiked
replicates
 Later eluting compounds absorbed to fiber more
 Overloaded fiber in high cal points reducing linearity in
several well retained compounds

53. HS vs. SPME
 Headspace more selective
for more volatile
compounds
 Solid Phase Microextraction
more sensitive for larger
molecules
 Overall SPME is more
sensitive for terpene analysis
Retention Time HS 2500 ng in vial SPME 1250 ng in vial
8.32 125 43
8.65 124 48
8.94 122 102
9.05 125 61
9.38 132 94
9.50 155 137
9.67 131 124
9.67 141 139
9.78 88 124
9.87 111 77
10.00 127 135
10.40 122 161
11.70 105 205
12.78 86 459
14.83 135 723
15.21 131 804
15.74 30 274
16.05 43 400
16.78 68 679
16.95 81 736
17.42 60 538
Area Counts

54. HS Analysis of Marijuana
 Homogenized
Marijuana
analyzed by HS
GC-FID
 Average of 5
replicates display
the Terpene
Signature
Below limit of
quantification ND= non detection
5mg of Marijuana
Compound ng/mg %RSD
alpha pinene 28 10
camphene 10 9
beta myrcene 36 8
beta pinene 26 11
delta 3 carene ND -
alpha terpinene ND -
o cimene 89 10
limonene ND -
p cymene ND -
eucalyptol 22 8
gamma
terpinene ND -
terpinolene ND -
linalool 119 9
isopulegol ND -
geraniol ND -
beta
caryophyllene 159 8
alpha humulene 58 7
nerolidol 1 ND -
nerolidol 2 65 11
(-)- guaiol ND -
caryophyllene
oxide ND -
alpha bisabolol ND -

55. Future Plans
 Utilize thermal desorption unit as a direct sampling
technique
 Rtx200MS column to analyze for terpenes and
cannabinoids
 Split mode to remove water from inlet and to avoid
overloading the mass spectrometer

56. Thanks to…
 Dorman Lab

57. Questions?
Rebecca Plessel - rxp5172@psu.edu