Chemical Assessment of Cannabis by GC-FID

Chemical Assessment of Cannabis by GC-FID

By:

Rebecca Plessel

Dorman Lab

Penn State University

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

Over 25.5 million

cannabis users

1.5 millionRegistered State-legal Medical Cannabis Patients

https://www.mpp.org/issues/medical-marijuana/state-by-state-medical-marijuana-laws/medical-marijuana-patient-numbers/

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

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

The Cannabinoids

Cannabidiol (CBD) Cannabigerol (CBG)

Cannabichromene (CBC)

Delta-9-tetrahydrocannabinol (THC)

Cannabinol (CBN)

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.

Why isn’t it FDA

approved?

http://www.dea.gov/druginfo/ds.shtml

https://www.drugabuse.gov/publications/drugfacts/marijuana-medicine

http://www.dea.gov/druginfo/ds.shtml

Schedule I

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

Other Schedule 1 Drugs

DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml

https://www.dea.gov/druginfo/ds.shtml

Schedule 2 Drugs

DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml

https://www.dea.gov/druginfo/ds.shtml

THC has various symptoms

http://headsup.scholastic.com/students/the-science-of-marijuana

Strouse, Cannabis and Cannabinoid Research 2016, 1(1), 38-43

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

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,

4 Studies

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

Marijuana Samples

Seized by University Park

Police

Marked for destruction

Provided by Lt. Stephanie

Brookes and Srgt. Monica

Himes for analysis

Sample Prep and Extraction 100mg plant material

Spike in 1-Naphthol

Solvate in 2ml Methanol

Sonicate for 15 minutes

Syringe Filter (0.45um,

PTFE)

Instrument

Agilent GC-dual FID

Equipped with a

Hydrogen generators,

and Nitrogen and

Helium tanks

Fitted with Gerstel

Rail MPS 2

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

Cannabinoid Peaks

CBC

(16.680)

CBD

(16.815)

THC

(17.334)

CBG

(17.658)

CBN

(18.041)

Mass Spectrum – THC

60 80 100 120 140 160 180 200 220 240 260 280 300 3200

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

m/z-->

Abundance

Scan 4153 (26.826 min): 02181603.D299

314

231

271

243

258

193174 2179177 107 137121 28555 161149 205

Mass Spectrum – CBD

60 80 100 120 140 160 180 200 220 240 260 280 300 3200

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

m/z-->

Abundance

Scan 4043 (26.197 min): 02181603.D231

246

314193174121

207107 2999177 137 159 27155 258219 286

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

ParsleySamples 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%

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

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

Conclusions If smoked, homogenized cannabis is the better

method of distribution

Provides a more consistent dosage that makes taking

this drug safer for patients

Future Studies

Use QuEChERS as alternative extraction

method

Exploring terpene and pesticide analysis in

cannabis

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.

Compounds There are 120 terpene in cannabis and only about 70

cannabinoids.

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

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

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

Terpene StandardsRestek 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)

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

HS GC-FID Method

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

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

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

Liquid Injection GC-MS

Liquid Injection GC-MS

Elution on

Rtx200MS

Retention Time Identification Confirmation

8.03 alpha pineneretention match to

standard

8.58 campheneretention match to

standard

9.15 beta pineneretention match to

standard

9.33 beta myrcene NIST match, no standard

9.42 delta 3 careneretention match to

standard

9.83 alpha terpineneretention match to

standard

9.87 d-limonene NIST match, no standard

10.06 ocimeneisomer, NIST match, no

standard

10.34 gamma terpineneretention match to

standard

10.42 p-cymeneretention match to

standard

10.50 ocimeneisomer, NIST match, no

standard

10.72 tepinolene NIST match, no standard

10.66 eucalyptolretention match to

standard

12.15 linaloolretention match to

standard

13.62 isopulegolretention match to

standard

14.73 geraniolretention match to

standard

16.25 beta-caryophylleneretention match to

standard

16.71 alpha humuleneretention 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-bisbololretention match to

standard

20.21 cayophyllene oxideretention match to

standard

20.91phthalate- lab contaminant NIST match

Restek Medical Cannabis Terpenes Standard #1

Restek Medical Cannabis Terpenes Standard #2

Cannabinoids on

Rtx200MS

CBC CBD THC CBG CBN

26- 28 minute elution period

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

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

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

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

SPME Results

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

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

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 -

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

Thanks to…

Dorman Lab

Questions?

Rebecca Plessel - [email protected]