Cannabinoid profiling of the Jamaican cannabis-Preliminary

findings

Carole Lindsay

University of the West Indies

Caribbean Toxicology Unit -Caritox

Order of presentation

• Background

• Aim

• Test method

• Results

• Summary of findings

• Acknowledgements

• References

Cannabis sativa L. • Family Cannabaceae. • Species Cannabis sativa L. is divided into several subspecies including

– C. sativa subsp. sativa, – C. sativa subsp. indica, – C. sativa subsp. ruderalis, – C. sativa subsp.spontanea, – C. sativa subsp. kafiristanca) [Hill,1983].

• Apply the name Cannabis sativa to all cannabis plants encountered (Flora of North

America, www.efloras.org (retrieved October 2014). • Extensive co-cultivation and cross breeding practices have effectively

crossed the boundaries between the various proposed taxonomic categories within Cannabis

• The term sinsemilla refers to a cultivation technique (that produces no seeds) and not to a genetic strain.

Some names used by local growers

Variety names are typically chosen by their growers, and often reflect properties of the plant such as effects, potency taste, color, smell, or the origin of the variety.

Purple skunk ice

Cannabis Products

• Legitimate cannabis products (<0.2 or 0.3% THC):

– agricultural crop for textile fibres

– cannabis seed,

– cannabis seed oil

– and the essential oil of cannabis.

• Illicit cannabis products :

– herbal cannabis (dried leaves and flowers- marijuana)

– Cannabis resin (hashish)

– and liquid cannabis (cannabis or hashish oil).

Production of Cannabis

• Herbal cannabis

– American continent - 55 per cent of global production in 2006,

– Africa - 22 per cent

• Cannabis resin

– Morocco world’s leading producer of cannabis resin.

– Afghanistan second largest producer

Cannabinoids Principle active ingredients of Cannabis are the cannabinoids.

They are produced by glandular trichomes that occur on most ariel surfaces of the plant (Dayanandan and Kaufman 1976; Turner et al; 1978) At least sixty unique to cannabis.

• Δ-9 Tetrahydrocannabinol (THC) is the main psychoactive cannabinoid (British Medical Association. 1997), but also has pharmacological properties . It occurs primarily as the acid THCA

• Cannabidiol (CBD) has pharmacological properties but is not psychoactive. (Mechoulam;2002)

• Cannabinol (CBN)

CBN is not directly synthesized by the cannabis plant it is a byproduct of the degradation of THC over time.

Cannabinoids exist primarily as cannabinoid acids (e.g., cannabidiolic acid[CBDA]), but as the plant material ages or is heated the acids decarboxylate into the neutral forms

(e.g., CBDA → CBD) (Meijer et al ;2003)

Source : http://www.postpoems.org/authors/orangejumpsuit/prose/972643

Cannabinoids as chemotaxonomic markers

The amounts of CBD and THC in an individual plant can be characterized both qualitatively and quantitatively (Hemphill et

al., 1980; Hillig, 2002; Mandolino et al., 2003).

Qualitative characterization

Small and Beckford (1973) recognized three :

1. Chemotype I - high THC/CBD ratio >>1.0, (THC drug)

2. Chemotype II - intermediate ratio (close to 1.0), (MIXED)

3. Chemotype III have a low THC/CBD ratio <<1.0). (CBD DRUG)

Others have included

Fibre –hemp type

Seed oil type

• Note: Ratio THC/CBD stable throughout the life of the plant

(Barni-Comparini et al., 1984; Vogelmann et al.,1988)

Cannabinoids as chemotaxonomic markers

Quantitative characterization

The plant can be characterized by the quantitative levels of

cannabinoids within its tissues.

Numerous biotic and abiotic factors affect cannabinoid production including:

• the sex and maturity of the plant,

• daylight length,

• ambient temperature,

• nutrient availablility,

• uv light intensity, .

• Variation among different tissues within a plant.

What types do we have in Jamaica?

Aim of Study

• The aim of this study is to determine the qualitative and quantitative patterns of the major cannabinoids in random cannabis plants collected from two parishes in Jamaica.

METHODOLOGY

Test Methods

The recommended methods for the identification and analysis of cannabis include:

Presumptive Tests

1. Colour tests

2. Immunoassays

3. Thin layer chromatography

Confirmatory tests

1. Gas Chromatography (FID)

2. Gas Chromatography – mass spectrometry –GCMS

3. High performance liquid chromatography HPLC (LCMS)

4. LCMSMS

Mass spectrometer The detector of choice

LCMSMS

The chromatographic system consisted of a Waters XEVO TQD

(LCMSMS)

Analyses were done on a Waters Acquity UPLC system equipped with an

autosampler and a Waters TQD detector. Mass Lynx software was used

to acquire data which was analysed by target Lynx software.

Location of Study Areas

Bagdale – St Elizabeth

Research Team – Caritox Laboratory

Plants were trimmed and

air dried.

Homogenized and stored (-

20°C)

Weighed amount extracted with

methanol:chloroform (9:1)

Vortexed sonicated and centrifuged

Supernatant was removed and Extraction repeated.

Pooled extracts dried under N2 at 30ºC

Reconstituted in mobile phase

Further dilutions were done

Internal standard was added

Then transferred to LC vials

Analyze by LCMS/MS

Decarboxylated by heating.

UNODC- extraction method; Grauwiler etal – LCMSMS method

Calibration

• Analytical reference standards and internal standard were purchased from Cerilliant Corporation in Texas ( An ISO accredited supplier)

• Calibrators were used to generate calibration curves and the concentrations of the cannabinoids were calculated by comparing peak areas in the samples with those on the standard curve.

Identification of cannabinoids Acceptance criteria

• Retention time (time taken for analyte to pass through the column and be detected.)

• Presence of the molecular ion. The charged intact molecule should be detected

• Ions produced after fragmentation of the molecular ion. (daughter ions)

• Ratio of the areas of the daughter ions.

Positive identification is only declared when all 4 criteria are met.

LCMSMS assay validation

• Linearity –R2> 0.995

• Limit of detection LOD – 0.0003%

• Limit of Quantification LOQ – 0.004%

• Recovery - 89%

• %CV- 3.1-4.9%

RESULTS

Typical Chromatogram of cannabis sample

Mean cannabinoid levels expressed as dry-weight % of cannabis plants collected in Mount Airy (A) Westmoreland

0

1

2

3

4

5

6

7

PSM#1 PSM#3 PSM#4 PSM#5

we

igh

t %

Cannabis type

THC

CBD

CBN

Total THC

Total CBD

Totral CBN

Mean cannabinoid levels expressed as dry-weight % of cannabis plants collected in Mount Airy (B) Westmoreland

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

mix 1 mix 2 mix 3 PS 4 PS 5 PS6 PS 7

we

igh

t %

Cannabis type

tHC

CBD

CBN

Total THC

Total CBD

Total CBN

Mean cannabinoid levels expressed as dry-weight % of cannabis plants collected in Mount Airy (C) Westmoreland

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

ICE #1 Mix#2

we

igh

t %

Cannabis type

tHC

CBD

CBN

Total THC

Total CBD

Total CBN

Mean cannabinoid levels expressed as weight % of cannabis plants collected in Mango Hall Westmoreland

0

0.5

1

1.5

2

2.5

3

3.5

Mix #1 Mix #2 ICE#3

we

igh

t %

Cannabis type

tHC

CBD

Total THC

Total CBD

Total CBN

Mean cannabinoid levels expressed as dry- weight % of cannabis plants collected in Bagdale Mountain ST Elizabeth

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

ICE #1 ICE # 6 ICE #7 ICE #9 ICE #11 MIX # 2 MIX #12 Mix #5 MIX#8 PS #3 PS #10 PS #13 PS #14

dry

-we

igh

t %

Cannabis type

THC

CBD

Tot THC

Tot CBD

Arithmetic means, standard deviations and ranges of the THC/CBD ratios and the dry-weight percentages of CBD and THC for “THC- Drug type” and “CBD- Drug type plants.

ST. Elizabeth

THC-Drug type (THC/CBD ratio >1)

CBD-Drug type (THC/CBD ratio <1)

Number of samples 10 3

THC/CBD ratio (SD) 194 (53) 0.66 (0.04)

range -ratio 80-248 0.6-0.7

Total THC% (SD) 4.1 (1.1) 1.5 (0.7)

range THC 2.3-5.8 1.0 -2.4

CBD % (SD) 0.02 (0.01) 2.2 (0.96)

range CBD 0.01-0.05 1.6-3.3

Arithmetic means, standard deviations and ranges of the THC/CBD ratios and the dry-weight percentages of CBD and THC for “THC- Drug type” and “CBD- Drug type plants.

Westmoreland

THC-Drug type (THC/CBD ratio >1)

CBD-Drug type (THC/CBD ratio <1)

Number of samples 10 6

THC/CBD ratio (SD) 74 (36) 0.35(0.2)

range -ratio 9-128 0.05-0.5

Total THC% (SD) 3.5(1.4) 1.07(0.6)

range THC 1.8-6.0 0.3-1.8

CBD % (SD) 0.1 (0.1) 3.6(1.5)

range CBD 0.02-0.36 2.2-6.4

Comparison of “THC- Drug type” data for St Elizabeth and Westmoreland.

ST Elizabeth THC-Drug type (THC/CBD ratio >1)

Westmoreland THC-Drug type (THC/CBD ratio >1)

Number of samples 10 10

THC/CBD ratio (SD) 74 (36) 194 (53)

range -ratio 9-128 80-248

Total THC% (SD) 3.5(1.4) 4.1 (1.1)

range THC 1.8-6.0 2.3-5.8

CBD % (SD) 0.1 (0.1) 0.02 (0.01)

range CBD 0.02-0.36 0.01-0.05

Comparison of “CBD- Drug type” data for St Elizabeth and Westmoreland.

St Elizabeth CBD-Drug type (THC/CBD ratio <1)

Westmoreland CBD-Drug type (THC/CBD ratio <1)

Number of samples 3 6

THC/CBD ratio (SD) 0.66 (0.04) 0.35(0.2)

range -ratio 0.6-0.7 0.05-0.5

Total THC% (SD) 1.5 (0.7) 1.07(0.6)

range THC 1.0 -2.4 0.3-1.8

CBD % (SD) 2.2 (0.96) 3.6(1.5)

range CBD 1.6-3.3 2.2-6.4

Comparison of “THC- Drug type” data for Indiana , St Elizabeth and Westmoreland.

Indiana-USA (Hillig ;2004)

ST Elizabeth THC-Drug type (THC/CBD ratio >1)

Westmoreland THC-Drug type (THC/CBD ratio >1)

THC/CBD ratio (SD) 205 (106) 74 (36) 194 (53)

range -ratio 52-727 9-128 80-248

Total THC% (SD) 6.3 (3.1) 3.5(1.4) 4.1 (1.1)

Range% THC 0.6-14.7 1.8-6.0 2.3-5.8

CBD % (SD) 0.04 (0.03) 0.1 (0.1) 0.02 (0.01)

range %CBD 0.01-0.16 0.02-0.36 0.01-0.05

Comparison of “CBD- Drug type” data for Indiana , St Elizabeth and Westmoreland.

Indiana-USA (Hillig ;2004)

ST Elizabeth THC-Drug type (THC/CBD ratio >1)

Westmoreland THC-Drug type (THC/CBD ratio >1)

THC/CBD ratio (SD) 0.05 (0.02) 0.66 (0.04) 0.35(0.2)

range -ratio 0.03- 0.17 0.6-0.7 0.05-0.5

Total THC% (SD) 0.24 (0.14) 1.5 (0.7) 1.07(0.6)

Range% THC 0.05-0.69 1.0 -2.4 0.3-1.8

CBD % (SD) 4.6(2.5) 2.2 (0.96) 3.6(1.5)

range %CBD 1.0-13.6 1.6-3.3 2.2-6.4

Summary of preliminary findings

• The two drug types were identified in St Elizabeth and Westmoreland.

• The THC-drug type appears to be more common

• Highest %THC so far detected is 6.0%

• Highest %CBD so far detected is 6.4%

• Of the two parishes Westmoreland had the better quality THC and CBD drug types

Considerations • Findings are preliminary . Larger data set is

required.

• Plants have been destroyed by the police

• US suppliers will not sell some reference standards (acids) to Jamaica.

• Though plants are grown outdoors we are not aware of any special farming practices eg application of fertilizers

• Variations in growing seasons, maturity of the plant, availability of water

The study continues

• It is hoped that all 14 parishes will be sampled

• The team will also analyse for terpenes, fatty acids, heavy metals and pesticides.

• Microbial assessment is currently underway (Dr Sherline Brown –UWI)

• Research could be enhanced with direct access to plant material and financial support.

Acknowledgements

• This study was supported by the MSc Forensic Science Programme, UWI , Mona

• This work could not have been undertaken without the valuable assistance of officers from the Narcotics Division of the Jamacia Constabulary Force and Professor McLaughlin in facilitating access to plant material.

• We are also indebted to the input made by our confidential advisor.

• Professor Norma McFarlane-Anderson my mentor

• Lastly, but by no means least, the success of this study was largely due to the input of the technical team at Caritox , Mr Garret Smith and Miss Tainia Taylor.

References

• British Medical Association. Therapeutic uses of cannabis. Amsterdam: Harwood Academic Press, 1997.

• Dayanandan, P., P. B. Kaufman. (1976). Trichomes of Cannabis sativaL. (Cannabaceae). American Journal of Botany 63: 578–591.

• de Meijer E.P.M., Bagatta M., Carboni M., Crucitti P., Moliterni V.M.C., Ranalli P. et al. (2003). The inheritance of chemical phenotype in Cannabis sativa L. Genetics. 63:335–46.

• Fetterman, P. S., AND C. E. Turner. (1972). Constituents of Cannabis sativa L. I. Propyl homologs of cannabinoids from an Indian variant. Journal of Pharmaceutical Sciences 61: 1476–1477

• Flora of North America. www.efloras.org (retrieved January 2009)

• Hemphill, J. K., Turner, J. C., Mahlberg, P. G. (1980). Cannabinoid content of individual plant organs from different geographical strains of Cannabis sativa L. Journal of Natural Products 43: 112–122.

• Hill, R.J. (1983). Marijuana, Cannabis sativa L., Regulatory Horticulture, Weed Circular No. 5, 9 (1-2), 57-66.

• Hillig, K. (2002). Letter to the editor. Journal of Industrial Hemp 7: 5–6.

• Mandolino, G., Bagatta, M., Carboni, A., Ranalli, P., de Meijer, E. (2003). Qualitative and quantitative aspects of the inheritance of chemical phenotype in Cannabis. Journal of Industrial Hemp 8: 51–72.

• Mechoulam, R., Hanus, L. (2002). Cannabidiol: an overview of some chemical and pharmacological aspects. Part I: chemical aspects. Chemistry and Physics of Lipids. 121: 35-43

• Small, E., Beckstead, H. D. (1973). Cannabinoid phenotypes in Cannabis sativa. Nature 245:147-148

• Turner, J. C., Hemphill, J. K., Mahlberg, P. G. (1978). Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. Cannabaceae. American Journal of Botany 65(10): 1103-1106