Detection of cannabinoids inCannabis sativa plants

THCA

THC

CBCA

CBC

CBDA

CBD

Thin Layer Chromatography (TLC)

Agilent

Olivetolic acid, OA

GPP

Divaric acid

Cannabigerovarinic acid, CBGVA

Cannabichromevarinic acid, CBCVA

Cannabidivaric acid, Cannabidivarinic acid, CBDVA

Δ9-Tetrahydrocannabivarinic acid,Δ1-Tetrahydrocannabivarolic acid, THCVA

(-)-Δ9-Tetrahydrocannabinol ,THC

Cannabidivarin , Cannabidivarol, CBDV

Cannabigerolic acid,CBGA

Cannabidiolic acid, CBDA

Cannabichromenic acid, CBCA

(-)-Δ9-trans-Tetrahydrocannabivarin,Δ9- tetrahydro-cannabivarol, THCV

(-)-trans-Cannabidiol- , CBD

Cannabichromene,CBC Cannabichromevarin, CBCV

(-)-Δ9-trans-Tetrahydrocannabinolic acid, THCA

TLC ofCannabinoids

Cannabichromene,CBC

(-)-Δ1-Tetrahydrocannabinol, (-)-Δ9-Tetrahydrocannabinol ,THC

Cannabigerol, CBG

Δ9- tetrahydro-cannabivarol, THCV (-)-trans-Cannabidiol- , CBD cannabinol,CBN

Extraction methods

➢ Solid-liquid extraction (plant material + solvent) (e.g.

Methanol, Methanol:H2O, ethanol, benzene, petrolium ether,

n-hexane ) (For LCMS or first step of extraction for GCMS)

➢ Liquid-liquid extraction (extract partitioning) (e.g. n-hexane,

n-hexane: ethyl acetate, (usually for GCMS)

ACQUITY QDa Detector

Alliance HPLC system Waters

6224 Accurate Mass Time of Flight (TOF)1200 HPLC system

Agilent

Extracted tissues

Stigma trichomes

Flower trichomes

Calibration curves for THC for0.6-37.5 ng in single injection

y = 1E+08x + 67134R² = 0.9953

4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

0

4500000

5000000

0.0000 0.0050 0.0100 0.0150 0.0200 0.0250 0.0300 0.0350 0.0400

THC : 0.6-37.5 ng in injection

Olivetolic acid, OA

GPP

Divaric acid

Cannabigerovarinic acid, CBGVA

Cannabichromevarinic acid, CBCVA

Cannabidivaric acid, Cannabidivarinic acid, CBDVA

Δ9-Tetrahydrocannabivarinic acid,Δ1-Tetrahydrocannabivarolic acid, THCVA

Cannabidivarin , Cannabidivarol, CBDV

Cannabigerolic acid,CBGA

Cannabidiolic acid, CBDA

Cannabichromenic acid, CBCA

(-)-Δ9-trans-Tetrahydrocannabivarin,Δ9- tetrahydro-cannabivarol, THCV

(-)-trans-Cannabidiol- , CBD

Cannabichromene,CBC Cannabichromevarin, CBCV

(-)-Δ9-trans-Tetrahydrocannabinolic acid, THCA

Cannabinoidspathway

(-)-Δ1-Tetrahydrocannabinol, (-)-Δ9-Tetrahydrocannabinol ,THC

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CBGA (ug) in 1 gram FW

Stigma Flower Small L. Medium L. Large L. Trichomes

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Stigma Flower Small L. Medium L. Large L. Trichomes

180

*

Whole “head” of three strains

0

50

100

150

200

250

300

350

CBG (ug) in 1 gram FW THC (ug) in 1 gram FW CBDA (ug) in 1 gram FW CBGA (ug) in 1 gram FW THCA(mg) in 1 gram FW CBD (ug) in 1 gram FW

Zach AD MEND1 2 3

Small leaf with trichomes Small leaf W/O trichomes

Collection of the Trichomes usingdry-ice and 300 microne mesh

Shaken trichomes (mostly)350 micron sieve

Capitate-stalked, crysolytic and capiate gland types

Trichome types in Cannabis

Glandular trichome types in Cannabis

15-30 micron 25 to 100 micron

150 to500 m

icron

*antherial glandular trichome, which has only been found on anthers

Capitate and bulbous gland

Dayanandan and Kaufman, 1976

Capitate, long and short cystolith trichomes

Dayanandan and Kaufman, 1976

Ring of cells surroundling long hair

350-100 double layers x3-- times-3

100 -50- double layers x2

Dry ice –fresh 50-100 micron

/

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501,1m

Very challenging to separate alltrichome types

Solution – enriched extracts - use your data wisely

Conclusions

➢Using RNA-seq data with metabolic data across multiple tissues and developmental stages could lead to discovery of new genes from the pathway

➢Regulators of cannabinoid pathway a yet to be discovered, when approach above could contribute

➢Developing proper tissue extraction techniques might refine the metabolic profile of specific tissues.

Although extensive research has beengoing in recent years, much remains

to be discovered Many thanks:➢ Ari Schaffer➢ Oded Sagee➢ Efraim Lewinsohn➢ Shahar Cohen➢ Rachel Davidovitz-

Rikanati➢ Einat Bar➢ Shahar Sroussi➢ Lena Esselson

ThankYou

Detection of cannabinoids inCannabis sativa plantsSlide Number 2TLC ofCannabinoidsExtraction methodsACQUITY QDa DetectorAgilentExtracted tissuesSlide Number 8Slide Number 9Flower trichomesSlide Number 11Calibration curves for THCfor0.6-37.5 ng in single injectionCannabinoidspathwayCBGA (ug) in 1 gram FWTHCA(mg) in 1 gram FWWhole “head” of three strainsSmall leaf W/O trichomesSlide Number 18Slide Number 19Slide Number 20Glandular trichome types in CannabisCapitate and bulbous glandCapitate, long and short cystolith trichomes350-100 double layers x3-- times-3100 -50- double layers x2Dry ice –fresh 50-100 micronSlide Number 27Slide Number 28Very challenging to separate alltrichome typesConclusionsAlthough extensive research has beengoing in recent years, much remainsSlide Number 32