cannabinoids in fibre-type cannabis sativa l. hplc … · edra, 2015. this work was aimed at the...

DEVELOPMENT OF A NEW EXTRACTION TECHNIQUE AND

HPLC METHOD FOR THE ANALYSIS OF NON-PHYCHOACTIVE

CANNABINOIDS IN FIBRE-TYPE CANNABIS SATIVA L.

Dr. Virginia Brighenti, Ph.D.

Department of Life Sciences

University of Modena and Reggio Emilia, Italy

PHYTO AND MORE LABUNIMORE

CANNABIS SATIVA L.

■ Cannabis sativa L. (hemp, Cannabaceae family) is well known, due to its history, pharmacology and social impact.

■ Fibre-type hemp remains at the moment underused in the pharmaceutical ambit, where drug-type C. sativa is employed as medicinal Cannabis.

■ Nevertheless, there has also been increasing interest in hemp varieties containing non-psychoactive cannabinoids.

■ Most of the European Union countries and Canada have recognized the value of fibre-type hemp and they have defined a legal limit of 0.3% Δ9-THC.


CANNABIS SATIVA L.


■ Cannabidiol (CBD) represents the most valuable compound, since it possesses a high anti-oxidant and anti-inflammatory activity, as well as neuroprotective, anxiolytic and anticonvulsant properties.

BIOLOGICAL ACTIVITY


BIOLOGICAL ACTIVITY


BIOLOGICAL ACTIVITY


■ Cannabigerol, Cannabichromene and Cannabidiolic acid have been found to possess promising antibacterial properties, even against multi-drug resistant bacteria.

Cannabigerolic acid (CBGA)

Cannabidiolic acid (CBDA) Cannabidiol (CBD)

Cannabigerol (CBG)

Δ

Δ

-CO2

-CO2

BIOLOGICAL ACTIVITY

■ Cannabinoids are biosynthesized in the acid form in plant tissues, where they can undergo a spontaneous decarboxylation process under the action of heat and light.


GCtechniques

vsHPLCtechniques

*Picture from «Cannabis. «Erba» Medica: norme, preparazioni galeniche, attualità e prospettive di cura», F. Firenzuoli, F. Epifani, I. Loiacono, Ed. Edra, 2015.

This work was aimed at the assessment and application of an efficient and reliable extraction technique in order to obtain extracts with a high content of non-psychoactive cannabinoids from several fibre-type hemp samples for their cannabinoids profiling, in order to identify potential candidates for the pharmaceutical industry


WORKFLOW

Development of the HPLC method

Optimization of the extraction

conditions

Identification of cannabinoids

Quantitative analysis

Validation of the analytical method

(ICH guidelines)




conditions




■ A fused-core (ore core-shell) stationary phase is characterized by 2.7 µm particles, which comprise a solid 1.7 µm diameter silica core that is encapsulated in a 0.5 µm thick layer of porous silica gel.

THE FUSED-CORE TECHNOLOGY


■ The innovative manufacturing process for fused-core particles produces a very narrow particle size distribution.

■ Traditional porous particles are not manufactured in a way to yield extremely narrow particle size distributions.

THE FUSED-CORE TECHNOLOGYDevelopment of the HPLC method


conditions





HPLC-UV/DAD METHOD

■ Column: Ascentis Express C18

(150 x 3.0 mm, 2.7 µm)

■ Mobile phase: 0.1% HCOOH in both H2O and ACN, gradient elution

■ Flow rate: 0.4 mL/min

■ Injection volume: 3 µL

■ Detection: UV/DAD at 210 and 220 nm



conditions




CBG

CBDA CBGA

CBD


CB

GA

CB

G

THC

A

Drug-type hemp

CB

N

THC

CBD-rich fibre-type hemp

CB

DA

CB

GA

CB

DC

BG

CB

DA

CB

GA

CB

D

CBG-rich fibre-type hemp

CB

G

Cannabigerolic acid (CBGA)

Cannabidiolic acid (CBDA) Cannabidiol (CBD)

Cannabigerol (CBG)

HPLC chromatograms of hemp extracts at 210 nm

REPRESENTATIVE CHROMATOGRAMSDevelopment of

the HPLC method


conditions





SELECTION OF THE EXTRACTION SOLVENT

a

b b bc

b,c bc adc b a

b,cb,c



conditions





SELECTION OF THE EXTRACTION TECHNIQUE

DM: dynamic maceration MAE: microwave-assisted extraction UAE: ultrasound-assisted extraction SFE: supercritical-fluid extraction

c

a a

a

b

b aac

bb a



conditions





Sample-to-solvent ratio: 1:40 (w/v)

Dynamic macerationwith EtOH

15 min (x 3) r.t.

FiltrationHPLC

analysis

Fibre-type hemp inflorescences

SAMPLE PREPARATION Development of

the HPLC method


conditions





IDENTIFICATION OF CANNABINOIDS

■ The HPLC-ESI-MSn analyses of cannabinoids were carried out with the same HPLC parameters aforementioned.

HPLC-ESI-MSn conditions:

■ Capillary voltage: 3.5 kV (+); 3.5 kV (-)

■ Nebulizer pressure (N2): 32 psi

■ Drying gas temperature: 350 °C

■ Drying gas flow: 10 L/min

■ Skimmer voltage: 40 V



conditions




The ion trap mass analyzer was used in the full-scan positive and negative ion modes, in the m/z range 200-1200. MS2 spectra were automatically performed with helium as the collision gas, using the SmartFrag function in the m/z range 50-1500.




conditions





CBDA

[M–H]–

[2M–H+Na]–

44

18



conditions





CBD

[M+H]+

56

HPLC-UV/DAD METHOD VALIDATION

The validation of the HPLC-UV/DAD method developed was performed to show compliance with ICH guidelines.

■ Linearity: r2 > 0.998

■ Sensitivity: 0.5 < LOD < 0.8 μg/mL ; 1.8 < LOQ < 2.5 μg/mL

■ System reproducibility: %RSDtR

< 1.4; %RSDPeak area

< 1.5

■ Extraction precision: 0.1 < SDmg/g

< 1.4

■ Accuracy: 74 < Recovery % < 91



conditions





QUANTITATIVE ANALYSIS

■ 9 fibre-type hemp inflorescences (C1-C9)

■ 2 samples of hemp oil

■ 2 samples of hemp balm

■ 1 hemp extract

pharmaceutical products

Samples:



conditions





CBD-rich fibre-type hemp

CB

DA

CB

GA

CB

DC

BG

CB

DA

CB

GA

CB

D

CBG-rich fibre-type hemp

CB

G

HPLC chromatograms of plant material extracts at 210 nm

CBDA: 0.9 – 46.8 mg/gCBGA: 0.1 – 9.8 mg/gCBG: 0.2 – 6.5 mg/gCBD: 0.1 – 23.9 mg/g

Amount of cannabinoids in fibre-type hemp inflorescences:



conditions




QUANTITATIVE ANALYSIS


CB

DA

CB

D

CB

DA

CB

DC

BD

Oil

Balm

Extract

CBDA: ≤ 3.5 mg/mLCBGA: < LODCBG: ≤ 0.7 mg/mLCBD: 3.5 – 78.6 mg/mL

Amount of cannabinoids in hemp oil:

CBDA: 44.7 – 80.4 mg/gCBGA: 2.0 – 3.9 mg/gCBG: ≤ 0.4 mg/gCBD: 7.6 – 193.7 mg/g

Amount of cannabinoids in hemp balm and extract:

HPLC chromatograms of pharmaceutical products at 210 nm



conditions





REFERENCE PAPER


■ A new and reliable RP-HPLC-UV/DAD, ESI-MS and MS2 method, based on the fused-core technology, was developed for the profiling of cannabinoids in fibre-type hemp and derivatives.

■ An efficient extraction procedure was optimized, based on dynamic maceration with ethanol at room temperature.

■ Thanks to the application to real matrices, the method proved to be a valuable tool for the selection of plant variety with a high content of bioactive compounds and for the quality control of hemp-based products.

CONCLUSION


ACKNOWLEDGMENTS

Prof. Stefania Benvenuti Ph.D.

Dr. Federica pellati, Ph.D.

Dr. Roberta Tardugno, Ph.D.

Marleen Steinbach, MD.

Tatiana Pedrazzi, MD

Bruna Wissmann Monteiro, MD

Davide Maran, MD


E-mail contact:[email protected]


mailto:[email protected]

cannabinoids in fibre-type cannabis sativa l. hplc … · edra, 2015. this work was aimed at the...

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