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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

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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.

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CANNABIS SATIVA L.

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■ 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

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BIOLOGICAL ACTIVITY

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BIOLOGICAL ACTIVITY

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■ 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.

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GC techniques

vs

HPLC techniques

*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

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WORKFLOW

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Quantitative analysis Validation of the analytical method (ICH guidelines)

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Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

■ 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

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■ 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 TECHNOLOGY

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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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

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

CBG CBDA CBGA CBD

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CBGA CBG THCA

Drug-type hemp

CBN THC

CBD-rich fibre-type hemp

CBDA CBGA CBD CBG CBDA CBGA CBD

CBG-rich fibre-type hemp

CBG

Cannabigerolic acid (CBGA) Cannabidiolic acid (CBDA) Cannabidiol (CBD) Cannabigerol (CBG)

HPLC chromatograms of hemp extracts at 210 nm

REPRESENTATIVE CHROMATOGRAMS

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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SELECTION OF THE EXTRACTION SOLVENT

a b b b c b,c b c a d c b a b,c b,c

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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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 a a c b b a

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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

Dynamic maceration with EtOH 15 min (x 3) r.t. Filtration HPLC analysis

Fibre-type hemp inflorescences

SAMPLE PREPARATION

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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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 Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis 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.

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Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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CBDA

[M–H]– [2M–H+Na]– 44 18

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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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

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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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:

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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CBD-rich fibre-type hemp

CBDA CBGA CBD CBG CBDA CBGA CBD

CBG-rich fibre-type hemp

CBG

HPLC chromatograms of plant material extracts at 210 nm

CBDA: 0.9 – 46.8 mg/g CBGA: 0.1 – 9.8 mg/g CBG: 0.2 – 6.5 mg/g CBD: 0.1 – 23.9 mg/g

Amount of cannabinoids in fibre-type hemp inflorescences: Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

QUANTITATIVE ANALYSIS

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CBDA CBD CBDA CBD CBD

Oil Balm Extract

CBDA: ≤ 3.5 mg/mL CBGA: < LOD CBG: ≤ 0.7 mg/mL CBD: 3.5 – 78.6 mg/mL

Amount of cannabinoids in hemp oil:

CBDA: 44.7 – 80.4 mg/g CBGA: 2.0 – 3.9 mg/g CBG: ≤ 0.4 mg/g CBD: 7.6 – 193.7 mg/g

Amount of cannabinoids in hemp balm and extract:

HPLC chromatograms of pharmaceutical products at 210 nm

Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis

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REFERENCE PAPER

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■ 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

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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

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E-mail contact: virginia.brighenti@unimore.it

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