CANNABINOIDS IN FIBRE-TYPE CANNABIS SATIVA L. HPLC … · Edra, 2015. This work was aimed at the...
Transcript of CANNABINOIDS IN FIBRE-TYPE CANNABIS SATIVA L. HPLC … · Edra, 2015. This work was aimed at the...
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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.
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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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GCtechniques
vsHPLCtechniques
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*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 TECHNOLOGYDevelopment 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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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
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 bc
b,c bc adc b a
b,cb,c
Development of the HPLC method
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
PHYTO AND MORE LABUNIMORE
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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 aac
bb a
Development of the HPLC method
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
PHYTO AND MORE LABUNIMORE
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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
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
PHYTO AND MORE LABUNIMORE
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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
PHYTO AND MORE LABUNIMORE
CBDA
[M–H]–
[2M–H+Na]–
44
18
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Development of the HPLC method
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
PHYTO AND MORE LABUNIMORE
CBD
[M+H]+
56
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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
PHYTO AND MORE LABUNIMORE
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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
PHYTO AND MORE LABUNIMORE
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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:
Development of the HPLC method
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
QUANTITATIVE ANALYSIS
PHYTO AND MORE LABUNIMORE
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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
Development of the HPLC method
Optimization of the extraction
conditions
Identification of cannabinoids
Validation of the analytical method
Quantitative analysis
PHYTO AND MORE LABUNIMORE
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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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