DEVELOPMENT OF A NEW EXTRACTION TECHNIQUE AND HPLC METHOD FOR THE ANALYSIS OF NON-PHYCHOACTIVE CANNABINOIDS IN FIBRE-TYPE CANNABIS SATIVA L.

Transcription

1 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

2 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. 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% Δ 9 -THC.

3 CANNABIS SATIVA L.

4 BIOLOGICAL ACTIVITY 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.

5 BIOLOGICAL ACTIVITY

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

7 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. Δ -CO 2 Cannabidiolic acid (CBDA) Δ Cannabidiol (CBD) HPLC techniques vs GC techniques -CO 2 Cannabigerolic acid (CBGA) Cannabigerol (CBG)

8 *Picture from «Cannabis. «Erba» Medica: norme, preparazioni galeniche, attualità e prospettive di cura», F. Firenzuoli, F. Epifani, I. Loiacono, Ed. Edra, 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 from several fibre-type hemp samples for their profiling, in order to identify potential candidates for the pharmaceutical industry

9 WORKFLOW (ICH guidelines)

10 THE FUSED-CORE TECHNOLOGY 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.

11 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.

12 HPLC-UV/DAD METHOD Column: Ascentis Express C 18 (150 x 3.0 mm, 2.7 µm) Mobile phase: 0.1% HCOOH in both H 2 O and ACN, gradient elution Flow rate: 0.4 ml/min Injection volume: 3 µl CBDA CBGA Detection: UV/DAD at 210 and 220 nm CBG CBD

13 REPRESENTATIVE CHROMATOGRAMS CBDA CBD-rich fibre-type hemp Drug-type hemp THC CBD THCA CBGA CBG CBG CBG-rich fibre-type hemp CBGA CBG CBN CBGA Cannabidiolic acid (CBDA) Cannabidiol (CBD) CBD CBDA HPLC chromatograms of hemp extracts at 210 nm Cannabigerolic acid (CBGA) Cannabigerol (CBG)

14 SELECTION OF THE EXTRACTION SOLVENT c b b b a b,c c b b,c a c d b b,c a

15 SELECTION OF THE EXTRACTION TECHNIQUE c a b a b a b a b a c a DM: dynamic maceration MAE: microwave-assisted extraction UAE: ultrasound-assisted extraction SFE: supercritical-fluid extraction

16 SAMPLE PREPARATION Dynamic maceration with EtOH 15 min (x 3) r.t. Filtration HPLC Fibre-type hemp inflorescences Sample-to-solvent ratio: 1:40 (w/v)

17 IDENTIFICATION OF CANNABINOIDS The HPLC-ESI-MS n analyses of were carried out with the same HPLC parameters aforementioned. HPLC-ESI-MS n : Capillary voltage: 3.5 kv (+); 3.5 kv (-) Nebulizer pressure (N 2 ): 32 psi Drying gas temperature: 350 C Drying gas flow: 10 L/min Skimmer voltage: 40 V The ion trap mass analyzer was used in the full-scan positive and negative ion modes, in the m/z range MS 2 spectra were automatically performed with helium as the collision gas, using the SmartFrag function in the m/z range

18 CBDA [M H] [2M H+Na] 18 44

19 [M+H] + CBD 56

20 HPLC-UV/DAD METHOD VALIDATION The validation of the HPLC-UV/DAD method developed was performed to show compliance with ICH guidelines. Linearity: r 2 > Sensitivity: 0.5 < LOD < 0.8 μg/ml ; 1.8 < LOQ < 2.5 μg/ml System reproducibility: %RSD tr < 1.4; %RSD Peak area < 1.5 Extraction precision: 0.1 < SD mg/g < 1.4 Accuracy: 74 < Recovery % < 91

21 Samples: QUANTITATIVE ANALYSIS 9 fibre-type hemp inflorescences (C1-C9) 2 samples of hemp oil 2 samples of hemp balm 1 hemp extract pharmaceutical products

22 QUANTITATIVE ANALYSIS CBDA CBD-rich fibre-type hemp CBGA CBG CBD CBGA CBG CBG-rich fibre-type hemp Amount of in fibre-type hemp inflorescences: CBDA: CBGA: CBG: CBD: mg/g mg/g mg/g mg/g CBDA CBD HPLC chromatograms of plant material extracts at 210 nm

23 CBD Oil Amount of in hemp oil: CBDA CBDA Balm CBDA: CBGA: CBG: CBD: 3.5 mg/ml < LOD 0.7 mg/ml mg/ml CBD Amount of in hemp balm and extract: CBD Extract CBDA: CBGA: CBG: CBD: mg/g mg/g 0.4 mg/g mg/g HPLC chromatograms of pharmaceutical products at 210 nm

24 REFERENCE PAPER

25 CONCLUSION A new and reliable RP-HPLC-UV/DAD, ESI-MS and MS 2 method, based on the fused-core technology, was developed for the profiling of 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.

26 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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