DEVELOPMENT OF A NEW EXTRACTION TECHNIQUE AND HPLC METHOD FOR THE - PowerPoint PPT Presentation

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 LAB UNIMORE

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% Δ 9 -THC. PHYTO AND MORE LAB UNIMORE

CANNABIS SATIVA L. PHYTO AND MORE LAB UNIMORE

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. PHYTO AND MORE LAB UNIMORE

BIOLOGICAL ACTIVITY PHYTO AND MORE LAB UNIMORE

BIOLOGICAL ACTIVITY ■ Cannabigerol, Cannabichromene and Cannabidiolic acid have been found to possess promising antibacterial properties, even against multi-drug resistant bacteria. PHYTO AND MORE LAB UNIMORE

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 HPLC Cannabidiolic acid (CBDA ) Cannabidiol (CBD) GC vs techniques techniques Δ -CO 2 Cannabigerolic acid (CBGA) Cannabigerol (CBG) PHYTO AND MORE LAB UNIMORE

*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 PHYTO AND MORE LAB UNIMORE

WORKFLOW Development of the HPLC method Optimization of the extraction conditions Identification of cannabinoids Validation of the analytical method (ICH guidelines) Quantitative analysis PHYTO AND MORE LAB UNIMORE

THE FUSED-CORE TECHNOLOGY Development of the HPLC method ■ A fused-core (ore core-shell) stationary phase is characterized by 2.7 µm Optimization of particles, which comprise a solid 1.7 µm diameter silica core that is the extraction encapsulated in a 0.5 µm thick layer of porous silica gel. conditions Identification of cannabinoids Validation of the analytical method Quantitative analysis PHYTO AND MORE LAB UNIMORE

THE FUSED-CORE TECHNOLOGY Development of the HPLC method ■ The innovative manufacturing process for fused-core particles produces a very narrow particle size distribution. Optimization of the extraction ■ Traditional porous particles are not manufactured in a way to yield conditions extremely narrow particle size distributions. Identification of cannabinoids Validation of the analytical method Quantitative analysis PHYTO AND MORE LAB UNIMORE

HPLC-UV/DAD METHOD Development of the HPLC method ■ Column: Ascentis Express C 18 (150 x 3.0 mm, 2.7 µm) Optimization of the extraction ■ Mobile phase: 0.1% HCOOH in both H 2 O and ACN, gradient elution conditions ■ Flow rate: 0.4 mL/min Identification of CBDA cannabinoids CBGA ■ Injection volume: 3 µL ■ Detection: UV/DAD at 210 and 220 nm Validation of the analytical method CBG CBD Quantitative analysis PHYTO AND MORE LAB UNIMORE

REPRESENTATIVE CHROMATOGRAMS Development of CBDA Drug-type hemp CBD-rich fibre-type hemp the HPLC method THC CBD Optimization of THCA the extraction conditions CBGA CBGA CBN CBG CBG Identification of cannabinoids CBG-rich fibre-type hemp CBG CBGA Validation of the Cannabidiolic acid (CBDA ) Cannabidiol (CBD) analytical method CBD CBDA Quantitative analysis Cannabigerolic acid (CBGA) Cannabigerol (CBG) HPLC chromatograms of hemp extracts at 210 nm PHYTO AND MORE LAB UNIMORE

SELECTION OF THE EXTRACTION SOLVENT Development of the HPLC method Optimization of the extraction c conditions b b b a Identification of cannabinoids Validation of the analytical method d c b b,c a c b b,c b,c a Quantitative analysis PHYTO AND MORE LAB UNIMORE

SELECTION OF THE EXTRACTION TECHNIQUE Development of the HPLC method Optimization of the extraction c conditions b a a Identification of cannabinoids Validation of the c b a a analytical method b a b a Quantitative analysis DM : dynamic maceration MAE : microwave-assisted extraction UAE : ultrasound-assisted extraction SFE : supercritical-fluid extraction PHYTO AND MORE LAB UNIMORE

SAMPLE PREPARATION Development of the HPLC method Optimization of the extraction conditions Dynamic maceration with EtOH HPLC Filtration 15 min (x 3) analysis Identification of r.t. cannabinoids Fibre-type hemp inflorescences Validation of the analytical method Sample-to-solvent ratio: 1:40 (w/v) Quantitative analysis PHYTO AND MORE LAB UNIMORE

IDENTIFICATION OF CANNABINOIDS Development of the HPLC method ■ The HPLC-ESI-MS n analyses of cannabinoids were carried out with the same HPLC parameters aforementioned. Optimization of the extraction HPLC-ESI-MS n conditions: conditions ■ Capillary voltage: 3.5 kV (+); 3.5 kV (-) Identification of ■ Nebulizer pressure (N 2 ): 32 psi cannabinoids ■ Drying gas temperature: 350 °C ■ Drying gas flow: 10 L/min Validation of the analytical method ■ 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 200-1200. MS 2 spectra were automatically performed Quantitative with helium as the collision gas, using the SmartFrag function in the m/z range analysis 50-1500. PHYTO AND MORE LAB UNIMORE

CBDA Development of [M–H] – the HPLC method Optimization of the extraction conditions [2M–H+Na] – Identification of cannabinoids 18 Validation of the analytical method 44 Quantitative analysis PHYTO AND MORE LAB UNIMORE

CBD Development of the HPLC method [M+H] + Optimization of the extraction conditions Identification of cannabinoids 56 Validation of the analytical method Quantitative analysis PHYTO AND MORE LAB UNIMORE

HPLC-UV/DAD METHOD VALIDATION Development of the HPLC method The validation of the HPLC-UV/DAD method developed was performed to Optimization of show compliance with ICH guidelines. the extraction conditions ■ Linearity: r 2 > 0.998 ■ Identification of Sensitivity: 0.5 < LOD < 0.8 μg/mL ; 1.8 < LOQ < 2.5 μg/mL cannabinoids ■ System reproducibility: %RSD t R < 1.4; %RSD Peak area < 1.5 Validation of the ■ Extraction precision: 0.1 < SD mg/g < 1.4 analytical method ■ Accuracy: 74 < Recovery % < 91 Quantitative analysis PHYTO AND MORE LAB UNIMORE

QUANTITATIVE Development of the HPLC method ANALYSIS Optimization of Samples : the extraction conditions ■ 9 fibre-type hemp inflorescences (C1-C9) Identification of ■ 2 samples of hemp oil cannabinoids ■ pharmaceutical products 2 samples of hemp balm Validation of the ■ analytical method 1 hemp extract Quantitative analysis PHYTO AND MORE LAB UNIMORE

QUANTITATIVE Development of ANALYSIS CBDA the HPLC method CBD-rich fibre-type hemp Optimization of CBD the extraction conditions Amount of cannabinoids in fibre-type hemp inflorescences: CBGA CBG CBDA: 0.9 – 46.8 mg/g Identification of cannabinoids CBGA: 0.1 – 9.8 mg/g CBG CBG-rich fibre-type hemp CBG: 0.2 – 6.5 mg/g CBD: 0.1 – 23.9 mg/g Validation of the CBGA analytical method CBD Quantitative CBDA analysis HPLC chromatograms of plant material extracts at 210 nm PHYTO AND MORE LAB UNIMORE

CBD Oil Development of Amount of cannabinoids in hemp oil: the HPLC method CBDA CBDA: ≤ 3.5 mg/mL Optimization of CBGA: < LOD the extraction ≤ 0.7 mg/mL CBG: Balm conditions CBDA CBD: 3.5 – 78.6 mg/mL Identification of CBD cannabinoids Amount of cannabinoids in hemp balm and extract: CBDA: 44.7 – 80.4 mg/g Validation of the CBD Extract analytical method CBGA: 2.0 – 3.9 mg/g ≤ 0.4 mg/g CBG: CBD: 7.6 – 193.7 mg/g Quantitative analysis HPLC chromatograms of pharmaceutical products at 210 nm PHYTO AND MORE LAB UNIMORE

REFERENCE PAPER PHYTO AND MORE LAB UNIMORE

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 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. PHYTO AND MORE LAB UNIMORE