Analysis of cannabinoids
0.01 g (±.0001) of crystals was dissolved in 1 ml of methanol (HPLC grade). Solution was sonicated for 2 min and vortexing for 10 sec. Samples before HPLC analysis were further diluted with methanol to the final concentration of 0.01 mg/ml.
Analysis of cannabinoids content was performed using Waters 2695 (Milford, MA, USA) separation module equipped with auto injector, sample cooler, vacuum degasser and column heater units. Separation of all cannabinoids was accomplished on YMC PRO C18 (150 x 4 mm I.D., S-3μm) RP column coupled with C18 precolumn maintained at 30 °C by a CTO-20AC column oven. Isocratic elution consisted of acetonitrile:water (FA 0.1%) (4:1) was done in 30 min. The flow rate was maintained at 0.8 ml/min. The cannabinoid CBD and CBD-A were monitored at 225 and 306 nm wave length respectively using dual absorbance detector Waters 2487 (Milford, MA, USA). The injection volume of 20 μl was injected using auto sampler at 10 °C. Data evaluation was
performed using Empower 2 software.
Quantification of CBD and CBD-A were obtained from linear regression equation of calibration curve of individual reference standards by plotting concentration versus the area ratio. The calibration range for CBD and CBD-A were linear from 5 to 500 μg/ml. Samples which contain CBD or CBD-A concentration higher than 40% were weaken diluting 10 times beforeinjection. Retention time of CBD-A was at 7.1 min and CBD at 8.1 min.
Analysis of terpenes
10 mg of homogenous sample was scaled and diluted with 1 ml of pentane containing 0.04 % of decane as internal standard. The tube containing the sample solution was placed in ultrasonic bath for 5 min and then mixed. 200 µL of prepared solution was diluted with 800 µL of pure pentane mixed and individually analysed by GC-FID.
An Agilent HP 6890 gas chromatograph equipped with FID was used for the analysis of terpenoids. Separation was accomplished on a Rtx-5 w/Integra-Guard capillary column (30 m length, 0.25 mm i.d. and 0.25 µm df). Injections were carried out in split mode using a general purpose split/splitless liner packed with glass wool. The program started at 50 °C, increased to 280 °C (at 15 °C/min) and held for 15 min for a total of 31 min. 2 µL of each sample was injected with helium as the carrier gas (constant flow mode, 1 ml/min) using a split ratio 1:10. Temperatures were applied 280 °C for the injector, 260 °C for the transfer line. Data was analysed using Chemstation v.D.02.00.275 (Agilent Technologies).
List of the target Terpenes:
- Compound RT
- Decane (IS) 5,165
- α-pinene 4,635
- Myrcene 5,095
- Limonene 5,538
- Linalool 6,176
- E-Caryophyllene 9,328
- Decane concentration in pentane 0,04% = 1 ml pentane contain 0.292 mg decane.
- 0.01g of sample dissolved with 1000 µL pentane containing 0.04 % decane.
- Extract with concentration of 10 mg/ml dissolved 5 times. Final concentration of the extract in solution 2.5 mg/ml (0.0025 g/ml).
- Final concentration of decane in solution 0.073 mg/ml.
- 0.073 mg of decane / 0.0025 g of extract = 29.2 mg/g extract
Peak Area of decane - 29.2 mg/g extract
Peak area of target compound - x mg/g extract
x= 29.2 × Peak area of target compound / Peak area of decane = amount of target compound mg/ g extract.