Analysis of cannabinoids

0.01 g (±.0001) of crystals were dissolved in 1 ml of methanol (HPLC grade).

The 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.

Chromatographic Analysis

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 wavelength respectively using dual absorbance detector Waters 2487 (Milford, MA, USA). The injection volume of 20 μl was injected using autosampler at 10 °C.

Data evaluation was performed using Empower 2 software.

Quantification of CBD and CBD-A were obtained from the linear regression equation of the 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 weakened diluting 10 times before injection.

The 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 the internal standard.

The tube containing the sample solution was placed in the 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

1. Decane concentration in pentane 0,04% = 1 ml pentane contain 0.292 mg decane.
2. 0.01g of sample dissolved with 1000 µL pentane containing 0.04 % decane.
3. 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).
4. Final concentration of decane in solution 0.073 mg/ml.
5. 0.073 mg of decane / 0.0025 g of extract = 29.2 mg/g extract
6. Calculations

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.