Cannabis: New text of the German Pharmacopoeia

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Chapter B: Revised Monograph:

Cannabis flowers

Cannabis flos

 

Definition

Cannabis flowers consist of the dried, flowering shoot apexes, whole or crushed, of the female plants of Cannabis sativa L. (Cannabaceae). The drug contains at least 90.0 and at most 110.0 % of the amount of cannabinoids stated on the label, such as ∆9-tetrahydrocannabinol and cannabidiol, and cannabinoid carboxylic acids, such as ∆9-tetrahydrocannabinolic acid and cannabidiolic acid, calculated as ∆9-tetrahydrocannabinol (C21 H30O2; Mr 314,5) or cannabidiol (C21H30O2; Mr 314,5), based on the dried drug.

 

Properties

Odour: characteristic of cannabis flowers.

 

Identity test

  1. The female inflorescences are left whole or are separated into their individual parts. The dense bracts and flowers of the overall inflorescences form a highly compressed panicle of a length and width of approximately 1 to 5 cm, with the dark-green bracts protruding out slightly. The light-brown to brown styles and remnants are generally up to 1 cm long. The flower husks are green to light-green and, like the bracts, are covered with dense yellowy-white hairs and stuck together with resin. The crumbled drug contains peduncle fragments, bracts and panicle sections, as well as individual flowers and flower organs. The individual flowers are 5 to 10 mm long, in some cases short-stemmed, and consist of the hood-like, green to light-green flower husk, the 1-2 mm whitish fruit node, which may contain a small brown ovule, and the brown style with two long, thin remnants. The bract fragments are dark-green to green; the peduncles are light-green. The bracts and all the flower organs, aside from the styles, are to some extent densely covered with glandular hairs that are sticky due to released resin.
  2. Testing is carried out under a microscope, using chloral hydrate solution R. The powdered drug (355) has the following features: Large glandular hairs having pluricellular stalks and pluricellular heads (A), isolated stalks (B) and isolated heads (C); pluricellular gland stalks from below (D); large, tapered covering hairs of different lengths and with very thick cell walls, isolated or on cuticles (E), in some cases together with cystoliths (F); bract fragments having short, wide cystolith hairs on the upper epidermis (G, H), the upper epidermis having polygonal or sinuate anticlinate cell walls, the cystolith hairs having very thick, in some cases verrucose cell walls, the cystoliths can be seen as racemose structures, the palisade parenchym is visible below the epidermis; bract fragments having fine, unicellular covering hairs (I); leaf fragments having sinuate or undulating, moniliform, thick anticlinate cell walls of the lower epidermis, anomocytic stomata; leaf fragments densely covered with contact points for the pluricellular stalks of the large glandular hairs; leaf fragments having a very high number of calcium oxalate twins in the mesophyll (J); the vessels within the leaf fragments have helicoidally thickened cell walls; the leaf epidermises may have small glandular hairs with a unicellular stalk and a unicellular to pluricellular head, or stalkless glandular hairs having cells arranged actinomorphically (N); peduncle fragments having covering hairs, helicoidal vessels and rows of crystal cells containing calcium oxalate twins; carpel fragments, the upper epidermis of which has cells with straight or slightly sinuate cell walls (K) and the lower epidermis of which has highly undulating anticlinate cell walls (L); fragments of the brown styles and remnants, densely covered with long, club-shaped papilla; infrequent tricolpate pollen grains with smooth exine.

 

 

  1. Testing is done with thin-layer chromatography (2.2.27).

Test solution: 0.1g powdered drug (710) is extracted for 10 min with 5 ml methanol R in an ultrasonic bath and then filtered through a folded filter. The filtrate is then filtered through a membrane filter made of regenerated cellulose having a nominal pore width of 0.45 µm. This solution is used as the test solution.

Reference solution: 5 mg each of cannabidiol RN and ∆9-tetrahydrocannabinolic acid RN are dissolved in 5 ml methanol R.

Stationary phase: Thin-layer chromatography plate with octadecylsilyl silica gel F254R (2 to 10 µm).

Application: 5 μl; in bands 8 mm x 2 mm.

Solvent: Mixture of 70 parts by volume methanol R, 15 parts by volume water R and 15 parts by volume 99 % acetic acid R.

Length of run: 6 cm.

Detection and evaluation of results

The plate is dried in air, then sprayed with vanillin reagent R and heated for approximately 15 minutes at 100 to 105 °C. The analysis is performed in natural light.

The sequence of zones in the reference and test solution chromatograms is shown in the information below. Other weakly coloured zones may be present in the test solution chromatogram.

 

Upper plate edge
 

 

 

 

 

_______

 

 

 

 

Cannabidiol: 1 purple zone

_______

 

 

 

 

9-tetrahydrocannabinolic acid: 1 purple zone

 

 

 

 

 

_______

 

 

 

 

1 purple zone (cannabidiol)

_______

 

1 purple zone

 

 

1 purple zone (∆9-tetrahydrocannabinolic acid)

Reference solution Test solution

 

 

Purity test

Foreign constituents (2.8.2): maximum 2 %.

Loss on drying (2.2.32): maximum 10 %, determined with 1 000 g powdered drug (710) by vacuum drying for 24 hours using molecular sieve R at 40 °C and a pressure between 1.5 and 2.5 kPa.

Cannabinol: maximum 1.0 %. Testing is carried out using liquid chromatography (2.2.29), as specified under Assay, using reference solution V.

The percentage content of cannabinol (C21H26O2) is calculated according to the following equation:

 

 

t           =          loss on drying of the drug in per cent.

 

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Assay

Testing is carried out using liquid chromatography (2.2.29).

Test solution: 0.500 g powdered drug (710) is shook for 15 min with 20 ml 96 % ethanol R and then centrifuged. The clear supernatant is transferred to a 50 ml graduated flask. The residue is treated twice, each time with 12.5 ml 96 % ethanol R. The organic solutions are combined and topped up to 50.0 ml with 96 % ethanol R. This solution is filtered through a membrane filter made of regenerated cellulose having a nominal pore width of 0.45 µm. 1.0 ml filtrate is topped up to 10.0 ml with 96 % ethanol R.

Reference solution I: 10.0 mg ∆9-tetrahydrocannabinol RN are dissolved in methanol R up to 100.0 ml (stock solution). By diluting this solution with methanol R, at least six calibration solutions Ia to If are produced, with a concentration range of 0.5 to 75 µg · ml-1; one calibration solution has the concentration 10 µg · ml-1.

Reference solution II: 30.0 mg ∆9-tetrahydrocannabinol RN are dissolved in methanol R up to 100.0 ml. By diluting this solution with methanol R, at least six calibration solutions IIa to IIf are produced, with a concentration range of 0.5 to 250 µg · ml-1; one calibration solution has the concentration 50 µg · ml-1.

Reference solution III: 10.0 mg cannabidiol RN are dissolved in methanol R up to 100.0 ml. By diluting this solution with methanol R, at least six calibration solutions IIIa to IIIf are produced, with a concentration range of 0.5 to 75 µg · ml-1; one calibration solution has the concentration 10 µg · ml-1.

Reference solution IV: 15.0 mg cannabidiolic acid RN are dissolved in methanol R up to 100.0 ml. By diluting this solution with methanol R, at least six calibration solutions IVa to IVf are produced, with a concentration range of 0.5 to 100 µg · ml-1; one calibration solution has the concentration 50 µg · ml-1.

 

Reference solution V: 10.0 mg cannabidiol RN are dissolved in methanol R up to 100.0 ml. By diluting this solution with methanol R, at least six calibration solutions Va to Vf are produced, with a concentration range of 0.1 to 10.0 µg · ml-1; one calibration solution has the concentration 1 µg · ml-1.

Reference solution VI: 10.0 mg ∆8-tetrahydrocannabinol RN are dissolved in methanol R up to 100.0 ml. 1.0 ml solution is mixed with 1.0 ml stock solution of reference solution I and topped up to 10.0 ml with methanol R.

The chromatography can be carried out as follows:

Precolumn

Material: stainless steel.

Dimensions: length: 5 mm; internal diameter: 3 mm.

Stationary phase: octadecylsilyl silica gel for chromatography R (2.7 µm).

Column

Material: stainless steel.

Dimensions: length: 0.15 m; internal diameter: 3 mm.

Stationary phase: octadecylsilyl silica gel for chromatography R (2.7 µm).

Column temperature: 40 °C.

Elution

Mobile phase A: aqueous solution of 85 % phosphoric acid R (8.64 g · l-1).

Mobile phase B: Acetonitrile R.

Flow rate: 1.0 ml · min-1.

 

Time [min] Mobile phase A

[vol.%]

Mobile phase B

[vol.%]

Notes
0-16 36 → 18 64 → 82 linear gradient
16-17 18 → 36 82 → 64 linear gradient
17-20 36 64 Equilibration

 

Test conditions

Detector: Spectrometer at a wavelength of 225 nm (for cannabidiol or ∆9-tetrahydrocannabinol) and 306 nm (for cannabidiolic acid or ∆9-tetrahydrocannabinolic acid).

Feeding system: sample loop.

Volume injected: 10 µl in each case.

Recording time: The test solution chromatogram is recorded for 20 minutes.

Relative retention (in relation to ∆9-tetrahydrocannabinol, tR approximately 8.3 minutes)

  • Cannabidiolic acid: approximately 0.48
  • Cannabidiol: approximately 0.57
  • Cannabinol: approximately 0.83
  • 8-tetrahydrocannabinol: approximately 1.04
  • 9-tetrahydrocannabinolic acid approximately 1.24

Suitability test

Resolution: at least 1.2 between the ∆9-tetrahydrocannabinol and ∆8-tetrahydrocannabinol peaks in the chromotogram for reference solution VI.

Precision: The reference solution II is injected six times and the areas of the peaks corresponding to ∆9-tetrahydrocannabinolic acid are determined. The test can only be evaluated if the individual values’ relative standard deviations from the average are no more than 1.0 %.

 

Evaluation

  1. The percentage content of ∆9-tetrahydrocannabinol (C21H30O2) is calculated according to the following equation:

 

 

Cr-a      =  ∆9-tetrahydrocannabinol concentration in the test solution in milligrams per millilitre, calculated using the calibration function of the calibration solutions Ia to If.

Gr-a    =  ∆9-tetrahydrocannabinol concentration in ∆9-tetrahydrocannabinol RN in per cent.

Cu      =  drug concentration in milligrams per millilitre.

t         =  loss on drying of the drug in per cent.

 

  1. The percentage content of ∆9-tetrahydrocannabinolic acid (C22H30O4) is calculated according to the following equation:

 

 

Cr-b      =   ∆9-tetrahydrocannabinolic acid concentration in the test solution in milligrams per millilitre, calculated using the calibration function of the calibration solutions IIa to IIf.

Gr-b    =   ∆9-tetrahydrocannabinolic acid RN content in per cent.

Cu      =   drug concentration in milligrams per millilitre.

t        =   loss on drying of the drug in per cent.

 

  1. The percentage content of cannabidiol (C21H30O2) is calculated according to the following equation:

 

 

Cr-c      =  cannabidiol concentration in the test solution in milligrams per millilitre, calculated using the calibration function of the calibration solutions IIIa to IIIf.

Gr-c    =  cannabidiol RN content in per cent.

Cu      =  drug concentration in milligrams per millilitre.

T        =  loss on drying of the drug in per cent.

 

  1. The percentage content of cannabidiolic acid (C22H30O4) is calculated according to the following equation:

 

 

 

Cr-d      =  cannabidiolic acid concentration in the test solution in milligrams per millilitre, calculated using the calibration function of the calibration solutions IVa to IVf.

Gr-d    =  cannabidiolic acid RN content in per cent.

Cu      =  drug concentration in milligrams per millilitre.

T        =  loss on drying of the drug in per cent.

 

Calculating the percentage contents

A + B = total content of ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabinolic acid, calculated as ∆9-tetrahydrocannabinol.

C + D = total content of cannabidiol and cannabidiolic acid, calculated as cannabidiol.

 

Storage

Tightly sealed, away from light, below 25 °C.

 

Note

Non-binding information on the relative content of cannabinoids in cannabis flowers:

Product group   Content of
I 9-tetrahydrocannabinol >> cannabidiol
II 9-tetrahydrocannabinol ≈ cannabidiol
III 9-tetrahydrocannabinol << cannabidiol

 

Labelling

The calculated percentage content of ∆9-tetrahydrocannabinol and cannabidiol must be stated on the container.

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