Health Canada Resource on THC analysis

ANNEX C

COMMUNITY METHOD FOR THE QUANTITATIVE DETERMINATION OF DELTA-9 THC (TETRAHYDROCANNABINOL) IN CERTAIN VARIETIES OF HEMP

1. Purpose and scope

This method permits quantitative determination of delta-9 tetrahydrocannabinol (delta-9 THC) in certain varieties of hemp Cannabis sativa L.) for the purpose of checking that the conditions laid down in Article 3 (1) of Regulation (EEC) No. 619/71 are fulfilled.

2. Principle

Quantitative determination of delta-9 THC by gas chromatography (GC) after extraction with a suitable solvent.

3. Apparatus.

-- gas chromatography equipment with a flame ionization detector,

--glass column 2,50 m long and 3,2 mm in diameter (1,8") packed with a suitable support impregnated with a stationary phase phenyl-methyl-silicon (e.g. OV 17 at 3 %).

4. Sampling and reduction of sample

Sampling

In a standing crop of a given variety of hemp, take not less than 500 plants, preferably at different points but not from the edges of the crop. Samples should be taken during the day after flowering has finished.

The pooled samples should be representative of the lot.

The plant material is then left to dry at ambient air temperature.

Reduction

Reduce the sample, obtained as described, to 500 stalks; the reduced sample should be representative of the original sample. Divide the reduced sample into two portions.

Send one portion to the laboratory which is to determine the delta-9 THC content. Keep the other portion for counter-analysis if necessary.

5. Reagents

-- petroleum ether (40)/65°), or a solvent of comparable polarity,
-- tetrahydrocannabinol (delta-9 THC), pure for chromatographic purposes,
-- solution of 0,1% (w/v) androstene-3-17-dione in ethanol, pure for chromatographic purposes.

6. Preparation of test sample

For the purposes of delta-9 THC determination, retain the upper third of the plants in the portion of sample received. Stems and seeds must be removed from the plant material retained.

Dry the material in an oven, without exceeding 40°C, to obtain a constant weight.

7. Extraction

Reduce the material obtained as described in point 6 to a semi-fine powder (sieve of 1000 meshes per cm2.

Take 2,0 g of well-mixed powder and extract with 30-40 ml petroleum ether (40-65°C). Leave for 24 hours, then shake in a mechanical shaker for one hour, and then filter. The extraction process is carried out twice under the same conditions. Evaporate the petroleum ether solutions to dryness. Dissolve the residue in 10.0 ml of petroleum ether. The prepared extract is used for quantitative analysis by gas chromatography.

8. Quantitative analysis by gas chromatography

(a) Preparation of assay solutions

The extraction residue dissolved in 10.0 ml of petroleum ether is subjected to quantitative analysis to determine the delta-9 THC content. This is performed with the aid of an internal standard and calculation of the peak areas.

Evaporate to dryness 1,0 ml of the petroleum ether solution. Dissolve the residue in 2,0 ml of a solution of 0,1% androstene-3-l7-dione in ethanol (internal standard with a retention time distinctly higher than that of other cannabinoids, and in particular twice that of delta-9 THC).

calibration ranges:
0,10, 0.25, 0,50, 1,0 and I,5 mg of delta-9 THC in 1 ml of a solution of 0,1% androstene-3-l7-dione in ethanol.

(b) Experimental conditions

Oven temperature:	240°C.
Injector temperature:	280°C.
Detector temperature:	270°C.
Nitrogen flow rate:	25 ml/min,
Hydrogen flow rate:	25 ml/min,
Air flow rate:	300 ml/min,
Volume injected:	1 ml of the final ethanol solution.

The relative retention time of delta-9 THC is calculated in relation to the andostene.

9. Expression of the results

The result is expressed in g of delta-9 THC per 100 g of the laboratory sample dried to constant weight. The result is subject to a tolerance of 0,03g per 100g.