Industrial Hemp Technical Manual - Standard Operating Procedures for Sampling, Testing and Processing Methodology

Basic Method For The Determination Of Thc In Hempseed Oil

1. Purpose

To measure the THC content in oil obtained by crushing the ripened fruits (seeds) of Cannabis sativa .

2. Scope

Because the oil may be intended for internal use as a food product, it is important that the level of THC allowed be kept at an extremely low level. Laboratories must validate the analytical procedure actually used, and must demonstrate that it meets the requirements of the performance standard published in Appendix B to this Manual.

3. Background Information/Rationale

In theory, the ripened seeds of Cannabis contain no detectable quantity of THC. However, because of the nature of the material, it is almost impossible to obtain the seeds free from extraneous THC in the form of residues arising from other parts of the plant which are in close proximity to the seeds. Although it is required for the seeds to be cleaned before any subsequent use, the resinous nature of some of the material makes complete cleaning extremely difficult.

4. Referenced Documents

[Method according to C. Giroud and L. Rivier, Toxicorama, 7 (4), 15 - 22 (1995)]

5. Safety

It is essential that all safety and security procedures in place in the laboratory be followed rigorously. Because of the nature of the samples, materials for analysis should be dealt with as if they were forensic specimens.

6. Equipment/Materials

As specified in the literature reference.

7. Reagents

As specified in the literature reference.

8. Procedure

8.1

An aliquot of hempseed oil (200mg), containing THC-D3 (50ng/200mg) as internal standard, is saponified using potassium hydroxide/water/ethanol (1:2:20, w/w/w) for 2 hours at 70 E C under nitrogen. After addition of 3ml of water, the THC is extracted using 3 successive portions of 3ml of petroleum ether/diethyl ether (1:1, v/v). The organic solutions are combined and evaporated under nitrogen.

8.2

The THC is quantified following formation of a methyl derivative, according to the following procedure.

After addition of 400µl of dimethylsulfoxide (DMSO) and 20µl of tetrabutylammonium hydroxide (TBAH, 55 - 60% aqueous solution) to the residue from the evaporation, agitation on a vortex-mixer at room temperature for 2 minutes, the extracts are methylated using 100µl of iodomethane (20 minutes at

30 E C). The extracts are then acidified with 700µl of hydrochloric acid (0.1M) and the methylated derivative is extracted into 2ml of isooctane. After centrifugation, the organic layer is removed and evaporated under nitrogen. The residue is taken up into 70µl of isooctane, and 1µl is injected into the GC-MS.

8.3

A multipoint calibration curve must be obtained using grape-seed oil (or other equivalent oil free from THC) containing a series of amounts of THC (for example, from 10 to 250 ng/20mg of oil). Analyses are performed each time on 4 aliquots of each specimen of hempseed oil, of which two are tenfold dilutions using the same oil as used to prepare the calibration curve.

8.4

The methylated THC is quantified by GC-MS operated in the SIM mode. The ions at m/z 328 and 331, representing respectively the base peaks from methylated THC and THC-D3, are used for the quantification.

9. Quality Control/Results Verification

Results are calculated as micrograms of delta9-THC per gramme of hempseed oil. The average is calculated from each pair of single measurements. The permissible limit for test error (ie, permissible difference between the individual values of a double measurement) under repetitive conditions is 0.03% absolute. If there is a greater difference between two single values, another measurement must be taken.

10. Method Validation

Validation of the analytical method in the hands of the actual analyst performing the analysis is mandatory. The validation procedure must be performed as specified in the laboratory's SOP for validation of analytical methods. Furthermore the method must be demonstrated to meet the performance standard in Appendix B to this Manual before being used for any analyses.

Office of Controlled Substances
Document Code : HECS-OCS-004
Revision Number: 002