INTRODUCTION:

No one known exactly when cannabis popularly known as marihuana or marijuana was discovered but it is certain that it is one of the oldest drugs known to man. According to the statistical information, cannabis is most widely abused illicit drug in the world today¹. It has been used for its euphoric effects for over 4000 years. Tetrahydrocannabinol (THC), the primary psychoactive drug, is found in the plant’s flowering or fruity tops, leaves and resin. The active principles of the drug are present in the resin which contains 30 derivatives of 2-(2-isopropyl-5-methyl phenyl)-5-pentylresorcinol, known as cannabinoids². The most important cannabinoids are cannabinol (CBN), Cannabidiol (CBD), Tetrahydrocannabinol and tetrahydrocannabinoic acid.

There are three cannabis products on the basis of THC level-

Herbal cannabis (Bhang):

Herbal cannabis is also known as ganja or marijuana. Herbal cannabis is a material obtained from the cannabis plant. The leaves and flowering tops of the female plant are harvested, dried and some times pressed into bricks or twisted into, in appearance to tobacco, although greenish rather than brown in colour.

Resin cannabis (charas):

The resin of the flowering tops of the cannabis plant is collected, dried and some times baked. This is cannabis resin also known as ‘hashish’ or ‘charas’. It may also be found in powder form.

Liquid hashish (Hashish oil):

Hashish oil also known as concentrate of cannabis extract, liquid hashish, this is obtained by a process of repeated extraction performed on herbal cannabis or cannabis resin. The end product is a viscous extract. It becomes more viscous on prolonged contact with air.

THC level in three illicit cannabis products are-

Herbal cannabis-0.5-5%

Resin cannabis – 2-10%

Liquid hashish – 10-30%

These values are only for guidelines to forensic chemist. Cannabis may have THC content out of the above-mentioned values.

MATERIALS AND METHODS:

Screening⁴:

For screening there are various steps followed as given below-

Sampling:

Cannabis products represent a special problem to the chemist in that the sample under examination are often enormous size and the chemical tests used require only a very small amount. Homogenization is not useful in such conditions. The analyst must ensure that the entire item is a controlled drug. The importance of visual examination, which plays a very minor role in the identification of powder drugs, cannot be overemphasized in the analysis of cannabis products. Sampling errors for quantitative analysis are reduced if large amounts of sample are used.

Physical Examination:

(a) Macroscopic Examination:

Morphological properties of individual cannabis plants are greatly affected by environmental factors such as space for growth, light, water and other conditions. Hereditary factors also affect plant growth. Thus plant growth depends on both environmental and hereditary factors.

The side branches are opposite on the main stem. The flowers are abundant and they are either male (staminate) or female (pistillate). Female plants are very leafy up to the top, whereas male plants have the leaves on inflorescence lesser and much further apart.

The male inflorescence is loosly arranged, much branched and many flowered, standing out from the leaves with individual flowering branches.

The female inflorescences do not project beyond the leaves. They are compact, short and contain lesser flowers.

(b) Microscopic Examination:

The very abundant trichomes, which are present on the surface of the fruiting and flowering tops of cannabis, are the most characteristic features to be found in the microscopic examination of cannabis products. Non glandular hairs (trichomes), numerous, unicellular, rigid, curved with a slender pointed apex and an enlarged base, usually containing a cystolith.

The glandular trichomes occur in three forms

- Sessile glands with one celled stalk

- Long multicellular stalk form

- The head in both forms is globular containing of eight to sixteen cells

IDENTIFICATION^3,4:

Colour tests:

(i) Fast blue B salt test

(ii) The rapid Duquenois test

(i) Fast blue-B salt test:

Sample (pasty mass or liquid), add petroleum ether then shake for one minute, add solid fast blue-B salt and then add 10 % solution of sodium bi carbonate (w/w) a purple red colour was obtained which indicate the possible presence of charas. This test can be performed in test tube.

(ii) The rapid Duquenois test:

Take sample in a test tube, add 2ml of the solution (0.5 drops of acetaldehyde and 0.4g of vanillin are dissolved in 20 ml of 95 % ethanol), shake for one minute, add 2 ml of conc. HCl, shake and stand for 10 min, if a colour develops add 2 ml of chloroform. If chloroform layer becomes violet i.e. cannabis product may be present.

Preparation of sample:

Extracts of the samples were used for qualitative and quantitative analysis. The extracts were prepared as 300 mg of an uniform sample weighed in a beaker were covered with 10 ml of petroleum ether and stirred at about 0^oC for 1h. After filtration into a small evaporating dish, the ether extract was dried under a stream of warm air and the dry residue was dissolved in a small amount of methyl alcohol, and the resulting solution was then poured into a 2 ml calibrated flask. If necessary, the solution was make up with methyl alcohol to the volume of 2 ml.

Thin layer chromatography of cannabis products:

Thin layer chromatography (TLC) was used for the qualitative analysis of the methanol extracts of the cannabis samples. The samples were spotted on the plate with the standard. The plates with the applied extracts were developed in a small chamber using the following solvent systems Pet ether: Diethyl ether (80:20) or Cyclohexane: Di - isopropyl ether: Diethyl amine (52:40:8).

Visualizing reagent:

After drying the TLC plate at room temperature spray the plate with the following reagent.

Fast blue-B salt: Dissolved 50 mg of Fast blue-B salt in 20 ml of 0.1 N sodium hydroxide solution. This solution should be freshly prepared. For proper colour development TLC plate may be made alkaline by spraying diethyl amine before the fast blue B salt solution.

Quantitation:

Gas liquid chromatography of cannabinoids:

Detector : FID

Column : 6ft (2m), I.D. 2 to 4mm

Packing : 3% OV-17 or SE-30 or OV-1

Carrier gas : Nitrogen at 30 ml / min

Instrumental conditions:

Injector temperature : 270^oC

Oven temperature : between 240-260^oC

Detector temperature : 300^oC

Internal standard :

N-tetradecane or n- docosane or other suitable n-alkene used as internal standard.

Preparation of solution for gas chromatography:

For qualitative GC analysis the extracts prepared for TLC may be used directly. Suitable injection volume may be 1-5 μl depending upon the actual concentration of the sample. For quantitation the main neutral cannabinoids a 10μl aliquot is taken of the same extract. After evaporating the solvent in vaccum the residue is redissolved in 10 ml of methanol: chloroform (1:1) containing 2 mg / ml n-tetradecane as internal standard

General formula used for calculation of any component of the cannabinoid

(Conc. Std) x (Area under peak for spl) / (Area of int. std for spl solu.)

%C =- -------------------------------------------------------X 100

(Conc. Spl) x (Area under peak for std) / (Area of int. std for std solu.)

RESULTS AND DISCUSSION:

Analysis of cannabis by the methodology discussed above is a powerful tool for forensic chemists. The area of field-testing may develop as a function of the changing pattern of the illicit drug traffic and scientific developments. It is therefore important to review different methodologies. Laboratories on the other hand, should take advantage of available literature and other expertise to review their current methodology of testing.

ACKNOWLEDGEMENT:

Authors are grateful to the Director LNJN, National Institute of Criminology and Forensic Science, Rohini, Delhi-110085 (India) for his constant inspiration and moral support.

REFERENCES:

1. M.A. Huestis, Cannabis Monograph, SOFT Committees on Driving Under Influence of Drug, 1994, pp 1-10.

2. Clarke’s Isolation and Identification of Drugs, 1986, 423.

3. C. Szulc, R. Zarzecka and A. Szuchnik, Forensic Science International, 46 (1990) 117-120.

4. Recommended methods for testing cannabis, manual for use by National Narcotics Laboratories, United Nations Newyork, 1987.

Received on 30.03.2009 Modified on 26.05.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 401-403