Forensic Science International, 4’7 (19901 165- 171 165

Elsevier Scientific Publishers Ireland Ltd.

AN IMPROVED THIN-LAYER CHROMATOGRAPHIC METHOD

FOR THE DETECTION AND IDENTIFICATION OF
CANNABINOIDS IN CANNABIS

KORLIMARLA LAVANYA and TULSIDAS R. BAGGI

Central Forensic Science Laboratory, B.P.R.&D., O.U. Campus, Ramanthapur, Hyderabad

500 013 IZndial

(Received March 20th, 19901

(Revision received May 22nd, 19901
(Accepted June 4th, 19901

Summary

An improved and alternate thin-layer chromatographic (TLC) method has been developed for
the detection and identification of cannabis. The method involves pre-chromatographic derivatis-
ation of the cannabinoids present in cannabis. The derivatised cannabinoids are subjected to
TLC. A pattern typical to the colour of the reaction products of each of the cannabinoids present
in cannabis is obtained. Derivatisation is done by the oxidative coupling of cannabinoids with 3-
methylWbenzthiazolinone hydrazone (MBTH) using acidic ceric ammonium sulphate (CAS) as
the oxidant. Substances like henna, mace and nutmeg do not interfere in the proposed method.
The reagent has not been reported to have carcinogenic properties.

Key words: Cannabis; Pre-chromatographic derivatization; Thin-layer chromatography; methyl-2-

benzthiazolinone hydrazone; identification

Introduction

Cannabis, commonly known as marihuana, is widely abused due to its psy-

chomimetic and euphoretic properties stemming from the depression of the
central nervous system [l]. The active principles of the drug are present in
the plant material and resin. They are the derivatives of 2-(2-isopropyl-5-
methyl phenyll-5-pentyl resorcinol and are known as cannabinoids [2]. These
are unique chemicals found only in cannabis. Cannabis is a controlled sub-
stance and comes under the purview of the Narcotic Drugs and Psychotropic
Substances Act [3]. Numerous TLC [4-g], GC [lO,ll] and HPLC [12-141
methods have been reported in the literature for the separation and identifi-
cation of cannabinoids. These cannabinoids are phenolic compounds having
vacant positions ortho and/or para to the phenolic functional group. Based on
the reaction of this functional group, reagents like anisaldehyde [15], 2,6-
dibromoquinone chlorimide, p-dimethylaminobenzaldehyde, diazotised sulfan-
ilic acid, diazotised p-nitroaniline, Fast blue salt B [16], 1-nitroso-2-naphthol

0379.0738/90/$03.50
0 1990 Elsevier Scientific Publishers Ireland Ltd.
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166

1171 and 3-methyl-2-benzthiazolinone hydrozone (MBTH) [18] have been devel-

oped for the detection of cannabinoids. All these reagents have been used
for spraying the developed TLC plates in order to post-chromatographically
derivatise the cannabinoids for visualisation. Of all these reagents, Fast blue
salt B is most widely used in forensic work. Various other diazonium dyes
have also been studied for their utility as spray reagents, but these have a
disadvantage of being potentially carcinogenic [19]. MBTH was reported by
one of the authors as a new visualisation reagent for the detection of canna-
binoids [18]. It was observed that this method of post-chromatographic deri-
vatisation with MBTH had certain drawbacks. The procedure involved was
cumbersome and the spots obtained on spraying were diffused. Therefore, an
improved and alternate method which is more specific, sensitive and far less
cumbersome was developed. The proposed pre-chromatographic derivatisa-
tion method separates the isomers of the derivatised products of cannabi-
noids. The method can possibly be utilised for the quantitation of
cannabinoids by densitometric measurement of the separated isomers.
The proposed method is based on the oxidative coupling of cannabinoids
with MBTH in the presence of acidic ceric ammonium sulphate (CAS). The
reaction mixture is made alkaline with the help of a buffer and the deriva-
tised products are separated by TLC. A pattern typical to the colour of the
reaction products of each of the cannabinoids is obtained thereby identifying
and confirming the presence of cannabis.

Materials and Methods

All the chemicals used were of analytical reagent grade. A 0.002 M solu-
tion of MBTH was prepared in distilled water. A 0.003 M solution of CAS
was prepared in 0.07 M sulphuric acid. The buffer solution was prepared by
dissolving 1.92 g of sodium hydroxide, 0.8 g of ethylenediamine tetraacetic
acid (disodium salt) and 3.2 g of boric acid in 200 ml of distilled water. Stand-
ard Ag-tetrahydrocannabinol (A~-THC), cannabinol, cannabidiol and cannabi-
gerol solutions were prepared in methanol. The standards were supplied by
the United Nations Narcotics Laboratory, Vienna.
The cannabis sample (or the sample suspected to be cannabis1 was ground
well and thoroughly mixed to obtain a composite mixture. The powdered
sample (100.mgl was triturated with benzene and passed through a short
column (12 cm x 1 cm I.D.1 of Florisil (Fluka, AG, 60 - 10 mesh) as per the
procedure given by Clarke [20] or it was extracted with light petroleum (60
-80%) as suggested by Aramaki et aL [8]. Other methods of extraction may
also be followed. The extract was evaporated to dryness and the residue
redissolved in methanol and made up to 10 ml.
Aliquots of the standard and sample solutions were taken in separatory
funnels. The MBTH solution (10 ml1 was added. The solutions were kept
aside for 5 min with occasional shaking. The buffer solution (20 ml) was then
added and the reaction mixture was extracted with chloroform (3 x 5 ml).
 167

The chloroform extracts were washed thrice with distilled water to remove
traces of CAS. The extracts were then filtered through anhydrous sodium
sulphate before evaporating them to dryness.
The dried residue of the extracts were redissolved in 1 ml of chloroform
and 5 ~1 each of these extracts were spotted on a pre-coated TLC plate (silica
gel G 60 pre-coated plate 10 x 20 cm of 0.25 mm layer thickness Merck Art
5626, E. Merck) pre-activated for 30 min at 105OC. The plate was then devel-
oped to a distance lo-cm from the starting point in an unsaturated tank in
the solvent system benzene (98)lmethanol (2). The development was done at
room temperature (27OC). The plate was removed, air dried and observed
under day and UV light. Characteristic coloured patterns are obtained for
cannabis and individual cannabinoids when observed under daylight. The
fluorescence of some of the spots at particular Rf values under short and
long wave UV light is a further confirmation to the presence of cannabis and
certain cannabinoids.

Results and Discussion

The Rf values of the cannabinoid derivatives along with their colours are
,given in Table 1. The products were observed under short and long wave

TABLE 1

CHROMATOGRAPHIC BEHAVIOUR OF MBTH-CANNABINOID DERIVATIVES

Cannubinoid R! Colour of the Fluorescence under UV light

value product in
day light

Long wave Short wave

A9-THC 0.69 Orange No fluorescence No fluorescence

red
0.56 Pink V. faint pink No fluorescence
fluorescence

Cannabinol 0.83 Maroon Bluish Bluish

fluorescence fluorescence
0.71 Yellowish Orange V. faint yellowish
pink fluorescence fluorescence

Cannabidiol 0.85 Pinkish Yellow Yellow

orange fluorescence fluorescence
0.28 Yellow No fluorescence No. fluorescence
orange

Cannabigerol 0.77 Yellow No fluorescence No fluorescence

0.43 Yellow No fluorescence No fluorescence
orange
168

UV light and the observations are included in Table 1. Chloroform extracts

when not washed thoroughly with distilled water, show blue coloured spots
on TLC development. These are due to the residual CAS present in the
extract.
Various solvent systems were tried for the separation of the derivatised
products. It was observed that efficient separation of the products occurred
by using a polar stationary phase and an essentially non-polar mobile phase.
The solvent system of ideal polarity for optimum separation was found to be
benzene (98llmethanol (21. Further studies showed benzene (49llacetic acid (11
and xylene (981lethanol (llldiethylamine (11 as the other mobile phases suita-
ble for separation.
An attempt to understand the reaction of cannabinoids with MBTH was
made. Phenols react with MBTH in the presence of an oxidiser to give char-
acteristic chromophores. Under the conditions of the reaction, MBTH loses a
proton and two electrons on oxidation to form an active coupling intermedi-
ate. This intermediate undergoes electrophilic substitution with phenols to
give coloured products. This coupling occurs in the vacant positions para
and/or ortho to the phenolic group [21].
The pharmacologically most active cannabinoids in cannabis such as THC,
cannabinol and cannabidiol have free positions ortho and para to the phenolic
group. Each of them, after the proposed derivatisation procedure result in
the formation of two coloured isomeric products. It has been stated that the
para derivatised products are generally red to yellow in colour and have
higher R, values than the ortho products [21]. It was observed that in this
case of electrophilic substitution the red-yellow coloured para isomers are
predominantly formed. The ortho isomers of the cannabinoid derivatives are
formed in very small quantities when compared to para products. Since the
ortho products are formed in small quantities, they are barely visible on the
TLC plate. To obtain large visible spots of ortho products, larger volumes of
the chloroform extract have to be spotted on the plates. When viewed under
day light, the ortho products of AS-THC and cannabinol do not have a larger
difference in their Rf values in samples of cannabis. Under long wave UV
light, the ortho isomer of cannabinol fluoresces while that of AS-THC does
not. The para isomers of most of the important cannabinoids are distinct in
their colours and can be unambiguously identified. The pattern obtained by
the cannabis sample using the proposed procedure is therefore characteris-
tic.
The sensitivity of the proposed method was tested by finding out the min-
imum detectable limits of each cannabinoid. It was observed that the pro-
posed method has a sensitivity comparable to the reported post-
chromatographic derivatisation method using Fast blue salt B and it was
several orders more sensitive than the other post-chromatographic derivatis-
ation procedures reported. The sensitivity of the proposed method as com-
pared to the post-chromatographic derivatisation methods using other
detection reagents reported by Korte and Sieper [16] and Baggi [18] is illus-
 169

TABLE 2

COMPARISON OF DETECTION LIMITS (rg) OF INDIVIDUAL CANNABINOIDS USING

VARIOUS REAGENTS

ReagenVMe thod Minimum detectable amount Ipg)

Cannabidiol Cannabinol THC Cannabigerol

Gibb’s reagent 1 1 1 _
Ghamrawy reagent 0.5 1 1 _
Duquenois reagent 0.5 10 1 _
Diazotised 0.5 1 _
p-nitroaniline
Pauly’s reagent 0.5 0.5 0.5
Fast Blue Salt B 0.01 0.01 0.01 _
Baggi’s method 5 2.5 5 5
Proposed method 0.05 0.01 0.03 0.08

trated in Table 2. Fast blue salt B is the only reagent which is slightly more
sensitive than the proposed reagent. However, the former has been reported
to show batch to batch variations [22] and there is a risk of the diazonium
salt containing residual unreacted potentially carcinogenic free amine [19] to
which the analyst is exposed. As no carcinogenicity has been reported in the
case of MBTH, the proposed procedure is advantageous over the existing
methods. Further, fast blue salt B reagent has to be freshly prepared before
spraying while the reagents used in the proposed method are stable for at
least a week. The derivatised products were found to be stable for several
days.
The Duquenois test which is commonly used as a preliminary test for the
identification of cannabis, shows interference from henna, mace and nutmeg.
These substances have also been reported to interfere with tests involving
diazonium dyes [22]. The chromatograms of cannabis, henna, mace and nut-
meg obtained by developing the plates in established solvent systems and
visualising them by iodine fuming, show similar patterns. These substances
do not show any interference in the proposed method, the pattern of canna-
bis being highly characteristic.

Conclusions

Cannabis contains cannabinoids which are detected and identified using

various rapid and simple techniques such as spot tests and TLC. The pro-
posed method utilises the TLC technique for the separation of pre-chromato-
graphically derivatised cannabinoids. Cannabinoids are oxidatively coupled
with MBTH using CAS as the oxidant to give chromophores capable of
extraction into chloroform. The extract, when subjected to TLC, gives a pat-
170

tern of colours characteristic of cannabis which is used for the identification

and conformation of its presence. The method is sensitive and specific, the
reagent has not been reported to be carcinogenic and the chromophores
obtained are stable for several days. Substances which have been reported
to interfere with the existing method of detection do not interfere with the
proposed method. The method is simple in that it does not involve spraying
with a reagent for visual detection. The proposed procedure can therefore be
used as an alternate method for the detection and identification of cannabis
in forensic exhibits.

Acknowledgement

The authors thank Mr. H.R.K. Murthy for technical assistance. One of the
authors (KL) thank UGC for the grant of fellowship. The authors also thank
the United Nations Narcotics Laboratory, Vienna, for the supply of standard
cannabinoid samples.

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