Two simple colour tests for cannabis

Sections

Abstract
Introduction
Methods
(A) "Meta" Duquenois test
(B) Field-test
Results and discussion
TABLE IV Red colour after one minute; green chloroform layer and red aqueous layer
TABLE V Violet colour after one minute; clear chloroform layer and violet aqueous layer
TABLE VIII Substances giving different colour reactions (Colours as stated)
(B) Field-Test
(C) Both tests

Details

Author: M. J. de Faubert MAUNDER
Pages: 37 to 42
Creation Date: 1969/01/01

Two simple colour tests for cannabis

M. J. de Faubert MAUNDER
Ministry of Technology, Laboratory of the Government Chemist, London, S.E.1.

Abstract

Two new colour tests for cannabis are described, one of which is recommended as a field-test, the other as an improvement on a conventional laboratory test. For the majority of samples, the two tests are adequate confirmation occupying, at most, three minutes analytical time.

Introduction

Most laboratory chemical methods for cannabis detection are very simple in application, and coupled with microscopy, are sufficiently specific for most purposes. Instrumental methods are often not necessary and largely used to indicate a "finger print" of chemical composition when evidence of a common origin is required, for instance, in conspiracy cases. However, no simple method for the rapid identification of cannabis under field conditions has received widespread acceptance to date. Two methods have been developed in this Laboratory, one of which is suitable as a field-test, the other as an improvement on a conventional laboratory test.

Two versions of the Duquenois test were described in his original publication [ (1)] and of these, his quantitative reagent has received wide acceptance for qualitative work, viz: add 2 ml of the reagent (0.4 g vanillin + 0.06 g acetaldehyde in 20 ml 95 % ethanol) to an evaporated petroleum ether extract (normally in a small evaporating basin), add 1 to 2 ml concentrated hydrochloride and observe the colour transitions. The test can be made more specific by the addition of 2 to 3 ml chloroform after 2 to 3 min, shaking, and noting the colour of the chloroform layer. This modification is also widely used.

The rapid Duquenois test [ (2)] uses the same reagents and is preferred to the conventional version because of its speed and simplicity, viz: add 2 ml of the reagent directly to the suspect material in a test-tube (without any prior petroleum ether extraction stage) add 2ml concentrated hydrochloric acid, observe the colour transitions, add 1 to 2 ml chloroform, shake and observe the colour of the chloroform layer. This modification lacks specificity without the chloroform addition stage. It was initially considered for field use and in these early investigations it was noted that a direct substitution of metaldehyde for acetaldehyde improved the keeping qualities of the Duquenois reagent, and allowed a solid form to be kept indefinitely with the added advantages of increased sensitivity and speed of response. To distinguish this modification from the rapid and standard versions, the prefix "meta" is used. It is now used to the exclusion of the other two in this Laboratory and was only discarded as a potential field-test after the superiority of the Fast blue B salt method [ (3)] became apparent. These two new tests are described and discussed below.

Methods

Commercial grade reagents are suitable

(A) "Meta" Duquenois test

Apparatus

Test tube(s)
Source of heat (see text)

Reagents

Ethanol *
Concentrated hydrochloric acid; d= 1.18 g/cm ³
Chloroform
Solid reagent - Powder and mix intimately 1 % metaldehyde in vanillin
Liquid reagent - 2 % solid reagent in ethanol *

Method

Place about 1 mg resin or 2 mg herbal sample in a test tube with 100 ± 50 mg of the solid reagent. Add 1 ± 0.5 ml ethanol and heat to dissolve the solid. It is not necessary to heat longer than the time needed to dissolve the reagent, which, if unshaken, is usually when the solution comes to the boil, say 10 seconds. In the laboratory heat is most conveniently applied from a steam bath, but a match or cigarette lighter provides adequate heat for an analyst's field kit. Without cooling, add 1 to 3 ml concentrated hydrochloric acid. If the acid is added dropwise, positive samples normally develop a strong violet colour during the addition. Allow up to a minute if needed for the colour to develop, add 1 to 3 ml chloroform, agitate the tube to mix and allow the phases to separate. A pink/mauve colour in the chloroform layer is a positive response.

Where duty restrictions apply, ethanol may he replaced by industrial methylated spirit.

(B) Field-test

Apparatus

Absorbent paper
2 dropping bottles
Micro spatula

Reagents

Low boiling petroleum ether (boiling range 40-60 °C) - PE
Water
Solid reagent Fast blue B salt, (3,3'-dimethoxybiphenyl-4,4'-bisdiazonium chloride). Dilute Fast blue B salt, 1:100 with solid anhydrous sodium sulphate

Method

Place 1 mg of the suspect material on an absorbent paper, add one drop of petroleum ether, allow to soak into the paper and dry naturally. Remove the suspect material from the paper (tip into a test tube if the" meta" Duquenois test is to be applied subsequently), place about 0.1 mg of the solid reagent on the absorbent paper at the original site of the suspect material and add one drop of water to it. A red to violet colour develops as the water spot expands over the area originally covered by the petroleum ether spot. When dealing with powdery or sticky materials, it is necessary to use two thicknesses of absorbent paper and sufficient PE to moisten the lower paper, and to apply the test to the lower paper. If the PE solution is not filtered in this manner, most powders will leave enough residue on the paper to give sufficient water soluble material for a false positive.

Results and discussion

(A) "Meta" Duquenois test

The colour developed by this modified reagent differs slightly in hue from the conventional reagent, but is essentially the same violet colour with a more pronounced red overtone. Good quality cannabis in any form will give an initial green colour on the addition of the acid, turning rapidly into a violet via many different hues.

The transitions are frequently too rapid to observe fully and an "immediate" violet colour is the result; complete light absorption (absorbance >10) within 10 sec is not uncommon with fresh samples. Differences in types of cannabis, concentration and temperature yield very variable colours from almost red, through mauve/violet to blue, At low concentrations of cannabis a red colour may predominate initially, whilst at very low levels or with old samples a pale green to olive green colour is more common. Regardless of the colour developed at this stage, the chloroform layer colour is invariably the same as given by the conventional reagent and is best described as a pink/mauve. Because of this consistency, a positive test is not recorded until this colour has been identified, and because it is almost impossible to describe in absolute terms it is best recognised by experience, as are the colour transitions in the acid solution.

Many different materials have been tested. All yielded a colour within one minute (see tables I - VIII) although in some cases this was little more intense than, and the same colour as, a blank. For a given material the colour range could be quite variable as indicated above, but rough standardisation of the conditions as described leads to a fairly confident reproduction of the generic colours listed in the tables. About half of the materials subsequently gave a coloured chloroform layer, and of these the majority were green with no risk of confusion with cannabis. A minority (see table VII) gave a red to blue chloroform solution which, without careful observation of the speed and sequence of colour development after the addition of the acid, may be difficult to distinguish from the cannabis colour. None of these materials gave precisely the same colour behaviour as fresh cannabis, but most could not be readily distinguished from the reaction with old, or trace amounts, of cannabis. Because of the wide commercial use of nearly all the materials in table VII, it is evident that this test should never be relied upon as the only positive evidence Other workers have examined other botanicals ( [ 4] , [ 5] , [ 6] ). and have reached a similar conclusion, but for the most part their criterion for selection was based on some botanical resemblance to the genus cannabis. As noted elsewhere [ (3)] , some quite common materials have a striking visual resemblance to both herbal and resin cannabis, and most of these appear in table VII.

Where it is difficult to obtain pure ethanol, adequate results are obtained with industrial methylated spirit, and provided a control is carried out at the same time, even mineralised methylated spirit with added dyestuff can be used with care. If the hydrochloric acid used is weak, or has deteriorated through excessive exposure to the atmosphere, poor colour development will result. Apart from strong sulphuric acid (>50 %) no satisfactory substitute was found; no solid acid produced a colour. Substitution of piperonal for vanillin did not offer any advantage, the colour being a little slower to develop but of about the same intensity. The concentration of metaldehyde in vanillin is not critical and can be varied between 0.1 and 10 %, the best results being obtained at about 1%.

Batches of the solid and liquid " meta " reagent have been stored successfully for over a year without deterioration, but the solid reagent does lose some activity when left unstoppered longer than a week, and the liquid darkens and throws out a precipitate if left in strong light longer than 2-3 months. The solid reagent when used as described is more suitable for detecting traces of cannabis. Thus a response is obtained from a 1 mm 2 piece of leaf even after the field test has been carried out on it. The liquid reagent is more suitable as a direct substitute for the conventional Duquenois reagent, or as a spray reagent for thin layer chromatograms.

Summary of the colours obtained with the " Meta " Duquenois test TABLE I Green colour after 1 min; clear chloroform layer and green aqueous layer


aconite	gum arabie	phlomis
angelica	gum barbary	podophyllum
anise	gum ghatti	quassia
banana (dried)	gum karaya	rape seeds
banana (dried skin)	gum tragacanth	rauwolfia
barberry root	horseradish	rue
bayberry	ipecacuanha	sabadilla
bearberry	jalap	safflower
blackberry leaves	khat	santal wood (yellow)
buchu	lavender	savory
calumba	lovage	scammony root
caraway	mandrake	senega
chicory	marjoram	spearmint
clove	marshmallow	stavesacre seeds
coca	mountain flax	stonecrop herb
cohosh (blue)	nettle	tansy
colchicnm	nux vomica	tarragon
coriander	olive stones (ground)	valerian root
dandelion (roasted)	onion	witchazel leaves
deer tongue leaves	palm kernel meal	wormwood
dill	pellitory root	yarrow
gentian	pepper (black alligator)
grass	perppermint herb

TABLE II Green colour after one minute; green chloroform layer and green aqueous layer


ailanthus	gamboge *	passion flower leaf
avens herb	golden seal	pepper (black)
balm herb	guaiacum gum	pilewort
basil	hyoscyamus	pulsatilla
belladonna	hyssop	pyrethrum
bladderwrack	Indian tobacco flower	rosemary
broom ( violet initially)	lily of the valley root	sage
bugleweed	lobelia	sanicle herb
burdock	mace	senecio
burner herb (greater)	marshmallow herb	shepherds purse
chervil	mate	silverweed
clivers herb	melilot herb	sloe leaves
coItsfoot leaves	melochia herb	southernwood herb
comfrey	mint	St. John's wort
cranesbill herb	molohia (jute)	strammonium
cummin	mugwort	tea plant
fennel	opoponax	thyme
fumitory	parsley	vervain

TABLE III Red colour after one minute; clear chloroform layer and red aqueous layer


black haw bark	hellebore (black)	quillaia bark
bryony	jasmin root	rupturewort
colocynth	logwood (Jamaican)	sarsaparilla
date stones (roasted)	mullein	snakeroot (black)
digitalis	opium	snakeroot (Virginian)
elecampane	pennyroyal	stargrass
fenugreek	poppy petals	starwort
gum benzoin*	quercetron bark	yohimba

TABLE IV Red colour after one minute; green chloroform layer and red aqueous layer


aloes	figwort	raspberry leaves
asefetida	grindelia herb	rhubarb (root)
balmony herb	holy thistle herb	saffron
blessed thistle	horehound	seaweed (edible)
boneset	horsetail	senna
capsicum	hops	skullcap herb
chamomile	jaborandi	squill
cistus	lupulin	sumach
coffee (instant)	meadowsweet herb	tobacco
curry powder	motherwort	turmeric
damiana	ragwort

TABLE V Violet colour after one minute; clear chloroform layer and violet aqueous layer


apocynum	cocoa	slippery elm bark
bayberry bark	cocillana bark	soya
betel nuts	cramp bark	squill (red)
boldo	derris	stropanthus
cascara	ephedra	tea (instant)
cassia bark	kola	wild cherry bark
catechu (black)	mustard (black)	wintergreen
catechu (pallid)	pimento	yeast
cinchona bark	sabal
cinnamon	savin

TABLE VI Violet colour after one minute; green chloroform layer and violet aqueous layer


agrimony	gum bdellium	squaw vine
cannella alb,	gum kino	tea
cardomon	gum olibanum
ginseng	gum thus

TABLE VII Violet colour after one minute; violet chloroform layer and violet aqueous layer


calamus	gum ammoniac	nutmeg
cannabis (flowering tops)	gum animi	orris
cannabis (leaf)	gum copal*	poison flag
cannabis (resin)	gum galbanum	sagapenum
cannabis (seed husk)	gum kowri	santal wood (red)
coffee (green)	gum myrrh	thuja
coffee (roasted)	gum sandarac	tolu
culvers root	henna	wood betony
ergot	lettuce opium	woodsage
ginger	liquorice

TABLE VIII Substances giving different colour reactions (Colours as stated)


Substance	Colour after 1 min	Chloroform layer	Aqueous layer
annato	green	orange	green
araroba	brown	orange	brown
butternut bark	black	brown	black
colophony	brown *	orange	brown
dragons blood	brown* (red anyway)	red	brown
kamala	green (orange *)	orange	green
laburnum	yellow (leached)	yellow	yellow
madder	orange	orange	orange

NOTES TO TABLES I-VIII

Green - any colour between yellow and turquoise. This category includes a nil colour equivalent to a blank.

Red - any colour between red, orange, brown.

Violet - any colour between red (pink) and blue.

- precipitate.

(B) Field-Test

This has been in use in this Laboratory for over a year as a first-action method, and been used by H.M. Customs as a field-test for almost a year. Experience by non scientific personnel has confirmed the high sensitivity and simplicity of the test; it has been applied to a wide range of natural products. In the early stages, a number of false positive results were recorded and traced to interference from powdery materials. The use of the double-layer-paper technique appears mandatory when dealing with powders. Because any single officer will have only occasional need to apply the test, it seems unreasonable to expect him to apply critical judgement in interpreting the quality of the colour stain produced to eliminate false positives, and the slight extra sophistication of the double paper test will allow him to apply the simple rule of thumb: "No red colour proves the absence of cannabis; any red colour is a strong ground for suspicion ".

TABLE IX Comparison of the response of substances to the " Meta " Duquenois test, the rapid [ (2)] and standard [ (10)] versions of the Duquenois test, and the Ghamravy [ (9)] test, after a positive result by the field-test


		Duquenois
Substance	Field test	" Meta "	Rapid	Standard	Ghamravy
Positive field-test and positive " meta " Duquenois
henna	†(pink)	‡	green	pale green	green-
			CHCl ₃clear		brown
nutmeg	†(pink)	‡	‡	slow	bright
		opalescent	CHCl ₃clear	too red	red
Positive field test and positive first stage of " meta " Duquenois
agrimony	†(pink)	‡	‡	nil	green-
		CHCl ₃green	CHCl ₃green		brown
mace	†(pink)	‡	‡	slow	bright
		opalescent &	CHCl ₃green	too red	red
Positive response to all 5 tests
(Cannabis, grown in England; flowering tops or Young shoots gave strong response, and large leaf NOT flowering tops were used as a better comparison between tests.)
female, flowering, 1962	†(strong)	‡	blue/green	‡	strong
female, fruiting, 1962	†(V.strong)	‡(strong)	‡	‡(strong)	V.strong
female, immature, 1968	†(slow)	‡(strong)	nil	weak-	weak
			CHCl ₃faint	blue/green
male, flowering, 1968	†(slow)	‡(weak)	nil	weak-	weak
CHCl ₃strong	CHCl ₃faint	blue/green

†positive response within one minute.

‡positive response of normal colour within one minute.

TABLE X Common azo dyes tested for colour response with cannabis


azo dye	Colour Index No.	colour developed	comment
diazo red RC	17780	orange	1
Fast-salt
black BS	37245	violet	2
black BTL	-	violet	3
black G	-	mauve	2
black K	37190	violet	2
blue B	37235	red/violet	1 see text
blue RR	37155	pale pink	3
blue V	-	pale orange	3
blue VB	37255	nil	3
blue VRT	37240	pale orange	3
Bordeaux BD	37170	orange/pink	3
brown V	37200	red brown	1
corinth LB	37160	orange	1
dark blue R	37195	violet	2
garnet GBC	37210	garnet	1
garnet GC	37215	red orange	1 see text
grey G	-	violet	2
navy blue 3RA	-	mauve	2
olive BR	-	olive	2
orange GC	37005	yellow	1
orange GGD	37045	yellow	1
orange GR	37025	yellow orange	1
orange R	37030	yellow	1
orange RD	37050	yellow	1
red AL	37275	orange/pink	3
red B	37125	orange	1 see text
red FRN	37075	yellow orange	1
red ITR	37150	orange	3
red TR	37085	orange	1
red 3GL	37040	orange	1
scarlet R	37130	yellow orange	1
violet B	37165	pink	3
violet F		orange/pink	3
violet LB	-	orange	3

1 - Colour and speed of reaction satisfactory; specificity may not be adequate.

2 - Too reactive, no specificity.

3 - Unsuitable.

A number of further "Fast" salts have been tested since the original publication to find a suitable substitute and only Fast garnet GR (2,3'-dimethylazobenzene-4'diazonium chloride) merited further study (see table X). The colour produced is a strong orange, but it can be weak on occasion with herbal cannabis or if the reagent is diluted too much with anhydrous sodium sulphate. If used undiluted it will produce a strong yellow background of its own and obscure weak posi- tives, thus incurring the same objection [ (3)] as Fast red B. Fast blue B remains the most satisfactory dye tested.

(C) Both tests

A few extra botanicals have been tested since the original field-test publication [ (3)] , agrimony being the only herbal material subsequently found to give a strong positive eraction. Although the colour developed on the paper is a paler hue, it could be mistaken for that given by cannabis. This false result would be" confirmed" by a subsequent " meta " Duquenois test if the chloroform addition stage is not used: the chloroform layer is green, and quite distinct. Henna is the only herbal material examined which cannot be eliminated by both tests. It will respond to the field-test if an excessively large sample is taken and it is sometimes difficult to convince non scientific staff that the very small sample size specific in " Method " should not be exceeded. The colour sequences obtained with the " meta " Duquenois test are not easily distinguished from cannabis, and the chloroform layer is the correct pink colour only turning a dull grey after several minutes. There are some superficial microscopic features in common with cannabis [ (6)] and in inexperienced hands, with small samples, the three different methods could lead to erroneous confirmation. The appearance of the sample is as much part of the test as the chemical/microscopic examination, and in view of the widespread use of henna as a drug and cosmetic dye, samples of unfamiliar appearance should be examined by gas liquid chromatography [ (7)] , or more simply by thin layer chromatography [ (8)] where henna does not produce spots. One large consignment of henna (weighing more than 10 kg) has recently been examined in this Laboratory. It was powdered and mixed in an approximate ratio of 1:2 with a light brown form of cannabis resin.

For the majority of samples submitted for analysis appearance, odour, the field-test and a subsequent " meta " Duquenois test are adequate confirmation of the presence of cannabis occupying, at most, three minutes analytical time. Microscopy can be completed on the residue from the chemical tests in a further 5-10 min, say, 10 min in all. A parallel sub-sample can be taken for thin layer chromatography and run at the same time as the other tests.

References

⁰⁰¹

P. Duquenois and H. N. Moustapha, J. Egypt. Med. Ass., 1938, 21, 224.

⁰⁰²

Methods of analysis for alkaloids, opiates and synthetic drugs, U.S. Treasury Department, Internal Revenue Service, Publication No. 341, Nov. 1956.

⁰⁰³

M. J. de Faubert Maunder, J. Assoc. Publ. Analysts., 1969, 7 (1), 21.

⁰⁰⁴

United Nations Secretariat, Document ST/SOA/SER.S/ (1960).

⁰⁰⁵

L. Grlic, Bull. Narcot., 1964, 16(4), 29.

⁰⁰⁶

G. R. Nakamura, Journal of the A.O.A.C., 1969, 52(1), 5.

⁰⁰⁷

B. P. Jackson and D. W. Snowdon, Powdered Vegetable Drugs, J. A. Churchill, Ltd., 1968.

⁰⁰⁸

L. T. Heasysman, E. A. Walker and D. T. Lewis., Analyst, 1967, 92, 450.

⁰⁰⁹

M. J. de Faubert Maunder, J. Pharm. Pharmac. 1969, 21, 334.

⁰¹⁰

M. A. Ghamravy, J. Egypt. Med. Ass., 1937, 20, 193.