An improved procedure for the field testing of cannabis

Sections

Abstract
Introduction
Solid reagent Dyestuff
Diluent
Test papers
Water (second liquid)
Solvent (first liquid)
Applications
Toxicological applications
Sampling tool
Packaging technique
Recommended field test method
Apparatus
Reagents
Method
Results and conclusions
Acknowledgement

Details

Author: M. J. de FAUBERT MAUNDER
Pages: 19 to 26
Creation Date: 1974/01/01

An improved procedure for the field testing of cannabis *

M. J. de FAUBERT MAUNDER
Department of Trade and Industry, Laboratory of the Government Chemist, London, S.E. 1 9NO, United Kingdom.

Abstract

Two alternative dyes have been found to have the same specificity as Fast Blue B when used as a field test for cannabis. A number of other dyes have been found yielding alternative colours to Fast Blue B. Other modifications have led to an over-all improvement in the speed and intensity of response, without loss of specificity.

Introduction

A simple and specific test for cannabis was introduced in 1968 and reported in 1969 [ [ 1] , [ 2] ]. It is increasingly being used as a field and toxicological test and has been incorporated in a commercially available kit [ [ 3] ]. The Merck Company [ [ 4] ] have also produced a kit that includes a test based on the same dyestuff - Fast Blue B. Their modification uses "sensitised" papers of the dyestuff presented as a pad at the end of a plastics handle. In operation, the test is both sensitive and simple but some false positives have been noted. These have arisen from the absence of a filtering stage [ [ 2] ], now regarded as essential.

A major disadvantage of this alternative approach [ [ 4] ] is the risk of contamination. The sample is dissolved in a depression on a plastics spot tile which must be cleaned after use. Lay opinion of cleanliness varies and cannabis resin is a tenacious material, difficult to remove completely even with polar solvents. This tenacity is relied upon for toxicological and trace testing. The alternative of disposable, but contaminated, spot tiles raises legal problems[ [ 5] ]. Disposal of restricted materials is not necessary in the procedures recommended here. A further disadvantage of sensitised papers is discussed by Woodhouse [ [ 6] ]. Long term storage is not possible. The sensitised papers replace a solid reagent, containing anhydrous sodium sulphate as a protective agent. Therefore, they are not stable in moist and contaminated atmospheres. Both alternative versions of the test [4,6] also require the use of two liquids.

The field test method has sufficient sensitivity to be used in toxicological studies. Woodhouse [ [ 6] ] and McCarthy and van Zyl [ [ 7] ] have applied the methods to monitor breath and lip swabs of suspected smokers. McCarthy also notes the well known phenomenon that certain types of tobacco yield a positive response. These are described as toasted tobaccos. It has been found in the Laboratory that all tobaccos tested after smoking give a weak pink to brown colour. However, cannabis when smoked in a cigarette or pipe partially sublimes and distils, so that an effective concentration of active components is found in the cigarette butt or pipe stem. Thus, in practice, the intensity of colour produced is significantly greater than that from unsmoked cannabis, and no confusion should arise with experienced investigators. Nevertheless, laymen would not be expected to make this distinction and all smoking residues should be submitted to laboratory examination. This has the added advantage of preserving both visual and chemical evidence for detailed study.

* The author wishes to thank the Government Chemist for permission to publish this paper.

Solid reagent Dyestuff

The safety of the Fast Blue B dyestuff is still sometimes questioned. This is essentially the risk of the diazonium salt containing residual unreacted, potentially carcinogenic, free amine. These doubts have led to a reduction in the number of manufacturers and suppliers. An alternative dye, Fast Red B was recommended in the first report [ [ 1] ], to alleviate this anticipated situation. The second report [ [ 2] ] discussed a further number of dyestuffs. Fast Garnet GC (GR) was selected from them as the best alternative available at that time.

During field trials with the cannabis test, it was found that non-scientists did not readily accept the substitute dye. This is a familiar phenomenen, particularly in pharmaceutical acceptance trials. The characteristic red/violet colour obtained with Fast Blue B had already become recognised as the normal or "correct" colour. Attention was therefore directed towards selection of a dye with a similar colour and response speed.

Although three were found to be interchangeable with Fast Blue B for field test purposes, the preferred dyestuff is Fast Corinth V (Colour Index Number 37220) [ [ 8] ]. This is produced commercially by coupling 2-nitro-4-toluidine with cresidine (2-methoxy-4-methylaniline) and re-diazotising. The reaction with cannabis is more rapid and sensitive than with Fast Blue B. The other two are an analogue of Fast Blue RR which had already been used as an alternative to Fast Blue B in chromatography [ [ 9] , [ 10] ]. Fast Blue RR itself had already been found unsuitable in a field test due to slow response speed and low colour intensity [ [ 1] , [ 2] ].

This analogue, Fast Blue BB, is slower to respond than Fast Blue B but the final intensity of response is better, being more vivid. The dye is considered to be a suitable substitute, but has the disadvantage of having a very similar name to, and is liable to be confused with, Fast Blue B. It is 4-Benzamido-2,5-diethoxyphenyl diazonium chloride, zinc Chloride. Another batch of dyestuff was not given a common name by the supplier [ 1] (1-diazo-4-benzoylamino-2,5-diethoxybenzene) which, for convenience, will be referred to in this paper as Fast Blue LGC. The speed of response is comparable to the first batch of Fast Blue BB: the final intensity is marginally superior. The final hue with both batches is very similar. Both batches are inferior to Fast Corinth V with respect to speed and colour intensity.

Quantitative measurements of the relative merits of dyestuffs are difficult because of the subjective nature of the final assessment. A further complication arises from the variable nature of cannabis itself. It was considered here that a definite response to all samples in a non-scientist's interpretation of "rapid" say, about ½ min. would be acceptable as an effective test. Only dyes providing an intense colour, of a different hue from the background colour (arising from dye self-coupling) were taken for further study.

1. A suitable reagent is available from Sigma Chemical Co., Cat. No. D4127.

To compare the proposed dyes, the following experiment was carried out, with Fast Blue B taken as the standard. This dye normally responds strongly to most cannabis samples within a few seconds - "instantaneously". A typical cannabis resin sample was diluted with petroleum spirit until a lµl sample responded on a filter paper to 1mg of the diluted solid reagent when moistened with one drop of 1 per cent sodium hydrogen carbonate solution. The petroleum spirit spot averaged less than 4 mm diameter. The time to respond was taken to be the first definite colour discernable in subdued daylight. The results are recorded in table I, from which it is confirmed that Fast Blue RR is a poor substitute for Fast Blue B, or the other dyes recommended here. Tables II and III list a series of dyes tested in this programme.

TABLE I

Summary of relative merits of reactive dyes in terms of response speeds

	Response speed in seconds for various aliquots
Dye (fast salt)	1µl	2µl	3µl
Blue B a	5 ^a
Corinth V	1.6
Garnet GC	15	2.6
Blue BB (batch 1)	25	10	10
Blue LGC (batch 2)	35	15	10
Red AV	60	5.2
Blue BR	over 60 (on drying)	20-40

^a Taken as reference standard.

TABLE II

Preferred azo dyes, in order of preference

Fast salt	Colour Index Number	Colour with cannabis
Corinth V	37220	red/violet - immediate
Blue LGC	37175	pink/violet - slight delay
Blue BB	37175	pink/violet - slight delay
Blue B	37235	red/violet - rapid response
Garnet GC (GR)	37215	red/orange - very suitable
Red AV	-	orange - very suitable
Garnet GBO	37210	garnet
Bordeaux GP	37135	pink/orange
Red B	37125	orange

Both batches of Fast Blue BB are preferred chromogens to Fast Blue B for thin layer chromatography of cannabinoids. Fast Corinth V is not suitable as a tlc chromogen because it yields similar hues for all the cannabinoids. This, however, is a practical advantage in a field test reagent for all samples of different age or origin yield the same hue. Where it is desirable to standardise on a single reagent for both tlc and the field/spot test, Fast Blue LGC is recommended.

TABLE III

Azo dyes not considered suitable

Fast salt	Colour Index Number	Colour
Black BS	37245	violet
Black BTL	-	violet
Black G	37260	mauve
Black K	37190	violet
Blue RR	37155	pale pink
BlueV	-	pale orange
Blue VB	37255	nil
Blue VRT	37240	pale orange
Bordeaux BD	37170	orange/pink
Brown V (VA)	37200	red brown
Corinth LB	37160	orange
Dark blue R	37195	violet
Golden orange CR	37065	yellow
Grey G	-	violet
Navy blue 3RA	-	mauve
Olive BR	-	olive
Orange G (GC)	37005	yellow
Orange GGD	37045	yellow
Orange GR	37025	yellow orange
Orange R	37030	yellow
Orange RD	37050	yellow
Ponceau L (Red PDC)	37151	yellow
Red AL	37275	orange/pink
Red FRN	37075	yellow orange
Red GG	37035	yellow orange
Red GL	37110	yellow orange
Red 3GL	37040	orange
Red KB	37090	yellow
Red KL	-	yellow
Red LTR (ITR)	37150	orange
Red RC	37120	yellow
Red RL	37100	yellow orange
Red TR	37085	orange
Red violet LB	-	orange
Scarlet GG	37010	yellow
Scarlet R	37130	yellow orange
Violet B	37165	pink
Violet F	-	orange/pink
Yellow GC	37000	yellow
4-diazo-2-chlorobenzene	-	yellow
4-diazo-diphenylamine .	-	pink orange
diazotised N, N-diethyl-3-toluidine	-	yellow
diazotised 2-chloro-N, N-diethylaniline	-	pink orange

Attempts to avoid the use of a solid reagent were not successful. The results of Woodhouse [ [ 6] ]were confirmed and ready-prepared test papers were not found to be practical for long-term storage: Replacement of the solid by a (third) liquid was no more successful. Some dyestuffs were not readily soluble in toluene; on health grounds, benzene had been ruled out for a field test. Simple aqueous solutions were not stable for extended periods. Addition of methanol [ [ 11] ] improved stability to in excess of one hour and addition of dilute hydrochloric acid to this, or the simple aqueous solution [ [ 12] ], extended stability to several days. Storage beyond a week was difficult, rendering the reagent unsuitable for use in a field test kit. However, the acidified reagents may be freshly prepared each week and used in a laboratory as a substitute for the solid reagent. Storage in dark glass bottles, or in the dark whether as a solid or as a liquid reagent, is essential to reduce free radical formation

Diluent

The solid diluent added to the dyestuff serves three main purposes. Firstly, it dilutes the highly reactive dye and effectively prevents the addition of too large a quantity. Secondly, it protects the highly reactive dye from corrosive and moist atmospheres. Thirdly, it acts as a mild light screen. Anhydrous sodium sulphate functions in all three capacities-inert diluent, desiccant and light screen. The absence of these three advantages may explain the poor storage properties of "sensitised" papers.

The diluent also provides a salting-out or pseudo-mordant effect. The colour is dispersed onto the paper fibres, rather than slowly precipitated as the fluid dries. The salting-out effect reduces the induction period, rendering the test more definitive in action.

For storage in excess of 3 months at ambient temperatures, some batches of anhydrous sodium sulphate were found to be unsatisfactory. Excessive amounts of residual moisture or alkalinity were identified as causes of premature decomposition. Each batch of anhydrous sodium sulphate must be tested for residual alkalinity. Only neutral batches can be used; "AR" quality is not always adequate. Suitable batches require a simple thermal dehydration, e.g. heat the solid (normally in the manufacturer's bottle) in an oven at around 100 °C for several hours, and cool in a confined space (normally achieved by replacing the closure).

The dye must be fully dispersed to avoid auto-reaction as far as possible. Some batches of dyestuff are difficult to triturate fully with the diluent at the 1 per cent level. In these cases, carry out a two stage dilution: ( a) Thoroughly triturate the dye with approximately twice its volume of diluent until homogeneous; then ( b) Triturate (mix) this strong reagent with the remaining diluent to a homogeneous 1% concentration.

Test papers

Some types of absorbent paper, particularly acid washed varieties, have been found to interfere with colour development: in some cases this was totally inhibited. Similarly, good quality neutral papers, such as Whatman No. 1, inhibited colour development if stored in an acid atmosphere. Only grades equivalent to Whatman No. 1 filter paper are now recommended for the field test.

Water (second liquid)

It was not always practicable to store the absorbent papers away from corrosive atmospheres. Such a condition arose in an acute form during the trial manufacture of composite test kits and was shown to arise from the LSD field test reagent then in use [ [ 13] ]. All kits containing concentrated hydrochloric acid in a reagent suffer from this feature. A simple modification of the LSD field test overcame the immediate problem [ [ 5] , [ 8] ]. Nevertheless, it was considered desirable to add an alkali to the water used as the second liquid reagent in the field test. Addition of a strong alkali increased the probability of the dyestuff coupling with a non-cannabinoid plant extract, but 1 per cent sodium hydrogen carbonate was found to give a solution of correct pH.

The buffer had sufficient alkalinity to permit the use of all grades of absorbent paper, including acid washed grades and adverse storage conditions were no longer important. Nevertheless, it is preferable to use only good quality paper as recommended above.

Incidental advantages were found to be improvement in the speed, intensity and permanence of colour development. The mildly alkaline solution also prevented mould and algae growth and could now be stored at elevated temperatures without the risk of deterioration.

Solvent (first liquid)

In hot climates, petroleum spirit of a low boiling range, 40-60 °C, was found to be unsuitable. Also, extended storage in polythene dropper bottles resulted in evaporation through the microporous material. Petroleum spirit of a boiling range 60-80°C was found to be a preferable solvent under all realistic storage conditions. The extended evaporation period in temperate climates is not noticeable in practice. However, petroleum spirit boiling above 100 °C is not recommended because residual solvent in the test paper inhibits colour development.

It had been suggested [ [ 1] ] hat any organic solvent could be used when the test is intended solely to prove the absence of cannabis. Cigarette lighter fluid was cited [ [ 1] ] as one alternative. However, solvents other than pure petroleum spirit are no longer recommended. This removes the element of discretion, particularly in lay hands.

Applications

Toxicological applications

Some suggestions have been made elsewhere [ [ 1] , [ 2] , [ 5] ] and the work of McCarthy and van Zyl and Woodhouse [ [ 6] ]has already been noted. In the hands of an experienced analyst, the field test is capable of detecting traces of cannabis (as its resin) adhering to the skin or any other contaminated surface. Solvent washing of a suspect's finger with, say, petroleum spirit onto a filter paper yields sufficient cannabinoidal material to yield a positive response. The skin of persons handling large quantities of cannabis, such as habitual smokers or smugglers, absorbs cannabinoids. These are not readily removed by simple wiping, or even by soap and water washing, and can be detected one day later with ease, and up to two days in the case of unwashed skin.

Sampling tool

It is often necessary for an analyst to examine a cavity or surface known, or suspected, to have been in contact with cannabis. A simple wiping of the surface will entrain sufficient material for the wiping paper to be used directly as the top paper of the field test. This technique preserves visual evidence as far as possible. The analyst proceeds with this screening process until satisfied that cannabis is absent.

It follows that all spatulas and other sampling tools must be wiped clean before, and after, each test.

Packaging technique

A positive response obtained as above will require confirmation. The partly extracted top paper may be retained for further examination by folding the paper so asto enclose the entrained material. An investigator may also submit the residue from the standard field test for confirmation in a laboratory. This is particularly important in situations where only minute samples are available for testing. Laboratory confirmation conveniently takes the form of a second chemical test, such as the "Meta" Duquenois test [ 2] , and then the minute residues are partly cleared and stained for direct microscopy. A permanent slide is made from these residues.

Recommended field test method

Apparatus

Whatman filter paper No. 1 (5.5 or 7 cm dia), or equivalent grade;
Two dropper bottles;
Micro spatulas, together with tweezers if desired.

Reagents

Commercial grade reagents are suitable if tested (see text).

Petroleum spirit (boiling range 60-80 °C)-PE;
One per cent sodium hydrogen carbonate in water;
Solid reagent - Dilute Fast Corinth V salt, 1:100 w/w with finely powdered solid anhydrous sodium sulphate.

Method

Clean spatulas and other sampling tools by vigorous wiping with a filter paper. Fold two filter papers into quarters and open partly to form a funnel. Place no more than 1 mg of suspect material in the bottom of the funnel and add one drop of PE. Allow to soak into the papers and dry naturally. Separate the papers, retaining the top paper and its residue for the "Meta" Duquenois test. Place no more than 1 mg of the solid reagent at the centre of the lower paper and add one drop of liquid reagent 2. A red to violet colour develops as the water expands over the area originally covered by the PE. The absence of a red to violet colour in the time taken for the second liquid reagent to dry, confirms the absence of cannabis.

Liquid samples may also be tested. A number of alcoholic beverages containing cannabis, either in suspension or solution, have been encountered. These may be loosely described as tinctures. An increasing number of oils are also noted, colloquially described as "Liquid cannabis" or "Hash oil". These have properties intermediate between tinctures and true cannabis extracts.

In the case of tinctures, one drop of the suspect material is placed on a test paper and allowed to dry in air as long as is necessary. In the case of the oils, the preferred technique follows the "Sampling Tool" method described above. A test paper is wiped across a contaminated surface, which is invariably available with this material; alternatively, a minute portion of the oil is added to the centre of a test paper. In either case, tincture or oil, fold the test paper with a second paper and proceed with PE as before. The intensity and speed of response is characteristically strong.

Results and conclusions

The addition of too much sample will, occasionally, produce weak false responses to some botanical materials, such as henna. Henna gives a characteristic green solution with PE due to the extraction of carotenes. Few cannabis or cannabis resin samples give a similar colour at the recommended level of I mg. This allows some preliminary diagnosis. An incidental advantage of the, now, mandatory double paper technique is that henna no longer responds to the test [ [ 2] ]. Henna is normally encountered as a compressed block simulating cannabis resin (generally from Morocco). Sufficient powder was entrained on the single paper to give a positive response, although the block was apparently not powdery. With the double paper technique, no response was noted until quantities of henna in excess of 100 mg were used. At the 1 mg sample size specified, the improved test is virtually specific for herbal cannabis.

The improved procedure has increased the sensitivity and specificity of the earlier version [ [ 2] ]. This, in turn, has allowed a simplified method to be specified. Laymen are no longer permitted the option of using a single paper: the double paper technique is now specified under all conditions, including laboratory spot testing.

The two alternative dyes have the same specificity as Fast Blue B. None of these respond to henna; Fast Black K responds. In situations where the familiar red to violet colour is not necessarily important, for instance, within a laboratory, at least two more dyes may be substituted for Fast Blue B.

Acknowledgement

The author wishes to thank Mr. G. F. Phillips for helpful editorial advice.

References

⁰⁰¹

de Faubert Maunder, M. J., J. Assoc. Public Analysts . 1969, 7, 24.

⁰⁰²

de Faubert Maunder, M. J., Bulletin on Narcotics , XXI, 4, p. 37.

⁰⁰³

BDH Chemicals Ltd., Catalogue number 32148.

⁰⁰⁴

Merck Ltd., Catalogue number 9500.

⁰⁰⁵

de Faubert Maunder, M.J., Med. Sci. Law , in press.

⁰⁰⁶

Woodhouse, E.J., Technical Report No. LWL-CR-08072, 1972, Midwest Research Institute.

⁰⁰⁷

McCarthy, T. J., and van Zyl, J. D., J. Pharm. Pharmacol ., 1972, 24, 489.

⁰⁰⁸

de Faubert Maunder, M. J., and Phillips, G. F., UK Patent Application 16148/72.

⁰⁰⁹

Beckstead, H.D., and French, W.N., "Methods of Analysis", Canadian Dept. of National Health, August 1968; published August 1971.

⁰¹⁰

Roser, R., and Tocci, P.M., J. Chromatog ., 1972, 72, 207.

⁰¹¹

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

⁰¹²

Gilhooley, R.A., 1968, Personal communication.

⁰¹³

Alliston, G. V., et al ., J. Pharm. Pharmacol ., 1971, 23, 72.

⁰¹⁴

de Faubert Maunder, M. J., J. Assoc. Public Analysts . 1970, 8, 42.

⁰¹⁵

de Faubert Maunder, M. J., Lecture to 13th Annual Meeting of the Society for Economic Botany, University of Mississippi, August 1972.

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