Recovery of cannabis constituents from the hands at autopsy
Author: Ann E. ROBINSON
Pages: 37 to 40
Creation Date: 1971/01/01
The successful demonstration of the presence of cannabis constituents on the hands of deceased persons, including cases where the body was found in water, is reported. The technique appears suited to epidemiological surveys of cannabis use.
Following the report by Stone and Stevens, 1969 ( [ 1] ), that cannabis constituents may be recovered from the hands of persons having smoked or handled the drug, it seemed worthwhile to put on record the successful recovery and demonstration of cannabis constituents (or smoke condensates) from the hands of some recent autopsy cases.
The analytical procedure adopted was based on chromatographic methods already established in the Department in connexion with the recognition and identification of cannabis, cannabis resin and the prescribable preparations. At the postmortem examination the pathologist separately swabbed each hand with cotton wool soaked in chloroform; each swab was transferred to a polypropylene container (Securitainer) and submitted for analysis. Loss of solvent from such swabs before analysis did not adversely influence the subsequent analysis. Each swab was eluted with solvent (chloroform or hexane fraction gave similar results) and the solvent was evaporated over a warm-water bath to leave a concentrated residue which was examined chromatographically with thin layer and gas chromatography systems. See Clarke and Robinson ( [ 2] ). The methods differed slightly from those used by Stone and Stevens,-the details of which were not available when the first case was presented. The method of collection of samples was evolved for convenience and seemed to yield extracts which did not require "cleaning up" on a column.
Dried thin-layer chromatograms on silica gel plates developed in either hexane-ether (8 : 2, by volume) or in chloroform-benzene (7 : 3, by volume) were sprayed with an alkaline solution of Fast Blue B to locate phenolic substances. Tincture of Cannabis (British Pharmaceutical Codex, 1948) was included on each chromatogram for reference purposes. Extracts from the hands of four autopsy cases all yielded chromatograms having Fast Blue B-positive spots of pink to purple hue, one of which in all cases corresponded with cannabinol. Other phenolic substances, mostly giving pink, rather than purple, spots were seen on the chromatograms at lower R 1 values. Some of these appeared to correspond with constituents of the tincture of cannabis, the latter being the most stable and convenient reference material for both the thin-layer and gas chromatographic procedures.
Gas chromatography of the concentrated extracts on a 6-foot column packed with 3.8 % W 98 on Diatoport S at 200°C in an F & M Biomedical 400 gas chromatograph fitted with a flame ionization detector yielded a number of peaks in every case in which T.L.C. results had been obtained. Comparison of the gas chromatograms with those of samples of tincture of cannabis derived from 2 different batches of extract of cannabis (i.e. from separate batches of plant material) and with alcoholic "solutions" of resins showed a number of peaks from the hand extract constituents which had retention times which corresponded with some of the substances in the samples of the tincture of cannabis and in some of the resins. Figure 1 shows gas chromatograms of (A) tincture of cannabis (manufacturer's batch No. 30); (B) a "good" cannabis resin; (C) extract from left hand of Case 3; sensitivity increased after 8 mins. to demonstrate the longer retention time components. Inspection of the chromatograms shows the presence of some peaks common to all three and some others which correspond with constituents of either the tincture or the resin.
Four autopsy cases have so far yielded positive evidence (i.e.T.L.C. and G.L.C. data) of the presence of cannabis constituents : these may have been derived either from handling cannabis or cannabis resin or from smoke condensates.
CURVE A CURVE B
Gas chromatograms of tincture of cannabis (Curve A), a "good" sample of cannabis resin (Curve B) and an extract from the left hand of Case 3 (Curve C). The instrument sensitivity was increased after 8 mins. for Curve C to demonstrate the longer retention time components. The peaks indicated are associated with cannabidiol (CBD,1), Δ 1-tetrahydrocannabinol (THC,2) and cannabinol (CBN,3).75
Thin layer chromatogram: (A) tincture of cannabis; (B) a "good" resin and (C) the extract from the left hand of Case 3 on silica gel developed in hexane-ether (80:20) and sprayed with freshly prepared 0.1% Fast Blue B in cold 3N sodium hydroxide solution.
The first case involved a girl of 19 yrs recovered from a river after 3 days immersion. Although she had been drowned, drug use prior to falling or jumping into the river was thought likely. Positive evidence for cannabis constituents on swabs from both hands was obtained despite the presence of finger print ink.
From the lipohilic nature of many of the identified constituents of cannabis, it might be expected that they would be found in the fatty tissue under the skin and that, as observed by Stone and Stevens (1), a soap and water wash would not prevent a positive result being obtained using a solvent extract or wash. It is perhaps surprising that cannabis constituents absorbed through the skin into the fatty tissue during life should not have been transported trougt the blood away from the point of contact and perhaps also have undergone (partial) metabolism in the liver in the time interval before death.
The second case involved two people found immersed after a car had been found overturned in shallow water. Of the two persons involved there was evidence of several cannabis constituents on the right hand of the female and to a lesser extent on her left hand while entirely negative results were obtained for her male companion. The period of immersion in this instance was only of the order of hours.
The hands of a deceased drug addict provided the third case. He was found collapsed in a house where several known drug users were staying and was dead on arrival at hospital. From the toxicological analyses the pathologist attributed death to morphine and methadone poisoning. Tests on both of his hands gave strong reactions in the thin-layer chromatographic systems and the swab from his left hand provided the gas chromatogram shown as curve C in Figure 1.
A further case was that of a murder victim who died of stabbing. Here again there was goodevi dence of cannabis constituents on his hands, slightly stronger responses being obtained for the right hand compared with the left.
Finally, it must be mentioned that negative results have been obtained for other cases in addition to the one referred to above.
There are, of course, limits to the inferences that may reasonably be drawn from these experimental observations. Unequivocal identification of individual substances cannot be achieved using T.L.C. spray location reagents which merely react with phenolic groups, as does Fast Blue B, and peaks on gas chromatograms for which no other parameter is known. The use of known reference substances where available undoubtedly assists but at the present time the conclusion must be that the evidence obtained is consistent with the recovery of substances which may be cannabis constituents or the condensed products of cannabis combustion.
Whether such results as these would be acceptable in a court of law as the only evidence offered in support of a charge of unlawful possession appears doubtful although there has not yet been a test case. Submicrogram quantities of the individual cannabis constituents are detectable both on the thin layer chromatogram with Fast Blue B reagent and by gas chromatography. Hence even if unequivocal identification was achieved by mass spectrometry, the amounts found in extracts from the hands of autopsy cases are so small as not to be comparable with a "normal dose ".
Thus, simple screening tests as described might be applied to investigate cannabis use among groups of individuals without fear of legal redress. This would allow the identification of users on an objective rather than subjective basis when a more accurate assessment of incidence might be achieved.
H. M. Stone and H. M. Stevens, J. For. Sci. Soc., 1969, 9, 31.002
E.G. C. Clarke and A. E. Robinson, Med. Sci, Law, 1970, in the press.
The author is grateful to Professor F.E.Camps, Dr. J.M. Cameron and Dr. H.R.M. Johnson for providing swabs from their autopsy cses.