Chapter 7 The Forensic Chemistry Of Alkaloids

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THE FORENSIC CHEMISTRY OF ALKALOIDS E . G. C. CLARKE The Royal Veterinary College. University of London London. England

I. Introduction ...................................................... I1 Poisoning by Alkaloids ............................................. A Introduction ................................................... B Pyrrolizidine Alkaloids .......................................... C Pyridine Alkaloids .............................................. D . Tropane Alkaloids .............................................. E Strychnos Alkaloids ............................................. F. Morphine Alkaloids ............................................. G. Colchicine ..................................................... H . Alkaloids of the Amaryllidaceae .................................. I. Indole Alkaloids ................................................ J Cinchona Alkaloids .............................................. K . Lupin Alkaloids L . Solanum and Veratrum Alkaloids .................................. M p-Phenethylamine and Ephedra Bases ............................. N . or-NaphthaphenanthridineAlkaloids ............................... 0. Erythrophleum Alkaloids ......................................... P. Aconitum and Delphinium Alkaloids ............................... Q . AlkaloidsoftheBuxaceae ........................................ R . Alkaloids of the Taxaceae .... ................................... S. Xanthine Derivatives ........................................... I11. Alkaloids as Drugs of Addiction ...................................... A Introduction ................................................... B . The Narcotic Analgesics ......................................... C. Stimulants or Psychoenergetics ................................... D Hallucinogens .................................................. IV ControlofAlkaloids ................................................ A . InternationalControl ............................................ B NationalControl ................................................ V Toxicological Analysis-General Considerations ........................ VI Extractionmethods ................................................ A . Introduction ................................................... B . Typesofsample ................................................ C Classification of Poisons ......................................... VII Identification Methods ............................................. A Introduction .................................................... B . Analytical Techniques ...........................................

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VIII. Tables of Analytical D a t a . . ......................................... Iiitroduction ...................................................... Table I. Solubility of Alkaloids ...................................... Table 11. Paper chromatography data. ............................... Table 111. Thin-layer chromatography data. ........................... Table IV. Ultraviolet spectrophotometry data ......................... Table V. Color Tests ............................................... StructuresofSynthetics ............................................ References ........................................................

560 560 561 561 566 570 573 576 579

I. Introduction Although the forensic chemistry of alkaloids has a long history, dating back t o the judicial murder of Socrates in B.C. 399’ or even before, it is only within comparatively recent times that the Law has seen fit to control and limit their use. As long as they were used solely for medicinal purposes, the Law had little interest in them. The isolation of the pure compounds from the original plant material during the 19th century, however, served to highlight their toxic and addictive properties, and it was these attributes that brought them into conflict with authority. Once a subject becomes of interest t o the Law, the question of detinition becomes of paramount importance. To confine the meaning of the word “alkaloid” to the original definition of “ a nitrogenous vegetable base” is to oversimplify the problem. Morphine is undoubtedly an alkaloid; so is codeine (although 90% of the codeine used is semisynthetic). Heroin is a simple derivative of morphine and levorphanol [( -)-3hydroxy-N-methylmorphinan] is a more distant one ; but pethidine and methadone cannot be regarded as derivatives of any vegetable base. Yet to the pharmacologist, the toxicologist, and the lawyer these are all members of the same group. They have a similar pharmacological action, they are isolated by the same analytical procedures, and controlled by the same laws. It is difficult t o draw a line of demarcation among them. The number of synthetic bases used in medicine increases almost dailynearly 1000 are mentioned in the current volume of the Extra Pharmacopoeia (1)-while plant alkaloids are falling into disuse as they are replaced by the cheaper and safer synthetics. Were one t o base one’s definition on therapeutic or toxicological importance, few vegetable bases would be included. I n order t o keep the subject within reasonable bounds, however, it has been decided to limit this chapter to the consideration of the plant alkaloids and their derivatives, purely synthetic substances being dealt with only if they illustrate some point of unusual forensic or analytical importance,

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11. Poisoning by Alkaloids

A. INTRODUCTION 1. Poisoning in Man Poisoning in man falls under one of the three headings: accident, suicide, or murder. Of these, accidents and suicides are distressingly common, and murder comparatively rare. I n England and Wales during the years 1958-61 there were a total of 1633 cases of accidental poisoning and 3955 suicides by poisoning, but only 6 cases of murder by this means ( 2 ) .At least, only 6 cases that were recognized as murder; there is, of course, no record of the number of cases of homicidal poisoning that were buried under the convenient epitaph of “death by natural causes.’’ Nowadays alkaloidal substances do not constitute a very important source of poisoning in man. During the years 1948-61 (2)a total of 15,045 people died of poisoning in England and Wales, deaths from carbon monoxide being excluded. Of this total, over half (8483) were due to barbiturates. Only 562 (3.7%) were due t o basic drugs; 429 of these were plant alkaloids (nicotine 123, morphine 94, strychnine 92, codeine 44, aconitine 26, atropine 18, quinine 10, ephedrine 8, heroin 5, cocaine 4, and ergotoxin,” hyoscine, quinidine, coniine, and theophylline 1 each), the remaining 133 being synthetic drugs such as analgesics, tranquillizers, and antihistamines. The number of deaths from alkaloidal poisoning has been decreasing for over a century. Taking the figures for England and Wales during 1837-38, 42% of all cases of poisoning were due to alkaloids, 37 % being due to morphine, which is here taken as including opium and laudanum (3).I n the years 1863-67 the figures were very similar, 42% due t o alkaloids, 39% due to morphine. By 1881 the totals had dropped t o 30% and 26%, respectively (4), while for the 10 years 1895-1904 out of a total of 8701 cases of poisoning, 1794 (21%) were due t o alkaloids, 1477 (17%) being caused by morphine ( 5 ) .I n America the same trend is noticed. I n the county of New York in the years 1841-43 75% of all poisoning was caused by alkaloids, 60% by morphine. Between 1866 and 1880 the figures had dropped to 28% and 20%, respectively, and in 1889 t o 1892 t o 14% and 12% (4).This steady decrease in the number of cases of poisoning caused by alkaloids is not entirely due to the increasing use of barbiturates, which did not become really popular until the period of World War 11. Other poisons have from time to time been fashionable. Paris green was the most popular poison in New York in the 1870’s, phenol (carbolic acid) in Great Britain a t the beginning of this century. ((

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The situation is rather different in the East. I n the Indian state of Uttar Pradesh for the years 1963 to 1965, out of 1355 cases in which poison was found, no fewer than 450 (33%) were due to alkaloids or substances containing them. This total included opium or morphine 248, datura or atropine 180, aconitine 12, and strychnine or nux vomica 10. Barbiturates only accounted for 73 cases (5.4%) (6).

2. Poisoning in Animals I n man poisoning is usually due to the ingestion of the pure alkaloid, or of some pharmaceutical preparation containing it ; it is seldom nowadays that the plant itself is involved, although such cases do occur from time to time. With animals, on the other hand, except in the case of strychnine, it is more frequently the plant that is eaten. Poisoning is nearly always accidental ; malicious poisoning is rare in spite of frequent accusations. It is difficult t o acquire any relevant figures, as authority is not interested in the deaths of animals unless they reach epidemic proportions or are caused by deliberate intent, and a full investigation is rarely made unless particular economic or sentimental factors are involved. Otherwise there is no inquest, seldom a postmortem, and rarely a toxicological analysis. But it is only from those cases in which an analysis is made that we can get any figures a t all, although for the reasons given above these may not form a representative sample. Out of 360 consecutive cases sent for analysis to a toxicologist in the south of England (7), 83 were found to be due to alkaloids or alkaloidcontaining material. Strychnine (76 cases) was by far the most common, the others comprising taxine (5 cases), solanine (2 cases), nicotine and theobromine (1 of each). Of cases submitted to the Royal (Dick) School of Veterinary Studies in Edinburgh from 1959-61, 10% were due t o strychnine (8).A similar figure was found in the cases submitted to the Wallaceville Animal Research Station in New Zealand in the years 1951-60 (9).These figures, however, do not present a true picture, as they deal only with cases arising from man-made conditions such as the use of strychnine as a rodenticide and take no account of the deaths of farm stock from the ingestion of such plants as ragwort (Senecio spp.),larkspur (Delphinium spp.), and Crotalaria spp. Such poisoning is most likely t o occur in remote rural areas where facilities for toxicological analysis are lacking, and therefore any diagnosis of poisoning always subject t o suspicion. There is often a tendency to argue post hoc, ergo propter hoc. A cow is found dead in a patch of hemlock, therefore it died of hemlock poisoning. Plants are frequently identified incorrectly ; and even if the plant contains an alkaloid it is not necessarily the cause of death. An

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animal which has died after consuming seed of the castor oil plant (Ricinuscommunis)has been poisoned by the phytotoxin ricin not by the alkaloid ricinine. I n spite of this, however, it must be realized that plants containing alkaloids are responsible for many hundreds of deaths in farm stock every year, and probably in a much larger number of cases for ill health and loss of condition due t o the ingestion of such plants in quantities insufficient t o cause death or even definite clinical symptoms.

3. Doping There is one example of animal poisoning which requires special mention-the doping or illicit medication of racing animals (10-13). By definition, doping is the administration of a drug t o an animal in order to affect its speed, stamina, courage, or conduct in a race. Horses are the animals most frequently subjected to this practice, but greyhounds, racing pigeons, and (possibly) bulls are sometimes doped, while athletes may dope themselves. Doping usually consists either of the administration of a stimulant to make an animal go faster (doping t o win) or of a sedative t o make it go more slowly (doping to lose or "nobbling"), but other procedures such as the use of local anesthetics to mask lameness, of tranquillizers t o control a highly spirited animal, and of sex hormones for a female in estrus are also employed. It is as stimulants that alkaloids most frequently find employment. Possibly caffeine has been used more frequently than any other drug. It is cheap, easy to obtain, and reasonably effective. A horse is more alert, gets away t o a better start, and responds more quickly t o its rider. Strychnine has also been used extensively for this purpose, but its act,ion seems less reliable. Both morphine and heroin, which act as stimulants in the horse, have also been widely used in the past. If the dose and the timing are both correct a horse doped with morphine will run far above its normal form. As these substances are alkaloids, the idea has arisen in certain circles that any alkaloid will do, and so such unlikely compounds as atropine, ephedrine, yohimbine, and quinine have been employed. Cocaine has also been used with limited success. The modern tendency however is to use synthetic drugs such as amphetamine," Alkaloids are not used as sedative drugs in the horse-barbiturates or chloral are usually employed-but codeine and quinine, the latter in contradistinction to its use as a potential stimulant in the horse, have

* The structures of the alkaloids mentioned in this chapter are given in other volumes of this series. The stri-cturesof the synthetic drugs discussed in this chapter are given at the end of Section VIII (p. 576).

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been used for this purpose in greyhounds, which are nearly always doped to lose. Racing pigeons are sometimes given amphetamine to delay the onset of fatigue. Bulls are reported to have been quietened with tranquillizers. The doping of athletes consists of self-medication with drugs of the amphetamine type. The proof of doping lies in the demonstration of the presence of the drug in the body fluids of the animal. Sweat, blood, saliva, and urine are available. The first is undesirable owing to likelihood of contamination, the second involves the risk, remote but theoretically possible, of damage to the animal while it is taken. Saliva is sometimes useful, as a drug may be present in unaltered form, although in small quantities and for a limited time only. It is also easy to obtain. Urine is more difficult to collect and usually contains a much larger quantity of the drug, but our knowledge of the metabolic processes of a horse is so limited that one seldom knows in what form it is likely to be encountered. B. PYRROLIZIDINE ALKALOIDS Pyrrolizidine alkaloids (14, 15) occur in a large number of plants including the genera Senecio and Erechtites (Compositae), Echium, Heliotropium, Trachelanthus and Trichodesma (Boraginaceae), and Crotalaria (Leguminoseae). Many of them are hepatotoxic, and give rise both in man and beast to the condition known as seneciosis, which has a worldwide distribution (16). I n man, poisoning may arise from eating bread made from flour contaminated with fragments of the leaves and stems of various species of Senecio, par‘ticularly 8. burchelli and 8. ilicifolius (the so-called “bread poisoning” of South Africa) (17,18), or from drinking “bush tea,” a concoction made in the West Indies from a number of plants including Crotalaria retusa (19)and C. fulva (20).This gives rise; particularly in children, to a partial or complete occlusion of the centrilobular veins of the liver causing a condition known as venoocclusive disease. The causative agent is monocrotaline (21). Poisoning in animals is usually due to eating plants belonging to the genera Crotalaria, Heliotropeum, or Senecio. This gives rise to a condition knowrLin different parts of the world as “walking disease,” “Winton disease,” ‘(Pictou disease,” (‘Molten0 disease,” or dunsiekte.” All species of farm stock are susceptible and, as in man, the chief pathological lesion seen is centrilobular necrosis of the liver. The condition has been produced experimentally on a number of occasions (22,23).Stock normally refuse to eat the growing plants as they seem to be unpalatable, but will consume them readily if they are made into hay, as when dried they ((

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lose their unpleasant taste, but not their toxicity. There is a considerable lapse of time between ingestion of the plant and the onset of clinical symptoms, which may not be seen until weeks or months after the animals have eaten the contaminated hay or been removed from the ragwortinfested pasture. It has been said that ragwort causes more poisoning among stock in Great Britain than all other poisonous plants put together (24). Members of the Senecio genus are not the only plants containing pyrrolizidine alkaloids to cause poisoning. Severe losses among stock have occurred in Australia due to eating Heliotropeum europeum, which contained heliotrine, lasiocarpine, and their N-oxides. Sheep are the animals most commonly affected. Cattle dislike the plant, and will only eat it if no other food is available, although, as with many other poisonous plants, animals introduced from another locality will eat it more readily than “resident ’) beasts (25).The effect is cumulative, and animals will often survive one year to succumb the next. The alkaloids cause typical liver lesions, the essential pathology being an atrophic hepatosis with megalocytosis of the parenchymal cells (26-28). The liver damage is apparently responsible for some malfunctioning of the copper storage mechanism, which leads to an accumulation of copper in the liver, very high values (over 1000 ppm) being found a t postmortem. Similar high copper values have been found in sheep poisoned by Echium plantagineum which contains echimidine and echiumidine (29).It is not certain what part copper plays in the syndrome, although death may be associated with an acute hemolytic crisis similar to that which occurs in chronic copper poisoning. It is noteworthy that pyrrolizidine alkaloids in which the ester chain has two or more hydroxyl groups on adjacent carbon atoms, e.g., monocrotaline, lasiocarpine, form complexes with copper, but there is as yet no evidence that this has any connection with the high copper content of the liver (30). Considerable losses have also been caused by various species of Crotalaria, which has already been mentioned in connection with “bush tea.” I n America, C . spectabilis, grown as a cover crop on sandy soils in the southern states, is the most toxic species. All parts of the plant are poisonous, but the highest concentration of the alkaloid (monocrotaline) is found in the seeds. Horses, cattle, pigs, and poultry have all been affected, the last named being particularly susceptible. Poisoning may arise from animals eating the green plant (31-33) or from contamination of grain by the seed ( 3 4 ) .As in cases of poisoning by Senecio or Heliotropeum spp., the fundamental lesion is damage to the liver, but ascites and gastric hemorrhage are also common findings a t postmortem (33-35). There may be an interval of weeks or months between removal from the

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plant and death (31).C. Sagittarius has caused poisoning in horses (36). C. giantstriata, although poisonous, is much less toxic than C. spectabilis. I n South Africa C. dura and C. globifera may give rise t o the condition known as “jaagsiekte” in horses ( 1 8 ) )while in Australia it has been shown that “Kimberley horse disease’’ is caused by C. retusa (37) or C. crispata (38). Trichodesma incanum which grows in central Asia is responsible for suiljuk,” a condition in cattle and horses which appears t o be similar t o seneciosis. Certain Cynoglossum spp. have also been shown to contain pyrrolizidine alkaloids. Poisoning of cattle by C. oflcinale has been reported (39))but it is not clear whether the alkaloids are implicated. An interesting point about the hepatotoxicity of the pyrrolizidine alkaloids is that male animals are considerably more susceptible to poisoning by them than female. It was found that the LD50 of retrorsine was 60 mg/kg for male rats and 180 mg/kg for females (40).Out of a herd of young cattle that had eaten silage containing ragwort, 40% of the bullocks died but only 27.5% of the heifers (41).I n an outbreak of poisoning by Heliotropeum europeum in Australia, 50% of the bullocks died, the heifers remaining clinically unaffected (15).Injection of testosterone into a spayed female rat gives it the susceptibility of the male, while injecting an estrogen into a castrated male gives it the resistance of the female (40). The pyrrolizidine alkaloids are not all poisonous. Schoental (42) has postulated that for an alkaloid to be toxic it must have a double bond between C-1 and C-2. The cyclic diesters are twice as toxic as the open diesters, and four times as toxic as the open monoesters. Esters of branched-chain acids are toxic while esters of straight-chain acids are not (43). The mechanism by which the alkaloids exert their hepatotoxic effect is still not clear. It is considered that the essential action is an alkylation, brought about by an alkyl-oxygen fission. and it has been shown that heliotrine appears to undergo a transformation of this kind in the sheep (44).The alkaloids interfere with a number of enzyme systems. I n vitro, lasiocarpine and heliotrine inhibit enzyme systems that need pyridine nucleotides for electron transfer ( 4 5 ) .The nicotinamide-adenine dinucleotide pyrophosphorylase activity of nuclei from rat liver that has been treated with heliotrine is reduced significantly below that of controls (46).It has recently been shown that in rats lasiocarpine inhibits RNA synthesis, causes a substantial reduction in tryptophan pyrrolase activity, and decreases the activity of RNA polymerase ( 4 7 ) . It is suggested that the alkaloids themselves are not hepatotoxic, but are converted in the liver to pyrrolelike derivatives which react with

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tissue constituents to form “bound pyrroles ” which either remain in the tissues or are excreted in the urine (48). It has been shown (49)that if heliotrine is incubated in rumen fluid in the presence of vitamin BIZit is converted to the nontoxic I-methylene derivative. This suggests that cobalt pellets might be used to protect sheep and cattle from chronic intoxication ; but so far this suggestion has not been confirmed (50).Treatment of poisoning is usually unrewarding, although some success has been claimed in the use of crystalline methionine for treating horses poisoned by Xenecio (51). A number of other toxic manifestations of the pyrrolizidine alkaloids have been recorded. Ingestion of C. spectabilis leads to alopecia of the dark-colored areas of skin in pigs (34).Young rats, suckled by mothers treated with lasiocarpine or retrorsine, may die with acute liver lesions even if the alkaloids have produced no apparent symptoms in the mother. This finding gives rise to the suggestion that liver disorders in children may result from drinking the milk of cows that have eaten plants containing these compounds in their fodder (52).Clark (53)has found that heliotrine is mutagenic in Drosophila. Schoental and Head ( 5 4 )have shown that it produces liver tumors in rats. Poisoning by the pyrrolizidine alkaloids has recently been reviewed by Bull ( 5 5 ) )who suggests that the condition should be called pyrrolizidine alkaloidosis. C. PYRIDINE ALKALOIDS The areca or betel nut, the fruit of Areca catechou, contains arecoline and other bases (56).It is cut into slices and chewed by the people of many races in India and Eastern Asia, usuaily after being mixed with lime and wrapped in leaves. Poisoning is rare, although it may be caused by the use of unripe nuts, the symptoms being flushing, perspiration, bronchial spasms, contraction of the pupils, diarrhea, dyspnea, and collapse. Some people are hypersensitive to betel nuts, and fatal cases have occurred after taking even small fragments of a nut (57).Arecoline in the form of the hydrobromide was formerly used ia human medicine as a cholinergic, and gave rise to occasional cases of poisoning through overdosage. It is seldom met with nowadays, though it is still used in veterinary medicine as an anthelmintic, either alone or in combination with acetarsol. Numerous cases of poisoning in dogs and cats have followed its use in inexpert hands, the combination with acetarsol being particularly dangerous. Lobeline, the chief alkaloid of Lobelia inJlata and other Lobelia spp. has a nicotinelike action and has had limited use as a respiratory stimulant

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and a smoking deterrent. Overdosage may cause nausea and vomiting, followed by convulsions and collapse. It has proved fatal when used as an abortifacient, a dram of the powderedleaves having caused death (57). I n addition to the classic case of Socrates, coniine from Conium maculatum (spotted hemlock or poison hemlock) has caused poisoning on many occasions. It has been used as a homicidal agent and has caused death accidentally through having been mistaken for parsley. Symptoms of poisoning have also followed inhalation of the vapor. Poisoning among animals is still fairly common as the young green shoots of hemlock come up in water meadows in the spring before the grass starts to grow. Ducks, sheep, cattle, and horses have all been poisoned (58, 59), but with large animals it is rarely fatal (60). Coniine is also said to occur in Aethusa cynapeum (fool’s parsley) which is recorded as having caused poisoning inpigs (61). Nicotine, the common alkaloid of Nicotiana tabacum is an extremely poisonous substance, the fatal dose for man being between 40 and 60 mg. It is the most common cause of poisoning by alkaloids in Great Britain a t the present time, probably because it is readily available. It is sometimes used as a suicidal agent, but most cases of poisoning arise accidentally through careless handling when employed as a horticultural insecticide. Poisoning may occur by absorption through the intact skin, the lungs, or the gastrointestinal tract. With large doses of poison death may occur in a few minutes. Nicotine has also been used as a homicidal agent on a number of occasions, the best known of these being the murder of Count Bocarmh by his brother-in-law, Gustav Fougnies, in 1850, an incident of great toxicological interest, as its investigation gave rise to the classic Stas-Otto process which was to hold the field for nearly a century (62). Smugglers carrying tobacco next to the skin have been poisoned by the nicotine absorbed. Children have been poisoned through being allowed to play with old tobacco pipes, and a,5-month-old baby by milk into which tobacco had been dropped by accident (63). Pigs have been poisoned after breaking into a field and eating a large quantity of tobacco plants (24) and poisoning from eating tobacco has also been recorded in the cat (64)and in the dog (65),but the most common cause of poisoning in animals is the use of solutions of nicotine as an insecticide. The poison may be absorbed through the intact skin or through wounds, or by animals licking themselves. The use of nicotine sulfate in the treatment of warble fly has resulted in the poisoning of cattle on numerous occasions (SS-SS), the nicotine being absorbed through the warble fly holes. Dosing with nicotine sulfate has also proved fatal in lambs (69).

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I n spite of its extreme toxicity, nicotine remains one of the safest horticultural insecticides, prcvided correct precautions are taken. As it is volatile it has no persistent effect and cannot give rise to lethal chain reactions such as those which may occur with the chlorinated hydrocarbons. It is reported that rabbits can consume 500 gm of fresh tobacco leaves in a week without ill effect (70).

D. TROPANE ALKALOIDS Poisoning by alkaloids of the atropine group (71,72)is fairly common, though rarely lethal. As d-hyoscyamine is almost completely inert, poisoning by atropine can be considered as being due to the 1-isomer. Atropine is usually considered to have a lethal dose in man of about 100 mg, although recovery has taken place after much larger quantities. Idiosyncracy to atropine, however, is fairly common, and cases are on record where administration of a therapeutic dose of atropine (e.g., for ophthalmic examination) has caused acute symptoms of poisoning (73). I n cases of sensitivity, fatal poisoning has been caused by less than 1 mg. Apart from this, poisoning may arise from overdosage (74, 75) and from the use of belladonna plasters on abraded skin (76, 7 7 ) . The usual symptoms are dilatation of the pupil and delirium. Hyoscine appears to be rather more toxic than hyoscyamine. The classic instance of poiqoning by hyoscine is, of course, the Crippen case : 25 mg were recovered from Mrs. Crippen’s body (78). There is a long history of poisoning associated with plants containing these alkaloids, during the middle ages particular interest having been centered on mandrake (probably Mandragoro oficinalis). Deadly nightshade (Atropa belladonna) also found considerable use by witches and professional poisoners. I n spite of the reputation which the plant had acquired, a case is on record in which deadly nightshade berries were sold in the streets of London in the last century as “edible nettleberries.” Two people died, and the man who sold the berries was convicted of manslaughter (79).Children are most frequently the victims of nightshade poisoning (80). Poisoning has been recorded on a number of occasions, particularly in South Africa, of children eating the seeds of Datura stramonium (81).Various species of Datura have been used as ordeal poisons. I n animals, poisoning arises from eating plants containing the alkaloids. Atropa belladonna poisoning has been recorded in pigs (82, 83), goats (84),and cattle (85).It is well known that rabbits can flourish on a

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diet of nothing but A . belladonna (86) and that the meat of such animals can be poisonous to man (87).A case is also recorded of atropine poisoning in a woman who had eaten an extract made from the livers of cattle which had apparently been grazing on deadly nightshade (88). Animals usually avoid Hyoscyarnus niger (henbane) owing to its unpleasant smell and taste, but they may eat it occasionally, particularly if it is fed in forage (89). Poisoning by Datura stramonium is also known (90,91),Pigs can tolerate small daily doses of datura seeds for long periods (92),while poultry have a very high resistance to it and can tolerate up t o 15 gm per day (93). Members of the genus Cestrum which are South American in origin, but are now cultivated as ornamental shrubs in various parts of the world, are reputed to contain alkaloids of the atropine type. They are known to have caused poisoning in stock (94) and their toxicity has been confirmed by experiment (95,96). Poisoning by cocaine is rare, and the chief forensic interest in this compound is as a drug of addiction. At one time, however, it was a fairly common suicidal agent. As ti local anesthetic it has now been almost completely replaced by synthetic compounds such as procaine and lignocaine which are without addictive properties, and whose toxicity, though by no means negligible, is considerably less than that of cocaine (97). Cocaine is not particularly stable as the ester linkage is easily hydrolyzed. Coca leaves kept for 40 years under museum conditions were found to contain no trace of the alkaloid. Rising and Lynn (98) mixed cocaine with the stomach contents of a sheep and found none remaining after 7 months.

E. Strychnos ALKALOIDS Strychnine (99, 100) has been used as a rodenticide in Europe since the 16th century. For almost as long it has found widespread use in medicine owing to its reputation, probably mistaken, as a tonic. As mentioned earlier, its ready availability and extreme toxicity led to its becoming one of the most common causes of suicidal and accidental poisoning, the latter being particularly common in children. I n spite of its intensely bitter taste i t has been used as a homicidal agent on numerous occasions. The lethal dose in man is usually given as 30 mg, but half this quantity could prove fatal. Death usually occurs within a few hours and has been known to take place in as little as 15 minutes after a massive dose (101). The longer the patient survives the greater the chance of recovery. About 7 0 % of the strychnine ingested is destroyed in the liver, most of the remainder being eliminated, mainly in the urine, within 24 hours. Strych-

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nine is very stable, and has been found in bodies exhumed years after death. Allergy to strychnine has been recorded (102). Owing to its widespread use as a vermin killer, strychnine has been and still is responsible for many deaths among domestic animals, especially dogs and cats, which appear to be particularly susceptible. Although the retail sale of strychnine, except under license for the destruction of moles, is now prohibited in Great Britain, such deaths still occur. This is prohably due t o the fact that during World War I1 the regulations governing the sale of strychnine were suspended in order to enable farmers t o deal with the plague of foxes brought about by the suspension of hunting. Many farmers laid in a stock of strychnine which they still possess. Brucine has only one-tenth of the toxicity of strychnine and poisoning by it is very rare. If it is met with in addition to strychnine in a toxicological analysis, it suggests that poisoning has been due to some preparation of nux vomica and not t o the pure alkaloid.

F. MORPHINE ALKALOIDS I n spite of the fact referred t o previously that morphine (103,104)and substances such as opium and laudanum which contain it were by far the most common cause of poisoning in man during the last century, strict control of the drug because of its addictive properties has greatly reduced the incidence of morphine poisoning, such cases as occur nowadays being usually associated with doct.ors, nurses, and midwives who have legitimate access to it. I n the East, however, opium still remains one of the chief causes of poisoning (57). Children are particularly susceptible t o poisoning by morphine, the fatal dose being in the order of 1 mg; and many cases have been caused by the use of “soothing syrups” containing opium t o quieten fractious infants. Such cases may still occur (105) although preparations of this kind are restricted by law to a morphine content of less than 0.02%. I n veterinary toxicology interest in morphine is limited to its use as a “dope” in racehorses. It is noteworthy that in the horse and the cat morphine acts as a stimulant. The forensic importance of the morphine alkaloids is centered on their use as drugs of addiction, and is discussed subsequently. Codeine, which has only one-quarter of the toxicity of morphine, is sometimes used as a suicidal agent ( Z ) , probably because it is easy to obtain, but as the majority of such suicides are caused by tablets in which the codeine is compounded with other substances such as aspirin or phenacetin, it is difficult to say if it is really the cause of death.

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G. COLCHICINE During the last century, poisoning by Colchicum autumnale (autumn crocus, meadow saffron) (106,107)was comparatively frequent. Witthaus ( 4 )collated references to 132 cases of human poisoning. Of these 1 18 were accidental, 6 suicidal, and 5 homicidal, the accidents being due to such diverse causes as inclusion of the leaves in salad, use of the drug as an abortifacient and gross errorsindispensing. Of these cases, 75% were fatal. The usual symptoms are nausea, vomiting, and hemorrhagic diarrhea. There is a latent period of about 4 hours between ingestion of the poison and onset of the symptoms. Death may be caused by as little as 5 mg of colchicine, and usually ensues within 24 to 36 hours, being due to asphyxia and circulatory collapse (97). Poisoning in livestock has been recorded on many occasions, and is due to animals grazing in meadows where C. autumnale grows. It is most likely to occur in spring and autumn. Cases due to inclusion of the plant in hay are also known. Horses and cattle are the animals most commonly affected (108-110). Colchicine is also found in various Gloriosa spp., G . superba being the best known. Decoctions of the plant have found considerable use in native medicine, particularly as abortifacients, and numerous fatalities are on record (81).Poisoning has occurred also through the tubers being mistaken for those of the yam (111).Alopecia is a prominent symptom.

H. ALKALOIDS OF THE AMARYLLIDACEAE Although a number of the Amaryllidaceae are poisonous, the agent responsible is not always an alkaloid. Agave lecheguilla, for example, which has caused heavy losses among sheep and goats in Texas and neighboring states, contains a photosensitizing agent and an abortifacient, both of which appear to be saponins (112,113). A number of both wild and cultivated species, however, contain alkaloids, of which lycorine is the most common (114,115).Of the cultivated species, various types of narcissus have been responsible for poisoning in both man and animals. Human poisoning has occurred through daffodil bulbs being mistaken for onions. Pigs have been poisoned by bulbs rooted up in a, park, and cattle when fed with bulbs owing to shortages of other foodstuffs in Holland during World War I1 (116). Some species belonging to the genera Amaryllis, Crinum, Haemanthus, and Nerine, which grow wild in South Africa, have been responsible for losses among sheep and goats (18, 81). The bulbs of Buphane disticha

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which contain buphanine and other bases have been used as a source of arrow poison. They have caused poisoning on a number of occasions when used as native medicine (81).

I. INUOLE ALKALOIDS 1. Xiinple Bases Tryptamine and its simple derivatives (117) are of interest mainly as hallucinogens, although the Australian grass Phalaris tuberosa, the cause of Phalaris staggers in sheep, contains N,N-dimethyltryptamine and its 5-hydroxy and 5-methoxy derivatives. The last named, given subcutaneously in a dose of 1-2 mg/kg, is lethal to sheep, producing symptoms similar to those of acute staggers (118-120).

2. Ergot Alkaloids Poisoning by ergot (121-123) is comparatively rare nowadays, but during the middle ages the mysterious scourge known as St. Anthony's Fire was responsible for many thousands of deaths, particularly in Eastern Europe where rye was a staple cereal. Poisoning arises from contamination of the grain-usually rye, but occasionally other cerealswith the sclerotia of Claviceps purpurea, and is most common in wet seasons. I n a heavy infestation as much as a quarter of the weight of the grain may be due to ergot, whereas grain containing as little as 1 % of ergot ma,ybe toxic. Since it became known that the condition was caused by ergot, outbreaks of chronic ergotism have become far less frequent, but they still occur from time to time. There was one in the south of France in 1952 (124) due to heavily contaminated bread. About 200 people were affected, several cases being fatal. The last outbreak in Great Britain appears t o have been in Manchester in 1988 (125).I n Russia during the years 192687 no fewer than 100,000 people are said to have been affected (126). It is usual to divide chronic ergotism into two types-gangrenous and convulsive-but there seems to be no clear line of demarcation between them as the symptoms, which include vomiting, diarrhea, peripheral pain, delirium, and hallucinations, vary from case to case, depending on the age and health of the individual and, it has been suggested, on the proportion of the various alkaloids present in the ergot (126). Acute poisoning is rare and is usually associated with gross overdosage with some ergot preparation taken in an attempt to procure abort' u1011.

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Ergot poisoning in animals is usually due to infestation of the growing grasses :perennial rye grass (Loliumperenne), cocksfoot (Dactylisglomerata),timothy (Phleumpratense),crested dogs tail (Cynosuruscristatus),oat grass (Avena pubescens), and Yorkshire fog (Holcus lanatus) are among British grasses susceptible to attack (127); while in America wheat grasses (Agropyron spp.), redtop (Agrostis alba), smooth bromegrass (Bromus inermis),reed grasses (Calamagrostis spp.), wild rye (Elymus spp.), reed canarygrass (Phalaris arundinacea),and bluegrasses (Poaspp.) have been implicated (128).The animals most frequently affected are cattle, but poisoning in sheep, horses, and pigs has also been recorded. Both the convulsive type of poisoning (129,130)and the gangrenous (131,132)occur; in some cases the symptoms recorded would fit either category (133). Although abortion in cattle grazing on ergotized pastures has been noted, there seems to be considerable doubt as to whether the quantity of alkaloid likely to be ingested is sufficient to cause i t (134))and it has not been possible to produce the condition experimentally (135). Sows fed on ergotiaed barley show a complete retardation of udder development and inhibition of milk secretion, but no other symptoms (136).

3. Alkaloids of Perganum harmala African rue (Perganum harmala )which contains harmine and similar bases (137, 138) has been suspected of poisoning cattle in New Mexico and its toxicity has been confirmed by experiment (139). Recent toxicological interest in the P-carboline bases depends not so much on their being a possible cause of plant poisoning, but because some of them (e.g., harman and norharman) may be met with in the most unlikely places, such as tobacco smoke (140))homemade wine (141))and postmortem material (142). It seems probable that they are artifacts formed by ring closure from tryptophan.

4 . G . Alkaloids of Gelsemiurn spp. I n the 19th century when tincture of gelsemium made from the root of Gelsemiurn sempervirens (143-145) was widely used in the treatment of neuralgia and similar conditions, cases of poisoning in man were fairly common (146,147).Death has been caused by 5 ml of the fluid extract. The preparation is still mentioned in the Extra Pharmacopoeia ( 1 ) . Poisoning is also said to have been caused by honey made from the flowers. A number of cases of poisoning in farm stock have occurred, the usual symptoms being weakness, incoordination, and convulsions. Morphine is said to be a specific antidote (148).Heavy mortality has been reported among turkeys, the birds becoming lethargic and incoordinate

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(149).Gelsemine also occurs in the Indian creeper Gelsemium elegans ; decoctions of this plant are frequently used for criminal purposes (52’).

5. Alkaloids of the Calabar bean Physostigmine (15U-152) is found in the seeds of Physostigma venenosum, the Calabar beau or ordeal bean of West Africa, where it has been used for centuries as a test for witchcraft. Poisoning has arisen from accident, as when the sweepings from a ship’s hold were dumped on a rubbish heap in Liverpool and eaten by children, one of whom succumbed after eating six seeds (73),or by overdosage (153),but such cases are rare and seldom fatal, although death has been caused by as little as 1.2 mg (154). Poisoning by synthetic substitutes for physostigmine, such as neostigniine, is rather more common (97).Physostigmine is an inhibitor of cholinesterase, so atropine or PAM (‘2 -pyridinealdoxime) may be used as antidotes. Animal poisoning seems to be unknown.

J. Cinchona ALKALOIDS Quinine is one of the least toxic alkaloids, but there is wide variation in tolerance to it, and allergy is not unknown ( 6 3 ) Nonfatal . cases of poisoning may be caused from its use as an antimalarial (155),but fatalities are usually due to its use as an abortifacient (156, 157). The toxic dose is difficult t o assess and may be anywhere in the region from 2 t o 20 gm. Deafness and blindness, which may be permanent, are symptoms commonly seen. Quinine was the most common cause of suicide in Bulgaria in the 1930’s (158). Poisoning in animals by quinine does not appear to have been recorded. It has, however, been used in doping : as a stimulant in the horse and as a sedative in the greyhound. Poisoning by quinidine is very rare and is associated with its clinical use (159).

K. LUPINALKALOIDS Since classical times lupins have been used as green manure, as a fodder crop, and as a source of meal for both man and beast. No cases of poisoning appear to have been recorded before the 19th century, although i t was realized that the seeds had harmful i)roperties, and special methods were used in preparing them for food (160). From 1860 onwards, however, numerous outbreaks of poisoning occurred in northern Europe, losses in

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sheep running into many thousands (161,162),but it seems that the condition, known as lupinosis and characterized by extensive liver damage, was not due to the alkaloidal content of the plant, but was probably fungal in origin. A similar condition has been noted in Western Australia during the last two decades (163-169). Poisoning in America, which has caused heavy mortality (270),is undoubtedly due to the alkaloids contained in the plant, which is particularly dangerous a t the seeding stage. Sheep are the animals most frequenkly affected. The symptoms are somewhat variable, but among those usually noted are nervousness, dyspnea, and ataxia, followed by convulsions, coma, and death. Unlike that of the hepatotoxin which gives rise to lupinosis, the effect is not cumulative and an animal may eat comparatively large quantities provided it does not consume a lethal dose a t any one time (171).Not all lupins are harmful. The most poisonous species are said t o be Lupinus sericeus, L. leucophyllus, L. argenteus, L. caudatus, and L. perennis, while certain European strains known as “sweet” lupins are practically nontoxic (162).During recent years, strains entirely free from alkaloids have been produced by selective breeding. Of the numerous alkaloids present in the lupin (172, 173),d-lupanine is the most widely distributed and is usually regarded as the most toxic (174).The alkaloids are excreted in the urine, Poisoning by cytisine (172,173)from Cytisus laburnum (syn. Laburnum anagyroides Medic.) is comparatively common. The alkaloid is found in all parts of the tree and children have been poisoned from eating the seeds or even from chewing twigs, but such cases are rarely fatal. I n spite of this, a decoction of laburnum bark has been used for homicidal purposes (78). Poisoning in animals is also well known and has been recorded in cattle ( 175,176),in pigs ( 17 7 ) ,and in horses (?a).Symptoms are usually excitement, sweating, and incoordination, occasionally followed by convulsions and death. Cytisine is excreted in the milk, and children may be poisoned by milk from a cow which itself shows no clinical symptoms. Cattle may be poisoned-by picking up laburnum seed in the grass (178). Cytisine is also found in Sophora secundiJlora (mountain laurel or mescal bean), which is found in the western United States, and has given rise to poisoning in cattle, sheep, and goats. Although the seeds contain the highest proportion of alkaloids, they pass through the digestive tract intact, and are thus harmless. Poisoning is usually caused by foliage or broken seeds (179).

L. Solanum AND Veratrum ALKALOIDS I n dealing with poisoning by Solanurn spp. it is convenient, in spite of the elaborate work which has been done in elucidating the structure of

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these compounds (180, 181), to consider “solanine” as a single entity. There is as yet no evidence as to whether the different solanine alkaloids vary in toxicity, although i t is suggested that the glycoalkaloid causes the irritant symptoms, the alkamine the nervous ones. The whole question is complicated by the fact that many of the Solanum spp. (including the potato) contain an active cholinesterase (182),but the part, if any, that this plays in the syndrome is as yet unexplained, although it has been shown to be present in an amount roughly proportional to the solanine content (183). Solanine poisoning is usually caused by potatoes. Normally, the inside of the tuber is comparatively harmless, containing from 5 t o 2 0 mg/100 gm of solanine, but the level is higher in the peel, in green or unripe potatoes, and particularly in the sprouts, where it may reach 500 mg yo. Poisoning in man has usually arisen when a population is living mainly on potatoes of doubtful quality under conditions of near starvation, or when the potatoes for some reason, climatic or otherwise, contain a much higher proportion of solanine than usual (184).The usual symptoms are vomiting, abdominal pain, diarrhea, and general malaise. The toxic dose in man is about 25 mg and although numerous outbreaks have been recorded, fatal poisoning is almost unknown. I n farm stock, on the other hand, many fatalities have been recorded, in cattle (185), horses (186), pigs (187-189), and sheep (190).Poisoning usually arises from green or sprouting potatoes ; the former have also caused poisoning in a dog (191), but seem to be harmless to poultry (192).Potato haulm has been known to cause poisoning in man (193)and cattle (194). Other Solanurn spp. are also harmful. Cattle (195, 196), horses (197), and sheep (198)have been poisoned by woody nightshade (S.dulcamara), while the berries of this plant have poisoned children (199, ZOO). S. elaeagnifolium (silver-leaved nightshade) has caused cattle losses in Western Texas (201).S. nigrum (black nightshade) has also proved toxic on a number of occasions (202-204) ; the plants are most toxic when the berries are green, although there is a cultivated variety which is completely nontoxic (128).Solanurn rostratum (buffaloburr)has caused losses in pigs, although they normally refuse t o eat it (205).S. carolinense may cause convulsions and death in sheep (204). Although a number of cases of poisoning by the veratrum alkaloids (180,181)in both man (206,207)and animals (208,209),usually due t o ingestion of the root or misuse of crude preparations of the drug, are on record, recent interest has centered on the teratogenic effects of Veratrum californicum in sheep (210-212). If ewes eat this plant on or about the 14th day of pregnancy the lambs are born with a cyclopean-type malformation (213).The exact agent has not yet been determined, as although

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veratramine has a teratogenic effect (214) the malformation caused is different from that caused by the growing plant. The Death Camas (Zygadenus spp.) which contain zygadenine, zygacine, and other bases (180, 181) are among the most dangerous plants growing in Western Canada and the United States, and are responsible for heavy losses every year among sheep on the spring ranges. The usual sequence of symptoms is salivation, nausea, vomition, ataxia, dyspnea, prostration, coma, and death. The different species of Zygudenus vary considerably in toxicity, Z . gramineus and Z . nuttalli being the most dangerous (128).

M. /3-PHENETHYLAMINE AND EPHEDRA BASES /3-Phenethylamine (215)is found in postmortem material where it has usually been formed by the deamination of phenylalanine. It also occurs in a number of plants, including the mistletoe (Viscum album), but the toxicity of the latter (39)is due to a polypeptide (216).Tyramine produced from tyrosine is also found in postmortem material, but its chief interest in toxicology lies in the fact that it may give rise to serious or even fatal poisoning if substances which contain it such as cheese (217-219), broad beans (220),or chianti (221)are consumed after taking one of the monoamine oxidase-inhibiting drugs such as phenelzine or tranylcypromine. Acacia berlandieri, a shrub growing in Texas and North Mexico, gives rise to a paralysis of the hindquarters known as “limberleg” or “guajillo wobbles,” in sheep and goats, particularly in times of drought when the condition may reach epidemic proportions. The plant contains tyramine, N-methyltyramine, and N-methyl-/3-phenethylamine (222,223).Hordenine has been suspected of playing some part in barley poisoning in stock. Mescaline is of interest only as an hallucinogen. Ephedrine is used in the treatment of asthmatic conditions. It is of low toxicity, but its prolonged use may lead to toxic psychosis with auditory and visual hallucinations (224). N. a-NAPHTHAPHENANTHRIDINE ALKALOIDS

Chelidonium majus, the greater celandine, contains the a-naphthaphenanthridine alkaloids chelidonine, homchelidonine, chelerythrine, and sanguinarine together with other bases (225).The plant has been known to poison cattle (226),but cases are rare; as the leaves and stems

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contain an acrid vesicant juice which causes intense conjunctivitis (24)it is not certain to what extent such poisonings are due t o the alkaloids present. Sanguinarine is also found in numerous other members of the Papaveraceae (227), among them Argemone mexicana (Mexican prickly poppy), the seeds of which, present as contaminants in wheat, have proved toxic t o poultry (228, 229). As sanguinarine is excreted in milk and is known t o increase intraocular tension, it has been suggested that milk from cattle grazing on fumitory type weeds may be responsible for endemic primary glaucoma in man (230). Papaver nudicale (Iceland poppy) has caused poisoning in sheep and cattle, which show nervous symptoms. The toxicity of the plant has been confirmed by experiment. Prostigmine may be used as an antidote (231, 232).

0. Erythrophleum ALKALOIDS Most of the members of the genus Erythrophleum contain alkaloids (233),and many have proved t.oxic to animals. I n particular, ironwood (E.chlorostachys)has caused poisoning in horses, cattle, sheep, and goats in tropical Australia, a few ounces of the leaves being sufficient t o cause death (234).

P. Aconitum AND Delphinium ALKALOIDS Aconite (235,236)has a long history and is reputed t o have been one of the ingredients of the euthanasic agent used by the inhabitants of the island of Ceos to get rid of their more elderly citizens. I n spite of its extreme toxicity-it is one of the most poisonous alkaloids known-poisoning by aconite has never been really common in Europe or America, although i t is met with more frequently in India where the roots of Aconitum chasmanthum are widely used in native medicine (57).Poisoning may arise from injudicious use of the Dincture, 5 ml of which may be fatal, or ingestion of the plant, the roots having been mistaken for horseradishand the leavesincluded in salad. Symptoms set in almost immediately, the most constant feature being a tingling of the tongue, mouth, and stomach, which spreads to all parts of the body and is accompanied by a burning sensation followed by numbness. These symptoms are almost diagnostic. The lethal dose is said to be as little as 2 mg of the pure alkaloid, but two medical students who took between

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5 and 10 mg in mistake for vitamin C recovered within a few hours with-

out treatment after experiencing the usual symptoms (Zl?7). Aconite is sometimes added to liquors in India to increase their intoxicating effect, and this has been known to lead to fatal poisoning. Numerous species of Aconitum occur in various parts of the world, varying considerably in their toxicity and alkaloidal content. Poisoning in livestock is not common, though horses have been known to crop the plant (238, 239) and cattle have been poisoned by plants thrown out of the garden in autumn ( 2 4 ) . Although the seeds of various species of larkspur (Delphinium spp.) (235, 236) have been used for medicinal purposes since classical times, there do not appear to be any cases of human poisoning on record. I n animals, however, the larkspurs are regarded as one of the most common causes of poisoning in the United States where they occur abundantly in the western ranges (240, 241). They are divided empirically into “tall” and “ low ” larkspurs, D. burbeyi being regarded as the most toxic of the former and D . nelsonii and D . tricorne of the latter. Poisoning is most common among cattle, as they eat the plant readily and appear to be particularly susceptible. Cases among sheep and horses sometimes occur. The symptoms shown include staggering and falling, with nausea, excessive salivation and frequent swallowing, death being due to paralysis of the respiratory centers; bloat is commonly seen (171).I n Britain the cultivated D. consolidum, thrown into a field with garden refuse, has been known to poison sheep (242).

Q. ALKALOIDS OF THE BUXACEAE I n spite of the extreme toxicity of the common box (Buxus sempervirens),an understanding of the alkaloids it contains is of comparatively recent date (243). Fatal cases have 6een recorded in most species of domestic animal, pigs being particularly susceptible (24, 244, 245). The chief symptom of poisoning is hemorrhagic enteritis.

R. ALKALOIDS OF THE TAXACEAE The yew (Tuxusspp.) contains the alkaloids taxine (246)and ephedrine, the former being responsible for the toxic properties of the tree. All species of yew, and all parts of the tree except the flesh of the berry, are

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poisonous. Toxicity is not reduced by drying, although taxine is thermolabile, being completely destroyed by heating a t fiOo for 1 hour (247),a fact to be remembered during toxicological analysis. Human poisoning is rare and is usually associated with decoctions of the leaves used as abortifacients. I n animals, however, it is comparatively common and the tree is considered the most dangerous in Great Britain ( 2 4 ) .All species of animal are susceptible and cases have been recorded in cattle (248,249),horses (250,251),pigs (251),and dogs (252).Cases usually occur through branches cut from trees being thrown where animals can find them, though they sometimes arise through branches being weighted down with snow, thus bringing them within reach of stock which cannot find alternative food, Animals fed on small quantities of yew can acquire a high degree of tolerance which may account for the fact that animals newly introduced into a locality where yew trees grow seem much more susceptible than animals living among them. Death occurs from heart failure accompanied by respiratory paralysis and is usually very rapid (247).The animal may be found lying dead under a tree with twigs of yew still in its mouth or falling down suddenly as if shot if attempts are made to drive it (253).

S. XANTHINEDERIVATIVES The toxicologist usually classes the xanthine derivatives caffeine, theobromine, and theophylline as alkaloids owing to similarity in methods of isolation and identification. There appear to be no fatal cases of poisoning in man by caffeine on record, but doses over 1 gm may produce alarming symptoms, including tachycardia and sensory disturbances. Theobromine and theophylline seem to be even less harmful, although the theophylline derivative, aminophylline, has caused poisoning in children on a number of occasions, some cases ending fatally (254,255). I n animals, several cases of poisoning have been reported. Dogs have been fatally poisoned from accidental ingestion of caffeine (256,257'),and there is an old record (258)of a horse being poisoned owing to the inclusion of 10 lb of dry tea on its corn. Poisoning due to theobromine has been noted on many occasions. During times of shortage, cacao meal containing up to 5% of theobromine has been used as food for pigs and poultry and has proved to be definitely harmful (259,260).Dogs have been poisoned by a proprietary food containing 15 grains of theobromine per pound (26'1).Aminophylline, dispensed in error, has proved very toxic t o pigs (262).

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111. Alkaloids as Drugs of Addiction A. INTRODUCTION Addiction may be defined as a state of intoxication, harmful t o the individual and to society, produced by repeated administration of a drug and leading to a compulsion to continue taking it in increasing dosage and to a state of physical or psychic dependence on its effects. Nowadays the term “drug dependence ” is frequently used instead of “addiction.” The causes of addiction are many and complex, but a t least two factors must be present-a personality defect in the addict and availability of the drug. I n addition to these there is frequently some precipitating factor such as an emotional crisis ; this is usually something that would be considered trivial by a normal person. Addiction to drugs may be associated with any or all of three phenomena: (i) tolerance; in order to obtain the same effect the dose must be continually increased: (ii) physical dependence on the drug, due to the physiological changes which are produced by its continued administration, and which are responsible for the unpleasant withdrawal symptoms when it is withheld; (iii) habituation or psychological dependence on the drug, a condition probably arising from the euphoria which it produces (263, 264). New drugs are tested for their addictive action by investigation of their ability (i)to cause addiction, (ii)to prevent the onset ofwithdrawal symptoms in a subject habituated to morphine, and (iii) to give rise to such symptoms when suddenly stopped or challenged by a dose of a narcotic antagonist (265). Drugs of addiction may be divided into three classes: the narcotic analgesics (e.g., morphine and heroin) ; the stimulants or psychoenergetics (e.g., cocaine and amphetamine) ; and the hallucinogens (e.g., mescaline and LSD25).

B. THENARCOTIC ANALGESICS The morphine-type analgesics constitute the largest and probably the most important class of drugs of addiction (263, 266). Although opium has been in use as a sedative and soporific for over 6000 years, addiction to i t does not appear t o have been a serious problem in classical or medieval times. The isolation of morphine in 1805 led to a steady increase in “ morphinism,” during the nineteenth century. Heroin (267)was first isolated in 1874, hut it was not until some 20 years later that i t came into

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widespread clinical use for the treatment of respiratory diseases. At first thought to be nonaddictive, and even used t o treat cases of “morphinism,” it was only slowly realized that it was a drug of addiction far more dangerous than morphine, or, in fact, than any drug discovered since. I t s use was forbidden in the U.S.A. in 1924, and is now prohibited in most countries, one of the few exceptions being Great Britain, where it is argued that its value in the treatment of terminal cancer outweighs its danger as a drug of addiction. It is debatable whether there is any real point in prohibiting heroin so long as morphine is available, as the latter may be converted into the former with the greatest of ease, using the simplest of equipment installed in kitchen, bathroom, or garage and reagents which are free from any control. Morphine used for the clandestine manufacture of heroin is usually derived from opium, though it can also be prepared from codeine (268),which has only one-tenth of the addictive action of morphine and is therefore not so strictly controlled. The enormous profits to be made from the illicit sale of heroin make its manufacture an extremely lucrative proceeding, and in order t o put a stop to it the authorities have made every effort t o stamp out the traffic in opium. For this purpose i t is essential to know where a contraband cargo of opium originated, and much work has been done on methods of differentiating between consignments of opium from different sources. It has, for example, been shown that the percentage of codeine is highest in opium grown in the Far East and lowest in that from Yugoslavia and Greece (269).Similarly, Russian opium has a higher narcotiiie content than that from India, while Indian opium contains more narcotine than Yugoslavian or Turkish (270). During the last 60 years repeated efforts have been made to modify tlie morphine molecule to give a compound which shall have the analgesic effect of morphine but without its addiction-forming properties ; but all such attempts have been unsuccessful, as the two attributes seem to be inseparably linked. A number of interesting compounds have, however, been prepared, and passing mention must be made of certain substaiices sometimes referred to as derivatives of oripavine (271-274) in which an ethylenic bridge has been added between positions 6 and 14 of the morphine molecule, and a new center created a t position 7 . Some of these co~npounds have unprecedented analgesic potency ranging up to 10,000 times that of morphine. The best known of them is etorphine ( h 1 . ~ ; 7,8 - dihydro - 7 ~ r- [l(R)- hydroxy - 1 - methylbutyl] - 06 - methyl - f i , I l endoethenomorphine), which has found considerable application in the control of large wild animals, an elephant or a rhinoceros being completely sedated and rendered immobile by the injection of as little as 2 ing of the compound when administered in a syringe fired as a dart (275).

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Acetorphine (M.183), its O-acetyl derivative, is more potent but less stable. Cyprenorphine (M.285), N-cyclopropylmethyl-7,8-dihydro-7cr'(1-hydroxy-l-methylethyl)-O~-methyl-6,14-endoethenonormorphine, is a narcotic antagonist resembling nalorphine in its action. Compounds with such potency as these pose quite a new problem for the forensic toxicologist, as the levels likely to be found in body tissues are far below anything that can be detected by existing techniques. I n addition to modifications of the morphine molecule, many purely synthetic analgesics have been produced, the first of these, pethidine (meperidine), having been synthesized in 1939 in an attempt t o make a substitute for atropine (276).As in the case of heroin, pethidine was a t first thought to be nonaddictive. It has been followed by a hundred or so other compounds of several different types, but, as with the morphine derivatives, none, with the possible exception of pentazocine, has been found to have analgesic without addictive properties. However, it seems that the two effects may not be entirely inseparable, as diphenoxylate, which has come into use as an antidiarrheal drug, has been found to possess the power to cause addiction but no analgesic action a t all (277). C. STIMULANTS OR PSYCHOENERGETICS 1. Cocaine Coca leaves (Erythroxylum coca) have been used in South America since the time of the Incas, if not longer. Although the habit of coca chewing is usually attributed to the extremes of altitude and climate, other causes, such as malnutrition and adverse social, educational, and economic factors, are probably of more importance (278).As with opium, use of the crude plant material does not produce so dramatic a syndrome as does the use of the pure alkaloid (279).Nevertheless, in spite of the often-repeated statement that the use of coca increases resistance to fatigue and is harmless and possibly beneficial in its natural environment, the fact remains that it undermines the physical and mental health of the population and thus leads to a deterioration of the very living conditions that caused it (280). Cocaine (281)when sniffed or injected gives rise to a state of euphoria often followed by paranoid delusions. Although there are no withdrawal symptoms, as it does not cause physical dependence, it probably does more harm to brain and body than any other drug (282).There is a good case for its total prohibition, as it no longer serves any essential medical purpose, and, unlike heroin, there is no readily available natural source from which it may be manufactured. Cocaine is frequently injected in conjunction with heroin.

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2. Amphetamine Both chemically and historically most drugs of the amphetamine type are related to ephedrine (215).The latter, the main active ingredient of the plant Ma Huang (Ephedrrc spp.), was used in China for some 5000 years before being introduced into clinical use in the West shortly after World War I, when it quickly found considerable application in the treatment of asthma and similar conditions. I t s success led to a search for a synthetic substitute, and a number were produced, amphetamine becoming the most widely used. I n addition to its original employment as a vasoconstrictor, amphetamine (particularly the d-isomer) was found t o be a powerful stimulator of the central nervous system, and became used for the treatment of depression, as an anorectic agent, and to delay the onset of fatigue, particularly under war conditions. It pr6duces a sensation of elation and excitability, leading to truculence and aggression, and has become highly popular among the lunatic fringe of teen-age society. Its harmful effects on young people were first noticed in Japan during the period of social confusion during the immediate postwar years (283), but have since become apparent in most “civilized” countries. There are numerous drugs of the amphetamine type on the market, very similar in structure and action. Analytical differentiation between them may sometimes present a problem (284). Before leaving the subject of the stimulant drugs, brief mention must be made of Khat (Cathu edulis), a plant grown in Ethiopia, and used by people of many races over a wide area of North Africa. Chewing it gives rise to a feeling of euphoria and depresses the appetite, and although it is not regarded officially as a drug of addiction, it nonetheless constitutes a definite social evil in that its consumption costs what is relatively a considerable proportion of the daily wage, and so leads to a lowered standard of living with consequent lowering of resistance to disease (285).The active principle is usually regarded as being d-norpseudoephedrine, but there is almost certainly some other factor involved, as the fresh plant is considerably more toxic than the total alkaloids which it contains (286).

I).

HALLUCINOGENS

From earliest times men have sought for a means to attain a transcendental state which will enable them to appear t o rise above their everyday earthbound existence and bring them closer to some ultimate reality beyond. I n certain primitive cultures, notably in Central and South America, this has been attempted through religious rites in which

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the participants eat or inhale substances obtained from certain plants which contain the drugs now called hallucinogens. Such plants include the cactus peyotl (Anhulonium lewinii) which contains mescaline (287), the Mexican sacred mushroom (Psilocybe mexicunu), which contains psilocin and its phosphate ester psilocybin (117,288),various Piptudeniu spp. which contain dimethyltryptamine and its 5-methoxy derivative bufotenine (289), and certain kinds of morning glory (Ipomoeu spp.) which contain lysergic acid alkaloids similar to, but apparently not identical with, lysergic acid diethylamide (290).It was the accidental discovery by Hofmann (291, 292) of the properties of the latter substance that initiated modern interest in hallucinogenic drugs. These may be divided into three groups (i)the mescaline group, (ii) the dimethyltryptamine group, and (iii) the lysergic acid amide group. These compounds vary greatly in activity, the effective dose of mescaline being 500010,000 pglkg, of psilocybin 100-200 pg/kg, and of lysergic acid diethylamide (lysergide, LSD25) only 1-2 pg/kg (293).It is mainly on the last of these substances that forensic interest is centered. It finds some legitimate application in the treatment of certain psychiatric states, but during the last few years it has come t o be used illicitly by numerous unbalanced people seeking for new sensations. The effects it produces can probably only be appreciated by those who have experienced them (294).Although it does not give rise t o physical dependence or tolerance, it is capable of causing profound and possibly irreversible mental disorders when used in inexpert hands. Owing to its extreme potency, only a minute dose (100-200 pg) is necessary. This makes its recognition in body fluids extremely difficult. Thin-layer chromatography (295,296)or gas chromatography (297)may be employed. The latter is the more sensitive.

IV. Control of Alkaloids Control of drugs may be national or international. The only substances under international control are, with the exception of Capnabis, basic nitrogenous compounds.

A. INTERNATIONALCONTROL By the beginning of the present century, the question of drug addiction had changed from a minor domestic issue to a major world problem. This change had come about from a number of causes, notably the marked

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improvement in transportation during the 19th century, which had made the conveyance of drugs from one part of the world t o another a simple matter; the pace of living inherent in an industrial society, which created an environment in which people tended t o turn more and more t o drugs for relief from the stresses of life ; and the ready availability of the active alkaloids extracted from the relatively inert opium and coca leaves (298). The first attempt a t international collaboration was the meeting in Shanghai in 1909 of the International Opium Commission, which had no executive powers, but which adopted resolutions recommending the suppression of opium smoking, and recognizing the dangers of manufactured drugs. This was followed by the International Opium Convention signed at The Hague in 1912 which formulated the basic principles for international control, and raised the level of the obligation to cooperate in the campaign against drugs from a purely moral one t o a duty under international law. After World War I, the control of drugs passed into the hands of the League ofNations which, under the Convention of 1925, set up the Permanent Central Opium Board, introduced a system of licensing and recording transactions in narcotic drugs, and required governments to furnish the necessary statistical information. This principle was carried further by the Convention of 1931 under which, in an effort to limit the manufacture of narcotic drugs, each country was required t o make in advance an annual estimation of the amount of each drug needed for medical and scientific purposes. The Convention of 1936 attempted t o suppress the illicit traffic in drugs by requiring member states to enact legislation imposing severe penalties on those who instigated, organized, or directed such traffic; but this attempt was only partially successful, as a number of states refused t o ratify the Convention (299). After World War 11, control of narcotic drugs passed to the United Nations, and in 1946 the Economic and Social Council set up the Commission on Narcotic Drugs to fulfill this function. When the Commission took over, there were 20 drugs, all (except Cannabis) derivatives of opium or coca; 20 years later, mainly owing t o the advent of the synthetic drugs, this number has been increased to nearly 100. One of the Commission’s first acts was to draft the Protocol of 1946, which amended the conventions of 1921, 1925, 1931, and 1936. This was followed by the Protocol of 1948 which gave it power to control synthetic drugs outside the scope of the earlier conventions. A further Protocol, aimed a t limiting the cultivation of the opium poppy, was signed in 1953, but owing to delay in ratification by the member states, did not come into force until 10 years later.

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Long before this time, however, it had become obvious that regulations based on a series of conventions, agreements, and protocols drawn up on an ad hoc basis over a period of half a century had become quite unworkable, and the increasing urgency of the question of drug addiction made it imperative that this collection of treaties should be replaced by a single instrument. This led t o the drafting of the Single Convention on Narcotic Drugs, 1961, which came into force in 1964. Stated in the briefest possible way, this convention (300)requires member states to control the legitimate trade in narcotic drugs by the licensing of imports and exports, and by the regulation of manufacture, distribution, and consumption, and to attempt to stamp out the illicit trade by such measures as the registration of addicts, and the prosecution of traffickers (301).

B. NATIONAL CONTROL As stated above, the Single Convention requires each signatory state to enact legislation to fulfill its obligations under the act. I n addition, most countries had additional laws for controlling the sale and use of drugs, including alkaloids. These vary greatly from country to country, and it is impracticable to consider them here except in the most general terms. The regulations imposed may include (1) total prohibition of possession or use, (2) restriction of possession or use to certain classes of individual, (3) limitation of sale to medical prescription only, (4) limitation of sale to those known to the pharmacist, and (5) regulations as t o labeling and packaging. It should be noted that alkaloids form only a very small fraction of the total number of drugs controlled. I n forming laws for the control of drugs, great care has to be taken t o word the regulations in such a way that not only are the proscribed compounds included and harmless compounds excluded, but that it is impossible to circumvent the rules by producing substances which, although pharmacologically active, are technically outside the scope of the regulations (302).As long as drugs are defined explicitly, using either chemical names, national approved names, or international nonproprietary names (INN), little or no difficulty arises; words such as “morphine” and cocaine’’ have an exact meaning. When, however, one comes t o the synthetic drugs, progress is so rapid that it is possible for a drug to get into circulation and do considerable harm before authority can catch up with it, give it a name, and bring it under control. Hence it is sometimes desirable t o attempt to control all possible compounds of a certain category by using such general phrases as “ derivatives of,’’ “ homologues of,” or “having the essential structure of.” It is when attempts are made t o (6

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cover a whole group of compounds, using such generic terms, that difficulty begins. The meanings of such words as “derivative” or “homologue,” although well known to chemists, are difficult to establish in law without further qualification. This was seen in a recent case in the English courts where there was considerable argument as to whether lysergide (lysergic acid diethylamide) was or was not covered by the reference in the poisons list to “ergot, alkaloids of; their homologues.” On the first occasion the jury disagreed. On the second the case was dismissed, the jury apparently having been completely bewildered by the learned arguments produced (302).An illustration of the type of situation which may arise is afforded by the British Drugs (Prevention of Misuse) Act (303)brought into use to control the amphetamine-type drugs. This prohibits the unauthorized possession or use of “P-aminopropylbenzene ; p-aminoisopropylbenzene ; any synthetic substance derived from either of the substances aforesaid by substitution in the side chain or by ring closure therein (or by both such substitution and such closure) except ephedrine, etc., etc.” Now, as will be seen, the act implicitly includes derivatives of amphetamine (P-aminopropylbenzene), such as methylamphetamine, which are substituted in the side chain ; but its wording excludes derivatives formed by substitution in the aromatic ring. Hence p-methoxyamphetamine, a potent hallucinogen (304)is not covered by the act, nor are the two extremely dangerous compounds TMA and STP (trimethoxyamphetamine and 4-methyl-2,5-dimethoxyamphetamine) (305).To control these would require special legislation.

V. Toxicological Analysis-General

Considerations

Toxicological analysis is one of the most highly specialized branches of analytical chemistry ; its difficulties are really only appreciated by those who practice it. The work of the toxicologist bears little relation to that of the clinical biochemist or of the analyst employed by the pharmaceutical manufacturers who work in a considerably more restricted field. If the toxicologist is looking for one particular named drug his task presents no particular difficulties. He has only to look up the subject in the literature and apply the relevant techniques. Should none be available, a t least he can obtain some of the compound in question and work out procedures for himself. All too often, however, he is faced with the task of searching in a mass of decomposing viscera for a few micrograms of any one of a thousand or more compounds, with no clue a t all as t o its identity ; he has to work with the urgency that is inseparable from all forensic invest,igations and with the knowledge that on the results of his analysis a man’s life,

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liberty, or reputation may depend. There is no room for error, nor opportunity for repetition, as his material once used can never be replaced. Not only is he unaware of the identity of the compound he is seeking, but also of the form in which he is likely to encounter it. I n common with many other compounds, the majority of alkaloids undergo metabolic changes in the body and are excreted in a form different from that in which they were ingested (306).These changes are brought about by enzymes, mainly in the liver, and in general may be regarded as taking place in two stages; firstly, a presynthetic stage, in which the compound undergoes oxidation, reduction, or hydrolysis ; secondly, a synthetic or conjugation reaction in which the metabolite thus formed combines with some substance present in the body. The most common conjugation mechanism is the formation of a glucuronide, but conjugation with sulfate, with glycine, or with g!utamine, as well as acetylation and methylation, also take place. If the original compound possesses a suitable group such as hydroxyl or amino in its molecule, it may be conjugated directly without undergoing the presynthetic stage. Frequently the drug follows a number of different pathways through the body and is excreted in several different forms. Taking codeine as an example, it may (i) be excreted unchanged, (ii) be conjugated with glucuronic acid to give “bound ” codeine, (iii) undergo 0-demethylation t o morphine, which may be excreted as such or as its glucuronide, and (iv) undergo N demethylation t o norcodeine, which may be excreted in the free or “bound” form. The different pathways are not all followed t o anything like the same extent; only traces of free morphine and free norcodeine are found in the urine, whereas “bound ” codeine may account for up to 50% of the dose (307). The fact that such a large proportion of the alkaloid may be present in the “bound” form, most frequently as glucuronide, has a practical bearing on the extraction techniques which must be employed. Conjugates of this type are water-soluble, and will remain in the aqueous phase during extraction with an immiscible solvent unless they are first hydrolyzed. This may be carried out by treatment with the appropriate enzyme, which, although it has the advantage of giving a cleaner product, has also the inherent drawback of specificity and is thus useless unless the type of conjugation is known, or by acid hydrolysis, which is nonspecific, but gives a very “dirty” extract and involves a risk of hydrolyzing ester linkages in the alkaloid molecule. I n toxicology, there is no such problem as a search for an unknown alkaloid, as there is nothing to differentiate poisoning by an “alkaloid” from poisoning by any other type of drug. The search for an alkaloid is simply part of the search for an unknown organic poison. It is now pro-

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posed to consider this problem with special reference to compounds containing basic nitrogen. This will, of course, include the techniques which would be employed in testing for a “known” poison; but i t must be emphasized that no drug is “known” until its presence has been established by analysis. A corpse clutching a labeled bottle provides presunil)tive evidence only. Before considering the question of analytical techniques, however, one further point must be made clear. The forensic chemist must be prepared to identify any unusual compound which he isolates from body tissues, not merely those of known and proved toxicity. Until the identity of such a compound is established beyond doubt it cannot be decided whether its presence is significant or not. To give a concrete example, some years ago the body of a man was taken fi-om a river where it had been immersed for several weeks. The pathologist was unable to decide on the cause of death and asked for a toxicological examination to be made. This disclosed the presence of a basic compound which when submitted to paper chromatography (citrate-butanol syst,em)gave an absorbing spot, weakly positive with iodoplatinate, a t Rf0.90. Elution of this spot,gave a substance which had a UV-maximum a t 264 mp, and gave a purple color with the Marquis reagent. Reference to the card index followed by comparison with a known sample identified the substance as bisacodyl, a harmless laxative, which could have played no part as the cause of death ; but until it was identified with certainty, it might have been some unusual toxic alkaloid, or else the metabolite of one.

VI.

Extraction methods

A. INTRODUCTION Before a compound can be identified it must be extracted and purified ; in the case of a “general unknown” this poses a problem of considerable difficulty. The analyst cannot choose a suitable extraction method until he knows the identity of the compound with which he is dealing, and yet at the same time he cannot begin to identify it until he has extracted it. The answer to this apparent paradox is to use a compromise method which will extract a t any rate some of any compound likely to be present,. It must be emphasized that such an extraction cannot be more than qualitative. Once the identity of the compound is known, a suitable extraction method for it can be chosen, and carried out on a further aliquot of material.

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The choice of the extraction method to be used depends on a number of factors, the most important of these being the facilities available, the urgency of the problem, and the nature of the sample.

B. TYPESOF SAMPLE The sample may consist of (i) a pure chemical substance, (ii) a pharmaceut>icalpreparation (tablet,capsule, or mixture), (iii)blood, urine, vomit, or stomach washings, or (iv) body tissues-liver, kidney, or brain, together with stomach and intestines and their contents.

1. Pure Chemical Substances These are rarely encountered in forensic toxicology. Dealing with them calls for no particular expertise; they afford almost the only case where classic procedures, such as preparation of a derivative and determination of the melting point, may be used.

2. Pharmaceutical Preparations There may sometimes be a particular urgency about these, as in the case when a child is brought into hospital unconscious, and identification of the tablets he may have taken must be made as quickly as possible so that correct therapy may be instituted. I n such a case much time may be saved by the use of some tablet identification scheme. A number of these are available (308-310), but they all suffer from the same drawbacks in that they are difficult to apply to the vast number of white tablets now available; they can never be up to date, as new drugs are continually coming onto the market, and old ones appearing in different form; and it is impossible to make them comprehensive, particularly with regard to tablets originating in another country. Nevertheless, they have great potential value as time savers, particularly as they can be used by office staff without technical training. Any tentative identification must, of course, be confirmed by chemical tJestsfollowing some simple extraction process.

3. Blood, Urine, Vomit, and Stomach Washings The reason for differentiating between these and other biological materials is that they may come from a living rather than a dead subject, and hence call for speed rather than detailed analysis. Fewer than 20

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drugs account for over ‘30% of hospital poisoning emergencies, so elaborate identification schemes are not necessary. The usual procedures in these cases are (i) to apply a series of spot tests to the urine to test for such compounds as aspirin, barbiturates, carbon monoxide, hydrocyanic acid, etc., which are the usual cause of hospital emergencies; (ii) to carry out simple extractions with immiscible solvents to isolate more complex compounds. The material is made acid with a few drops of concentrated hydrochloric acid, extracted with an equal volume of ether, then made alkaline with ammonium hydroxide and extracted with an equal volume of chloroform. The first extract (acid-ether) will contain the acidic drugs (aspirin, barbiturates, glutethimide) and the neutrals (meprobamate, carbromal), but is unlikely to contain any alkaloids. The alkaline chloroform fraction will contain any basic compounds present, the substances most likely to be met with under these conditions being the phenothiazine tranquillizers (promazine, chlorpromazine), imipramine, chlordiazepoxide, amphetamine, codeine, quinine, and the ergot alkaloids. The last named group include lysergic acid diethylamide (LSD); they are unlikely to be present in sufficient quantity to be found in blood or urine, but might be detected in stomach washings or vomit. The chloroform is evaporated, the residue taken up in a drop or two of dilute acetic acid and spotted on filter paper. The spots are examined under UV-light (preferably 2537 b)and tested with (1) iodoplatinate solution, ( 2 )p-dimethylaminobenzaldehyde solution, and (3) the Marquis reagent. Quinine and the ergot alkaloids show a bright blue fluorescence, but care must be used in interpreting this, as blue fluorescence is commonly found in crude extracts from body fluids. The iodoplatinate reagent gives a dark blueblack spot with most of the bases likely t o be met with here. p-Dimethylaminobenzaldehyde gives a blue color with the ergot alkaloids, and a bright yellow with compounds containing a primary aromatic amino group. The Marquis reagent gives a bluish purple with codeine (and morphine), a red-purple with most of the phenothiazine derivatives, and an orange color with amphetamine and several other synthetic sympathomimetic drugs. The residue may now be submitted to thin-layer chromatography and UV-spectrophotometry in order t o confirm any provisional identification made at this point, or to obtain further data on which such an identification may be based.

4 . Body tissues and visceral contents Analyses of these provide by far the largest part of the toxicologist’s work and must be considered in rather more detail in the following sections.

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C. CLASSIFICATION OF POISONS It is usual to divide poisons, from the analytical point of view, into five groups : ( 1 ) Volatile poisons (e.g., hydrocyanic acid, phosphorus)

( 2 ) Toxic anions (e.g., oxalate, fluoride) (3) Toxic metals (e.g., arsenic, barium)

(4) Solvent-extractable nonvolatile organic poisons (phenobarbitone, strychnine) (5) Miscellaneous poisons (conjugates ; quaternary ammonium compounds)

The vast majorit,y of alkaloids occur in group (4),although some may be found in groups ( 1) and ( 5 ) . The choice of the sample ta be analyzed will depend 011 what is available. Assuming it to be the whole cadaver, there is much to be said in the case of an acute poison in favor of stomach contents, as it will contain the drug in unaltered form and possibly in high concentration. When death has been delayed, intestinal contents may be more useful, while the liver, as the chief detoxicating organ of the body, has the power of concentrating many drugs. Analysis of blood, urine, and bile will also often furnish useful information. The decision as to whether a single sample should be analyzed first, or analysis of a number of samples carried out concurrently, will depend on the urgency of the case and the facilities available. 1. I’olntile Popisons

These may be isolated by steam distillation. It is usual to make two such distillations, the first from acid solut,ion, the second from alkaline. About 50-1 00 gm of tissue is homogenized, mixed with an equal quantity of water, acidified with tartaric acid and steam-distilled until a volume equal to the original sample taken has been collected. This distillate will contain neutral and acidic compounds such as hydrocyanic acid, phenol, and phosphorus. The tissue slurry is now made alkaline with sodium hydroxide solution and again subjected t o steam distillation. This second distillate will contain basic compounds. The tip of the condenser should be immersed in dilute hydrochloric acid to trap the more volatile bases. The plant alkaloids most likely to be present are nicotine, coniine, arecoline, ephedrine, and sparteine, but a number of synthetic drugs, notably amphetamine and other sympathomimetic bases, also occur in this fraction. Pethidine may also be isolated by steam distillation. The

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distillate is made alkaline with sodium hydroxide, and extracted with ether or chloroform. The extract is dried with sodium sulfate, and examined in a way similar to that described for the extracts of section 3.

2. Toxic Anions and Metals These groups do not contain any alkaloidal substances, and need not be further considered.

3. Solvent-Extractable Nonvolatile Organic Poisons This group is the most important from the point of view of basic nitrogenous compounds, as the vast majority, over 90% of them, occur here, and much work has been done in devising methods for their isolation, but it must be admitted that no really satisfactory answer has yet been discovered to what is commonly agreed to be the biggest problem in analytical toxicology. Most extraction procedures depend for their success on the fact that alkaloidal bases are usually soluble in ether or chloroform, but insoluble in water, while their salts are soluble in ethanol or water, but not in the fat solvents. Such processes, then, consist essentially of two stages, the first being the preparat,ion of a protein-free aqueous extract, and the second the extraction of this solution a t alkaline pH with an immiscible solvent. Most of these methods, however, are modifications of the classic Stas-Otto process (62,311),which is still in use after over a century. By general consent it is long and tedious, yields a somewhat impure product, and gives rather poor recoveries, yet with some modification it still remains the method of choice under certain circumstances. A version of it is described below. I n an attempt to overcome the inherent drawbacks of the Stas-Otto process, numerous other methods have been described. One of the most popular of these is that of Daubney and Nicko1:s (312-314) which removes proteins by precipitation with ammonium sulfate. Abernethy et al. (315) extract buffered homogenized tissue with acetonitrile and ether. Alha and Lindfors (316)extract with acetone instead of ethanol. Berman and Wright (317)and Valov (318)use tungstic acid as a protein precipitant, while Stewart et al. (319) use trichloracetic acid. A promising method recently described by Stevens (320)uses aluminum chloride. Gettler and Sunshine (321) and Feldstein and Klendshoj (322)extract the filtrate from the residue left after steam distillation, relying on the heat t o have denatured most of the protein. Tompsett (323) also boils the minced tissue with dilute hydrochloric acid, subsequently absorbing any alkaloids present in a cation exchange column.

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Once the clear protein-free extract has been obtained, it is extracted with an immiscible solvent. This is usually done in several stages. The most common practice is to extract the acid aqueous phase with ether to remove the acidic and neutral drugs, then to make it alkaline and extract with chloroform or ether to remove the bases. These two extracts are commonly called the acid-ether and the alkaline-chloroform extracts, and may be further subdivided as follows. The acid-ether extract is shaken with a solution of sodium bicarbonate to remove the " strong" acids such as aspirin, then with sodium hydroxide solution to remove the "weak" acids, such as the barbiturates, neutral drugs such as the carbamates, and a few weak bases such as caffeine, remaining in the ether layer. The alkaline chloroform extract may be subdivided by adding a few drops of hydrochloric acid to it and evaporating to dryness. The residue is dissolved in water, and extracted with chloroform, which removes the drugs whose hydrochlorides are soluble in chloroform. (Fraction D1; this includes aconite, cocaine, papaverine, hydrastine and many synthetic drugs, notably the phenothiazines and most of the antihistamines and narcotics.) The aqueous phase is now made alkaline with sodium hydroxide solution, and again extracted with chloroform. This gives Fraction Dz, which contains most of the organic bases. The aqueous phase is now acidified, made alkaline with either ammonium hydroxide or sodium bicarbonate, and extracted with ethyl acetate or a chloroformpropanol ( 5 :1) mixture. This gives Fraction Ds, which contains the amphoteric drugs, notably those such as morphine which contain phenolic groups. Although the fractionation of the acid-ether extract is essential, as it divides the drugs which it includes into different classes which are investigated by quite different methods, e.g., by different schemes of chromatography, in the case ofthe bases it is doubtful whether any useful purpose is served by such subdivision, unless it is particularly required to separate, say, phenothiazines from other alkaloidal compounds. There are methods of paper and thin-layer chromatography that can be used for screening all nitrogenous bases, and preliminary subdivision is of little help. The more complicated a system becomes, the more time is wasted and the more material lost. A few practical details are worth mentioning in connection with solvent extraction. Quite apart from the question of separating compounds on the basis of the solubility of their hydrochlorides, chloroform is the solvent of choice for bases, as the majority of alkaloids are much more soluble in chloroform than they are in ether (see Table I).It is certainly more prone to form emulsions, but these may usually be avoided by using a relatively large volume, 5 or 10 times as great as that of the aqueous

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phase, by the use of a rolling extractor (324)or by the addition of a little ethanol. Finally, when several extractions are being made, a solvent that is heavier than water is more convenient to use than one that is lighter. A number of authors have advised the use of continuous extraction. For the extraction of the aqueous phase when obtaining the acid-ether and alkaline-chloroform fractions this is of doubtful value, as, although it may extract marginally more of the drug, it is certain to extract considerably more impurities. Mention should be made, however, of a scheme for the continous extraction of minced tissues with ethanol described by Curry and Phang (325);this has proved most useful in extracting difficult compounds. During recent years there has been a growing tendency for toxicologists to abandon the two-stage extraction process, and to extract biological material directly with immiscible solvents. This is particularly convenient with fluids which would have to be evaporated to dryness before processes such as the Stas-Otto could be applied. Urine may be extracted without preliminary treatment (326), but blood is best deproteinated with tungstate before extraction (327). With tissue homogenates direct extraction is less satisfactory; drugs tend to be occluded in the tissue particles, and emulsions which are difficult to break frequently form. Some authors, however, use direct extraction for stomach contents (328). As it is obviously impracticable to give experimental details for all the various methods referred to above, it is proposed to limit detailed instructions to a modified Stas-Otto extraction for tissues and visceral content, a direct extraction for use with urine, and a tungstate precipitation method for use with blood. It is again emphasized that these procedures are designed for use when there is no clue at all as to the nature of the drug present. Should there be any evidence pointing to its identity, they may be simplified accordingly. The Stas-Otto type extraction is carried out as follows: 100 gm of tissue is homogenized with 200 ml of ethanol made acid with a little acetic acid. The resulting homogenate is heated on the water bath for at least 1 hour a t a temperature not exceeding 60°, cooled, and filtered. A further 100 ml of ethanol is added to the residue, the process repeated, and the two filtrates combined. The ethanol is removed a t low temperature by vacuum distillation or by heating in an open dish in a current of warm air. Hot ethanol, 50-100 ml, is added little by little to the syrupy residue, each portion being stirred and decanted before the next is added. The process is continued until the residue appears dry and granular. The extract is filtered and evaporated as before. If the residue appears dark and greasy, the extraction with hot ethanol should be repeated. The final residue is now extracted with 25 ml of 0.01N sulfuric acid; the choice of

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acid is important here if one wishes t o extract as few basic drugs as possible into the acid-ether fraction. Great care must be taken a t this stage, as it is here that most of the losses occur. Continuous stirring by hand with a glass rod is probably more efficient than mechanical shaking or stirring. Heating is not advisable for fear of hydrolysis. After filtration, the aqueous extract is ready for the next stage, which consists of shaking or rolling it with two 50 ml portions of ether. If there is any tendency to form emulsions, the volume of ether can be increased, and a little ethanol added ; but as a rule emulsions are unlikely here. The two extracts are combined, washed with 10 ml of saturated sodium chloride solution [water might remove hydroxybarbiturates (329)],and extracted with 20 ml of 5% sodium bicarbonate solution saturated with sodium chloride to remove ‘‘strung” acids, then with 20 ml of 1N sodium hydroxide solution to remove “weak ” acids, and finally dried with anhydrous sodium sulfate and evaporated to give the “neutral” Fraction C. The sodium bicarbonate extract and the sodium hydroxide extract are both acidified with a few drops of HCI and extracted with equal volumes of ether, which is dried with anhydrous sodium sulfate and evaporated to give Fractions A and B, respectively. As has been stated previously, extract A will contain the salicylates, B will contain the barbiturates, and C the neutral drugs and the weak bases such as caffeine and benzocsine. The original aqueous phase remaining after the extraction with ether is made alkaline with concentrated ammonium hydroxide solution added drop by drop and extracted twice with 100 ml of chloroform to which 10 ml of ethanol has been added. The two extracts are combined and extracted with 50 ml of 0.1N sulfuric acid. The aqueous phase is separated, made alkaline with ammonium hydroxide, and reextracted with two 50 ml volumes of chloroform. The chloroform is dried with sodium sulfate, and carefully evaporated t o dryness afher addition of one or two drops of dilute hydrochloric acid to prevent the loss of volatile compounds. This gives Fraction D. It will be noted that no attempt has been made to subdivide this fraction. As has been said before, there is little advantage t o be gained from doing so, and every unnecessary manipulation leads t o delay and loss of material. It is admitted that this process is tedious and time consuming, but it is based on a method that has been in use for many years, and about which much information is available. It does not pretend to be quantitative, but should be capable of isolating a t least some of any alkalkoid likely to be present. If one were looking for a single known substance many steps could be omitted. Similarly, if one were only concerned with acidic drugs such as barbiturates one would not use this method. The tungstate precipitation method (318,327)is much better for this purpose.

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Direct extraction of urine or blood is of particular value to the hospital biochemist when the subject is stil1,alive and every minute counts; it is also useful as a rapid screening method in routine toxicological analysis. A measured volume of urine is acidified with hydrochloric acid and added to 5 volumes of ether. The extraction may be made by shaking or rolling; the latter is essential for horse urine which forms emulsions extremely easily. Hensel(326) uses large centrifuge tubes ;simple inversion 100-200 times produces adequate partition. The layers are rapidly separated by centrifuging and may be transferred by means of bulb pipettes. After the ether layer has been separated, the aqueous phase is made alkaline with ammonia and extracted with 5 times its volume of chloroform to remove basic drugs. Blood may be treated in the same way, but the result is less satisfactory. It is more usual to remove the protein first. This may be done with sodium tungstate (318, 327) as follows: To 10 ml of blood add 2 ml of 10% w/v sodium hydroxide solution, 50 ml of water, and 20 ml of 10% wjv sodium tungstate. Then acidify by adding 2 N sulfuric acid slowly with continuous stirring or shaking. Immerse the vessel in boiling water for 10 minutes, filter, cool, and make up to 100 ml with water. Extract with 200 ml of ether. Then make the aqueous layer alkaline with ammonium hydroxide, and extract it with 5 times its volume of chloroform. The acid ether extract may be subdivided to give the strong acid, weak acid, and neutral fractions as described previously. The method is not particularly satisfactory for bases, but will often given an indication as t o what is present. 4. Miscellaneous Poisons

a. Conjugates. As has been stated above, many alkaloids undergo conjugation in the liver to form water-soluble complexes such as glucuronides which cannot be extracted by immiscible solvents. Thus, except in the case of stomach and intestinal contents, the free drug obtained by the ordinary extraction processes is only a small fraction of the total drug present. To increase the yield it is necessary t o break down the conjugate. This may be done by either acid hydrolysis or by enzyme action. The former may be carried out by mixing the material (urine or tissue homogenate) with half its volume of concentrated hydrochloric acid, and heating on a boiling water bath for 1 hour. It is then cooled, extracted with ether, made alkaline with ammonia, and extracted with chloroform (or chloroform: propanol 5: 1 if morphine is suspected). This method would almost certainly destroy any alkaloids containing ester groups. As an alternative, therefore, the sample may be hydrolyzed a t 37" a t

E. G. C. CLARKE

554

pH 4.5 for 20 hours with P-glucuronidase. This will serve to break down glucuronides and will yield a cleaner extract, but would have no effect on sulfate conjugates. h . Quaternary ammonium compounds. These are invariably watersoluble, and remain in the aqueous phase. No really satisfactory method for their extraction has been described, although several have been suggested. I n one of these urine, acidified with HCI, is allowed to percolate down a column containing Dowex 500 x 12 (200-400 mesh). The alkaloids are eluted with more concentrated HCl(323). I n another, which is suitable for combining with the modified Stas-Otto process described above, the aqueous phase after extraction of the D fraction is acidified with dilute acetic acid, evaporated t o dryness under reduced pressure, and the residue extracted with methanol. The methanol extract is evaporated and submitted t o paper chromatography (330).

VII. Identification Methods

A. INTRODUCTION Once the alkaloid has been isolated, it must be identified. The methods available are ( 1 ) the classic color and crystal tests, ( 2 ) chromatography, and (3) spectrophotometry. 1. The Classic Methods Until about 20 years ago the only way in which an alkaloid isolated from cadaveric material could be identified was by means of color and crystal tests. Both of these date back for well over a century, the color test being the older of the two. It has the great advantage of speed and simplicity and is ideally suited to rapid screening procedures. It has recently secured a new lease on life owing to the way in which it may be used for locating compounds on paper and thin-layer chromatograms. Few color tests are specific, but many have the advantage that they will work satisfactorily in the presence of a high proportion of impurity. Color tests for many alkaloids have been described (331-334). Crystal tests are more specific than color tests, but need purer material, and call for more skill and experience on the part of the operator. They are extremely delicate, having sensitivities well down in the microgram range, and can thus be used on the material eluted from paper or thinlayer chromatograms. Although largely outmoded, they are still of considerable value in confirming a final identification, as a great deal of

7.

THE FORENSIC CHEMISTRY O F ALKALOIDS

555

information about them is available (332,335,336).They are of particular value in differentiating between microgram quantities of optical isomers (337,338).This is a point which may be of considerable forensic importance as, for example, in the case of the N-methylmorphinan analgesics, where the Z-isomers have addictive properties, and are under international control as narcotic drugs, while the d-isomers, which are useful antitussives, are nonaddictive, and thus free from control.

2. Chromatography a. Paper chromatography. The introduction of paper chromatography (333,339-342)was one of the greatest single advances in analytical toxicology. Coming as it did in the 1950’swhen the post-war “pharmaceutical explosion ” was beginning to swamp the facilities then available, it provided not only a simple method of provisional identification, but in addition a means of obtaining material pure enough for subsequent spectrophotometric analysis. Although now very largely replaced by thin-layer chromatography, it still remains a most valuable tool for the forensic chemist. It is extremely simple, requiring neither expensive equipment nor skilled personnel, and is thus within the reach of any laboratory in the world. I n addition to the R, value, the appearance in UV light, and the behavior with the conventional spray reagents, paper chromatography can furnish much additional information of analytical value owing to the ease with which a variety of chemical reactions such as diazotization can be carried out directly on the paper, b. Thin-layer chromatography. Thin-layer chromatography (343-346) has come to the fore during the last 5 years or so. Compared with paper chromatography it has the great advantages of increased speed and sensitivity ; in addition most materials used will stand high temperatures and corrosive acids. On the other hand it is not quite so simple, the plates are fragile and cannot be filed for reference, and silica gel (the medium most commonly used) is strongly absorbent in UV light and hence masks absorbing spots. Ready-made plates and films can be purchased, but are expensive if used in any quantity. Even plates produced in the laboratory with a commercial spreading device tend t o make thin-layer chromatography more expensive than paper, but quite satisfactory results can be obtained with homemade equipment using microscope slides or old 3 x 3-inch projector plates. The speed with which a result can be obtained makes the technique of great value to the hospital biochemist. As with paper chromatography many simple reactions may be carried out directly on the plate. c. Gas-liquid chromatography. Gas-liquid chromatography is the latest chromatographic technique to have found application in toxicology (347,

556

E. G . C. CLARKE

348).Originally introduced for the identification of volatile compoundsa field in which it, remains unsurpassed-it has now found much wider application (349, 350). Numerous systems, varying in support material, liquid phase, and type of detector, are in use. It differs from paper and thin-layer chromatography in that the apparatus is costly and cannot be improvised. On the other hand it is in many instances considerably more sensitive. Fractions can be trapped and submitted to other analytical techniques, but the only direct information afforded is a retention time. 3 . Spectrophotometry a. Ultraviolet. During the last 15 years UV spectrophotometry (351354) has become a standard analytical technique. Earlier attempts to use it (355)were disappointing as extracts obtained directly by the Stas-Otto process were too impure to give satisfactory results, and it was not until the advent of paper chromatography that it became possible t o obtain samples sufficiently free from interfering substances t o be of any use for spectrophotometric purposes. The technique is of particular value in helping to identify substances which show a definite shift of peak with change of pH, but considerably less so when one has a whole group of related compounds (such as the amphetamines) which have a weak uncharacteristic absorption; but even in such cases the UV curves can be of some use when considered in relation to other analytical data. Tables of maxima arranged in sequential order are of considerable help in making a provisional identification, but actual comparison of the spectrum with that obtained from an authentic sample is of much more value. The apparatus is fairly expensive, but it is now (at any rate in the manually operated form) to be found in most laboratories. b. Infrared. IR spectrophotometry (354,356,357)came into use some years later than UV. Although it calls for more expensive equipment, rather more material, and a very high degree ofpurity, it is probably the most satisfactory method of final identification, as the spectra are far more characteristic than those given in the UV. The very complexity of the spectra itself poses a problem, as although various methods of coding have been described none is really satisfactory, and at the best can only allow a very tentative identification t o be made. For anything more than this a facsimile of the spectrum of the authentic compound, or better still the actual curve run on the same instrument as the unknown, is needed ; but even in the absence of such a curve a great deal can be learned about the structure of a compound from an examination of its IR spectrum.

7.

THE FORENSIC CHEMISTRY O F ALKALOIDS

557

B. ANALYTICAL TECHNIQUES Under ideal conditions the identification of an unknown alkaloid may be carried out by submitting the extracted material to gas chromatography, trapping the emerging fractions, and finding their IR, spectra, then programming the latter and feeding them to a computer. I n a few laborakories such a scheme is actually in operation. Although a computer cannot yet be considered as a normal part of laboratory equipment, most of the toxicological laboratories in Europe and North America are fully equipped for instrumental work. Many hospital laboratories on the other hand do not possess anything more sophisticated than a UV spectrophotometer, while in Asia and Africa, where the majority of cases of poisoning by alkaloids occur, only the simplest equipment is available. I n order to be of the widest use, the scheme given below is based on paper and bhin-layer chromatography with the help of UV spectrophotometry if it is available.

I . Paper chromatography Paper chromatography is carried out by the citrate-butanol system (341,358).This system has been chosen because it has proved itself over the years to be the most satisfactory for general screening purposes, and data for several hundred bases, both natural and synthetic, are available (332, 336). Sheets of Whatman No. 1 paper are dipped in a 5% solution of sodium dihydrogen citrate, roughly blotted, and hung up to dry a t room temperature. They may be stored indefinitely. The solvent is made by dissolving 4.8 gm of citric acid in a mixture of 130 ml of water and 870 ml of n-butanol. It may be used over a period o f a month or more provided that water is added from time t o time t o keep the specific gravity a t 0.843-0.844. This monophasic solution replaces the diphasic solution originally described (341)as it gives more reproducible results, but as the system is not equilibrated, reproducibility is also affected by such factors as temperature, size of tank, number of sheets of paper, and time of running (358).The Rfvalues given in Table I1 were obtained running four sheets 14 x 6 inches a t a time for 5 hours in a tank 8 x 11 x 155 inches deep and containing 500 ml of solvent, but any size paper and tank may be used provided suitable corrections are applied. After evaporation to dryness (in the presence o f a drop of dilute HCl t o avoid loss of volatile bases) the residues from the C (neutral) and D (alkaline-chloroform) extracts as well as that from the alkaline distillate fraction are dissolved in a few drops of 2N acetic acid and spotted on the paper. The amount of alkaloid needed for each spot is 10-25 pg. If only a trace of material is available, it may be dissolved in ethanol and spotted

558

E. G . C. CLARKE

in several aliquots, allowing each to dry before the next is added. For very dilute solutions a continuous flow spotting device may be employed (359). Four papers should be spotted, or four spots run on the same paper, together with adequate controls. The paper is allowed t o develop for a t least 5 hours by the ascending method. At the end of this time it is removed, dried, and examined under UV light (2537 A), absorbing or fluorescent spots being marked; in this connection i t should be noted that a certain amount of material showing blue fluorescence is nearly always extracted from body tissues. The first paper (or spot) is sprayed with iodoplatinate solution, the second with bromocresol green, the third and fourth being kept in reserve. The Rf value is measured, and compared with those given in Table 11. If a tentative identification is possible, the remaining spots may be eluted for confirmatory tests such as UV spectrophotometry, or, alternatively, color reactions such as the Marquis test may be carried out directly on the paper. If it is necessary to elute a spot which has been sprayed with iodoplatinate, it should be cut out, and moistened with successive drops of 10% sodium sulphite solution, 10% barium chloride solution, and 0.880 ammonium hydroxide solution, drying between each addition and finally eluted with chloroform (360).The barium chloride is added to convert the citrate to its insoluble barium salt and thus prevent its elution. The citrate-butanol system can be run as a thin layer system, using plates spread with a slurry made from cellulose powder and a 5 yosolution of sodium dihydrogen citrate and the same solvent as given above. Results are reasonably comparable with those obtained by paper chromatography (361).

2. Thin-Layer Chromatography A further aliquot of the same extracts is submitted t o thin-layer chromatography, using the method described by Sunshine (362).Glass plates 20 x 20 cm are coated with a slurry made by mixing 30 gm of silica gel G with 60 ml of water, to give a layer 250 p thick, and dried in the oven a t 110" for 1 hour. The figures given in Table I11 were obtained using tanks 21 x 21 x 10 cm, the ends of which are covered with paper t o assist evaporation; 100mlof methanoland 1.5mlof 0.880ammoniumhydroxide are placed in the tank which is allowed to stand for 1 hour before use in order to equilibrate. After two runs the solvent should be changed and the tank reequilibrated. The test material is dissolved in 2N acetic acid and 1 p1 spotted on the plate, which is allowed to develop for 4 hour, by which time the solvent front will have risen about 10 cm. After drying,

7.

THE FORENSIC CHEMISTRY OF ALKALOIDS

559

the plates are sprayed, the most satisfactory reagents being acidified iodoplatinate solution and a 1yoaqueous solution of potassium permanganate. The results obtained should be compared with those given in Table 111. 3. U V Spectrophotornetry

One of the spots on the paper or thin-layer chromatograms is eluted, the eluate dissolved in 0.1N sulfuric acid, and its UV spectrum determined. The wavelength of the principal peak is compared with those given in Table IV, which gives these figures arranged in descending order. No practical details are given here, as these will, Qf course, depend on the type of instrument available.

4 . Color Tests Only two color tests, those ascribed t o Marquis (363)and Vitali (364), need be used as part of a routine screening procedure, although any suitable test may be employed for confirmatory purposes. The Marquis test is carried out by placing a drop of the test solution on a white tile, allowing it to dry and moistening it with the reagent, which is made by adding a drop of 40% formaldehyde solution to 1 ml of concentrated sulfuric acid. The colors obtained with a number of alkaloids are given in Table V. It must be remembered, however, that many synthetic drugs not included in this table also give colors with this reagent ; for example, most of the phenothiazine tranquillizers give a reddish purple, while the benzhydryl ether antihistamine drugs give a bright yellow. The test may be carried out directly on the residue from the alkalinechloroform extract, or on the spot on a paper or thin-layer chromatogram, or on the material eluted from such a spot. Colors from crude extracts always tend to be duller than those obtained from pure materials owing to the charring of the impurities by the sulfuric acid, I n the case of chromatograms it is convenient to pour the reagent over the sheet resting on a sheet of white opal glass; paper chromatograms must be absolutely dry. For spots eluted from chromatograms a microtechnique (365) may be employed. Glass rods about 20 cm long and 0.5 cm in diameter are heated in the middle and pulled out until the diameter a t the thinnest point is about 0.1 cm. They are broken a t this point and the ends ground flat. When the narrow end of such a rod is allowed t o touch the surface of a liquid it brings away a small pendant “microdrop.” This is transferred to a piece of opal glass, allowed t o dry, and a microdrop of the Marquis reagent added. This technique may be used for a wide

560

E. G.

C. CLARKE

variety of color and microcrystal tests (332,336).As thevolumeof a microdrop is about 0.1 p1i t follows that 500 different tests can be carried out on one conventional drop (0.05 ml) of solution. Vitali’s test is carried out by placing a microdrop of the test solution 011 a piece of opal glass, allowing it to evaporate, adding a microdrop of fuming nitric acid, and evaporating to dryness on a boiling water bath. After cooling, the residue is moistened with a microdrop of a 5% w/v solution of potassium hydroxide in ethanol. The colors obtained after adding the fuming nitric acid and after adding the ethanolic potassium hydroxide are given in Table VI. It will be noted that many compounds give indeterminate shades of yellow, orange, or brown which have little diagnostic value. Among distinctive colors given on addition of the fuming nitric acid by synthetic drugs not included in the table may be mentioned the green color given by imipramine and its cogeners, the red color given by antazoline, and the purple flash turning to yellow given by most of the phenothiazine tranquillizers. On the addition of ethanolic potassium hydroxide, diphemanil, aminocrine, cyclopentolate, and adiphenine give a purple color not unlike that given by the atropine alkaloids. Vitali’s test cannot be carried out satisfactorily on paper or thin-layer chromatograms.

VIII. Tables of Analytical Data INTRODUCTION Table I shows the solubility of various alkaloids in ether and chloroform. Table I1 gives data for the paper chromatography of a number of alkaloids on the citrate-butanol system. The first column shows the R, value, the second the name of the alkaloid, the third the appearance under UV light (2540A), while the fourth and fifth columns indicate the most satisfactory location reagents. Table 111gives data for thin-layer chromatography with the methanolammonia system. The first column gives the R, value, the second the name of the compound, the third the most satisfactory location reagent, while the last column gives references to literature where additional chromatographic information on the substance in question may be found. Table IV gives UV spectrophotometric data, the first column giving wavelength of the principal peak, the second the name of the compound,

7.

56 1

THE FORENSIC CHEMISTRY OF ALKALOIDS TABLE I SOLUBILITY OF ALKALOIDS Base

Chloroforms

Etheru

2 5 110 0.7 0.5 1.5 1 1220 5 1.6 1.2 5

50 187 500 3.5 18 100 245 6250 250 56 250 5500

Aconitine Brncine Cinchonine Cocaine Codeine Heroin Hydrastine Morphine Narcotine Quinidine Quinine Strychnine a

Milliliters of solvent to dissolve 1 gm of base.

the third the solvent used, and the fourth the wavelengths of secondary peaks. Table V gives colors obtained with the Marquis reagent and with Vitali’s test. I n the latter case the first column shows the color formed on the addition of fuming nitric acid, the second the color shown when a solution of potassium hydroxide in ethanol is added to the residue left after evaporation of the acid. Table VI gives details of the various location and color reagents employed. TABLE I1

PAPER CHROMATOGRAPHY DATA Location reagents

Rf

Compound

0.00 0.00

Agmatine Histamine

0.00 0.02 0.02 0.03 0.03

Pethidine Pseudomorphine Trigonelline Cytisine Ecgonine

UV light

1

Iodoplatinate Iodoplatinate (white) Dragendorff Bright blue Iodoplatinate Absorbs (strongly) Iodoplatinate Blue Iodoplatinate Iodoplatinate -

-

2 Broincresol green -

Marquis Marquis Dragendorff Dragendorff -

562

E. G . C . CLARKE TABLE 11-continued ~

Location reagents

Rf

Compound

UV light

1

__ 0.03 0.04 0.05 0.0.5 0.05 0.06 0.07 0.07 0.08 0.08 0.08 0.10 0.11 0.11

Ergothionine Tubocurarine Choline Oxycanthine Tropine Ariabesine Connessiiie Nicot,ine Berbamine Hydroxylupanine Tomat ine Pilocarpine Cephaeline Dehydroemetine

Absorbs Absorbs

0.11

L ysergamide

Blue

0.12 0.12 0.12 0.12 0.12

Chelerythrine Cycleanine Demissine Normorphine Serotonin

0.13 0.14 0.14

Sparteine Hydrastinine Monocrotaline

0.14 0.14 0.15 0.15 0.16

Morphine Morphine N-oxide Arecoline Solanine Bufotenine

Iodoplatinate Iodoplatinate Iodoplatinate Absorbs (strongly) Iodoplatinate Iodoplatinate White (white) Iodoplatinate Bright blue Iodoplatinate Iodoplatinate (white) Absorbs (strongly) Iodoplatinate Absorbs Iodoplatinate Iodoplatinate Absorbs Iodoplatinate Iodoplatinate White

0.16 0.16 0.17 0.18 0.18 0.18 0.18 0.19 0.19 0.19 0.19 0.19

Codeine or-Isolupanine Brucine Apocodeine Lycorine Neopine Norcodeine Canadine Codeine N-oxide Cotarnine Emetine Lupanine

Absorbs (strongly) Iodoplatinate Iodoplatinate Absorbs (strongly) Iodoplatinate Blue Iodoplatinate Pale green Iodoplatinate Absorbs (strongly) Iodoplatinate Absorbs Iodoplatinate Yellow Marquis Absorbs Iodoplatinate Pale green Iodoplatinate Iodoplatinate Iodoplatinate

Iodoplat inate Iodoplatinate Iodoplatinate Absorbs (strongly) Iodoplatinate Iodoplatinate Absorbs (strongly) Iodoplatinate Iodoplatinate Absorbs (strongly) Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplat,inate Blue Iodoplatinate Iodoplatinate

Orange Dark blue

2 -

Dragendorff Dragendorff Bromcresol green Bromcresol green Bromcresol green Brorncresol green Bromcresol green Dragendorff Bromcresol green Marquis Bromcresol green Dragendorff Potassium permanganate p-Dimethylaminobenzaldehyde Visible orange spot Dragendorff Bromcresol green Marquis Potassium permanganate Bromcresol green Bromcresol green Potassium permanganate Marquis Marauis Dragendorff Marquis p-Dimethylaminobenzaldehyde Marquis Bromcresol green Bromcresol green Marquis Bromcresol green Marquis Marquis -

Marquis Marquis Bromoresol green Bromcresol green

7.

563

THE FORENSIC CHEMISTRY O F ALKALOIDS

TABLE 11-continued Location reagents

Rf

Compound

UV light

1

Absorbs (strongly) Iodoplatinate Absorbs Iodoplatinate

0.20 0.20

Dihydrocodeine Homatropine, ,\T-methyl Hyoxcine, N-methyl Perloline

Bright yellow

Iodoplatinate Iodoplatinate

0.21 0.22

Bulbocapnine Anileridine

Blue Pale green

Iodoplatinate Iodoplatinate

0.22 0.23 0.23 0.23 0.25 0.25 0.25

Isolupinine /3-Colubrine Hordenine Hyoscine Atropine, N-methyl Berberine Ergometrine

Absorbs Absorbs Absorbs Absorbs Faint yellow Bright blue

0.25 0.26

Iodoplatinate Iodoplatinate

0.26 0.27 0.27 0.27 0.27 0.28

Absorbs (strongly) Mescaline 5-Methoxydimethyl. White tryptamine Absorbs (strongly) Sinomenine Blue Boldine Pale green Harmalol a-Isosparteine Blue Scoulerine 5-Methoxytryptamine White

0.29 0.30 0.30 0.30 0.30 0.30 0.30 0.31 0.31

Demecolcine Harmol Homatropine Nalorphine Staphysine Strychnine Strychnine N-oxide Procaine Psilocin

Pale yellow Bright blue Absorbs Absorbs Dark blue Absorbs (strongly) Absorbs (strongly) Blue Absorbs (strongly)

Marquis Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

0.32 0.33 0.33 0.33 0.33 0.34 0.35 0.35

Thebaine Apomorphine Bicuculline Heroin Norharman j-Phenethylamine Ethylmorphine Theobromine

0.20 0.20

-

-

-

Iodoplatinate Iodoplatinate Iodoplatinrtte Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

2

Marquis

Dragendorff Potassium permanganate Marquis p-Dimethylaminobenzaldehyde Bromcresol green Dragendorff Marquis Dragendorff Dragendorff Marquis p-Dimethylaminobenzaldehyde Nitrogen dioxide p-Dimethylaminobenzaldeh yde Marquis Marquis Bromcresol green Bromcresol green Dragendorff p-Dimethylaminobenzaldehyde -

Marquis

-

Marquis Marquis Dragendorff Dragendorff Bromcresol green p-Dimethylaminobenzaldehyde Absorbs (strongly) Iodoplatinate Marquis Blue Iodoplatinate Marquis Blue Iodoplatinate Marquis Absorbs Iodoplatinate Marquis Iodoplatinate Bromcresol green Bright blue Absorbs Iodoplatinate Bromcresol green Dark blue Iodoplatinate Marquis Brominelammonia Blue

E. G . C. CLARKE

564

TABLE 11-continued Location reagents Compound

UV light

2

1

-~

0.36 0.36 0.37 0.37 0.37 0.37 0.37 0.38 0.38 0.38

Tryptamine

Blue

Iodoplatinate (white?) Aquaticine Iodoplatinate Absorbs Atropine Iodoplatinate Dark blue Gelseminine Iodoplatinate Norpseudoephedrine Absorbs Bromcresol green Harmine Bright blue Iodoplatinate Hyoscyamine Absorbs Iodoplatinate Cocaine Absorbs (strongly) Iodoplatinate Dark blue Iodoplatinate Cryptopjne Dimethyltryptamine Blue Iodoplatinate

Marquis

Dragendorff Dragendorff Bromcresol green Ninhydrin Marquis Dragendorff Dragendorff Marquis p-Dimethylaminobenzaldehyde Iodoplatinate Dragendorff Iodoplatinate p-Dimethylaminobenzaldeh yde Bromcresol green Ninhydrin Iodoplatinate Bromcresol green Iodoplatinate Marquis Iodoplatinate Marquis Iodoplatinate Iodoplatinate Bromcresol green Iodoplatinate Marquis Iodoplatinate Marquis Iodoplatinate Bromcresol green p -Dimethylamino benzaldehyde

0.39 0.39 0.39 0.40 0.40 0.40 0.40 0.40 0.40 0.40

Hydrastine Tryptamine, N-methyl Norephedrine Pseudoephedrine Harmaline a-Allocryptopine Atropine N-oxide Benzoylecgonine Benzylmorphine Chelidonine Gelsemine Methylergometrine

0.40 0.41 0.42 0.43 0.45 0.45

Narcotine Cinchonidine Harman Physostigmine Ephedrine Methysergide

Bright blue Blue Bright blue Dark blue Absorbs Blue

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

0.46 0.46 0.46 0.47 0.47 0.47

Ephedrine, N-methyl Narceine Quinine Cinchonine Delphinine Lysergide

Absorbs Absorbs (strongly) Bright blue Bright blue Pale yellow Blue

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Marquis Iodoplatinate

0.48 0.48 0.49 0.51

Hydroquinine Quinidine Papaverine Amphetamine

Bright blue Bright blue Pale green

Iodoplatinate Iodoplatinate Iodoplatinate Bromcresol green Marquis

0.38 0.38

Blue Blue Absorbs Absorbs Green Dark blue Absorbs Absorbs Dark blue Absorbs Bright blue

-

~

Marquis Bromcresol green Marquis Dragendorff Bromcresol green p-Dimethylaminobenzaldehyde Bromcresol green Marquis Bromcresol green Bromcresol green

-

p-Dimethylaminobenzaldehyde Bromcresol green Bromcresol green

565

7. THE FORENSIC CHEMISTRY O F ALKALOIDS TABLE 11-coritinued Location reagents Compound

UV lighb Absorbs Absorbs (strongly) Absorbs (strongly) Bright blue Pale blue Pale blue Bright blue Absorbs

0.56 0.56 0.58 0.58 0.58 0.60 0.60 0.60 0.61 0.62 0.63 0.63 0.63

Apoatropine Cassaine Tropacocaine Hydroquinidine Pseudoyohimbine Yohimbine E thylhydrocupreine Physostigmine N-oxide Coniine Methylamphetamine Corynanthine Theophylline Thiocolchicoside Ethylnarceine Levomethorphan Levorphanol Taxine Aconitine Cyprenorphine Etorphine Dihydroergotamine

Absorbs Absorbs Absorbs Absorbs Pale blue

0.64 0.64 0.65 0.65

Ibogaine Sempervirine Caffeine Ergosine

Blue Dark blue Blue

0.65

Ergotamine

Blue

0.68 0.68 0.68 0.72 0.72 0.73 0.74 0.78 0.78 0.79 0.80

Acetorphine Veratridine Veratrine Deserpidine Rescinnamine Methoserpidine Methadone Ethylpapaverine Reserpine Aimaline " Dihydroergotoxin "

Absorbs Pale green Blue Pale Blue Blue White Absorbs Yellow Pale green Blde Green

0.82 0.83

Lobeline Colchicine

Absorbs Yellow

Rf 0.51 0.51 0.51 0.53 0.53 0.54 0.55 0.55

-

Absorbs Blue Blue Brown Absorbs (strongly)

-

-

1 Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Dragendorff

2

Bromcresol green Bromcresol green Marquis

-

Bromcresol green Iodoplatinate

Iodoplatinate Bromcresol green Iodoplatinate Bromcresol green Iodoplatinate Marquis Bromine/ammonia Marquis Iodoplatinate Marquis Iodoplatinate Bromcresol green Iodoplatinate Bromcresol green Iodoplatinate Marquis Iodoplatinate Iodoplatinate Marquis Iodoplatinate Marquis p-Dimethylaminobenzaldehyde Iodoplatinate Marquis Marquis Bromine/ammonia p-Dimethylaminobenzaldehyde p-Dimethylaminobenzaldehyde Iodoplatinate Dragendorff Marquis Marquis Iodoplatinate Marquis Iodoplatinate Marquis Iodoplatinate Marquis Iodoplatinate Iodoplatinate Marquis Iodoplatinate Marquis Iodoplatinate Dragendorff p-Dimethylaminobenzaldehyde Iodoplatinate Marquis Marquis -

566

E. G . C . CLARKE TABLE 11-continued Location reagents

Rf

UV light

Compound

0.83 0.84 0.84

Ketoyobyrine Cinnamylephedrine “Ergotoxin”

0.87 0.90 0.92 0.95

Jervine Solanidine Colchiceine Piperine

2

1

Marquis Marquis p-Dimethylaminobenzaldehyde Marquis Marquis

Bright blue Iodoplatinate Absorbs (strongly) Iodoplatinate Blue Iodoplatinate -

Brown Absorbs

Iodoplatinate Iodoplatinate Marquis Marquis

-

-

TABLE I11 THIN-LAYER CHROMATOGRAPHY DATA

Rf

Location reagent

Compound

References for additional information

~~

339,366 367 368

Berberine SparZeine Ergothionine Brucine Histamine Pseudomorphine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate Iodoplatinate Iodoplatinate p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate Iodoplatinate, very acid Iodoplatinate Iodoplatinate Iodoplatinate

Connessine Homatropine

Iodoplatinate Iodoplatinate

339 366, 370, 371

N-Methyltryptamine Ecgonine Normorphine

Iodoplatinate Iodoplatinate Potassium permanganate

296 373

0.00 0.00 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.04 0.05

Hydrastinine Tubocurarine Agmatine Atropine, N-methyl Choline Cotarnine Homatropine, N-methyl Hyoscine, N-methyl Sempervirine Trigonelline Psilocybin Isosparteine Colchiceine

0.07 0.08 0.1 1 0.12 0.13 0.13 streak 0.14 0.15 streak 0.16 0.17 0.17

-

339 339,366,369

-

339

-

339,366 296, 339,369, 370

-

339,366, 367,371, 372

-

366

7.

567

THE FORENSIC CHEMISTRY O F ALKALOIDS

TABLE III-continued

Rf

Compound

Location reagent

0.18 0.18 0.18 0.20 0.21 0.21 0.21 0.22 0.23 0.23 0.23 0.23 0.24 0.24 0.24 0.25 0.25 0.25 0.26 0.27 0.27 0.28

Apoatropine Atropine Hyosc yamine fi-colubrine Atropine N-oxide Benzoylecgonine Norcodeine Strychnine Codeine N-oxide Mescaline Morphine N-oxide Strychnine N-oxide Levomethorphan Levorphanol Sinomenine Dihydrocodeine 5-Methoxytryptamine Serotonin Coniine Staphysine Tryptamine Ephedrine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate lodoplatinate lodoplatinate lodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate

0.28 0.30 0.31 0.32 0.32 0.32 0.32 0.32

Methylamphetamine Pseudoephedrine Physostigmine N-oxide Apocodeine Bufotenine Cytisine Hordenine 5-Methoxydimethyltryptamine N-Methylephedrine Neopine Narceine /3-Phenethylamine

Iodoplatinate Potassium permanganate Potassium permanganate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

0.32 0.32 0.33 0.33 streak 0.34 Dimethyltryptamine 0.34 Hydroxylupanine 0.34 Morphine 0.34 0.35 0.35

Psilocin Berbamine Codeine

lodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

References for additional information

371 339,346,366, 370 366,370,371 367 373 339, 346, 366, 367,370 -

296 296

-

296 339, 346, 366, 368, 371, 3 74 284,295,374 -

339 296,375

366,376 368 296 -

295, 339, 346, 369, 370-372, 376,377 296 295, 339, 346, 366, 369, 370.376. 377

568

E. G . C. CLARKE

TABLE 111-continued

Rf

Compound

0.35 0.35 0.36 0.36 0.36 0.37 0.37 0.38 0.38 0.39 0.39 0.40 0.40 0.40 0.40 0.41

Harmalol Tropacocaine Dehydroemetine Ethylmorphine Monocrotaline Benzylmorphine Methadone a-Allocryptopine Harmaline Isolupinine Oxycanthine Anabasine Bulbocapnine Cycleanine Tropine Thebaine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

0.42 0.44 0.44 0.44 0.45 0.45 0.45 0.46 0.48 0.48 0.48 0.48 0.48 0.49 0.50 0.50 streak 0.50 0.51 0.51 0.52 0.52 0.52 0.52 0.53 0.53 0.54

Hydroquinine Aquaticine Hydroquinidine Pseudoyohimbine E thylnarceine Heroin Lupanine Cryptopine Amphetamine Cinchonine Norpseudoephedrine Perloline Pethidine Gelsemine Norephedrine Ethylhydrocupreine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Potassium permanganate Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate

Gelseminine Cephaeline Cinchonidine Emetine Harmol Quinine Solanine Arecoline Cassaine Demissine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate Iodine Potassium permanganate Iodoplatinate Iodoplatinate

Location reagent

References for additional information

366,372,373 366 346,370

-

371 295, 339, 366, 369, 376,377

295, 346,366,370 339,366,376 284,374 339,346,366 370 339 -

339,366 366 339,366

-

569

7. THE FORENSIC CHEMISTRY OF ALKALOIDS TABLE 111-continued

Rf

Compound

Location reagent

0.54 0.55 0.55 0.55 0.55 0.55 0.56

Hyoscine a-Isolupanine Lobeline Physostigmine Quinidine Yohimbine Demecolcine

0.57 0.57 0.58 0.59 0.59 0.60

Apomorphine Nicotine Lycorine Bicuculline Veratridine Cocaine

0.60

Lysergamide

0.60 0.60 0.61 0.61 0.61 0.62 0.62

Pilocarpine Scoulerine Hydrastine Procaine Veratrine Ajmaline Colchicine

0.62 0.62 0.62

Nalorphine Narcotine Thiocolchicoside

0.62 0.63 0.63 0.64 0.65 0.66

Tomatine Caffeine Dihydroergotamine Condelphine Ibogaine Lysergide

0.66 0.66

Methysergide Papaverine

p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodine Potassium permanganate p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate Iodoplatinat,e Iodoplatinate Iodoplatinate p-Dimethylaminobenzaldehyde Iodoplatinate Iodoplatinate

0.67 0.67 0.67 0.68 0.68 0.68

Chelerythrine Cinnamylephedrine Ergometrine Canadine Ergotamine Harmine

Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate

Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate Iodoplatinate p-Dimethylaminobenz aldehyde Iodoplatinate Iodoplatinate Potassium permanganate Iodoplatinate Iodine Iodoplatinate

References for additional information

346,366,370, 371 366 366,370 339,370 339, 346,366,370

-

~

366, 370 339, 346,370 -

346, 366, 370, 372, 373, 378 339, 366, 370 -

346,366 339

-

366

295,339,366,369, 376 -

339,346,374 295,296 295,296,297 296 295, 339, 346, 366, 369, 370,376,377 295,296 295, 296 -

570

E.

a. C.

CLARKE

TABLE 111-continued

Rf

References for additional information

Location reagent

Compound

0.68 0.68 0.68 0.70 0.70 0.70 0.70 0.71 0.71

Jervine Norharman Solanidine Corynanthine Harman Methylergometrine Taxine Aconitine “Dihydroergotoxin ”

0.72 0.72 0.72 0.72 0.72 0.73 0.73 0.75 0.75 0.77 0.77 0.77 0.78

Acetorphine Chelidonine Ergosine E thylpapaverine Etorphine Anileridine “Ergotoxin ” Cyprenorphine Deserpidine Methoserpidine Rescinnamine Reserpine Piperine

Iodoplatinate Iodoplatinate Iodine Iodoplatinate Iodoplatinate Iodoplatinate Iodine Iodoplatinate p-Dimethylaminobenzaldehyde Gdoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Iodoplatinate Potassium permanganate Potassium permanganate Potassium permanganate

339

-

296

-

366, 370 295, 296

339 296

-

339,366

TABLE IV

UV SPECTROPHOTOMETRYDATA Maximum peak 284 234 245 237 258 258 272 214 258 255 258 234

Compound Acetorphine Aconitine Ajmaline or-Allocryp topine Amphetamine Anilderidine Apomorphine Arecoline Atropine Atropine, N-methyl Atropine N-oxide Benzoylecgonine

Solvent 0.1N Hydrochloric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid

Secondary peaks

275 290 288 252, 264 252,264 252, 264 251,263 252,264 275,281

7.

571

THE FORENSIC CHEMISTRY OF ALKALOIDS

TABLE IV-continued Maximum peak

284 228 292 282 265 277 268 272 270 235 236 234 284 280 284 268 332 236 288 303 230 220 283 281 257 256 257 312 315 251 285 277 252 210 253 246 247 247 279 269 258 274 295 249 250

Compound Benzylmorphine Berberine Bicuculline Boldine Brucine Bufotenine Bulbocapnine Caffeine Chelerythrine Cinchonidme Cinchonine Cocaine Codeine Apocodeine Codeine N-oxide Coniine Cotarnine Cryptopine Cyprenorphine Cytisine Dehydroemetine Deserpidine Dihydrocodeine Emetine Ephedrine Norpseudoephedrine Pseudoephedrine Ergometrine Ergotamine Ethylhydrocupreine Ethyl morphine Ethyl narceine Ethyl papaverine Gelsemine Gelseminine Harman Norharman Harmine Heroin Histamine Homatropine Hordenine Hydrastine Hydrastinine Hy droquinidine

Solvent

0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Hydrochloric acid 0.1N Sulfuric acid 0.1N Hydrochloric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Hydrochloric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid

Secondary peaks

264, 343 327 302 300 296 306 322 316 316 275 -

253 283 232 282 272

-

251,263 250,262 251,263 317,347 31 0 252,280 299,366 300 320 252, 264 306, 363 317,345

572

E. G . C. CLARKE TABLE IV-continued

Maximum peak 250 257 257 258 277 278 279 249 288 269 259 269 257 257 226 284 285 23 1 285 276 312 284 260 257 247 241 215 343 227 222 267 251 250 268 264 214 254 280 284 271 260 279 280 262 220

Compound Hydroquinine Hyoscine Hyoscine, N-methyl Hyoscyamine Ibogaine Levomethorphan Levorphanol Lobeline Lycorine Mescaline Methadone Methoserpidine Methylamphetamine N-Methylephedrine Methylergometrine Morphine Normorphine Pseudomorphine Nalorphine Narceine Narcotine Neopine Nicotine Pethidine Physostigmine Physostigmine N-oxide Pilocarpine Piperine Procaine Psilocin Psilocybin Quinidine Quinine Reserpine Sinomenine Sparteine Strychnine Taxine Thebaine Theophylline Thiocolchicoside Tryptamine Tubocurarine Veratrine Yohimbine

Secondary peaks

Solvent 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.01N Hydrochloric acid Methanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid Ethanol 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid 0.1N Sulfuric acid

316, 345 251, 263 251, 263 252, 264 -

-

238 -

253, 265, 292 252, 263 251, 263 313 -

260 -

291 -

251,263 303 301 -

312 274, 279 -

317,346 317, 346, -

370 273,287 293 272,277, 288

7.

573

THE FORENSIC CHEMISTRY OF ALKALOIDS

TABLE V COLOR TESTS~ Vitali 2

1

Marquis

Compound ~

Acetorphine Ajmaline a-Allocryptopine Amphetamine Anabasine Anileridine Apoatropine Apocodeine Apomorphine Atropine Atropine, N-methyl Atropine N-oxide Benzylmorphine Berbamine Berberine Bicuculline Boldine Brucine Bufotenine Bulbocapnine Canadine Cephaijline Chelerythriie Chelidonine Cinnamylephedrine Codeine Codeine, N-oxide

Bluish gray + yellowbrown Purple Purple Orange + brown

Red Yellow

-

Slowly orange

-

-

Purple Purple

4

black

-

Yellow-brown Red-brown

-

Red-purple

-

(Yellow) yellow-green Orange (Green) purple 3 green

-

Greenish brown Brown Faint orange

-

(Orange) orange Green Red-brown Purple Purple

Norcodeine Colchiceine Colchicine B-Colubrine

Purple Pale yellow Yellow

Conessine Corynanthine Cotarnine Cryptopine Cycleanine Cyprenorphine

Yellow + orange Slowly brown, gray rim

Dehydroemetine Delphinine Demecolcine

Faint orange

-

-

Blue

4

green -

Bluish gray + yellowbrown Faint orange Faint yellow Yellow

Faint yellow Faint yellow Brown Yellow Brown Orange Greenish brown Deep brown Pale yellow Yellow (Orange) orange Yellow

-

Yellow Yellow Pale yellow Dull purple Deep purple Dull purple 4 yellow

Yellow Brown Faint yellow Faint orange Faint brown Purple Brown Gray-brown Purple Purple Purple Orange Orange Dark brown Brown Dark brown Red Brown Dark brown Brown Brown Brown Brown

-

Orange Purple + dark brown Pale orange Brown Red Orange

-

Yellow Yellow Yellow Brown

-

Faint yellow

-

Yellow

4

purple

-

Red-purple Brown Yellow Brown Faint orange Brown

-

Red-purple

574

E. G . C. CLARKE TABLE V-continued Vitali

Demissine Deserpidine Dihydrocodeine Dihydroergotamine Dimethyltryptamine Emetine “Ergotoxin” Ergometrine Ergosine Ergotamine Ethylmorphine Ethylnarceine Ethylpapaverine Gelsemine Gelseminine Harmaline Harmalol Harman Norharman Harmine Harmol Heroin Hordenine Hydrastine Hyoscine Hyoscine, N-methyl Hyoscyamine Ibogaine Jervine Ketoyobyrine Levorphanol Lobeline Lycorine Lysergamide Lysergide Mescaline Methoserpidine 5-Methoxydimethyltryptamine .5-Methoxytryptamine

I

Marquis

Compound

Faint yellow Gray-green Purple Gray-brown Dull orange Pale yellow Gray-brown Gray-brown Gray-brown Gray-brown Yellow + purple + black Brown + green + blue Blue + brown

-

Brown Faint yellow Dull orange Yellow Yellow Dull orange Dull orange Dull orange Dull orange Yellow Yellow -

-

(Yellow) brown-green (Yellow) Green Green Orange Orange Violet Brown(+ green

-

Purple + yellow

-

Yellow Green Yellow Faint yellow Yellow Yellow

-

-

-

-

-

Gray + pale orange Red-brown Faint gray

-

Red-purple

-

Yellow Yellow

Greenish brown brown

--f

deep

Red-purple Orange Brown Red-brown Yellow Dull purple Brown-purple Dull purple Brown-purple Brown Orange Brown Bright yellow Bright yellow Brown Purple-brown Red Red Red Orange Orange Bright orange Brown Purple Purple Purple Red-brown -

Red-orange Orange -

-

Brown Gray Orange Gray Brown

Methylamphetamine Orange Methylergometrine Gray-brown N-Methyltryptamine Dull orange

Yellow

2

-

Yellow Brown Orange-brown Dull purple Brown Yellow

Brown Purple-brown Purple-brown Brown Purple-brown Brown

Yellow

Red-brown

Yellow-brown Yellow

Brown-purple Red-brown

-

7.

575

THE FORENSIC CHEMISTRY O F ALKALOIDS

TABLE V-continued Vitali Compound Met hysergide BIorphine Morphine A-oxide Normorphine Pseudoinorphinc Nalorphine Narceine Nnrcot ine Neopine Oxycanthine Papaverine Perloline Pethidine P-Phenet hylainine Physostigmine Physostiginine ,V-oxide Piperine I’roraine Psilocin Psilocybin Rescinnarnine Reserpine Scoulerine Seinpervirine Serotonin Sinomenine Solanidine Solanine Staphysine Strychnine Strychnine N-oxide Taxine Thebaine Thiocolchicoside Toinatine Tryptainine Tubocurarine Veratridine Veratrine Yohimbine Pseudoyohimbine

Marquis Faint gra,v Violet Purple Purple Green Purple BIWWII+ deep brown + green Bluish violet, fading Blue - v iolet

(Yellow) pale yellow Dull orange Orange

Yellow Yellow Yellow Yellow Orange Yellow Yellow

Dull purple Orange Orange Orange Brown Orange Orange

Red + yellow Pale yellow Brown Faint yellow (Yellow) pa.le yellow

Yellow Orange Orange Brown Colorless

-

Yellow Yellow Red-brown -+ green-brown Yellow -

Greenish brown Dull orange Gray-green Gray-green + brown

Gray-green Brown, slowly Orange + green + blue (Yellow) purple Yellow + purple Orange-brown

-

2

1

-

-

Purple Yellow

-

Green-brown Yellow Red-brown Purple flash + orange Yellow Pale orange Gray-green Yellow

-

Faint yellow

-

-

Dull purple Orange Red-brown Red-brown Brown Brown Brown Dull orange Brown Red-purple

-

Faint yellow Bright orange Bright orange Faint brown Orange Purple brown

Yellow-brown Red + orange Yellow (Pale yellow) bright yellow Brown Faint orange Dull orange Yellow Red-brown Pale purple-brown Orange - brown Red-orange + brown Faint orange Bright purple Greenish gray Yellow Red-purple Greenish gray Yellow Red-purple

A color shown in parentheses indicates the color of the residue before the addition of the reagent. Q

576

E. G. C. CLARKE

TABLE VI REAGENTS Bromcresol green spray. 0.5% in ethanol. Bromine/ummonin. Expose the chromatogram to bromine vapor for 2 minutes, hold it in the steam from a boiling water bath for 1 minute, then heat in the oven a t 110°-120" for 5 minut,es. With caffeine, theobromine, theophylline and other xanthine derivatives a rose pink spot develops which becomes reddish purple when exposed to ammonia. p-Dii?~ethylomii~obentrrldehyde spray. 1 gm of p-dimethylaminobenzaldehydeis dissolved in 100 ml of ethanol and 10 ml of concentrated hydrochloric acid added. Drrrgendorff rengent. (a)Dissolve 0.86 gin of bismuth subnitrate in 40 ml of water and add 10 ml of glacial acetic acid. (b) Dissolve 8 gm potassium iodide in 20 ml of water. Mix 1 volume of (a),1 volume of (b), 4 volumes of glacial acetic acid, and 20 volumes of water. Zodine. 1yo in carbon tetrachloride. (Alternatively, chromatograms may be exposed to iodine vapor.) Zodoplatinnte sprny (for paper chromatograms). Add 10 ml of 5 q ! platinum chloride solution to 240 ml of 2y0 potassium iodide solution, and dilute with an equal volume of water. Iodoplatinrrte sprtry (acid; for thin-layer chromatograms). Add 10 ml of 50,: platinum chloride solution and 5 ml of concentrated hydrochloric acid to 240 ml of 2:4 potassium iodide solution. lodoplntinnte qmtcy (strongly acid, for weak bases). Mix 1 ml of 5Yb platinum chloride solution, 9 ml of 10% sodium iodide solution, 2 ml of water, and 3 ml of concentrated hydrochloric acid. Mtrrquis recigent. 1 ml of formalin solution in 10 ml of concentrated sulfuric acid. The reagent is poured over the chromatogram (which must be thoroughly dry) supported on a sheet of white opal glass. Ninhydrin spruy. 0.5% in acetone. Heat papers a t 100" for 5 minutes after spraying. Pota,ssiumpermnnganute. 1 in water.

STRUCTURES OF SYNTHETICS Acetorphine

HO-C-CH~

I

CH2CHzCH3

7.

THE FORENSIC CHEMISTRY OF ALKALOIDS Amphetamine

6

CHz-CH-NH2

AH3

Cyprenorphine

HO-C-CH3

I

CHI

Diphenoxylate

Etorphine HO

0@ b C H 3

CH30

HO-C-CH~

I

C3H7

577

578

E. G . C. CLARKE

Methadone

p-Methoxyaniphetamine

0

CHz-CH-NHz

OCH3

AH3

Nalorphitie

Pentazocine

~

N

~

C

H

~

-

C

H/CH3= ‘CH3

OH

Pethidine OCzH6

I

C

7.

THE FORENSIC CHEMISTRY O F ALKALOIDS

579

STP CHZ-CH-NHZ

1

i

CH3

CH3

TMA

OCH3

REFERENCES 1. R. G. Todd, ed., “Extra Pharmacopoeia (Martindale).” Pharm. Press, London, 1967. 2. “Registrar General’s Statistical Review of England and Wales.” H.M. Stationery Office, London, 1948-1961. 3. R. Christison, “A Treatise on Poisons.” A. & C. Black, Edinburgh, 1845. 4. R. A. Witthaus, “Manual of Toxicology.” Wm. Wood & Co., New York, 1911. 5. J. D. Mann and W. B. Brend, “Forensic Medicine and Toxicology.” Griffin, London, 1914. 6. S. N. Tewari, “Annual Reports of the Chemical Examiner to the Government of Uttar Pradesh.” Superintendent, Printing and Stationery, Allahabad, 1963, 1964, 1965. 7. A. B. Orr, Vet. Record 64, 339 (1952). 8. P. J. Barden and H. Paver, Vet. Record 73, 992 (1961). 9. E. L. J. Staples,J. Sci. Technol. 10, 129 (1964). 10. L. F. Addis-Smith, N e w Zealand Vet. J . 9, 121 (1961). 11. E. G. C. Clarke, Medico-LegaZJ. 30, 180 (1962). 12. A. Mackay, N e w Zealand Vet. J . 9, 129 (1961). 13. €3. Schubert, Acta Vet. Scand. Suppl. 21 (1967). 14. N. J. Leonard, in “The Alkaloids” (R. H. F. Manske and H. L. Holmes, eds.), Vol. 1, p. 107. Academic Press, New York, 1950. 15. N. J. Leonard, in “The Alkaloids’’ (R. H. F. Manske, ed.), Vol. 6, p. 35. Academic Press, New York, 1960. 16. K. R. Hill, Proc. Roy. Soc. M e d . 53,281 (1960). 17. D. G. Steyn, OnderstepoortJ. Vet.Sci. A n i m a l I n d . 1,219 (1933). 18. D. G. Steyn, “The Toxicology of the Plants of South Africa.” South African Central News Agency, Johannesburg, 1934. 19. K. L. Stuart and G. Bras, West I n d i a n Med. J. 5, 33 (1956).

580 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

E.

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