U.S. Army Medical Department, Office of Medical History
Skip Navigation, go to content

HISTORY OF THE OFFICE OF MEDICAL HISTORY

AMEDD BIOGRAPHIES

AMEDD CORPS HISTORY

BOOKS AND DOCUMENTS

HISTORICAL ART WORK & IMAGES

MEDICAL MEMOIRS

AMEDD MEDAL OF HONOR RECIPIENTS External Link, Opens in New Window

ORGANIZATIONAL HISTORIES

THE SURGEONS GENERAL

ANNUAL REPORTS OF THE SURGEON GENERAL

AMEDD UNIT PATCHES AND LINEAGE

THE AMEDD HISTORIAN NEWSLETTER

Chapter 4, Part 4

Medical Science Publication No. 4, Volume II

PHARMACOLOGY AND TOXICOLOGY OF DRUG ADDICTION AND ALCOHOLISM*

MAJOR EDWARD C. KNOBLOCK, MSC

The problems of drug addiction among the troops and populace of a country has in the past led to wars between nations (the First and Second Opium Wars between England and China) and during the recent Korean incident, intelligence reports indicated that the Communists were exploiting the drugs traffic as a means of gaining dollars to finance their operations in Korea and elsewhere.

A full report of these operations by the Communists was prepared by the CIC, OSI, CID and other investigating agencies of the Far East Command for presentation before the United Nations as evidence of Communist activity in drug traffic. Part of this evidence included the data obtained by checking the wholesale shipments of heroin and opium to Chinese ports; it was pointed out in this report that the wholesalers apprehended in Japan were either Koreans or Chinese with Communist affiliations.

One of the problems encountered by the investigating agents involved the apprehension of the narcotic users who often resorted to very clever ruses to conceal the sources of their supply. Even though Special Regulations and Administrative Memoranda amply prescribed the prohibition against the possession of narcotic substances and implements used in the administration of such substances, the user of narcotics quickly became aware of these regulations and made it a point to get his drugs directly from the "pushers" who were usually found in the gay quarters of Japanese and Korean cities. It became increasingly difficult for investigating agents to apprehend the users in possession of drugs or implements for injection. Consequently, the sale of narcotics continued at a rate consistent with the number of susceptible troops within an area and was concentrated within those cities in Japan and Korea which were centers for troop and harbor activities.

The CID laboratory and the 406th Medical General Laboratory in Tokyo were the organizations whose facilities supported the investigating agents; in 1952 the resources of the 1st Medical Field Labora- 


*Presented 27 April 1954, to the Course on Recent Advances in Medicine and Surgery, Army Medical Service Graduate School, Walter Reed Army Medical Center, Washington, D. C.


171

tory in Korea were added to this service. Close liaison was maintained between these laboratories, and this sharing of information was found to be mutually valuable when cases involving the services of both laboratories were being considered. The medical laboratories were charged with the analyses of blood and urine specimens taken from suspected drug users and for the toxicologic examinations of autopsy material; the CID laboratory identified samples of suspected narcotics that were brought in by the field investigators. With such an arrangement, the interlaboratory sharing of information was a necessity.

The drugs most commonly identified in the laboratories were heroin, morphine, opium, various types of barbiturates, amphetamines, marihuana, and a wide variety of alcoholic beverages. The bulk of the toxicologic examinations performed by the medical laboratory involved the search for these substances. All the autopsies in which the examining pathologist could not determine the cause of death were followed by toxicologic analyses. Those specimens collected from the remains of suspected drug addicts were also submitted to the medical laboratory, all of which required considerable work in order to insure that the investigating authorities received the fullest support. These investigations led to an improved system of identifying the types of opiates whose description will be included in this report.

Aside from assuring proper analytical technics the practical considerations in establishing an operational toxicology laboratory included a procedure which assured the adequate sampling of all necessary specimens and tests for carrying out the examinations requested, an adequate procedural protocol and description of the conditions surrounding the circumstances of death, as well as a system providing that the specimens be subjected to a legally responsible chain of custody from the time of collection until the completion of the examination. Without fulfilling this latter requirement the performance of a toxicologic examination is valueless since the report itself cannot be considered as adequate evidence in the courts. Direct transportation of specimens to the testing laboratory by a trustworthy courier without recourse to intermediate channels provides a satisfactory link in the chain of custody from examining physician to the laboratory. Complete examination of the specimen container and a satisfactory seal are also necessary. Should courier service not be available, the use of registered mail addressed to the attention of the toxicologist is also satisfactory. The general requirements for submitting specimens for toxicologic examination as outlined by TB Med 237, Department of the Army Technical Bulletin, satisfactorily cover the general aspects of the sampling procedure and transfer of samples.


172

During the years 1950, 1951, and 1952 the various examinations by the 406th Medical General Laboratory in regard to drugs and alcohol are summarized in the following table:

1950

1951

1952

Total autopsy cases examined

171

201

215

Narcotics findings:

 
Morphine

 

1

 

18

 

13

 
Codeine

 


-----

 


1

 


-----

 
Nicotine

 


-----

 


1

 


-----

 
Barbiturates

 


14

 


36

 


8

 
Ethanol

 


69

 


58

 


63

 
Methanol

 


-----

 


13

 


1

Total clinical cases

-----

404

618

Narcotics findings:

 
Morphine

 

-----

 

102

 

67

Barbiturates

-----

85

55

The toxicology and pharmacology of each of the drugs encountered will be discussed as related groups.

Toxicology of Alcoholism

The detection of alcohol consumption presented the analytical laboratory with its primary forensic problem since automobile accidents, accidental drownings, fights, and miscellaneous disturbances associated with intoxication were not infrequent. Alcohol taken by mouth is partly absorbed by the stomach but primarily by the small intestine. Foods high in the amino-acids, glycine and alanine lower the rate of food absorption (1)-a reaction similar to that which occurs when foods and aqueous beverages are diluted with alcohol. Alcohol distributes itself into tissues and secretions by diffusion. Those tissues with the highest water content are found to contain the highest concentration of alcohol. Approximately 90 percent of the ingested alcohol is metabolized within the body with 2 percent being expired through the lungs and 8 percent eliminated by the kidney
(2). Maximum blood concentrations in man occur within 10 to 20 minutes after an intravenous injection of alcohol, but it takes 25 to 35 minutes for the same dose to reach maximum alcohol concentrations in the cerebrospinal fluid (3). The rate at which the blood-alcohol concentration drops from the maximum level, within certain limits, is linear and appears to be independent of the amount injected (4).

Alcohol is a normal constituent of the body although its life in tissues is short and the concentration is small (5). It is normally oxidized in the body by an enzyme system using alcohol which is primarily found in the liver. Mirsky and Nelson (6) have shown that liver damage retards the oxidation of alcohol and that the liver oxidizes 90 percent of the absorbed alcohol.


173

Ethanol is normally oxidized to form acetaldehyde which upon further oxidation is converted to acetic acid that may then enter the tricarboxcylic acid cycle where the oxidation is completed with the formation of carbon dioxide and water. If some chemical such as antabuse (tetraethyl thiuramdisulfide) or another enzyme inhibiting agent interfered with the cycle involving the oxidation of the alcohol, the intermediate products will accumulate and produce their own effect. Habituation apparently does not accelerate the metabolism of alcohol which continues at a relatively consistent rate of 10 cc. per hour.

Ethanol acts upon the sensory areas of the cortex and extends to the motor areas as the blood concentration increases. The first effect is the so-called "social stage" which is the depression of the inhibitory centers of the cortex. It is this observed effect that prompts some persons to believe alcohol to be a stimulant even though its pharmacologic effect is always that of a depressant. The conditioned reflexes are slowed and coordination impaired under the influence of alcohol. Many attempts have been made to correlate the activity of the individual with the blood alcohol level and the mean of these attempts would approximate the following:

Blood alcohol

Reaction

0-55 mg./100 ml. blood

Normal

55-100 mg./100 ml. blood

Querulous, false euphoria

100-250 mg./100 ml. blood

Irregular gait, lack of coordination, mental confusion

250-500 mg./100 ml. blood

Respiratory depression, stupor, possible death

The effect of alcohol on the cortex reaches its peak while the blood alcohol level is increasing. During this time the cortex cells are adjusting to the effects of the alcohol. Tolerance may be increased by repeated doses as shown by the response of the habitual drinker compared with that of the uninitiated.

Alcohol in large quantities affects not only the cortex but continues to the medulla as well as exerting a strong depressant action on the respiratory centers. Deaths due to acute alcoholic poisoning are the result of respiratory paralysis (7).

The technics employed to detect the presence of alcohol in the blood are reliable; most of these methods depend upon the oxidation of alcohol by a standard potassium dichromate solution. The chromous ion formed may be measured or the residual dichromate may be titrated with a standard thiosulfate solution.

Alcoholic beverages include the wines (12 percent), fortified wines (25 percent), beer (2.0-6 percent), whiskeys (45-55 percent), and various forms of distilled wines (cognac, brandy, etc.). These drinks


174

produce intoxication dependent upon their alcoholic content, but the increasingly unfavorable after-effects that result from mixing different types of liquors may possibly be the result of synergistic action between the various impurities in the liquors (8). Little is known about this particular phenomenon, however.

Methyl alcohol (methanol) oxidizes much more slowly in the body than does ethanol and its oxidation produces formaldehyde and formic acid which are extremely toxic. Methanol is also a depressant of the central nervous system; it evokes, besides, cerebral edema, neuritis and a specific toxicity for the ganglionic cells of the retina with the resultant blindness observed in methanol poisoning. Methanol also is hepatotoxic and may produce fatty degeneration of the liver (9).

The beers and liquors examined in Japan during the 3-year period 1950-1952 (inclusive) showed no sample to be outside the limits allowed for medicinal beverages, as specified by the United States Pharmacopoeia, even though Japanese law allows 0.2 percent methanol to be present in distilled beverages. The same cannot be said for those brandies and other beverages that were obtained in Korea, the composition of which ranged from mixtures of chloroform and water, ethylene glycol, pure methanol, to methanol-containing brandies. These liquors were all attractively packaged but constituted a real threat to the life of the consumer-if he had the fortitude to swallow them. Deaths recorded herein as due to methanol poisoning occurred exclusively in Korea although the consumption of methanol in one drinking party aboard a ship accounted for six of the deaths due to methanol poisoning in 1951.

Toxic Effects Produced by Use of Barbiturates

Drugs used for their sedative and hypnotic effect are often brought to the attention of the laboratory as a result of accidental overdosage or as a result of suicides. Usually these drugs are classed under four headings (long, intermediate, short, and ultrashort) depending on their duration of action upon the cerebral cortex and thalamus. The duration of depression and the intensity of action depends upon the structure of the drug, the amount used and the mode of administration. The various classes of the barbiturates are derived from barbituric acid (malonylurea) and derive their individual properties from the various attached prosthetic groups all of which cover a wide range of organic structures; the substitutions usually being made in the 5, 51, and 1 positions.

The barbiturates affect the sensory and motor areas of the brain, which is the basis for their physiologic action in producing sleep and alleviating convulsions. Therapeutic doses of barbiturates produce only slight respiratory changes but large doses markedly depress respi-


175

ration. When death is caused by these drugs, it is the result of respiratory failure or of the complications associated with a decreased rate of respiration. Therapeutic doses do not affect the circulation, but large doses produce peripheral vasodilation and a fall in blood pressure. With removal of the drug, the blood pressure returns to normal.

Barbiturate poisoning is characterized by shallow and slow breathing. Mental confusion, ataxia, clammy skin, reduced reflexes and constricted pupils are noted in early stages of this type of poisoning. As the dosage is increased, a fall in body temperature, dilation of the pupils and coma are noted. Severe poisoning usually results from an intake that ranges between 5 and 10 times the therapeutic dose. Accidental poisonings have occurred when persons who were accustomed

to the drug took repeated doses during a period of blurred consciousness in order to induce sleep. Various psychogenic factors, usually associated with insomnia, are largely responsible for habituation to the barbiturates since sleep is the most common effect these drugs produce. Although there may be a certain degree of transient tolerance in man, possibly motivated by a strong psychogenic factor (10) which may result in a form of habituation, withdrawal symptoms such as occur in the alcoholic and opiate addict are not generally shown with barbiturate abstinence.

Toxicologic search for the presence of barbiturates is most successful when the urine is analyzed since the slow- and intermediate-acting barbiturates are largely eliminated by this route. The short- and ultrashort-acting drugs are largely detoxified by the liver (11) and are


176

not eliminated in the urine. For this reason it is very important that the toxicologist be apprised of the type of drug the patient or deceased is or was suspected of using. At autopsy the brain is the organ of choice for examination. Chemical methods of examination are very reliable and the method of Goldbaum (12) using the ultraviolet spectrophotometer was found most adaptable to the work done in the Far East Command.

Deaths due to barbiturates were associated with suicides, accidental drowning, concurrent administration with heroin, carbon monoxide asphyxiation from space heaters in hotels, and other conditions where sedation prevented the response of the individual to a danger stimulus.

Marihuana-Its Source, Physiological Effect and Detection

The drug prepared from the flowering tops of Cannabis satira was brought to the attention of the 406th Medical General Laboratory shortly after United Nations troops entered Korea. The Koreans cultivate this plant from the hemp fiber which forms a large part of domestic fabrics. The ready access of these plants allowed free traffic for the marihuana smoker. Several samples of this drug were submitted for laboratory identification but the laboratory was never apprised of any widespread use of this narcotic.

Cannabis (known popularly as marihuana, hashish, and bhang) is produced as a narcotic by removing the flowering tops of this hemp plant before the fruits have developed. At this time the highest concentration of the natural resin is to be found in the plant. The active agents which have been isolated from cannabis are an alcohol, cannabinol, and a volatile oil. The alkaloids of cannabis are apparently not pharmacologically active.

Cannabinol is highly toxic and has a strong narcotic effect manifested by a perversion of time, distance and sound perceptions. It produces different effects in different personality types. Some are offered a complete escape from reality, sensuous dreams are experienced by others, and in others there is a complete release of inhibitions. Habitual doses are likely to result in facial bloating, ataxia, moral degeneration and mental stupor. For the occasional smoker the mental effects are the most pronounced and peripheral effects are negligible.

Smoking of the dried plant is the most common route of administration and the habitué usually desires company in his venture. Apparently no added physiologic tolerance to the narcotic develops with continued use and only the psychic factors appear important upon withdrawal of the narcotic from the user (13).

The most satisfactory method used to identify marihuana is the microscopic examination of suspected cigarettes. The characteristic


177

spiculated stem of cannabis is easily identified by the botanist. Chemical tests have been described but were not used for laboratory examinations since a qualified botanist was available.

Amphetamines-Their Possible Toxicologic Effects

Various types of stimulants were often brought into the laboratory. In Japan these are generally produced in 1 ml. or 2 ml. sterile ampules containing 10 mg. amphetamine for injection which are marked under a variety of names such as "Methylpropamin," "Agotin," "Neoagotin," "Zedrin," "Hospitan," "Metabolin," "Koripron," and a variety of other names. These preparations were readily available from the Japanese pharmacies and some instances of habitual use were reported among the United Nations personnel.

Amphetamine, 1-phenyl-2-aminopropane, is most commonly called by its trade name Benzedrine. Its high volatility makes it most useful as a vasoconstrictor when inhaled through special vaporizers (inhalers). Taken parenterally or orally, the amphetamine derivatives stimulate the central nervous system and are useful in treating acute effects of barbiturates or opiates by stimulating brain circulation which assists in removal of the depressing drug. This stimulant action produces in many persons a mild euphoria, brighter spirits and loss of fatigue; it was sought in Japan by the students and workers who spent many tiresome hours daily at a job. However, the basic need of the body for rest is in no way affected by the use of this stimulant and its prolonged use can lead to a general weakening of the body.

As evidenced by the manner in which amphetamine is often packaged, the favorite route-also the most effective (14)-is the subcutaneous injection of the drug. Danger from continued dosage of amphetamine lies in the development of hypertension, gastrointestinal disturbances, vexation and restiveness. Toxic doses may show dilation of the pupils, palpitation, chills and collapse which respond to treatment by the short-acting barbiturates.

The drug may become habit-forming with the development of psychic dependence. In recent years the use of Benzedrine inhalers and similar non-prescription items has been largely replaced in the United States by Benzedrex which is N-Methyl-ß-cyclohexylisopropylamine. Benzedrex retains the nasal constrictive properties but lacks the stimulating properties for the central nervous system shown by Benzedrine.

Although amphetamine was readily available and its misuse occasionally reported to the laboratory, no widespread use of this substance among United Nations personnel was reported. No acute or fatal cases were reported to the laboratory.


178

Alkaloids

Description of Several Alkaloids

Opiates were the class of drugs about which the laboratory was most deeply concerned. The detection and analysis of these narcotics required more work than was needed to find and study the other drugs of interest to the Army toxicologist. The primary opiate found on the drug market was heroin (diacetylmorphine) which has the strongest addicting properties of the opiates. As stated in the early paragraphs of this report, the drug was readily available to the addict, both in Japan and Korea, and investigating agents were often stymied in their work by not being able to catch the suspect in possession of the drug or the implements used to administer it. Since the opiates are in part excreted in the urine as free or conjugated derivatives of alkaloids, produced by the body's detoxication processes, laboratory personnel were assigned the task of developing a satisfactory procedure for the identification and isolation of opiates found in urine samples taken from suspects. A satisfactory and reliable chromatographic purification procedure as well as an adaptation of a hydrolytic process for urinary extraction were developed and are to be described in this report.

Opium, the sun-dried latex of the unripe fruit of Papaver somniferum, has a total alkaloid content approximating 20 percent. For economic reasons, mainly cheap labor, the opium poppy is grown in the Orient, but this plant will grow equally well in the United States.

Two principal alkaloid types may be derived from opium-(1) the phenanthrene derivatives include morphine, codeine and thebaine which act primarily on the central nervous system, and (2) the 1-benzylisoquinoline derivatives, which include narcotine and papaverine (the more commonly mentioned of this class alkaloids) that exert little effect on the central nervous system. A total of 23 alkaloids have been isolated from opium but the most important are those that have been named.

Morphine is the main opium alkaloid, composing approximately 9 percent of opium. Although the structure of morphine has been definitely established, attempts to synthesize the drug have not been successful. Morphine contains the phenanthrene nucleus with a phenolic hydroxyl group and a secondary alcohol hydroxyl group which are subject to alkylation or esterification with numerous organic radicals. Oxidation and reduction of the secondary alcohol produces additional derivatives which have been studied extensively (15). This structural variation of morphine has recently produced an excellent morphine antagonist, N-allyl morphine (Nalline) which is specific in counteracting the analgesic and depressant effect of morphine (16).


179

The tertiary nitrogen atom of morphine gives the molecule basic properties which permit it to be readily converted to such salts as the sulfate, hydrochloride, etc.

Morphine acts primarily on the medullary centers of the brain as evidenced by the depression of the respiratory, cough and vasomotor centers as well as the stimulation of the vomiting centers. When death occurs as a result of acute morphinism, respiratory failure is the primary cause of death. This was the principal clue in a number of deaths suspected to have been caused by the administration of dope. The principal pharmacologic response by the body to this drug is the relief of pain, but in some individuals it elicits a feeling of euphoria and may produce sleep. Morphine has strong addicting power and produces violent withdrawal symptoms in many of its addicts.

Diagnosis of the addict is by physical examination of the body with emphasis on such evidence as the presence of needle marks over the large blood vessels, pupillary constriction, dulling of sensory response, lethargy, drowsiness along with a record of association with narcotics users or finding the victim in an area where these drugs are sold. These findings justify strong suspicion of an opiate overdosage.

Heroin, a synthetic drug, is diacetylmorphine and is more strongly addicting than morphine. It readily produces euphoria in many individuals but is also approximately five times as toxic as morphine. Its manufacture is forbidden by law in the United States but it is not difficult to produce. Korean dope peddlers were producing high-grade heroin, 90 to 95 percent pure, from opium, using small alcohol burners and reaction flasks which could all be assembled in a box the size of an orange crate. Heroin traffic was found to be particularly vicious when analysis of samples showed the active drug content to range from 5 to 99 percent. Among the many adulterants with which this opiate was prepared, sugar, starch, bicarbonate of soda, sand and


180

ground rice husks were commonly used. Because the manufacture of the drug was not standardized, each sample most likely contained one of a wide variety of possible dosages (normal dose is 4 to 10 mg.). When coupled with the fact that a harmful adulterant could be used, this meant that the addict was flirting with death with each injection. The principal method of drug administration was by hypodermic needle, although heroin and opium were smoked in some cases and taken orally in others.

Heroin by deacetylation in the body is converted to morphine and follows the pattern of morphine metabolism. The principal substances recoverable from the urine are conjugated products which may be obtained by the use of suitable hydrolytic procedures.

Codeine composes approximately 0.3 percent of opium. Structurally it is methyl morphine. This alkaloid is obtained principally by the synthetic process of morphine methylation. Codeine is widely used in medicine and is less stringent than morphine in its action on the medullary centers. It is likewise much less addicting. Codeine has approximately one-third the activity of morphine on the cough center and approximately one-twentieth of morphine's activity on the higher centers of the brain. This property leads to wide prescription of codeine in cough syrups and complicates the work of the toxicologist since morphine and codeine react similarly with numerous test re-


181

agents. However, there are sufficient specific reactions, as will be described in this report, to permit identification of either alkaloid.

Laboratory Analysis

The following is the process for isolating and identifying the alkaloids as developed by personnel of the 406th Medical General Laboratory:

Alkaloids. With few exceptions, every toxicologic specimen received is routinely examined for the presence of these narcotics. A new method for the extraction of alkaloids from body fluids has been developed and the results gained in hundreds of extractions have shown the method to be superior to any used in the past.

Detecting the Presence of Alkaloids by the Analysis of Urine Samples. Gross and Thompson (17) have shown that acid hydrolysis will permit the extraction of that portion of morphine from urine present in a conjugated form. An amount of 50 to 100 ml. is placed in a 500 ml. Erlenmeyer flask and subjected to hydrolysis by the addition of concentrated (10 percent by volume) hydrochloric acid and by autoclaving the sample at 15 pounds pressure for 30 minutes. The specimen is cooled, transferred to a 250 ml. centrifuge bottle where 25 to 50 ml. of chloroform is added. The bottle is stoppered, shaken for 3 minutes and centrifuged for about 15 minutes at 3,000 rpm in an International size II centrifuge. With the aid of a 50 ml. volumetric pipette and rubber bulb, as much of the chloroform layer as possible is removed and filtered through filter paper. The extraction is repeated a second time and the chloroform is combined and evaporated in a small beaker. The residue is examined for barbiturates and other acid and neutral substances.

Experience has shown that hydrolysis produces a dark brown discoloration in many of the normal constituents of urine. This material is extractable with chloroform from an acid solution so that the residue in most cases is not entirely suitable for examination. Where the presence of barbiturates is suspected, a portion of urine is extracted without hydrolysis and this residue is examined for that type of preparation. The aqueous solution remaining in the centrifuge bottle is made alkaline by adding 10 percent sodium hydroxide solution and the extraction is repeated with chloroform, in two portions. The residue will contain alkaloids other than morphine. For the extraction of morphine, the aqueous solution is first made acid with hydrochloric acid and then basic with ammonium hydroxide solution. Two extractions are made with a 9:1 chloroform-alcohol mixture.

Paper Chromatography of the Opium Alkaloids. The difficulties attending the purification and identification of opium alkaloids in tissues and body fluids have long plagued the toxicologist. The amounts of alkaloids present in toxicologic specimens are extremely


182

small and must be isolated in a relatively high state of purity before chemical identifications can be made. Because the isolation technics are imperfect and the chemical tests subtle, the experience and skill of the toxicologist is often the deciding factor between a positive and negative alkaloid identification. Since the finding of narcotic alkaloids raises questions of grave nature, there is a reluctance to report as positive any but the most conclusive chemical tests; doubtful or borderline reactions are reported as negative. The application of the paper chromatography technic has done much to minimize these difficulties.

The general methods and principles of paper partition chromatography can be described briefly as follows: A small drop containing the solute under investigation is placed near one edge of a strip or sheet of filter paper. After the spot has dried, the paper is placed in a sealed container. The atmosphere within the chamber consists of air saturated with water and the vapors from an appropriate solvent. After the paper has come to equilibrium with the atmosphere, the end of the paper containing the spot is brought into contact with the solvent. The solvent flows past the spot by capillary action and up the length of the paper. By this time the solute will have moved a distance along the paper, the amount of this movement depends upon the relative solubility of the solute in water and in the solvent (i. e., the partition coefficient). The movement of the solute zone has been explained conveniently as follows: "The cellulose fibers have a strong affinity for the water present in the solvent phase but very little for the organic liquid. The paper itself is thought of as an inert support holding a stationary aqueous phase. As solvent flows through a section of the paper containing the solute a partition of this compound occurs between the mobile organic phase and the stationary water phase. Thus, some of the solute leaves the paper and enters the organic phase. When the mobile liquid reaches a section of the paper containing no solute, partition again occurs. This time, solute is transferred from the organic phase to the paper phase. With continuous flow of solvent, the effect of this partition between the two phases is the transfer of a solute from the point of its application on the paper to a point some distance along the paper in the direction of solvent flow (18)."

General Chromatographic Procedure. Rectangular sheets of filter paper measuring 28 x 32 cm. are cut from larger sheets of No. 1 Whatman paper. A pencil line (base line) is drawn 4 cm. from the longer edge of the paper, paralleling that edge. On points along this line, at 3 cm. intervals, 0.005 to 0.01 ml. drops of the samples to be chromatographed are placed using 0.1 ml. Mohr pipettes. The paper is dried at room temperature. When volumes greater than 0.01 ml. are to be chromatographed, the solution is applied to the paper in aliquot portions allowing each aliquot to dry before further additions are


183

FIGURE 1. Chromatographic apparatus.

made. The paper is then bent perpendicularly to the base line into a cylinder and fastened at the end opposite the base line with paper clips using a paper bridge to separate the edges which close the cylinder. The paper cylinder is lowered, base line end downward, into a Smillie jar (fig. 1). The jar is clamped shut, using stopcock grease to effect an air-tight seal. Employing a pipette, the aqueous phase of the two-phase, water-solvent mixture is introduced through opening A onto cotton surrounding delivery tube B. This arrangement fa-


184

cilitates saturation of the atmosphere. Enough solution is applied to saturate the cotton, any excess falling into beaker C. Opening A is stopped and the apparatus is untouched for a period of 4 or 5 hours during which time the moisture content of the paper reaches equilibrium with the inclosed atmosphere. At the end of this period, 50 ml. of the solvent phase of the two-phase mixture is introduced into the bottom of the jar via funnel D and delivery tube B. The solvent immediately begins its capillary climb up the paper. After approximately 13 hours, the solvent front has advanced to within 2 or 3 cm. of the top of the paper cylinder.

The paper is then removed and laid flat and the solvent front boundary is marked immediately with pencil. The paper is air dried at room temperature by clipping to an electric fan. Using an atomizer, the paper is sprayed with a reagent to show the location of the alkaloid areas. In establishing the Rf value, a point is selected in the alkaloid spot where the greatest color density is observed. The distance between this point and the base line divided by the distance between the base line and the solvent front boundary is the Rf value of that spot.

Paper Chromatography of Pure Opium Alkaloids. Before toxicology studies could be initiated, it was necessary to establish a workable procedure using pure alkaloids. While more than 25 alkaloids have been isolated from opium, about 98 percent of the total alkaloid content of opium is made up of 6 alkaloids-namely, morphine, narcotine, papaverine, thebaine, codeine, and narceine. Accordingly, of the naturally occurring opium alkaloids, only these 6 compounds were considered in this study. In addition, 3 synthetic morphine derivatives were included in the series-heroin, donine, and apomorphine.

Stock Solutions. Solutions of the 9 alkaloids and their hydrochloric and acetic acid salts were prepared. The free bases were made up as 0.5 percent solutions and the salts in concentrations equivalent to 0.5 percent solutions of their corresponding free bases. Fifty percent ethanol was employed as the solvent for all of the alkaloid salts except narcotine acetate, which was prepared with 95 percent ethanol. Of the free bases, narcotine, thebaine, heroin, and apomorphine were dissolved in chloroform; papaverine, codeine, and dionine were prepared with 50 percent ethanol solutions; morphine was made up with isoamyl alcohol; and narceine with 10 percent ammonium hydroxide.

Chromatographic Developing Solvents. The following solvents were employed experimentally in an attempt to find a suitable chromatographic developing agent:

    n-butanol
    n-butanol, acetic acid
    n-butanol, ammonium hydroxide


185

    n-butanol, chloroform
    n-butanol, chloroform, acetic acid
    n-butanol, chloroform, ammonium hydroxide
    n-butanol, saturated sodium bicarbonate
    n-butanol, phosphate buffer (serial pHs from 4.5 to 8.5)
    n-butanol, hydrochloric acid
    n-butanol, sodium hydroxide
    isoamyl alcohol
    isoamyl alcohol, acetic acid
    isoamyl alcohol, ammonium hydroxide
    isoamyl alcohol, pyridine
    butyl acetate
    butyl acetate, acetic acid
    butyl acetate, ammonium hydroxide
    toluene
    toluene, acetic acid
    toluene, ammonium hydroxide
    chloroform
    chloroform, acetic acid
    chloroform, ammonium hydroxide
    pyridine
    aniline
    ethanol (various concentrations), acetic acid
    ethanol (various concentrations), ammonium hydroxide
    ethylene glycol monoethyl ether

Of these, only butanol-acetic acid and isoamyl alcohol-acetic acid gave well-positioned, clear-cut separations. Because its capillary climb up the paper is slower and therefore more suitable for laboratory time arrangements, the isoamyl alcohol solvent was selected in preference to the butanol solvent. It is prepared as follows: isoamyl alcohol (100 ml.), glacial acetic acid (10 ml.) and water (50 ml.) are shaken together thoroughly in a separatory funnel and the mixture allowed to stand for 3 days at room temperature before being separated into its solvent and aqueous phases. The 3-day standing period enables the constituent solutions to reach an equilibrium with respect to the formation of isoamyl acetate.

Spot Developing Reagents. Preliminary trials with a number of reagents giving color reactions with opium alkaloids led to the selection of a modified Dragendorff's reagent and a potassium iodoplatinate solution as the reagents most nearly filling the requirements of this study. They are prepared in the following manner:

Dragendorff's Reagent. A mixture of bismuth subnitrate (2.5 gm.), water 20 ml.), glacial acetic acid (5 ml.), and potassium iodide (4 gm.-previously dissolved in 10 ml. of water) are mixed. The precipitate that forms on standing for about 3 hours is removed by filtration. The filtrate is the stock solution. Just before using, one part of the stock solution is mixed with two parts of glacial acetic acid and three parts of water.


186

Potassium Iodoplatinate Reagent. 1 ml. of 10 percent platinic chloride and 25 ml. of 4 percent potassium iodide are mixed and enough water added to make the volume of the resulting solution equal 50 ml.

The sensitivities of these two reagents were tested against 10, 20 and 50 microgram spots of the 9 alkaloids which had been developed chromatographically as described above. All quantities of each of the alkaloids were applied to the paper in 0.01 ml. volumes. The results are shown in table 1.

Table 1. Reagent Sensitivity for Alkaloids
 

 


Alkaloid

Quantity of Alkaloid

10 mu

20 mu

50 mu

D 1

P 2

D

P

D

P

Morphine

Minus

Plus

Minus

Plus

Plus

Plus.

Narcotine

Plus

Minus

Plus

Plus

Plus

Plus.

Papaverine

Plus

Plus

Plus

Plus

Plus

Plus.

Thebaine

Plus

Plus

Plus

Plus

Plus

Plus.

Codeine

Plus

Plus

Plus

Plus

Plus

Plus.

Narceine

Minus

Minus

Minus

Minus

Plus

Plus.

Heroin

Plus

Plus

Plus

Plus

Plus

Plus.

Dionine

Plus

Plus

Plus

Plus

Plus

Plus.

Apomorphine

Minus

Plus

Plus

Plus

Plus

Plus.

1Dragendorff's reagent.
2Potassium iodoplatinate reagent.

It is seen from the table that when potassium iodoplatinate was used all of the alkaloids but narcotine and narceine could be detected in 10 microgram quantities, and that the narcotine spot was visible at the 20 microgram level. With Dragendorff's reagent, morphine, narceine, and apomorphine could not be discerned at a 10 microgram level. Fifty microgram spots of all alkaloids were visible when either reagent was used. The platinate is the reagent of choice because it offers a better contrast between the spot and its background and is more sensitive in its reaction with morphine, the alkaloid of greatest interest to the personnel of the 406th Medical General Laboratory.

Rf Values. The nine opium alkaloids were chromatographed in the form of free bases, acetates, and hydrochlorides. More variation was observed with the alkaloid salts than with the free bases. The results obtained with the latter, using both the butanol-acetic acid solvent and the isoamyl alcohol-acetic acid solvent are reported in table 2.


187

Table 2. Comparison of Solvents for Alkaloids
 

Alkaloid

Butanol-acetic acid solvent Rf (A)

Isoamyl alcohol acetic acid solvent Rf (B)

B/A

Morphine

0.41

0.12

0.29

Narcotine

0.76

0.62

0.82

Papaverine

0.74

0.61

0.82

Thebaine

0.65

0.43

0.66

Codeine

0.48

0.20

0.42

Narceine

0.67

0.46

0.69

Heroin

0.61

0.36

0.59

Dionine

0.57

0.30

0.53

Apomorphine

0.65

0.33

0.51

The spot patterns obtained with both solvents are similar, the general difference being the lower Rf values produced with isoamyl alcohol. That this lowering of Rf values is not proportionate throughout is seen by noting the B/A ratios given in the table. Morphine and codeine migrate at less than the average retarded rate. The reverse is true of narcotine and papaverine.

The values given for the butanol solvent represent averages of five determinations, three of which were run simultaneously, but in different chambers. The average variation between the Rf values of the alkaloids of the two nonsimultaneous trials is 7.2 (0.9 to 15.8) percent. In the simultaneous trials the deviation from the mean never exceeded 3.2 percent and the average is only 1.9 percent. Variation is even less if one considers the relative position of the spots, i.e., the relation of each alkaloid spot to all others on the same sheet. Such a consideration can be appreciated mathematically by comparing the Rf of each spot with the average Rf of all spots. Here, the deviation from the mean does not exceed 1.9 percent (average: 1.2 percent).

The values given for the isoamyl alcohol solvent were obtained from a single trial. Experience has shown that there is considerable variation in the Rf values obtained over a period of months. The following factors are known to influence Rf values: (1) the paper employed, (2) temperature, (3) quantity of material being chromatographed, (4) extraneous substances, (5) degree of saturation of the paper with water and solvent, (6) starting point with relation to solvent, (7) height of chromatograph, and (8) volume of starting spot. In these experiments temperature was the only factor not controlled. Indeed, it was found in many subsequent determinations where morphine, codeine, and heroin were employed as standards for toxicologic determinations that temperature had a definite influence on the Rf


188

values obtained. The values reflected the effects of seasonal room temperature changes, higher temperatures giving higher Rf values. A summary of 92 trials employing the isoamyl solvent against morphine, codeine, and heroin gave respective mean Rf values of 0.151 (values ranging between 0.12 and 0.19), 0.243 (0.20-0.30), and 0.382 (0.31-0.47). These figures emphasize the necessity of simultaneously employing standards where unknown samples are being determined, especially when controlled temperature conditions do not prevail.

Two-dimensional Chromatography. It is seen in table 2 that several of the Rf values fall within close range of each other. Compounds which group together in one-dimensional chromatographs can often be separated by two-dimensional chromatography. With this technic a spot is placed near the corner of a square of filter paper and chromatographed in the usual manner. After the solvent has run its course, the various compounds have aligned along one edge of the paper. When dry the paper is rotated 90 degrees and the spots rechromatographed with a different solvent, the new solvent flows through the horizontal row of spots and transports them vertically to another area of the paper commensurate with the Rf value of the compound in that solvent. The completed sheet shows the spots distributed in characteristic positions throughout the paper. By plotting the Rf values of one solvent as abscissae against the Rf values of another solvent as ordinates, it is possible to approximate the positions of spots as they would appear if these two solvents were used in a two-dimensional chromatograph (19).

This has been done in figure 2 where isoamyl alcohol-acetic acid solvent is used in the first phase of the two-dimensional scheme and isoamyl alcohol-ammunition hydroxide solvent* is used in the second phase. The points placed along the abscissa and ordinate show the expected positions of the alkaloids had they been developed in a one-dimensional system by the respective solvents. Experimental results closely approximate the calculated results shown diagramatically. While this two-dimensional system is not ideal (e. g., papaverine and narcotine still remain unseparated), much of the crowding found in the one-dimensional schemes is eliminated.

Chromatography of Alkaloids Extracted From Toxicologic Specimens. The final dry extracts obtained from tissues by the Stas-Otto procedure or from blood or urine by a new procedure developed in the 406th Medical General Laboratory and outlined in the section of this paper that deals with toxicology, are transferred from 50 ml. beakers to 13 x 100 mm. lipless test tubes (Wassermann type) using 2 ml. of boiling ethanol. The ethanol is removed by evaporation on 


*This differs from the isoamyl-acetate acid solvent only in that concentrated ammonium hydroxide is substituted for glacial acetic acid.


189

a water bath. Three or four drops of ethanol are added to the tube and, while still in the water bath, the solution is drawn up and down a 0.1 ml. Mohr pipette until a volume of 0.06 ml. is reached. Four 0.005 aliquots of this are applied to the paper forming a single spot. Each aliquot is allowed to dry before making the next addition, thus keeping the area of the final spot at a minimum. The remaining two-thirds of the material is saved for chemical testing. Morphine, codeine, and heroin standards (free bases) serve as controls. A 0.005

FIGURE 2. Diagrammatic representation of two-dimensional paper chromatograph of opium alkaloids.

ml. volume of 0.5 percent solution of each of the aforementioned alkaloids is applied to a single point on the base line. The spots of the various extracts to be tested are located at 3 cm. intervals on the base line, chromatographed in the manner previously described with the isoamyl alcohol-acetic acid solvent, and sprayed with the iodoplatinate reagent.

A comparison of the results obtained by chromatography and by chemical tests (Marquis, Frohde, and Mecke) during the months of August, September, October, and November 1952, on a total of 221 cases is shown in table 3.


190

Table 3. Comparison of Chemical and Chromatographic Detection of Alkaloids
 

Chemical results

Chromatographic results

Negative

173

146

Questionable

20

30

Positive

28

45

All but a small number of the cases in table 3 involve blood and urine specimens. A good part of the discrepancy between the number of positive cases found by chemical and chromatographic methods results from the manner in which the urine was processed. Gross and Thompson (17) found that hydrolysis* of urine from morphinized dogs increased the yield of morphine four or five times. This is due to the release of morphine from a combined form not recovered by usual extraction procedures. In order to take advantage of this extra source of morphine, the hydrolysis procedure was incorporated in the extraction process. A comparison of hydrolized and unhydrolized urines soon verified the advantages of hydrolysis as was evidenced by the greatly increased sizes and intensities of the developed morphine spots. However, hydrolysis introduces a serious problem-that of increasing the impurities in the final extracts. These impurities are dark brown and mask the chemical color reactions to the extent that, even while greater amounts of morphine are present, a more positive conclusion could have been reached had the urine not been hydrolized. These impurities do not present any difficulties in performing the chromatographic process since they do not affect the progress of morphine migration and they rise to positions on the paper high above the morphine spot area.

The "questionable" cases of table 3 require some discussion. Almost all of those so classified from chemical tests were listed as positive chromatographically. On the other hand, those cases which were considered "questionable" chromatographically result from chromatographs where only very weak morphine spots were observed, where morphine spots were "off color," or where the Rf values were slightly different from that expected for morphine. The very weak spots represent quantities of morphine less than 10 micrograms. Chemical confirmation of such minute amounts of morphine cannot be expected when it is remembered that the remaining two-thirds of unchromatographed extract must be divided to make the several chemical tests. 


*A suitable aliquot of urine was hydrolized with 10 percent by volume of concentrated hydrochloric acid in the autoclave by heating at 15 pounds pressure for 30 minutes.


191

The ideal chromatographic procedure would be specific for opium alkaloids and nothing else. The achievement of such an ideal is not likely because of the non-specificity of alkaloid isolation methods, Rf values and spot detection reagents. That the procedure developed here is not ideal is evidenced by the appearance of occasional miscellaneous spots. The distribution of the extraneous spots from extracts of blood, urine, stomach contents, liver, and brain from 201 negative cases is given in table 4.

Table 4. Distribution of Extraneous Chromatographic Spots
 

 


Specimen

 


Total cases

Cases where miscellaneous spots were found

Percent of cases showing miscellaneous spots

Blood

152

7

5

Urine*

86

18

21

Stomach Content

20

10

50

Liver

19

8

42

Brain

10

3

33

*Includes both hydrolized and unhydrolized samples.

The high incidence of extraneous spots found in stomach contents extracts is to be expected since a large variety of alkaloids are found in the plant foods commonly ingested. Blood and urine extracts showed the fewest spots, not only in the number of cases but in the number of spots per case. Undoubtedly some of these unidentified spots represent alkaloids of pharmacologic interest. Fortunately, few of these spots fall in the morphine area and, when they do, they show a violet coloration rather than the characteristic dark blue of morphine. These extraneous spots are quite often diffuse and leave trailing streaks in their wake. They are easily recognized by an experienced technician.

Of particular interest are those extraneous spots found in positive morphine extracts. In the sodium hydroxide-chloroform extract of most urine samples showing a strong morphine spot, a second spot is found in the codeine range. The persistent appearance of this spot in association with morphine suggests that it represents a metabolic end product of this drug possibly codeine. The conversion of codeine to morphine in tissues has been demonstrated (18); that the reverse process might take place is not a fanciful suggestion. Quite often a third spot is found located between the codeine and heroin areas. A fourth spot is sometimes observed in the range of Rf 60 to Rf 70. This spot is most commonly seen in hydrolized specimens. Attempts at identification of these materials are being made with the hope that


192

something may be learned about morphine metabolism. There is some evidence that addicts metabolize morphine in a different manner than novices (21, 22), and it is possible that the appearance of the spots associated with morphine in some cases and not in others may reflect the degree of addiction of the subject. An alternative explanation may be that because the urine samples are collected at different times after the drug is administered they represent various stages of morphine metabolism.

While heroin is undoubtedly the offending drug in most subjects, this alkaloid has never been observed on the chromatographs, thus supporting the view that heroin is rapidly converted to morphine in the tissues.

These results show the validity of applying the paper partition chromatographic technic to toxicologic problems. They also point out the need for an improved method of purifying extracts.

Chromatographic Purification of Alkaloids Extracted from Toxicologic Specimens. After establishing the presence of suspected opium alkaloids by chromatography using one-third of the final extracts, the remaining two-thirds of the material is purified chromatographically before attempting a chemical identification (when very large spots are observed on the preliminary chromatograph, only half of the remaining material is used for purification). Instead of spraying this second chromatograph, small squares of paper are cut from areas corresponding to the spot areas found on the preliminary sheet. These areas are located by clipping out a strip of paper containing chromatographed standards for morphine, codeine and heroin, spraying this strip, and then replacing the strip to act as a guide. The areas to be extracted can be even more perfectly located by cutting a 1 mm. strip up through the center of the chromatographic path, spraying this and replacing it in the sheet. Areas on both sides of the color band on this narrow strip are removed for extraction. The squares of paper are rolled into a cylinder and placed in Wassermann-type test tubes. The paper is wetted with 2 drops of concentrated ammonium hydroxide and 2 ml. of chloroform is added. The chloroform is boiled for a few seconds in a water bath and the chloroform extract transferred in 1 ml. aliquots to a second test tube contained in the water bath, allowing the first portion of chloroform to evaporate before adding the second. Spraying of these small pieces of paper after extraction shows the alkaloid has been removed. Similar extractions employing ethanol proved unsuccessful; substances interfering with the chemical tests are extracted with ethanol. As a check against the possibility of one spot having "wandered" into the "lane" of an adjacent extract, the sheet from which the squares have been cut is sprayed with the iodoplatinate reagent. In the experience of this


193

laboratory such migrations have not occurred, but it is felt that this precaution must be taken to obviate legal criticism.

The residue remaining after the chloroform is removed is colorless and of such minute quantity that only by close inspection can it be seen at all. For chemical testing, 5 drops of ethanol are added to the tube and the tube warmed briefly in a water bath. Aliquots of this are dried on a spot plate or microscope slide for testing. The resulting reactions are comparable to those seen with tests on pure alkaloid standards; background colors and charring are absent; the colors persist and show the classical transformations.

The chromatographic purification of alkaloids has been applied routinely in this laboratory only during the last 3 weeks of December 1952, and, while there have not been sufficient cases to establish the absolute validity of the method, the results obtained thus far have been highly encouraging. Spots have been graded as to size and density so that a spot which is just discernible receives a rating of 1/2 plus, a dense spot with dimensions of about 11/2 x 2 cm. is classified as 3 plus, and other spots between 1/2 and 5 cm. are graded accordingly. It is estimated that a 1/2 plus spot of morphine contains about 5 to 10 micrograms and a 3 plus spot 50 to 75 micrograms of the alkaloid. The chromatographically purified extracts were given a grade derived from the preliminary chromatograph, thus adjusting for the amount of extract used. Thirty-four urine extracts that showed preliminary chromatograph spots in the morphine area, with grades ranging from 1/2 to 5 plus, were purified as described above. All but l of 2l purified residues with a 3 plus rating or greater gave positive chemical reactions. The single exception was obtained from an extract which showed a violet spot rather than the characteristic blue of morphine. Five 21/2 plus, three 2 plus, and two 11/2 plus urine residues were all positive. One 1 plus urine residue was positive, another negative. A single 1/2 plus urine residue gave negative results. A purified extract from brain (1 plus) showed good positive chemical reactions. A codeine spot (3 plus) from a stomach contents extract was purified chromatographically and identified chemically. The advantages of paper purification become obvious when these figures are compared with those given for unpurified extracts in table 4.

More recently it has been found that morphine can be recovered directly from the sprayed spot. When the paper is sprayed lightly, only a small amount of the morphine combines with the platinate. This lightly sprayed spot is treated with ammonium hydroxide and extracted as previously described. The residue thus obtained is indistinguishable from that recovered from an unsprayed area. This modification of the procedure eliminates the preliminary chromatograph,


194

and saves not only time, but working material. In addition, it enables perfect delineation of the spot to be extracted.

Paper chromatography has not been applied to quantitative analyses of the alkaloids in the 406th Medical General Laboratory, but the above studies indicate that the technic could be readily adapted to such procedures.

References

1. Cori, C. F., Villiaume, E. L., and Cori, G. T.: J. Biol. Chem. 87 : 19, 1930.

2. Haggard, H. W., and Greenberg, L. A.: J. Pharmacol. 52 : 167, 1934.

3. Harger, R. N., Hulpiew, H. R., and Lamb, E. B.: J. Biol. Chem. 120 : 689, 1937.

4. Newman, H. W., Lehman, A. J., and Cutting, W. C.: J. Pharmacol. 61 : 58, 1937.

5. Harger, R. N., and Gross, A. J. L.: Am. J. Physiol. 112 : 374, 1935.

6. Mirsky, I. A., and Nelson, N.: Am. J. Physiol. 127 : 308, 1939.

7. Krantz, J. C., and Carr, C. J.: The Pharmacologic Principles of Medical Practice, 2nd Edition, p. 420. Williams and Wilkins Company, Baltimore, 1951.

8. Ibid. 7, p. 431-432.

9. Newman, H. W., and Lehman, A. J.: J. Pharmacol 62 : 301, 1938.

10. Ibid. 7, p. 557.

11. Williams, R. T.: Detoxication Mechanisms, Second Impression, pp. 214-222. John Wiley & Sons, Inc., New York.

12. Goldbaum, L. R.: J. Pharmacol. and Exper. Therap. 94 : 66, 1948.

13. Loewe, S.: J. Pharmacol. and Exper. Therap. 84 : 78, 1945.

14. Ibid. 7, p. 614.

15. Burger, A: Medicinal Chemistry, Vol. 1, pp. 157-159. Interscience Publishers, Inc., New York.

16. Clark, R. L., Pessolano, A. A., Weijlard, J., and Pfister, K. S.: J. Am. Chem. Soc. 75 : 20, 1953.

17. Gross, E. G., and Thompson, F.: J. Pharmacol. & Exper. Therap. 68 : 413, 1940.

18. Block, R. J., Le Strange, R., and Zweig, G.: Paper Chromatography. A Laboratory Manual, p. 4. Academic Press, Inc., New York, 1952.

19. Consden, R., Gordon, A. H., and Martin, A. J. P.: Biochem. J. 38 : 230, 1944.

20. Adler, T. K., and Shaw, F. H.: J. Pharmacol. and Exper. Therap. 104 : 1-10. 1932.

21. Pierce, I. H., and Plant, O. H. J.: J. Pharmacol. and Exper. Therap. 46 : 201, 1932.

22. Zauder, H. D.: J. Pharmacol. and Exper. Therap. 104 : 11-19, 1952.