Some applications of paper chromatography in the detection of opium alkaloids

Sections

Heroin and Procaine Mixture
Tobacco and Heroin Mixture
"Codenon"
Estimation of Morphine and Codeine by Paper Chromatography
Detection of Morphine in Urine Stains
Summary
Acknowledgement

Details

Author: George R. Nakamura
Pages: 17 to 20
Creation Date: 1960/01/01

Some applications of paper chromatography in the detection of opium alkaloids

George R. Nakamura
With the technical assistance of Alejandro B. Mondala, Master Sergeant, Military Police Corps, U.S. Army, Koichi Funakoshi, Sonoko Ito and Satoru Kusafuka U.S. Army Military Police Laboratory

The proximity of this laboratory to sources of illicit narcotics in the Far East has afforded this organization a great deal of experience in examining and identifying these dangerous drugs in various forms.

Since heroin is cheaply converted from morphine, resulting in much increased potency, it is the most popular underground vehicle in the narcotics trade in the Far East, if not throughout the world. This laboratory has standardized its analytical procedure so that at least three principal criteria are satisfied before a conclusion of positive identification for heroin is rendered. The criteria are that the classic colour tests ( [ 1] ) of Mecke, Marquis & Freude give characteristic reactions for heroin; that the characteristic ultra-violet absorption spectra of heroin ( [ 2] ) are demonstrated by spectrophotometry; and that the characteristic crystalline derivative of heroin ( [ 3] ) is obtained in reaction with platinic chloride. In addition to these criteria, or in substitution for the second and third criteria, particularly when the heroin sample contains interfering substances, chromatographic detection is attempted.

Interfering substances present in heroin samples make spectrophotometric and platinate derivative determinations difficult. Paper chromatography has proven to be a simple and effective tool for the separation of a mixture of opium alkaloids and other drugs, tobacco products or various nondescript substances occurring in narcotic examination. Some of the latest applications of chromatographic methods for forensic studies, including the detection of dangerous drugs, have been reviewed by McBay ( [ 4] ).

The procedure employed by this laboratory for the detection of opium alkaloids has been essentially the same as those described by Mannering et al ( [ 5] ) and Dobro & Kusafuka ( [ 6] ). The ascending type of chromatography was performed in a cylindrical jar, 46 cm in diameter, fitted with a ground-glass lid. This tank accommodated a 40 sq cm sheet of Toyo Roshi No. 51 paper, rolled and stapled in such a manner that the edges did not touch each other.

Samples were applied onto a base line of the chromatogram by the use of capillary pipettes which were made from clean glass tubing and disposed of after each use. One of the solvent mixtures found most efficient by Mannering et al ( [ 5] ) and standardized by this laboratory for separating a mixture of heroin, morphine, codeine and other opium alkaloids consisted of 10 parts of 1-butanol, 5 parts of water and 1 part of glacial acetic acid. The top layer was used as the working solvent, and the lower layer was used to saturate the atmosphere.

The solvent mixture was allowed to irrigate the chromatogram overnight for eighteen hours. The paper was then dried in the open air and sprayed with potassium iodoplatinate reagent. The violet spot corresponding to the position of standard heroin was removed from the chromatogram, wetted with saturated sodium bicarbonate solution and eluted by extraction with chloroform. The chloroform extract was dried and re-dissolved in acidic or alcoholic solution for ultra-violet spectra determination or the colour tests, or both.

While the iodoplatinate reagent served as a sensitive tool to probe for opium alkaloids on the chromatogram, it had limited use in identification inasmuch as some other compounds, including non-alkaloids, also gave positive reactions. Various substances which are of interest in forensic examination were selected at random and tested for their reactions with the iodoplatinate reagent. These compounds were chromatographed under standard conditions and then sprayed with iodoplatinate. Results, presented in table 1, made it apparent that only a negative reaction could be used as a basis for conclusion.

Heroin and Procaine Mixture

Occasionally illicit heroin powder submitted to this laboratory was contained in used penicillin vials. Contamination of penicillin or penicillin-procaine had been noted in such heroin samples by this laboratory. When unusually large amounts of procaine were found, their presence was attributed to adulteration as an analgesic. Inasmuch as procaine absorbed ultra-violet light with much greater sensitivity than heroin, its interference in ultra-violet spectra determination of heroin was appreciable. This laboratory employed paper chromatography as a method (7) for separating procaine from heroin, using the standard solvent mixture containing 10: 5: 1 1-butanol, water and acetic acid resolved heroin and procaine. A butanol solvent containing ammonia proved to separate heroin and procaine even better, but the alkaline medium caused heroin to de-acetylate partially to morphine, resulting in two separate spots. The respective spots were removed and eluted under standard conditions and examined by spectrophotometry and colour tests.

TABLE 1

Reactions of iodoplatinate reagent with chromatographed compounds Solvent mixture: l-butanol 10, acetic acid 1, and water 5 Positive reacting compounds

 

Colour

Typical Rf *

 

Colour

Typical Rf *

Aminophylline
Light grey
.10
Nicotine
Blue
.49
Aminopyrine
Violet
.80
Papavarine
Violet
.74
Amodiaquin.
Purple
.55
Pentolinium
Purple
.65
Amphetamine
Blue
.68
Primaquine
Purple
.76
Atropine
Violet
.62
Procaine
Purple
.56
Brucine
Blue
.52
Promazine
Purple
.76
Choline
Blue
.10
Promethazine
Violet
.79
Chlocyclizine
Violet
.75
Quinidine
Violet
.79
Chorpheniramine
Blue
.76
Quinine
Violet
.79
Chloroquine
Blue
.52
Strychnine
Violet
.55
Chlorpromazine
Purple
.78
Tetracaine
Purple
.78
Cocaine
Violet
.54
Thiamine
Blue
.11
Diacetylmorphine
Violet
.62
Thonzylamine
Purple
.65
Dimenhyrinate
Violet
.79
Thorazine
Purple
.78
Doxylamine
Purple
.74
Tolazoline
Purple
.58
Homatropine
Blue
.54
Transentine.
Violet
.80
Methimazole
Purple
.77
Trimeprazine
Purple
.84
Morphine
Blue
.41
Tripelennamine
Violet
.75
Narcotine
Violet
.72
Yohimbine
Purplish pink
.66

* The Rfs of these compounds do not represent values which are absolute, but values which are relative to each other.

Negative reacting compounds *

Acetazolamide
Dextrose
Lactose
Phenylbutazone
Acetylsalicylic acid
Dibucaine
Liothyronine
Pipradol
Amobarbital
Diethylstibestrol
Marihuana
Prednisone
Ascorbic acid
Digoxin
Meclizine
Prednisolone
Antipyrene
Digitoxin
Menadione
Progesterone
Barbital
Diphenylhydantoin
Methanamine mandelate
Propadrine
Belladonna
Disulfiram
Methylhexabital
Reserpine
Benzocaine
Ephedrine
Methyltestosterone
Riboflavin
Bishydroxycourmarin
Ergonovine
Mephenesin
Rutin
Bromisovalum
Ergotamine
Meprobamate
Saccharin
Caffeine
Ethinamate
Menadione
Secorbarbital
Cantharidin
Ethisterone
Nicotinic acid
Streptomycin
Cascara sagrada
Glucoronic acid
Nitrofurantoin
Sulfisoxazole
Cholesterol
Glutethimide
Penicillin
Tocopherol
Chlorthiazide
Hydralazine
Phenobarbital
Wafarin
Colchicine
Isoniazid
Phenol
Zoxazolamine

* Yellow and brown colour reactions were considered negative.

Penicillin did not interfere with heroin in any of the three criteria determinations. Its presence was, however, detected by ultra-violet determination in hot acetate buffer, pH 4.6 (8). The absorption maximum peak was at 322 millicrons.

Tobacco and Heroin Mixture

Lately, many cigarette samples which have been examined in this laboratory have shown no visible trace of powder on the tobacco fragments, but have rendered positive reactions with the three colour tests. Hitherto, the occurrence of heroin powder was apparent enough, so that it could be shaken free from the tobacco.

The presence of nicotine and a large amount of nonspecific substances found in the aqueous extract of tobacco caused troublesome interferences in the identification of heroin. Heroin and nicotine were separated by paper chromatography under standard conditions. To keep the browncoloured material at a minimum in the extract, a mild extraction procedure was used. The tobacco fragments were placed in a fluted filter paper and water was allowed to run rapidly through the fragments and paper. The filtrate was collected, saturated with sodium bicarbonate and extracted with 9: 1 chloroform-ethanol mixture. The extract was then concentrated for chromatographic application.nnn

Iodoplatinate spray reagent gave a bluish coloured spot for nicotine at a Rf position on the chromatogram close to that for morphine (table 1). However, nicotine was differentiated from morphine by colour tests and ultra-violet spectrophotometry. Heroin stained violet with iodoplatinate and moved more rapidly than nicotine in this particular solvent mixture. For identification, the heroin spot was eluted and examined.

A rapid micro-filtration technique was employed when heroin was mixed with a small amount of tobacco fragments or other non-specific debris. An equal amount of water was added to the fragments contained in a test tube or an evaporating dish. A small cotton piece was placed in the mixture. The tip of a medicine dropper was pressed into the cotton and the clear liquid was drawn into the dropper pipette. This technique proved to be useful when a small amount of liquid was required to be filtered rapidly. The clear filtrate was concentrated or used per se for chromatographic applications.

"Codenon"

A liquid product contained in sealed ampoules and labelled with the trade mark "Codenon" was submitted to this laboratory for analysis. The user declared that the product was purchased from a pharmacy in Korea. Chromatographic examination showed that a sample of" Codenon" could be resolved into two iodoplatinate positive components with Rf values corresponding to papaverine and heroin standards respectively. When the spots were eluted and subjected to spectrophotometric examination and the colour tests, the presence of papaverine and heroin was confirmed. In another case, a batch of ampoules labelled " Codenon "was submitted to this laboratory for analysis; the liquid content of these ampoules was found to contain morphine as the only active principal.

Estimation of Morphine and Codeine by Paper Chromatography

In March 1959, this laboratory received eight compressed bars which resembled crude soaps wrapped in cellophane and with the trade mark "999" on both the bars and the wrappings. These bars were purported to have been shipped from or through Hong Kong to Japan through underground channels. The courier bearing these bars was intercepted by armed forces investigators at Yokota Air Base, Japan.

An analytical method using paper chromatography showed that these bars contained an average of 85% morphine and 7% codeine (estimated as free bases). The procedure for the separation and estimation of morphine and codeine was adapted from Knight's procedure (9) for the micro-analyses of purine bases and pyrimidine nucleotides using paper chromatography. The solvent mixture used for the best possible separation of morphine and codeine was that formulated by Asahina & Ono (10). It consisted of 50 parts of 1-butanol, 15 parts of water, and 9 parts of 28% ammonia. The chromatogram was scanned for morphine and codeine spots with a germicidal lamp equipped with a filter (peak at 2,537 angstrom units) contained in an ultra-violet light box assembly built for the U.S. Army by the Sirchie Fingerprint Laboratory, U.S.A., described as electric light assembly MX-1291/PAQ.

The chromatogram spots were removed and eluted with 3 ml of 0.1-N hydrochloric acid. The paper used was Toyo Roshi No. 51, manufactured by a Tokyo firm of the same name, which gave the same average blank reading, if not slightly lower, as Whatman No. 1.

Inasmuch as morphine and codeine have comparatively weaker ultra-violet absorption than purines and pyrimidines at their respective maxima, this adaption method suffered by comparison by having a lower sensitivity range. The accuracy for this chromatographic analysis for morphine and codeine was in the range of 100 to 350 microgrammes (figure 1) in which a linear curve was obtained for each alkaloid under standard conditions. In this range, 96% to 98% recovery could be obtained for chromatographed morphine and codeine. Asahina & Ono (10) described a paper chromatography method by which the absorbance of morphine spots on chromatogram strips was measured directly in a specially devised accessory for the Beckman DU spectrophotometer. They obtained readings in 10 to 60 microgramme range for the determination of chromatographed morphine. Investigation is being conducted in this laboratory for further refinements of the method, using paper chromatography and ultra-violet spectrophotometry for the rapid separation and estimation of morphine and codeine.

FIGURE 1: The standard curve plotted by relating the absorbance of chromatographed morphine and codeine to a concentration range of 100 to 350 microgrammes. The curve is linear for both alkaloids in this range under standard analytical conditions.

Full size image: 11 kB, FIGURE 1: The standard curve plotted by relating the absorbance of chromatographed morphine and codeine to a concentration range of 100 to 350 microgrammes

Detection of Morphine in Urine Stains

Following the modified purview of military laws, urine specimens for narcotic examination could no longer be collected from suspect individuals without their consent. Thus, this laboratory received undershorts and trousers bearing urine stains presumably of the suspect users. The urine stains from the garments were subjected to aqueous or dilute hydrochloric acid extraction and then the extracts were chromatographed according to the procedure of Mannering et al (5) for the detection of opium alkaloids. In addition, the iodoplatinate positive spots removed from the chromatograms were examined by ultra-violet spectrophotometry as well as by colour tests.

While about thirty-two pairs of shorts have been examined to date, three pairs were demonstrated to contain morphine in urine stains. The amount found was estimated to be 100 to 250 microgrammes in each urine stain. As may be surmised, these positive tests for morphine were obtained where the concentration of urine stain was high. In each of the three cases, the presence of morphine was confirmed by ultra-violet spectral curve study. In one case, the stains from three pairs of shorts belonging to an individual suspect were combined to obtain positive results.

Summary

This paper describes a few experiences in which the paper chromatographic method was applied to the separation of opium alkaloids and certain non-specific substances in mixtures. The most common mixtures examined in this laboratory have been heroin in tobacco fragments and in procaine mixtures. Various compounds of forensic interest were chromatographed and tested with potassium iodoplatinate reagent for specificity study. Results are presented in table 1. Heroin has also been detected by paper chromatography in liquid samples contained in ampoules labelled "Codenon". Paper chromatographic techniques were applied to the estimation of morphine and codeine components of a compressed bar. Morphine has been detected in urine stains found on men's shorts by the use of paper chromatography.

Acknowledgement

This work had the support and encouragement of George R. Bird, chief of the U.S. Army Military Police Laboratory, Far East, who has contributed more than thirty years of his career in civilian and military law enforcement to forensic laboratory development.

Bibliography

001

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