Physico-chemical methods for the identification of narcotics (cont.) Part Vb - Paper chromatographic data for narcotics and related compounds

Sections

1.Introduction
2. Materials and methods
3.Results and discussion
Conclusions
Acknowledgements

Details

Author: Klaus Genest, Charles G. Farmilo
Pages: 15 to 24
Creation Date: 1960/01/01

Physico-chemical methods for the identification of narcotics (cont.) Part Vb - Paper chromatographic data for narcotics and related compounds

Klaus Genest
Charles G. Farmilo
Organic Chemistry and Narcotic Section, Food and Drug Directorate Department of National Health and Welgare, Ottawa (Ontario)

The Bulletin on Narcotics presents in this issue the continuation of the Canadian series of articles on the " Physico-Chemical Methods for Identification of Narcotics" which appeared in volumes V (1953), VI (1954), VII (1955) and XI (1959) of this journal.

CONTENTS

1.Introduction

This study was originally undertaken to develop methods for the rapid qualitative and quantitative separation of morphine, codeine, thebaine, papaverine and narcotine from opium, and has been extended to include both manufactured and synthetic narcotics which are related to the opiates for purposes of cataloguing the routine qualitative and quantitative features for their identification by paper chromatography. A thorough review on paper chromatography of narcotics has previously been given in part Va [ 7] .

2. Materials and methods

The methods were essentially those described in a recent paper by Nadeau et al. (15). A more detailed description of the material, equipment, sources of supply, etc., can be found in United Nations document ST/SOA/SER.K.58, by Farmilo et al. [ 6] .

The results reported in the present paper were obtained by the technique of ascending paper chromatography. Cylindrical jars (12" X 18") and paper cylinders (18" X 14") shaped from Whatman No. 3 MM paper were used. In experiments to determine sensitivity, Whatman No. 1 paper has been employed.

A. Paper Impregnation

Paper impregnated with 0.5 M KH 2PO 4 (pH4.2) and with (NH 4) 2SO 4 (2%, pH 5.3) has been employed.

B. Solvents

Isobutanol: glacial acetic acid: water (100 : 10 : 24) was mixed and shaken until clear. This solution was used as the mobile phase. A small portion of the mobile phase was withdrawn and shaken with water (100 ml) until water saturated. This aqueous layer was drawn off and used for equilibrating the chromatographic chamber. The isobutanol solvent system was used for most experiments with opium and synthetic narcotics. The times of equilibration and developing were 16 and 18 hours respectively. For the separation of papaverine-narcotine, the following solvent was employed: diethylether (peroxide-free) saturated with 0.1 M acetic acid. Equilibration of the paper required 2 hours, while the development stage required 4 hours. In two dimensional chromatography of opium to separate papaverine and narcotine, development of the chromatogram was carried out with acetic acid (0.1 M) saturated ether along the second dimension, after ordinary development with the isobutanol : acetic acid: water along the first dimension.

C Standard Solutions

Standard alkaloid stock solutions were prepared by dissolving the narcotics (10 mg) in methanol (10 ml) and stored at 4°C. After several days' storage, some solutions became green coloured. Coloured solutions of narcotine when chromatographed show fluorescent bands in addition to those found from freshly prepared solutions. For this reason, solutions of standards were discarded when they became discoloured, and fresh solutions were prepared.

D. Opium Samples

Extracts of opium samples were prepared by triturating opium powder (20/40 sieve size) (0.5 g) with glacial acetic acid (2.2 ml) and water (2.8 ml). The opium triturate was allowed to stand for one-half hour, with occasional stirring, then filtered through a fluted filter paper (Whatman No. 2, 7 cm diameter). This filtrate (5 microlitres) was applied directly to the paper for chromatography.

E. Examination in Ultraviolet Light

After drying at room temperature, the chromatograms were examined under ultraviolet light at two wave lengths, 2537 and 3660 . Using Mineralites® and an Aristogrid, cold grid type® ultraviolet source (22" X 26" X 6") equipped with filtres.

F. Detection and Identification by Chemical Reagents

Potassium-iodo-platinate was chosen as the spray reagent for detecting and identifying alkaloids on chromatograms. The reagent was prepared as follows: 1 g PtCl 4 2 HCl. 6 H 2O was dissolved in water (10 ml) and mixed with a solution of KI (10 g) in 250 ml and then diluted with water to 500 ml. Even spraying of the surface area of the chromatogram produces coloured spots varying from blue to violet on a brownish pink background. The colours of the spots of alkaloids differ depending on the kind of salt used to impregnate the paper. The coloured areas were outlined with ball-point ink while the paper was still moist. The centre of greatest colour density of each area was also marked for R f measurements. Immediate marking of the stained areas was found necessary, since some of the colours fade and shrink rapidly, especially on ammonium sulphate salted papers.

3.Results and discussion

A. Opium Alkaloids and Opium

In table I, a colour code for interpretation of the letters on the chromatogram pictures is given.

TABLE I

Colours of lumincscent areas of chromatograms *

Pure colour

Shade or tint

Other designations

R = Red
Ol = Olive
NC = No well defined colour
O or Or = Orange
Gy = Grey
Weak fluorescence
Y = Yellow
Br = Brown
A = Absorbance
G or Gr = Green
P = Pink
Vl & L = Very light and light
B = Blue
Aqu = Aquamarine
BGVL = Very light blue green
V = Violet
 
B (Ol) = Blue area with olive zone inside
W = White
   

Code employed for recording results in figures 1 to 6.

It will be observed that the fluorescent patterns of natural opium have characteristic features of colour and intensity which can be described qualitatively, and aid in making comparisons between samples of opium. The first step in characterizing the opium by means of chromatography consists in carefully describing the fluorescent regions according to table I. It will be observed on some illustrations that outlined regions do not have a code letter beside them, indicating that no adequate description of the colour could be given in simple terms. In these instances, the intensity of the colours was described in relative terms as +,++,+++, etc.

Table II illustrates the limits of detection of the opium alkaloids, morphine, codeine, thebaine, papaverine, narcotine and narceine, using the physical and chemical methods of detection directly on the paper chromatogram. In column 1, the names of the alkaloids are shown, column 2 lists the salt solution with which the paper is treated, including the pH value. Columns 3 to 8 list the lowest concentration of alkaloid in microgrammes (gamma) which were detectable on Whatman 3 MM and No. 1 paper chromatograms using ultraviolet light for the examination. Columns 3 and 4 show the results

TABLE II

Limits of detection of alkaloids in microgrammes

 

UV, mu

3660(t)*

3660(r)*

2537(r)

Spray with K-Iodo platinate

Alkaloid

Salt

3MM

1x

3MM

1

3MM

1

Colour

3MM

1

Colour

               
*
     
Morphine
M/2KH 2PO 4, pH 4.2
60
>150
>150
>150
30 30
dark spot I (A)
5 1
blue
 
2% (NH 4) 2SO 4, pH 5.3
60
>150
>150
>150
20 20
on light background
5 2
blue
Codeine
M/2KH 2PO 4, pH 4.2
>150
>150
>150
>150
20 45   5 3
blue-violet
 
2% (NH 4) 2SO 4, pH 5.3
>150
>150
>150
>150
20 45
ditto (A)
5 4
blue-violet
                       
Thebaine.
M/2KH 2PO 4, pH 4.2.
>150
150 150 150 2 3   4 4
violet-blue
 
2% (NH 4) 2SO 4, pH 5.3
>150
150 150 150 3 10
ditto (A)
5 4
violet-blue
                       
Papaverine
M/2KH 2PO 4, pH 4.2.
1 0.5 1 0.5 0.5 0.5
yellow-green (F)
3 3
violet-blue
 
2% (NH 4) 2SO 4, pH 5.3
0.5 0.5 0.5 1 0.5 0.5
spot on darker background
5 3
violet-blue
                       
Narcotine .
M/2KH 2PO 4, pH 4.2.
7.5 1 7.5 1 5 1
blue-grey (F)
7.5 5
violet-blue
 
2% (NH 4) 2SO 4, pH 5.3
10 3 10 5 10 3
spot on darker background
0.5 0.5
brown-pink **
Narceine
M/2KH 2PO 4, pH 4.2
>150
>150
>150
>150
30 30
bluish spot (F)
30 30
violet-red
 
2% (NH 4) 2SO 4, pH5.3
>150
>150
>150
>150
30 30
on darker background
7.5 7.5
(Purple) ditto

* t = Transmitted. r = Reflected.

** At concentrations > 30 pg a violet-blue nucleus which is surrounded by the brown-pink area becomes visible.

I Probably due to absorption of the ca. 285 mµ maximum by phenanthrene-type compounds indicated by (A) in table. The isoquinoline types fluorescence indicated by (F).

x Whatman number of paper used.

for transmitted light of 3660 ? wavelength through 3 MM and No. 1 paper. In columns 5 and 6, the concentration values detected by means of reflected ultraviolet light of 3660 ? wavelength are shown. In columns 7 and 8, the limits of detection of alkaloids by means of reflected ultraviolet light of 2537 ? wavelength are listed. In column 9, the observed colour or other response of the compound to the ultraviolet radiant energy is shown. In columns 10 and 11, the concentration limits of the drug detected by reaction with potassium iodoplatinate solution sprayed on the paper are shown. In column 12 the colours of the complex formed between the reagent and alkaloids on the paper are given. The background colour of papers salted with phosphate was brownishpink, and with ammonium sulfate the background colour strikes pink and fades to a mottled grey or white. The fading requires time and was found to be accelerated in daylight. In table III, R franges for five major alkaloids of opium for papers salted with (NH 4) 2SO 4 and KH 2PO 4, respectively, are listed. The R f values were compiled from spots of single compounds, mixtures of compounds and of opium samples covering a concentration range of 2 to 250 γ per spot.

Figure 1 shows a chromatogram of pure alkaloids and opium extracts after iodoplatinate spray. The conditions for this chromatographic experiment were as follows: paper treated with M/2 potassium dihydrogen phosphate, 18" X 24" size, 16 hours' equilibration and 8 hours' developing time. The distance travelled by the solvent front was about 12 inches (30 cm). Isobutanol : glacial acetic acid: H 2O : 10: 1: 2.4 was the solvent used for development. The alkaloids were detected by the iodoplatinate reaction, and by ultraviolet light examinations. Below the starting line, the point of application of the alkaloidal solutions indicated by the symbols M= morphine, C = codeine, T= thebaine, P = papaverine, N = narcotine, are shown from left to right. The sixth group of spots on the chromatogram lie above the point where a mixture of these pure alkaloids was applied. Narcotine and papaverine show one spot in the chromatogram. The locations of the fluorescent and stained areas of luminescent constituents and alkaloids of opium are distin-

TABLE III

Alkaloid R f ranges

Solvent

Iso-Bu OH : AcOH : H 2O (10 : 1 : 2.4)

     
Paper impregnation .pH
(NH 4) 2SO 4(2%)5.3
 
KH 2PO 4(0.5 M) 4.2
 
Alkaloid
Number of spots measured
R f range
Number of spots measured
R f range
Morphine.
25
0.09-0.14
25
0.20-0.36
Codeine.
25
0.17-0.30
25
0.43-0.50
Thebaine
25
0.59-0.70
25
0.69-0.79
Papaverine
25
0.76-0.84
25
0.78-0.87
Narcotine
25
0.76-0.84
25
0.78-0.87

guished by weak pencil lines and heavy ball-point ink lines. Starting at the solvent front and working down the chromatogram toward the origin line, the following fluorescent colours are observed: green (mixture of papaverine and narcotine); area of absorbance (2537 ?), thebaine (only slight concentration); below this is a region of blue-green (BGr) fluorescence in samples 224 D and 803; while sample 711 shows an orange(Or)region. Sample 224 D shows an absorbance followed by a green (G) fluorescence, while none is present in the samples 711 and 803. The next fluorescent region is blue-green. The codeine absorbance spot follows, and is present in all samples 711, 224 D and 803 which are listed in decreasing order of concentration. The next region is an orange or brown fluorescent colour, followed by a heavy absorbance and stained spot which corresponds to morphine. The samples arranged in order of increasing morphine content are 711, 803 and 224D. All samples show another region of absorbance at about R f 0.14 which does not stain with the iodoplatinate reagent. The region adjacent to the origin line commonly shows a blue fluorescent zone with a brown centre.

Figures 2 and 3 are photographs of the same chromatogram of pure alkaloids and opiums as follows: sample 711, U.S. customs seizure, No. 63424, originating in Inner Mongolia; sample 224 D, a Turkish druggist opium; sample 803, a sample of chocolate cake opium, probably of Turkish origin. The chromatogram was developed on ammonium sulphate treated paper using isobutanol, acetic acid, water as mobile solvent. Figure 2 is a photograph to illustrate the kind of fluorescent pattern obtained using ultraviolet light (3660 ?). This type of photograph is called a luminogram. Figure 3 is the same chromatogram photographed with visible light after reaction with potassium iodoplatinate reagent. Figure 3 shows that the order of the R f values is the same regardless of paper treatment with phosphate or sulfate salts. The R f values are different and depend on the salting technique. The background colour and the colours of the individual spots of alkaloids are different. Figures 4 and 5 illustrate chromatographic comparison of thirteen opium seizure samples. UN 79c * (seizure, El Paso, Texas), U.S. customs seizure 63415, seizure in U.S.A. No. 2, seizure in U.S.A. No. 8, UN 112A, * seizure in Guatemala, seizure in USA 1754, UN 112B * seizure in Guatemala, UN 97 * seizure by Mexican Government in Mexico, UN 98 * seizure at Linoloa by Mexican Government, Houston seizure in U.S.A., chocolate cake opium seizure in U.S.A. The object of this illustration is to compare the chromatographic appearance of the separated constituents of the seizure No. 1754 with other seizures of apparently similar origin. Figure 4 shows the ultraviolet light picture (luminogram) of the chromatogram treated with potassium iodoplatinate shown in figure 5. Figure 6 is a picture of a two-dimensional chromatogram of an old Indian sample No. 13 from the East Punjab region of India. The paper was saturated with ammonium sulphate and the sample applied in the usual manner. The chromatogram was developed in the second dimension with peroxide-free ether saturated with acetic acid (0.1 N).

This sample is not included in the "List of authenticated samples in the United Nations Distribution Centre" published by the United Nations Division of Narcotic Drugs as document ST/SOA/SER.K/82 (5 January 1959).

Discussion

The object of the study of chromatography of natural opium is to achieve a rapid separation of the major opium alkaloids, morphine, codeine, thebaine, papaverine and narcotine for qualitative and quantitative comparison in the process of determining the orig n of opium samples.

Munier & Machebœuf [ 12] , [ 13] devised the solvent system, isobutanol : acetic acid : water, which is used as the basis of the chromatographic separation of the major opium alkaloids. The system is not quite ideal since papaverine and narcotine are not separated. The separation of papaverine and narcotine was achieved by the method of Krogerus [ 9] . These authors have also shown that the salt with which the chromatographic paper is treated, and the apparent ionic strength of this medium are of importance in the resolution of opium aklaloids. The influence of the ions must be considered, as well as acidity. Resplandy [ 17] showed that ammonium sulphate when used as mobile phase decreased R f values more than any other salt investigated. Our preliminary investigations with acetate and McIlvaine buffer confirmed the findings of other workers. In the range between pH 4.0 and 5.0, a higher R f is generally found with a higher pH (1-5, 10, 11, 16). Comparing the values in table III indicates that the inhibiting effect of ammonium sulphate on R f appeared to overcome the accelerating effect of increased pH. The ratio of the R f values of papaverine and morphine may be used as an indication of the spread or resolution obtained with a given solvent system, changing only the salt with which the paper is treated. Using the butanol : acetic acid : water system with sodium acetate, phosphate-citrate, ammonium sulphate and sodium phosphate salted papers the following

TABLE IV

Spread ratios for opium alkaloids by chromatography on different salt stabilized media

Salt

Acetate

Phosphate citrate

Phosphate

Sulphate

pH.
4.19 5.10 4.23 5.35
Ratio.
1.48 2.00 2.74 6.66

The ammonium sulphate system has the largest spread between the papaverine, the leading spot on the chromatogram, and morphine, the major trailing spot. Thus, 6.66 is the largest spread ratio and indicates the system in the set, best suited for resolving the largest number of alkaloids.

Reproducibility of R f values is dependent upon the concentration of the substance whose R f is being determined. The fluctuations in R f values for substances from mixtures of pure drugs and raw opium may be seen in table III. The concentration ranges vary from about 2 to 250 µg of alkaloid per spot. The change in R f is also apparently affected by the naturally occurring resins and other material present in opium, which accounts for the apparent spread in R f values in table III.

One very important feature of paper chromatography is the ease with which mixtures of ultramicro amounts (less than 100 microgrammes) can be resolved quantitatively. The sensitivity of the method for qualitative identification is limited only by the smallest amount that can be detected by physical and chemical tests applied directly to the paper.

The limits of detection, as shown in table II using transmitted ultraviolet light (3660 ?), vary from 0.5 to 150 µg for compounds which fluoresce like the isoquinoline alkaloid, papaverine, and those which absorb ultraviolet radiation like the hydro-phenanthrene alkaloid, codeine. When the wavelength of the light for the detection is closer to the absorption maximum of the phenantherene alkaloids (2850 ?) the limit of detection is improved from 3 to 5 times. Using 2537 ?) light, 3 µg of thebaine were detected. It should be observed that the molar extinction coefficient value for thebaine at the maximum is about 5 times that of morphine and codeine, and that the increase in detectability on ammonium sulphate paper is of the same order. In table V some sensitivities of opiates detectable with ultraviolet light and potassium iodoplatinate which were listed in table II are compared with those obtained by Mannering et al [ 10] . These authors used Whatman No. 1 paper sprayed with Sorenson buffer (sodium monohydrogen phosphate mixed with potassium dihydrogen phosphate) and developed the chromatogram with an isoamyl alcohol-acetic-acid-water saturated solvent. A "Menlo Fluoretor" with a maximum transmission of 3250 ? was the ultraviolet source used by Mannering et al. for detection of alkaloids on the paper along with an iodoplatinate spray.

Comparison of observed limits of detection with those obtained by Mannering et al. phosphate-treated papers

TABLE. V

Comparison of observed limits of detection with those obtained by Mannering et al. phosphate-treated papers

Author

Our value

Mannering et al.

Our value

Mannering et al.

Our value

Alkaloid name
Ultraviolet - Wavelength, A
       
2537 3250 3660
K-iodoplatinate
   
Morphine
30 50 150 2 1
Codeine
45 25 150 2 3
Thebaine
3 15 150 2 4
Papaverine
0.5 1 0.5 3 3
Narcotine
1 5 1 5 5
Narccine
30 20 150 35 30

The sensitivities found in this laboratory are in general of the same order as those obtained by Mannering et al. The amount of drug detected using ultraviolet light remains about the same for isoquinoline alkaloids, papaverine and narcotine for all light sources, with the exception of narceine which decreases remarkably in sensitivity as the wavelength shifts from 3250 to 3660 ?. The minimum amounts of alka- loids detected by the chemical reaction on the paper with potassium iodoplatinate by Mannering et al. [ 10] and by ourselves are quite comparable. Slight differences in sensitivities with which the chromatograms were treated may account for the difference in concentration of the salts. In Mannering's work, the buffer was sprayed on and dried, and in our work the papers were dipped.

Büchi & Schumacher [ 4] , on the other hand, report a lower sensitivity - namely, 10 µg for morphine, codeine, thebaine, papaverine and narcotine. They used the Zaffaroni-modification (19) of the iodoplatinate spray whereas, in Mannering's and our studies, the spray as suggested by Munier & Machebœuf [ 12] has been employed.

Samples of opium may be compared with one another by means of paper chromatography. The qualitative comparison of the composition may be carried out, and the approximate quantities of their constituents estimated. The results of experiments with two means of detection and estimation, by ultraviolet light examination, and by spraying with potassium iodoplatinate reagent have been discussed. For opium origin determinations, samples of opium whose similarities of origin have been determined by other physicalchemical methods may be compared on a single sheet. The unknown placed on the centre spot of the origin line is flanked with samples of known origin, and similarities of composition may then be determined chromatographically. Figure 5 shows sample U.S. seizure 1754 to contain 4 major alkaloids in sufficient quantity to produce colours with potassium iodoplatinate. Samples 168, 167 and 162 do not contain 5 major alkaloids as do the other samples. Samples 168, 167, 162, 166 and 172 Florida seizure have approximately similar codeine concentrations. Morphine content of 168, 166 and 172 are also approximately equivalent, while those of 167 and 162 are slightly higher. The thebaine content of 172, 167, 162 and 166 are similar. The thebaine concentration of 168 is highest. Comparing the intensifies of the papaverine-narcotine spots, it was observed that 172, 167 and 162 are similar, while the narcotine spot in 166 and 168 are least and most intense respectively. The observations of luminescence colours, and their intensities, have been summarized in table VI. The samples all compare closely in the colours observed, and in

TABLE VI

Comparison of luminescence colour patterns of five opium samples on a chromatogram - Figure 5

 

Sample number

Luminescence colours

168

172

167

162

166

Green
Gr
Gr
Gr
Gr
Gr
Brown
BrL
BrL
BrL
Br
Br
Absorbance
A
A
A
A
A
Blue
BL
BL
B
BL
BL
Yellow
YL
-
-
-
-
Blue
B
B
B
B
B
Blue (Brown)
B (Br)
B (Br)
B (Br)
B (Br)
B (Br)
Absorbance
A
A
A
A
A
Brown (Green)
Br Gr)
Br Gr)
Br Gr)
-
Br (Gr)
Blue (Brown)
B (Br)
BL (Br)
BL (Br)
BVL
BVL

their intensities. Samples 172 and 167 are very similar. The approximate quantities of opiates present in nine samples, considered to be of similar origin, were compared and summarized in table VII.

TABLE VII

Comparison of alkaloids present in the nine opium samples shown in figure 5

 

Sample number and approximate quantity of opiate

Alkaloid name

165

171

824

830

168

172

167

162

166

Papaverine / narcotine
5+
1+
4+
3+
6+
3+
3+
2+
1+
Thebaine
1+
3+
3+
3+
2+
2+
2+
1+
1+
Unknown
2+
2+
3+
3+
0+
0+
0'
0
1+
Codeine
1+
1+
6+
2+
4+
3+
3+
2+
3+
Morphine
1+
1+
6+
4+
2+
2+
3+
3+
2+

The conclusion from a comparison of the luminescence pattern and approximate relative alkaloidal composition is that samples 167 and 172 are practically identical, and closely similar to 168 and 162.

B. Manufactured and Synthetic Narcotics

A second objective of the chromatographic study was to compare the R f values of substances related chemically and physiologically to those of the opiates. Paper chromatographic studies of manufactured and synthetic narcotics can be found in our review of the literature [ 7] . The paper chromatographic behaviour of most of the narcotics, under international control, and of related compounds which may be found in connexion with narcotics, were studied and catalogued with the chromatographic systems described in section 2 of this article.

Tables VIII and IX give a survey of these results obtained in the Iso-BuOH : AcOH : H 2O system on paper salted with phosphate and sulphate. The name of the compounds accompanied by (N) designates a narcotic under international control. The international non-proprietary names were used throughout wherever applicable. The paper chromatographic properties of a compound were characterized by the R f values shown in column 3, tables VIII and IX. * R x values (R f relative to a standard compound) have been used in the narcotics field by Goldbaum [ 8] and Nadeau [ 14] (codeine = R COD), and by Thomas & Roland [ 18] (morphine = R MOR). The R COD and R MOR values are given in columns 4 and 5, respectively. Further characterization of a compound has been obtained by noting the colour of the stained spot after the potassium iodoplatinate spray (column 6), and by examining the chromatogram prior to spraying under the ultraviolet light of 2537 ? and 3660 ? (columns 7 and 8). The R f, R MOR and R COD values represent averages of 8 or 10 spots obtained from 4 or 5 different chromatograms. Fifty microgrammes of each compound have been used for each spot. The reproducibility of the R f values was +0.02.

Tables VIII and IX may be found on pp. 22 and 23.

Tables VIII and IX illustrate the possibility of separating narcotics and related compounds of widely different chemical structure but similar pharmacological properties. The compounds are arranged according to increasing R f value.

Conclusions

A method for the chromatographic examination of opium and its alkaloids and related compounds has been devised. The method, including reagents, materials, chemicals and equipment for the procedure, has been described. Typical patterns obtained on chromatographing the pure alkaloids and opium samples have been shown, and the main factors controlling the R f values have been investigated. R f values for the major alkaloids of opium under different conditions of development, paper treatment, etc., have been reported. The effect of ion composition and pH of the supporting paper has been tabulated. R f values for 64 narcotics and related compounds in two different media have been listed. The limits of detection of the major alkaloids of opium have been investigated, and the minimum quantities (microgrammes) detected by physical and chemical means have been discussed. The method for obtaining the approximate alkaloidal composition of a number of opiums has been discussed in relation to an opium origin determination.

Acknowledgements

The authors wish to acknowledge the receipt of gifts of drugs from various pharmaceutical manufacturers and the preparation of thebenine, morphothebaine and codeinone supplied by Mr. E. G. Clair. The authors thank Dr. L. I. Pugsley, Dr. R. A. Chapman, and Mr. K. C. Hossick for their continued interest in this research.

Full size image: 23 kB

FIGURE 1. CHROMATOGRAM OF PURE ALKALOIDS AND OPIUM EXTRACTS AFTER K-IODOPLATTINATE SPRAY Figure 1. CHROMATOGRAMME D'ALCALOIDES PURS ET D'EXTRAITS D' OPIUM APRÈS PULVÉRISATION D'IODOPLATINATE DE POTASSIUM

Full size image: 24 kB

FIGURE 2. CHROMATOGRAM OF PURE ALKALOIDS AND OPIUM EXTRACTS WITH TRANSMITTED UV LIGHT

Figure 2. CHROMATOGRAMME D'ALCALOIDES PURS ET D'EXTRAITS D'OPIUM EN LUMIÈRE ULTRAVIOLETTE TRANSMISE FIGURE 3. CHROMOTOGRAM OF PURE ALKALOIDS AND OPIUM- SPRAYED WITH K-IODOPLATINATE

Figure 3. CHROMATOGRAMME D'ALCALOIDES PURS ET D'EXTRAITS D' OPIUM APRÈS PULVÉRISATION D'IODOPLATINATE DE POTASSIUM 65 171 824 830 168 172 167 162 166

Figure 4. Chromatogram of opium samples of different origins under transmitted uv light Figure 4. CHROMATOGRAMME D'ÉCHANTILLONS D'OPIUM D'ORIGINS DIVERSES EN LUMIÈRE ULTRAVIOLETTE TRANSMISSE

Full size image: 22 kB

Figure 5. Chromatogram of opium samples from sources (origin). same as fig. 4, but after K-iodoplatinate spray Figure 5. Chromatogramme d'échantillons d'opium d'origines diverses effectué dans lesmêmes conditions qu' a la figure 4, mais après pulvérisations d'iodoplatinate de potasium

Full size image: 32 kB

Figure 6. TWO-DIMENSIONAL CHROMATOGRAM OF EAST PUNJAB INDIAN OPIUM

TABLE VIII. - Paper chromatographic data of narcotics and related compounds

Solvent: Iso-BuOH : AcOH : H 2 O (10: 1 : 2.4) - Paper: Whatman No. 3 MM, salted with 0.5 M KH 2 PO 4

 

Observations with transmitted ultraviolet light, A wavelength

 

Name of compound

N*

R f

R codeine

R morphine

Colour with K-iodo-platinate

3660

2537

Remarks

Meconic acid
  0.04 0.08 0.18
-
B
B
 
Pholcodine
N
0.08 0.16 0.24
V
-
A
 
Pseudomorphine
  0.09 0.18 0.26
V
-
B
 
Dihydromorphine
N
0.30 0.61 0.88
B
AL
AL
 
Thebacon
N
0.32 0.65 0.94
VL
AL
A
 
Morphine-N-oxide
N
0.33 0.67 0.97
-
A
A
 
Morphine
N
0.34 0.69 1.00
B
AVL
A
 
Oxycodone
N
0.34 0.69 1.00
VL
AL
A
 
Hydrocodone
N
0.46 0.94 1.30
BV
AL
A
 
Metopon
N
0.46 0.94 1.40
B
-
-
 
Nalorphine
  0.48 0.98 1.40
BV
AL
A
 
Codeine
N
0.49 1.00 1.40
BV
AL
A
 
Dihydrocodeine
N
0.56 1.14 1.65
BV
AL
A
 
α-Monoacetylmorphine
N
0.56 1.14 1.65      
Fluorescence at higher
Cryptopine
  0.56 1.14 1.65
V
OI
O
R fthan stain
Morphothebaine
  0.58 1.18 1.70
V
VL
B(AL)
 
Sinomenine
  0.58 1.18 1.70
V
A
A
 
Methylketobemidone
  0.65 1.32 1.91
BV
-
A
 
Ethylmorphine
N
0.66 1.34 1.94
V
AL
A
 
Apomorphine
  0.68 1.39 2.00
V→B
A
BG
Grey-B at daylight
Diacetylmorphine
N
0.73 1.49 2.14
V
-
-
 
Thebenine
  0.78 1.59 2.14
V
A
A
Becomes Br at daylight
Hydroxypethidine
N
0.79 1.61 2.32
V
-
-
 
Ketobemidone
N
0.80 1.63 2.35
V
-
AL
 
Cotarnine
  0.80 1.63 2.36
RV
Ora
ORa
a(R =0.5)
Narceine
  0.80 1.63 2.36
BV
AL
A
 
Acetoketobemidone
  0.80 1.63 2.36
V
-
-
 
Propylketobemidone
  0.81 1.65 2.38
V
-
-
 
Opianic acid
  0.82 1.67 2.41
-
-
A
 
Cocaine
N
0.83 1.70 2.44
V
A
A
 
Thebaine
N
0.85 1.73 2.50
RV
AL
A
 
Hydromorphone
N
0.87 1.78 2.56
BV
AL
A
 
Papaverine
  0.88 1.80 2.59
V
YG
Y
 
Narcotine
  0.88 1.80 2.59
V
BG
B
 
Dimethylthiambutene
N
0.88 1.80 2.59
V
-
A
 
Diethylthiambutene
N
0.89 1.82 2.62
V
-
A
 
Ethylmethylthiambutene
N
0.90 1.84 2.64
V
-
A
 
Codeinone
  0.91 1.85 2.68
V
YL(A)
YL(A)
Becomes Br at daylight
Pethidine
N
0.91 1.85 2.68
V
-
-
 
Alphaprodine
N
0.91 1.85 2.68
V
-
-
 
Levorphanol
N
0.92 1.88 2.70
V
-
-
 
Dextrorphan
  0.92 1.88 2.70
V
-
-
 
Ethoheptazine
  0.93 1.90 2.73
V
-
B
 
Benzylmorphine
N
0.94 1.92 2.76
V
AL
A
 
Acetylmorphenol
  0.94 1.92 2.76
-
-
VL
 
Diacetylmorphol
  0.95 1.94 2.79
-
-
BVL
 
l-2-Methoxy-N-methylmorphinan
N
0.96 1.96 2.82
V
-
-
 
Methylmorphenol
  0.96 1.96 2.82
-
-
VL
 
Dioxyline
  0.96 1.96 2.82
BrVL
Y
Y
 
Ethylnarceine
  0.98 2.00 2.88
VL
-
A
 
Xanthaline
 
0.98†
2.00 2.88
BrV
A
A
Y at daylight
Racemethorphan
N
0.98 2.00 2.88
BV
-
-
 
Dextromethorphan
  0.99 2.02 2.91
BV
-
-
 
Levomethorphan
N
0.99 2.02 2.91
BV
-
-
 
Phenadoxone
N
0.99 2.02 2.91
RVL
-
-
 
Methadone
N
1.00 2.04 2.94
V
-
AL
 
l-Isomethadone
N
1.00 2.04 2.94
V
-
-
 
d-Isomethadone
N
1.00 2.04 2.94
V
-
-
 
Dipipanone
N
1.00 2.04 2.94
V
-
-
 
Alphaacetylmethadol
N
1.00 2.04 2.94
V
-
-
 
Alphamethadol
N
1.00 2.04 2.94
V
-
-
 
Betaacetylmethadol
N
1.00 2.04 2.94
V
-
-
 
Betamethadol
N
1.00 2.04 2.94
V
-
-
 
Ethylpethidine
  1.00 2.04 2.94
V
-
-
 

N* = Internationally controlled narcotic. † Tailing.

TABLE IX. - Paper chromatographic data of narcotics and related compounds

Solvent: Iso-BuOH : AcOH : H 2 O (10 : 1 : 2.4) - Paper: Whatman No. 3 MM, salted with (NH 4 ) 2 SO 4 (2%)

  Observations with transmitted ultraviolet light, A wavelength  

Name of compound

N*

R f

R codeine

R morphine

Colour with K-iodo-platinate

3660

2537

Remarks

Pholcodine
N
0.03 0.13 0.23
BV
-
-
 
Pseudomorphine
  0.03 0.13 0.23
BV
-
B
 
Meconic acid
  0.10 0.42 0.77
-
B
B
 
Morphine
N
0.13 0.54 1.00
B
AL
A
 
Dihydromorphine
N
0.14 0.58 1.08
B
AL
A
 
Thebacon
N
0.16 0.67 1.23
Br V→RV
AL
A
 
Morphine-N-oxide
  0.18 0.75 1.38
P (fading)
AL
A
 
Metopon
N
0.20 0.83 1.54
V
AL
A
 
Oxycodone
N
0.22 0.92 1.69
VL
AL
A
 
Codeine
N
0.24 1.00 1.85
BV
AL
A
 
Nalorphine
  0.24 1.00 1.85
VB
AL
A
 
Hydrocodone
N
0.26 1.08  
BV
AL
A
 
Morphothebaine
 
0.32 ††
1.33 2.46
V
VL
B(AL)
 
α-Monoacetylmorphine
N
0.32 1.33 2.46
V
-
AL
 
Methylketobemidone
  0.33 1.37 2.54
V
-
AL
 
Dihydrocodeine
N
0.33 1.37 2.54
BV
AL
A
Fluorescence at higher R f than stain
Cryptopine
  0.34 1.42 2.61
V
OI
O
 
Sinomenine
  0.40 1.67 3.08
V (Br halo)
A
A
 
Apomorphine
  0.44 1.83 3.39
V
A
BG
 
Ethylmorphine .
N
0.45 1.88 3.46
BV
AL
A
 
Diacetylmorphine
N
0.50 2.08 3.85
AQU→V
-
-
 
Thebenine
  0.58 2.41 4.47
RV †
V
AL
Becomes Br at daylight
Ketobemidone
N
0.58 2.41 4.47
AQU→V
-
AVL
Fluorescence at higher R f than stain
Cotarnine
  0.63 2.62 4.85
RV
Or
Or
 
Hydromorphone
N
0.64 2.66 4.92
B(V)→V
AL
A
 
Thebaine
N
0.67 2.79 5.16
V
AL
A
 
Cocaine
N
0.67 2.79 5.16
V
AL
A
 
Hydroxypethidine
N
0.70 2.92 5.38
V
-
-
 
Acetoketobemidone
  0.71 2.96 5.46
AQU→V
-
-
 
Narceine
  0.75 3.12 5.77
Br(V)
AL
A
 
Ethoheptazine
  0.76 3.17 5.85
V
-
-
 
Levorphanol
N
0.77 3.21 5.92
V
-
-
 
Dextrorphan
  0.77 3.21 5.92
V
-
-
 
Pethidine
N
0.77 3.21 5.92
V
-
-
 
Propylketobemidone
  0.77 3.21 5.92
V (W halo)
-
-
 
Papaverine
  0.78 3.25 6.00
V
YG
YG
 
Narcotine
  0.78 3.25 6.00
PBr(V)
B
B
 
Codeinone
  0.78 3.25 6.00
V
GY(V )
GY(A)
Becomes Br at daylight
Alphaprodine
N
0.81 3.37 6.22
AQU→V
-
-
 
Benzylmorphine
N
0.84 3.50 6.46
V
AL
A
 
Ethylpethidine
  0.86 3.58 6.61
VL
-
-
 
Dimethylthiambutene
N
0.87 3.62 6.70
RV
-
A
 
Dioxyline
  0.87 3.62 6.70
BrVL
Y
Y
 
Ethylmethylthiambutene
N
0.89 3.70 6.85
RV
-
A
 
Diethylthiambutene
N
0.90 3.75 6.92
RV
-
A
 
l-2-Methoxy-N-methylmorphinan
N
0.91 3.80 7.00
AQU→V
-
-
 
Dextromethorphan
  0.92 3.83 7.07
AQU→V
-
-
 
Levomethorphan
N
0.92 3.83 7.07
AQU→V
-
-
 
Racemethorphan
N
0.92 3.83 7.07
AQU→V
-
-
 
Opianic acid
  0.92 3.83 7.08
-
VVL
A
 
Acetylmorphenol
  0.93 3.87 7.15
-
-
VL
 
Diacetylmorphol
  0.94 3.92 7.23
-
-
VL
 
Alphamethadol
N
0.94 3.92 7.23
RV
-
-
 
Betamethadol
N
0.94 3.92 7.23
RV
-
-
 
Ethylnarceine
  0.94 3.92 7.23
V
-
A
 
Methadone
N
0.96 4.00 7.38
V
-
-
 
d-Isomethadone
N
0.96 4.00 7.38
RV
-
-
 
l-Isomethadone
N
0.96 4.00 7.38
RV
-
-
 
Xanthaline
 
0.98 †
4.08 7.55
BrV †
A
A
Y at daylight
Methylmorphenol
  0.98 4.08 7.55
-
AL
A
 
Phenadoxone
N
0.98 4.08 7.55
RV(B)→RV
-
-
 
Alphaacetylmethadol
N
1.00 4.16 7.70
RV
-
-
 
Betaacetylmethadol
N
1.00 4.16 7.70
RV
-
-
 
Dipipanone
N
1.00 4.16 7.70
RV
-
-
 

N* = Internationally controlled narcotic. † Tailing †† Elongated spot.

References

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002

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003

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004

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005

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006

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007

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008

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009

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010

MANNERING, G.J.; DIXON, A. C.; CARROLL, N. V.; COPE, O. B., Paper chromatography applied to the detection of opium alkaloids in urine and tissues. The Journal of Laboratory and Clinical Medicine, 1954, 44, 292-300.

011

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012

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013

MUNIER, R., Bases azotées (Alcaloides, amines, vitamines) et acides organiques hydrosolubles. Bulletin de la Société de Chimie de France, 1952, 852-873.

014

NADEAU, G., Note on the identification of alkaloids by paper chromatography. Clinical Chemistry, 1956, 2, 347-352.

015

NADEAU, G.; SOBOLEWSKI, G.; FISET, L.; FARMILO, C. G., Separation and identification of major alkaloids from opium by partition chromatography on paper. Journal of Chromatography, 1958, 1, 327-337.

016

PFEIFER, S., Zur Papierchromatographie der wichtigsten Mohnalkaloide (Paper chromatography of the major alkaloids of poppy). Scientia Pharmaceutica, 1956, 24, 84-92.

017

RESPLANDY, A., Chromatographie sur papier d'alcaloides par des solutions d'électrolytes. Comptes rendus hebdomadaires des séances de l'Académie des Sciences, 1954, 238, 2527-2529.

018

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019

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