A method of isolating porphyroxine from opium

Sections

A. Extraction
B. Purification by selective extraction
C. Chromatographic separation on columns
TABLE I

Details

Author: K. SZENDREI
Pages: 51 to 54
Creation Date: 1968/01/01

A method of isolating porphyroxine from opium

K. SZENDREI
Faculty of Pharmacy, Pharmacognostical Laboratory, Szeged, Hungary1

During recent years definite proof has been found of the existence of porphyroxine, an important opium alkaloid long considered impossible to isolate. After many fruitless attempts [1] , [2] ,[ 3] , [4] , [5] Klayman [6] in 1956, then in 1962 Pfeifer and Teige [7] and Genest and Farmilo [8] isolated in fact independently this "mysterious" alkaloid. Although the characteristics of the substances isolated by the authors mentioned are somewhat different, isolation can be considered as a completely established fact, since Pfeifer and his collaborators have already succeeded in determining also the structure of porphyroxine [9] .

Although isolation and the determination of structure have only been achieved in the last ten years, porphyroxine has played a considerable part since its discovery (1825) particularly in the detection and identification of opium. After Merck discovered it, and gave it a name by reason of its very marked colour reaction, other authors set about utilizing this reaction in the identification of opium. Since 1948 experiments have been conducted, with the encouragement of the United Nations, for determining the geographical origin of opium and in the process at present employed porphyroxine plays an essential role.

Although it is true that porphyroxine has already been isolated several times, the quantities isolated up to the present were so small that the Laboratory had no authentic substance for determining the origin of opium. Moreover, the true pharmacological effects of porphyroxine are still unknown. In experiments carried out previously such contaminated preparations were used that the results achieved do not give any certain indication of the real pharmacological action of porphyroxine.

A detailed analysis of the above-mentioned methods of isolation shows that they are fairly complicated (for example Klayman's method [6] . The simplest method is that of Pfeifer and Teige, but even they used paper chromatography for the final purification, a method which gives us a relatively small amount of the substance. It is therefore reasonable to conclude that the difficulties of isolating porphyroxine in a large quantity arise in fact from the complicated nature of the methods employed, together with its known sensitivity. As part of our plan for the isolation of a larger quantity of the substance we first investigated the possibilities of simplifying methods.

Note: The original of this article is in French.

1 This study was carried out at the United Nations Narcotics Laboratory at Geneva during a research course on a fellowship granted by United Nations Technical Assistance.

A. Extraction

It was Fulton [5] who conducted systematic research on the question of the solubility of porphyroxine in different solvents. His data, compared with those of Pfeifer and Teige, Genest and Farmilo, etc., have already been partially superseded, for instance with regard to its solubility in ether. We therefore took later results into consideration also.

It is clear that porphyroxine is decomposed in an acid solution, but it is known from experiments that this decomposition is slight in a case where the influence is of short duration. Hence we carried out the extraction by means of dilute acetic acid, which is a very good solvent of opium alkaloids.

The opium was homogenized in 100 g portions in a Turmix apparatus with 35 ml of concentrated acetic acid by shaking for 1-2 minutes. The mixture was then diluted with 350 ml of water and again shaken for 1-2 minutes. After 10 minutes' centrifugation the supernatant was filtered through verro fibre silk. The precipitate was then washed again twice with 75 ml of 10% acetic acid.

B. Purification by selective extraction

In the following experiment we tried to eliminate absorbent but non-alkaloidal substances. For that purpose we used the exchange of phase method, well known in alkaloid isolation processes, bearing in mind the fact that ether was one of the best solvents in such a case.

The acetic extract was alkalinized immediately by concentrated ammonia and extracted by ether purified in a liquid-liquid extractor until there was a negative reaction from the porphyroxine. After dehydration we evaporated the solvent by a rotary evaporator in a vacuum.

  1. The chromatogram of the extract from a sample of Japanese opium. Adsorbent - silica gel. The porphyroxine was revealed by heating for 10 minutes before the pulverization of the Dragendorff reagent.

We thus obtained from a Japanese opium sample the dark brown resinous extract of 17.2% which still contained all the important opium alkaloids (see fig. 1) and also a large quantity of impurities.

Subsequently we tried to separate, as far as possible selectively, the porphyroxine from the impurities and other alkaloids. In order to obtain it we made a series of extractions from an acetic solution of the extract, by a series of organic solvents (petrol ether, cyclohexane, benzene, chloroform and ether) on the one hand, and on the other hand by a gradual increase in the pH value of the aqueous phase. The composition of all the fractions obtained was controlled by thin-layer chromatography (see fig. 2) as well as the porphyroxine content obtained by the method of Genest and Farmilo [10] .

  1. Control of preliminary extraction tests. (O = original extract, pe =petrol ether, cy = cyclohexane, bzl = benzene, chlf = chloroform, R = residue).

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The results of these analyses all showed that both factors - the pH value of the aqueous phase and also the polarity of the organic solvent in question - have a considerable influence on the solubility of porphyroxine. From many series of experiments we have concluded that the simplest process is the following: the extract is dissolved in 10 % acetic acid in the proportion of 1:10. This solution must be immediately extracted five times by half-volume ether; it is then possible to eliminate not only most of the narcotine and papaverine but also most of the other impurities. Then the pH is regulated to 7 by 25% ammonia and the extraction is repeated until a negative reaction is obtained from the porphyroxine (7-10 times). The fractions containing porphyroxine are evaporated after dehydration in a vacuum.

C. Chromatographic separation on columns

For chromatographic separation the following adsorbents have hitherto been used:

Florisil (Klayman, Genest and Farmilo);

Ion exchanger resin (Klayman);

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Acid aluminium oxide (Genest and Farmilo);

Paper (Pfeifer and Teige);

A thin layer of silica (Pfeifer and others).

In no case were the authors able to obtain pure porphyroxine by a single chromatographic separation. Later Genest and Farmilo carried out systematic research on the elution of porphyroxine on different adsorbents [10] . They found that out of eight adsorbents controlled (acid, neutral and basic alumina, lactose, florisil, celite, kieselguhre and calcium sulphate), elution on a column of acid alumina is the most complete. That is why in our preliminary tests we compared the latter adsorbent with silica which is also of an acid nature. We also prepared parallel columns 13 mm in diameter and 100 mm wide from acid alumina (Fluka) and silica gel (Merck) using a chloroformic suspension of the adsorbents. 1.1 g of the extract obtained by the phase exchange already referred to was dissolved in chloroform and put on columns. Both columns were eluted by the chloroform, thus obtaining fractions of 5 ml. The composition of the fractions was controlled by chromatography on a thin layer (see figs. 3 and 4) and the efficacy of the elution by the quantitative method of Genest can be much more selectively eluted from a silica gel column; it is crystallized directly from the fractions containing it in the largest quantity. After these preliminary tests we made the separation again on silica eluted by chloroform. The fractions were controlled by chromatography on a thin layer and those containing the porphyroxine were concentrated in a vacuum and the residue was crystallized from methanol. The porphyroxine is crystallized in the form of prismatic crystals. These crystals are washed in chloroform and the substance is recrystallized several times from methanol.

A method of isolating porphyroxine from opium 53

  1. Control of chromatographic separation on silica column.

(The figures indicate the fractions.)

  1. Control of chromatographic separation on acid alumina column.

(The figures indicate the fractions.) and Farmilo. From these results we were able to show that by exceeding complete elution the porphyroxine

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By means of this method (see the table I) it was possible in four parallel tests to isolate the porphyroxine from two different samples of opium with an average yield of 25 per cent. The substance obtained has a melting point of 237 °C and is found to be identical (melting point, infra-red spectrum, chromatographic control) with that isolated by Pfeifer and others.2

TABLE I

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2 The author expresses his sincere thanks to Professor Dr. S. Pfeifer for the authentic porphyroxine placed at his disposal.

BIBLIOGRAPHY

001

Merck, E., Ann. Pharm, 21,201, 1837, Annual report on Progress of Physical Sciences 18, 379, 1839.

002

Hesse, O., Liebig Ann. Chem. 153, 47, 1870.

003

Rakshit, J. N., J. Chem. Soc. (London) 115 (1), 455, 1919.

004

Rajagopolan, S., Current Sci. 12, 24, 1943; J. Org. Chem. 10, 175, 1945.

005

Fulton, C. C., E/CN.7/117, 1948; Bulletin on Narcotics IV, No. 1, 15, 1952.

006

Klayman, D. L., Thesis, Rutgers University, 1956.

007

Pfeifer S., and Teige J., Pharmazie 17, 692, 1962.

008

Genest, K., and Farmilo, C. G., Analytic Chem. 24, 1464, 1962; J. Pharm. Pharmacol. 15, 197, 1963; ST/SOA/SER.K/141.

009

Pfeifer, S., Banerjee, S. K., Dolejs, L., and Hanus V., Pharmazie 20, 45, 1965.

010

Genest, K., and Farmilo, C. G., ST/SOA/SER.K/142