Quantitative Determination of Morphine in Opium by Paper Chromatography and Spectrophotometry

by Haruyo Asahina, & Masako Ono - National Hygienic Laboratory, Tokyo, Japan 1956

Quantitative Determination of Morphine in Opium by Paper Chromatography and Spectrophotometry

INTRODUCTION

Paper chromatography was used with good results by the authors for the determination of morphine in opium tincture.*

By this method, we now estimate the morphine content in opium. As only a small quantity of opium is employed, it is possible to determine the morphine content of opium from one poppy capsule - i.e., between 0.03 and 0.5 g of opium per poppy head, and to compare the morphine content in opium from successive lancings of the same poppy capsules. We have confirmed the decline in morphine content with successive lancings.

PROCEDURE

Extraction

The extraction of morphine with hydrochloric acid was found to be satisfactory. The other opium alkaloids and the coloured substances were easily separated from morphine in the paper-chromatographic step.

About 0.1 g of air-dried opium was accurately weighed and 1 cc. of N/10 hydrochloric acid (precisely measured) was added, and the mixture was rubbed with a glass rod to a homogeneous paste. The resulting suspension was centrifuged at 2,000 r.p.m. for thirty minutes and 0.003 cc. of the supernatant solution was measured by a micro-pipette with accuracy.

Chromatography

Chromatography of an opium tincture made to contain 1% morphine was carried out in the same container, in order to obtain a calibration curve. In this case, 0.001, 0.003, 0.006 cc of opium tincture (10 γ, 30 γ, 60 γ of morphine) was applied to the paper.

Solvent : n-butanol 50, 28% ammonia 9, distilled water 15 parts by volume. The top layer of the mixture, 40 cc. for each container, was used.

Paper : Toyo No. 51, 2.5 cm × 40 cm.

Container : inner diameter 9.5 cm., height 45 cm.

Starting line : 7 cm from the lower edge of paper.

At the start of the chromatography, about 0.5 cm. of paper dipped in the solvent.

Height of development: approximately 25 cm. above the starting line.

Measurement of Absorbance by Beckman Spectrophotometer

The maximum absorbances of the morphine spots on the paper of both the opium tincture and the opium extract were determined at 286 mµ.

Recently we have devised a simple apparatus for passing the filter-paper which is more convenient than the one previously used. In place of the sample chamber, an accessory of our making (fig. l) is attached tightly to the Beckman spectrophotometer DU by using the part of the accessory for fluorescence.

FIGURE 1. Front view (scale ½)

* See Document ST/SOA/SER.K/40, 25 August 1955.

Through A of our accessory, the filter paper is passed horizontally (fig. 2). The strips of filter paper on which the chromatograms are developed are 2.5 cm wide. The height of A is 2.5 cm - that is, the width of the strip of filter-paper. The thickness of the slot A is just a little more than that of the filter-paper.

After development with solvent in the chromatography, the strip of filter-paper is fastened to the end of a celluloid plate (length about 25 cm, width exactly 2.5 cm) with cello-tape (fig. 3), and this plate is used to pull the strip of filter-paper through the apparatus.

FIGURE 2. Side view

FIGURE 3.

Pulling the celluloid plate slowly by hand, the place of maximum morphine absorbance on the filter-paper is searched for, while the absorbance of the other part of the filter-paper (where there is no spot) is set at zero.

Calculation

From the calibration curve of the opium tincture and the absorbance of the morphine spot of the opium extract thus obtained, the quantity of morphine in 0.003 cc. of the opium extract was determined, and from it the content of morphine in the opium was calculated.

TABLE A

Variation of weight and morphine content of opium from individual capsules

Variation of weight and morphine contentof opium from individual capsules

TABLE B

TABLE B (continued)

- Number of capsules in different classes

TABLE 2

Weight of opium from successive lancings

(In grammes)

Opium from Successive Lancings

The variation of weight and morphine content of opium samples from successive lancings was examined. The following 15 air-dried opium samples from Papaver somniferum were produced during 1954-55.

TABLE 1 - Origin of opium sample

(Compare with Tables 2 to 7)

TABLE 3

Percentage of Weight form each lancing to total weight

TABLE 4

Morphine content of opium from successive lancings

(Percentage)

Reference

Method used for morphine in opium

A : Paper chromatography and spectrophotometry.

B : Slightly modified Japanese Pharmacopoeia VI using 1g. of opium.

TABLE 5

Calculated morphine quantity

(In milligrammes)

Two Collections from each Lancing

The above samples 14 and 15 represent collections from the same capsules of the Ikkanshu variety, No. 14 the Kiritori collection and No. 15 the Asabuki collection.

In the Kiritori collection the unripe capsule is incised, and immediately afterward the latex which flows out is taken from the capsule. Its meaning is "cutting" (kiri) and "taking from" (tori).

The asabuki collection is made from the same lancing, and is the juice collected early the following morning. Its meaning is "flowing out" (buki) "at dawn" (asa).

The cultivators commonly mix the opiums obtained in these two collections, but they were kept separate in these trials.

TABLE 6

Percentage of morphine quantity from each lancing to total morphine

TABLE 7

Calculated morphine content of opium from all thelancings

Methods of Successive Incision

We have also made a comparison of opiums obtained by two different methods of successive incision, setsuzoku-kiri and hantai-kiri. The cultivator commonly incises every second day until no more latex flows from the capsule. This usually requires four lancings. Vertical incisions are made by means of a knife having three edges.

The ribs of the poppy capsules are popularly called" mountains ". In both methods, two adjacent ribs or" mountains" are incised at the first lancing. Setsuzoku-kiri (adjacent cutting), means that at the second lancing three ribs adjacent to the first two are incised, and so on around the capsule at the third and fourth lancings. Hantai-kiri (opposite cutting)

FIGURE 5. Poppy head : first lancing

means that at the second lancing three ribs adjacent to each other but opposite to the two already cut are incised. The third lancing is then made by incising three "mountains" between those cut in the first and second lancings, and the fourth lancing is then opposite to the third. Setsuzoku-kiri is usually used, as it is less laborious.

In these trials, poppy plants of the Ikkanshu variety were cultivated in two separate areas. The soil of area B was more fertile than that of area A.

FIGURE 6. Knife for incision

The opium produced in the more fertile area had a higher morphine percentage. Differences between the results by the two methods of successive incision in these two trials are not considered sufficiently great to establish that they have any significance.

This year we shall study this problem in greater detail.

COMPARISON OF ASSAY RESULTS

In the following table, results of morphine determinations assayed on various samples of opium of different origin by our method are compared with results obtained on the same samples with the Japanese Pharmacopoeia VI method.

FIGURE 7. Cross-section o, poppy head

Order of lancings

Opium from Papaver setigerum DC

Papaver setigerum DC is included in the Japanese Opium Law. We obtained some seed by the courtesy of Dr. Charles G. Farmilo, Organic Chemistry and Narcotic Section, Food and Drug Laboratories, Department of National Health and Welfare, Ottawa, Canada, and cultivated this poppy experimentally at the Kasukabe Experiment Station of Medicinal Plants attached to the National Hygienic Laboratory, Saitama, Japan.

The opium from Papaver setigerum gave the following results on analysis:

Morphine content by our method of paper chromatography and spectrophotometry for morphine in opium, 5.1%;

Porphyroxine-meconidine absorbance, Beckman B, 1 cm cell, 1% solution, 0.235.

The presence of codeine and other opium by-alkaloids was demonstrated by the paper chromatogram.

CONCLUSIONS

The quantity of opium fell off rapidly with later incisions in a substantial number of samples.

As shown in Table 4, 11 opium samples out of 15 showed a marked decline in the morphine content. Of these, only one sample, No. 1, contained less than 15% of morphine in the first lancing.

In opium samples Nos. 2, 6, 10 and 12, the change of morphine content was not regular. These had comparatively low morphine content in the first lancing, and the morphine content of the second incision was higher than that of the first incision (Nos. 2, 6, 10).

When the first morphine content was high, a large and steady decline occurred, as previously shown by Annett and Machiguchi; but when it was low, the second morphine content increased in comparatively small degree.

The quantity of morphine in opium from successive lancings of the same poppy capsules was calculated and is given in Table 5. The figures generally show a definite diminution of morphine quantity with repeated incisions, in most of these opium samples.

It was thus possible to demonstrate that the chief reason for the lower morphine content of Japanese opium as compared with that of Turkey, Yugoslavia, Greece, and Bulgaria, where it is customary to bleed each capsule only once, is the successive scarifications of the poppy capsules in Japan.

For example, the first morphine content of Japanese opium (Ikkanshu), Nos. 14, 15, was more than 20%. But usually in our country, the capsules are incised repeatedly, four times on different days, and all the opium so obtained is mixed together. The mixed opium sample, Nos. 14, 15, contained 17.5 % of morphine, whereas the morphine content of the first opium from these capsules was 24.8% and 21.4% respectively.

See United Nations document ST/SOA/Ser. K/40.