Quantitative Determination of Morphine in Opium by Paper Chromatography and Spectrophotometry

Sections

INTRODUCTION
PROCEDURE
COMPARISON OF ASSAY RESULTS
CONCLUSIONS

Details

Author: Haruyo Asahina, Masako Ono
Pages: 39 to 44
Creation Date: 1956/01/01

Quantitative Determination of Morphine in Opium by Paper Chromatography and Spectrophotometry

Haruyo Asahina
Masako Ono
National Hygienic Laboratory, Tokyo, Japan

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

	No.	Opium weight	Morphine content	Morphine quantity			No.	Opium weight	Morphine content	Morphine quantity
		mg	%	mg				mg	%	mg
1		262.3	13.7	35.9		42		296.3	10.5	31.1
2		105.0	12.4	13.0		43		272.1	10.5	28.6
3		219.0	12.4	27.2		44		227.3	15.3	34.8
4		173.9	11.3	19.7		45		154.2	4.5	6.9
5		229.2	15.0	34.4		46		160.9	11.3	18.2
6		171.8	11.2	19.2		47		277.7	11.9	33.0
7		226.9	5.2	11.8		48		81.9	11.4	9.3
8		94.4	7.1	6.7		49		298.1	18.1	54.0
9		115.1.	12.1	13.9		50		88.3	11.5	10.2
10		184.0	12.5	23.0		51		231.7	12.4	28.7
11		88.2	10.2	9.0		52		124.7	11.3	14.1
12		281.5	10.9	30.7		53		201.0	13.2	26.5
13		130.0	10.2	13.3		54		163.7	12.3	20.1
14		223.9	13.8	30.9		55		92.4	9.3	8.6
15		218.6	12.5	27.3		56		184.3	13.4	24.7
16		54.4	11.3	6.1		57		285.1	13.6	38.8
17		275.7	12.2	33.6		58		219.5	12.9	28.3
18		121.2	15.3	18.5		59		186.7	9.7	18.1
19		186.4	9.7	18.1		60		80.0	17.7	14.2
20		420.8	15.5	65.2		61		59.8	12.0	7.2
21		220.0	9.8	21.6		62		34.8	13.9	4.8
22		136.4	12.9	17.6		63		173.5	9.4	16.3
23		270.1	12.6	34.0		64		95.5	14.3	13.7
24		267.3	12.8	34.2		65		138.1	9.5	13.1
25		469.5	6.4	30.0		66		134.0	6.6	8.8
26		243.4	12.4	30.2		67		172.0	8.9	15.3
27		202.7	13.7	27.8		68		77.7	9.1	7.1
28		192.0	11.0	21.1		69		52.3	13.3	7.0
29		240.9	14.5	34.9		70		154.1	9.2	14.2
30		139.4	8.2	11.4		71		162.6	9.3	15.1
31		106.0	7.0	7.4		72		154.5	10.3	15.9
32		363.4	11.3	41.1		73		158.0	7.8	12.3
33		122.6	5.1	6.3		74		31.7	16.3	5.2
34		215.3	12.7	27.3		75		36.6	12.1	4.4
35		126.9	9.9	12.6		76		223.6	12.3	27.5
36		244.4	11.7	28.6		77		283.7	12.6	35.7
37		247.2	7.9	19.5		78		79.9	14.5	11.6
38		291.9	7.4	21.6		79		126.2	11.8	14.9
39		271.1	13.7	37.1		80		163.4	8.7	14.2
40		279.4	15.7	43.9		81		85.9	7.7	6.6
41		278.6	9.3	25.9		82		305.2	10.9	33.3

Variation of weight and morphine contentof opium from individual capsules

TABLE B

Poppy plant	Japanese origin, Ikkanshu variety
Crop season	1954-55
Cultivator	Kasukabe Botanical Garden
Method of morphine determination	paper chromatography and spectrophotometry

TABLE B (continued)

- Number of capsules in different classes

Opium weight		Morphine content		Morphine quantity
Below 100 mg	16	Below 5%	1	Below 10 mg	16
100-200	30	5-10	24	10-20	28
200-300	32	10-15	49	20-30	17
Above 300	4	Above 15	8	30-40	17
	___		___	40-50	2
TOTAL	82	TOTAL	82	Above 50	2
					- -
				TOTAL	82

TABLE 2

Weight of opium from successive lancings

(In grammes)

Lancing
Opium sample	1st	2nd	3rd	4th	5th
1	0.7642	0.2958
2	0.6773	0.0668
3	0.7105	0.3996	0.0742
4	1.0974	0.6213	0.0608
5	0.6590	0.5194	0.0744
6.	0.3746	0.1989	0.0351
7	0.1488	0.2484	0.0577
8	0.0314	0.0098	0.0202
9	0.0400	0.0253	0.0253
10	0.0721	0.0214	0.0176
11	3.3026	5.4959	2.7627	3.1152	0.0969
12	0.1782	0.1179	0.1614
13	0.2789	0.3300	0.1319
14	22.7462	14.6107	6.7535	5.2203
15	16.6977	15.6183	14.9554	12.3396

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)

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Opium sample	Cultivator	Seed obtained from
1	Kasukabe Botanical Garden	Rome, Italy
2	"	Adelaide, Australia
3	"	Amsterdam, Netherlands
4	"	Torino, Italy
5	"	Braunschweig, Germany
6	"	Amsterdam, Netherlands
7	"	London, England
8	"	Adelaide, Australia
9	"	Amsterdam, Netherlands
10	"	Torino, Italy
11	Drug company in Kyoto	Pakistan
12