THE SEPARATION AND ESTIMATION OF MORPHINE, PSEUDOMORPHINE, AND RELATED SUBSTANCES.*

BY A. K. BALLS.

(From the Department of Pharmacology, University of Pennsylvania, Philadelphia.)

(Received for publication, October 28, 1926.)

This paper aims to present a number of analytical methods, worked out in this laboratory, for studying systems in which mor- phine is gradually oxidizing. In order to approach a quantitative study it is necessary to possess a scheme of analysis for such rapidly changing systems whereby not only the residual morphine may be estimated, but also some information may be obtained concerning the amount of morphine-derived substances existing at various oxidation levels. The problem of devising such a scheme differs from the usual one of isolating and estimating morphine itself, not only by its object, but also by taking into account the properties of several other substances which must be estimated in the presence of each other.

When a solution of morphine undergoes gentle oxidation, some of the substances formed resemble morphine in many chemical properties. In particular they are capable of further oxidation, hence are reducing agents; their behavior with many reagents of alkaloid chemistry is like that of morphine; they are precipitated by the complex acids of tungsten and molybdenum; and they are comparatively insoluble in solutions near the neutral point.

The first of these oxidation products is theoretically pseudo- morphine, formed by the removal of 1 equivalent of hydrogen per morphine. The occurrence of this substance is a possibility in any morphine oxidation, and its presence has been reported in a great number of cases, such as oxidation by gold or silver salts, by oxygen in alkaline solutions, by hydrogen peroxide, by tyro-

* This work was made possible by a grant from the Committee on Drug Addictions, New York City.

543

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Morphine and Pseudomorphine

sinase-like ferments, and by many other means. Because of the probable formation of pseudomorphine at the very outset of morphine oxidation, its consideration in these methods was regarded as important, and the properties of pseudomorphine were first studied and have been given in a previous paper.

Besides pseudomorphine, a large number of other substances formed during the early course of the oxidation, are precipitated by phosphotungstic and silicotungstic acids, and apparently nearly as completely as morphine itself. If then silicotungstic acid be added to partially oxidized morphine in solution under proper conditions, it will precipitate pseudomorphine, morphine, and a heterogenous group of substances resembling morphine and pseudomorphine in many ways, but containing less hydrogen. To distinguish these substances from pseudomorphine and to classify them (for convenience) as a group, we shall call them “higher oxidation products of morphine,” without regard to the fact that such a group is comprised of many substances probably of widely differing structure, some of which are still active re- ducing agents.

The quantity of these substances is, however, a measure of the amount of morphine oxidized beyond the pseudomorphine stage, and yet not far enough to loose all chemical resemblance to the parent alkaloid. Precipitability by silicotungstic acid becomes for the time being the criterion of this group, but it is not entirely an artificial one, because a very large proportion of the complex bodies formed on oxidation is precipitated in this way. This procedure has the advantage of almost instantaneously removing a large part of the morphine-derived substances from the further effect of the osidant. It allows a mixture of continuously changing composition, in which the constituents sought are unstable, to be fixed at a given time and separated afterward.

The separation has been worked out to allow the determination of morphine-like substances at three levels of oxidation; namely, residual morphine, pseudomorphine, and the empirical class of higher oxidation products. From such analytical data, and in systems originally containing known amounts of morphine and oxidant, five variables may be evaluated as aids in following the reaction.

From the mixture of silicotungstates, pseudomorphine is sepa-

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A. K. Balls

rat,ed first, and its removal allows an accurate determination of morphine, which is not possible, at least by any of the commonly described methods, in the presence of pseudomorphine. The morphine is next removed from the higher oxidation products and may be determined by any of the numerous accepted procedures. The precipitation method described is selected because it has given very satisfactory results.

The behavior of morphine in biological material such as animal tissues cannot be followed so easily. The estimation of morphine under such circumstances has received much attention and an extensive literature describes the work. The methods all depend ultimately upon the isolation of morphine in some comparatively pure form, and the presence of pseudomorphine would never interfere, because if present, it would be excluded by the various processes of protein removal, clarification, and alkaloid extraction which in one form or another invariably accompany all our mor- phine methods. Only a few attempts have been made, however, to measure the pseudomorphine. Marme (1) sought to do this, apparently using methods resembling those for morphine, to which, for this purpose, objection has just been taken. Donath (2) was able to estimate both bases, but in urine only, and hardly with satisfying accuracy. In this laboratory a method of deter- mining both morphine and pseudomorphine when together in biological material has been developed, but in such a complex mixture no way of estimating the amount of “higher oxidation products” has been successful. This is because proteins, pro- teoses, and some peptones are also precipitated with silicotungstic acid, while the prior removal of these interfering substances would here, as in the standard methods for morphine, carry off prac- tically all the pseudomorphine, and much of the more highly oxidized group.

An account of the materials used in testing these methods is given below, followed by descriptions of the details in determining the individual -bases. The scheme of separating the mixed silico- tungstates is next outlined, and finally a description of the modification adapted to biological mat’erial is given.

Preparation of Materials.

Morphine.-Pure morphine was prepared from the commercial hydrochloride by a method designed primarily to eliminate pseudo-

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546 Morphine and Pseudomorphine

morphine and the other oxidation products. The acetic acid solution was warmed with decolorizing carbon, and the base precipitated first by ammonia and secondly after solution in caustic potash, with acetic acid, adjusting the reaction to about pH = 9.0. Kolthoff (3) has determined the isoelectric point of morphine at pH = 8.96. The free base was redissolved in hydro- chloric acid and salted out of this solution by adding about 5 volumes per cent of the concentrated acid. At this concentration the morphine hydrochloride exhibits a maximum insolubility. The hydrochloride was then dissolved and crystallized out of pure methyl alcohol, washed with ethyl alcohol, and dried under reduced pressure. A practically anhydrous morphine hydro- chloride was obtained, with a specific rotation [a], = -96.4 ho.4 at 27” on a 2 per cent solution (calculated to a basis of 3Hz0 of crystallization). On further recrystallization from water this rotation did not change appreciably. [o(& = ‘95.9 (2 per cent solution).

The free base was prepared from this hydrochloride by precipi- tation with ammonia, solution in boiling absolute alcohol, and separation from this solution by chilling. Washed with alcohol and vacuum-dried, it was a white, feathery, beautifully crystalline substance containing 6.22 per cent water, which corresponds to slightly more than 1 mo. of water of crystallization.

Pseudomorphine.-The preparation of pseudomorphine hydro- ,chloride has been described in a preceding paper.

H&her Oxidation Products.-A solution of the higher oxidation products of morphine was made by treating the slightly alkaline solution of commercial hydrochloride with successive additions of 3 per cent hydrogen peroxide for 24 hours on the water bath. On testing the dark colored solution, it was found to contain neither morphine nor pseudomorphine. It was then acidified with hydrochloric acid, evaporated to dryness, to remove hydrogen peroxide and free acid, dissolved in water with a small amount of potassium bicarbonate and filtered to remove a small amount of insoluble matter.

The morphine-derived substances in this solution were precipi- tated by silicotungstic acid, but the precipitate is more readily soluble in water than that formed from morphine itself. They were partially precipitated by strong acids, completely and readily

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A. K. Balls 547

soluble in even very weak alkali. On evaporating the acid solu- tion, these dark colored substances were partially soluble in the higher alcohols, chloroform, and benzyl alcohol; nearly insoluble in the liquid hydrocarbons, lower alcohols, and ether. From alkaline solutions, they were not removed by any of these solvents. These substances resemble morphine in their qualitative properties only by precipitability with most of the alkaloidal reagents, such as the derivatives of tungstic and molybdic acids, picrolonic acid, trichloroacetic acid, salts of lead, mercury, gold, platinum, and the like. They are obviously more acid in character than either morphine or pseudomorphine.

The precipitate with silicotungstic acid, when dried at 120” contains about 64 per cent silica and tungsten oxide, which is less than the value for the corresponding morphine precipitate.

TABLE I. Determination of Morphine as Silicotungstate.

I Morphine (free base). I

Gsmple Nq. Taken.

0m.

1 0.0815 2 0.1630 3 0.0818 4 0.0505

Found. Calc~l~eri,from

Calculated from dried precipitate.

- I

0m. 0m.

0.0807 0.0807 0.1622 0.1640 0.0818 0.0815

Calculated from loas on igniting.

0m.

0.0807 0.1594

Determination of Morphine as Silicotungstate.

Bertrand (4) showed that morphine silicotungstate dried at 120” has a constant composition: SiOz.Hz0*12W03*4 morphine + 3H,O. As a means of estimating morphine it is open to two objections, appreciable solubility and a tendency to form colloidal suspensions. These difficulties are overcome by using solutions of small volume containing a strong electrolyte, in which the freshly formed precipitate is coagulated by shaking.

The morphine solution, if possible not more than 10 cc. in volume, is placed in a small test-tube, acidified with hydrochloric acid, and a few crystals of potassium chloride are added. An excess of acid, even amounting to 2 or 3 per cent, does no harm. An approxi-

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548 Morphine and Pseudomorphine

mately 10 per cent excess of silicotungstic acid is added (as a IO‘ per cent solution in water). A yellowish pink precipitate forms at once. The tube is stoppered and vigorously shaken for several minutes, then chilled by immersing in cold water. The precipi- tate coagulates and may be filtered onto a Gooch mat. Some of the filtrate is used to wash out the tube. The morphine silico- tungstate is washed with cold water containing 2 or 3 cc. of hydro- chloric acid per liter, but on account of the solubility of the pre- cipitate an excess of wash water is avoided. Alcohol should not be used as a washing fluid. The crucible is dried at 120’ until weight is constant (usually 2 or 3 hours). The method has the advantage that only 0.2810 of the weighed precipitate is morphine (as free base).

As a check determination the crucible may be ignited, and the weight of the residual oxides of tungsten and silicon determined. Due to the hygroscopic nature of freshly ignited silica, this pro- cedure needs care.

Morphine silicotungstate 31520 (as dried at 120”) X 0.2510 = free base. Ignited oxides X 0.401 = free base. Loss on ignition of the silicotungstate X 0.9406 = free base.

Working with pure morphine solutions, the met.hod gives most excellent results, provided the volume of liquid is kept small.

Determination of Pseudomorphine.

No quantitative methods for the estimation of pseudomorphine have been described. The exceptional insolubility of many salts of this base makes several good methods possible. In this labora- tory pseudomorphine has been determined in three forms, the free base, the silicotungstate, a,nd the sulfate.

Determination as the Silicotungstate.-The precipitation of pseudomorphine silicotungstate can be carried out in the same manner as that of morphine silicotungstate, but the greater insolubility of Dhe pseudomorphine compound makes it unneces- sary to reduce the volume of the solution so much. The solution may be warmed. The precipitate is weighed on a Gooch mat after drying at 120”, but it may be safely washed with alcohol as well as with acidulated water, which facilitates the drying. The weighed precipitate may also be ignited as a check determination. The

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A. K. Balls 549

precipitate when dried at 120” contains no water of crystallization, and the factors for calculating the free base are as follows:

Pseudomorphine X 0.283 = free base. Ignited oxides X 0.399 = “ “ Loss on ignition X 0.970 = “ “

Determination as Free Base.-Pseudomorphine has a rather wide isoelectric range, and since the free base is highly insoluble, it may be precipitated by adjusting the reaction of the solution to a pH of about 7.8. The separated base is hard to filter unless electrolytes are present; a convenient method of obtaining the proper conditions is to make the solution decidedly alkaline with KOH or NH40H, and then add a large amount of solid ammonium chloride. After this dissolves, the solution should be acid to

TABLE II.

Determination of Pseudomorphine as Silicotungstate.

Pseudomorphine.

Taken.

l7m. c/m. 0.0218 0.0218 0.0436 0.0432 0.043G 0.0429 0.0572 0.0884

Found.

phenolphthalein, but still alkaline to litmus. The base precipi- tates very slowly and is crystalline. It may be filtered in a Gooch crucible, washed with 30 per cent alcohol, and dried to constant weight. Water cannot be used as a wash liquid, since the base comes through the filter in colloidal condition when all the salts are removed. Under these conditions the solubility of the base is about 2 mg. per 100 cc.; so the method is not recommended for small amounts.

Determinckion of Pseudomorphine as Mixture of Silicotungstate and Free Base.-If silicotungstic acid be added to the alkaline solution of pseudomorphine, and the reaction adjusted as described for the free base, a mixture of the free base and the silicotungstate separates. This precipitation is quantitative. In order to deter-

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550 Morphine and Pseudomorphine

mine the pseudomorphine it is filtered off, washed with 30 per cent alcohol, and the loss, on ignition, in the weight of the dried precipi- tate is found. Considerable variations in the pH of the solution do us no h&m, but merely change the ratio of free base to silico- tungstate precipitated. Since the presence of ammonium salts may be objectionable, other substances having a buffer effect may be used to adjust the reaction. Glycocoll is satisfactory, and a solution of potassium dihydrogen phosphate works very well. With the last named substance, the solution is made slightly but distinctly alkaline to litmus. The advantage of this method lies in its ability to separate pseudomorphine from morphine, the higher oxidation products of morphine, and protein hydrolysis products.

TABLE III.

Determination of Pseudomorphine as Mixed Precipitate of Free Base and Silicotungstate.

Pseudomorphme (free base).

Taken. Found.

Buffer solution contained.

07%

0.0626 0.0626 0.0624 0.0626 0.0624 0.0626

0:;638 0.0607 0.0625 0.0622 0.0606 0.0614

NH&l

KHzPOa

Glycocoll.

When considerable silicotungstate occurs in the precipitate the loss on ignition must be corrected by the amount of combined water present. This may be calculated from the weight of the oxides remaining in the crucible after ignition.

Free base = loss on ignition of mixed precipitate - (0.013) X (weight of oxides remaining after ignition).

Determination as sulfate.-Pseudomorphine sulfate is only slightly soluble in strong alcohol, and even less so in ace0one. From either of these media, but particularly from the latter, it is quantitatively precipitated in the presence of sulfuric acid. The method is useful in separating pseudomorphine from alkaloids,

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A. K. Balls 551

particularly morphine, which give soluble sulfates. The precipi- tate is brought into a Gooch crucible, washed with acetone, and dried to constant weight at 100”.

Pseudomorphine sulfate X 0.852 = free base.

Separation of Morphine, Pseudomorphine, and Higher Oxidation Products of Morphine.

After acidifying an oxidizing morphine solution and treating with silicotungstic acid, these substances are at hand as t,he silico- tungstates. They are thus quickly removed from the reaction, and may be separated at leisure. When all three classes of con- stituents in this precipitate are to be estimated, it is necessary to work with two parallel samples, on one of which the total weight of precipitated silicotungstate is determined, while on the other the morphine and pseudomorphine are found. When the last two are known, the quantity of the higher oxidation products (as silico- tungstates) can be calculated by difference. It is necessary then to determine morphine and pseudomorphine in the presence of each other and of the higher oxidation products.

The separation of pseudomorphine has been done by using the ammonium chloride or the potassium phosphate buffer solution previously described, which yields a mixture of the free base and the silicotungstate. Neither morphine nor its higher oxidation products interferes. Small amounts of protein and other foreign substances precipitable by silicotungstic acid are likewise harmless, but of course do prevent the parallel estimation of the higher oxidation products. The case where large amounts of protein are present, as in tissue, is discussed later.

The filtrate from the pseudomorphine contains the morphine which must be separated now from its oxidation products. This can be done by taking advantage of the more pronounced acidic character of the latter group and extracting the morphine from a comparatively alkaline solution.

Considerable objection has been made to the method of remov- ing morphine by extraction, and it is liable to serious error unless the reaction of the solution is carefully regulated and kept constant during the extraction. Morphine is most easily extracted at its isoelectric point, approximately pH = 8.9. A variation from 8.9

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552 Morphine and Pseudomorphine

TABLE IV.

Extraction of Morphine from Solutions of pH = 9.1 in a Continuous Extractor.

Morphine (base).

Added. Recovered.

om. 0%

0.0815 0.0815 0.0815 0.0800 0.0815 0.0820 0.0815 0.0517 0.0515 0.0790

-

i Extraction time. Solvent.

min. 60 CHCL + GHaOH. 50 “ “ 40 60 40

Amy1 alcohol. “ “

Butyl “

TABLE V.

Estimation of Pseudomorphine in Presence of Morphine and Its Oxidation Products.

Taken .............. Recovered. .........

Taken. ............. Recovered. .........

Taken .............. Recovered. .........

Taken .............. Recovered. .........

Taken .............. Recovered. .........

Taken .............. Recovered. .........

Taken ............... Recovered. .........

Morphine Pseudomorphine (free base). (free base).

07%

0.2

0.18

0.15

0.15

0.15

0.10

0.10

om. 0.0626 0.0660

0.0626 0.0660

0.0626 0.0631

0.0624 0.0600

0.0624 0.0612

0.0624 0.0616

0.0626 0.0632

Higher oxidation roducts calculated as silicotungstate

x 0.36.

om.

0.05

0.05

0.03

0

0.03

0

0.015

to 9.1 is quite allowable, but the solution retains considerable morphine if the variation becomes larger. This is very noticeable

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A. K. Balls 553

with amyl alcohol, which is one of the best solvents for the alka- loid. A variation on the acid side is of less moment with the lower alcohols and chloroform as solvents, due to the solubility of morphine salts in the former, and the practice of mixing ethyl alcohol with the latter. Variation on the acid side of the iso- electric point will allow the oxidation products to be extracted also; while the older methods of using an excess of ammonia or soda hold back morphine because the solution is far too alkaline. The necessity for a heavily buffered solution, containing for instance much phosphate, is also quite apparent.

TABLE VI.

Results of Described Method for Estimating Morphine, Pseudomorphine, and Their Silicotungstic Acid-Precipitable Oxidation Products in Vati-

ous Synthetic Mixtures.

Taken .............. Recovered. .........

Taken .............. Recovered. .........

Taken .............. Recovered. .........

Taken ............. Recovered. ........ -

Morphine Pseudomorphine (free base). (free base).

om.

0.0163 0.0164

0.0515 0. osos

0.0165 0.0173

0.0815 0 .os32

-

-

Higher oxidation mducts calculated as silicotungstate

x 0.36.

om. am.

0.0624 0.0860 0.0596 0.0576

0.0624 0.0130 0.0612 0.0142

0.0624 0.0860 0.0618 0.0846

0.0124 0.0860 0.0112 0.0875

It is not generally realized that butyl alcohol and alcohol containing chloroform will extract large quantities of morphine from even strongly acid solutions, at least if continuous extraction is employed. With butyl alcohol as a solvent, 10 cc. of a (nearly) 1 per cent morphine hydrochloride solution, containing 1 per cent sulfuric acid, were extracted for 30 minutes in a continuous extrac- tor. By this time 72 per cent of the morphine had been removed. In a similar experiment amyl alcohol removed only 9 per cent of the morphine in the same time. The last traces of the alkaloid are removed only with difficulty, however, and there seems no possibility of basing an analytical procedure on this behavior.

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554 Morphine and Pseudomorphine

For the separation of morphine from its oxidation products, the filtrate from the pseudomorphine (evaporated if necessary) is treated with 2 or 3 gm. of disodium phosphate, and then sufficient potassium hydroxide to bring the reaction to a pH = 9.0 or 9.1. Because the solutions are frequently highly colored, this is best tested by bringing a drop of the solution in contact with a drop of dilute brom-thymol blue on a spot plate. Comparison with a known buffer solution makes a sufficiently accurate adjustment easy. The solution is placed in a continuous extractor, and may be extracted with amyl or butyl alcohol (under vacuum) or with chloroform containing about 5 per cent ethyl alcohol. Since extractors vary greatly in their efficiency, no time of extraction can be specified. With the “lighter than water” extractor used in this laboratory, 0.1 gm. of morphine dissolved in 10 to 15 cc. of solution is completely removed by amyl alcohol in 35 minutes. The solvent is evaporated on the water bath, being replaced with water containing a few drops of hydrochloric acid. Usually traces of waxy substances are present and can be removed by shaking the acid solution once or twice in a separatory funnel with pure chloroform or benzene. The acid solution is then evaporated, nearly to dryness, filtered if necessary, and the mor- phine in this small volume of liquid is determined as already described by the silicotungstate precipitation.

Determination of Morphine and Pseudomorphine in Tissue ‘.

As stated previously only the determination of morphine and pseudomorphine is considered, not that of the higher oxidation products.

The method was worked out on ground beefsteak containing a considerable amount of fat. It is not claimed to be applicable to material differing greatly from this. Weighed amounts of the powdered bases were added to the meat, and then thoroughly incorporated by grinding with sand.

The separation of these bases from tissue is rendered difficult by the similarity between proteoses and pseudomorphine in their behavior toward precipitants. It may be made from small amounts of proteose, peptone, and the like, by precipitating the free pseudomorphine at its isoelectric point, or by precipitating the mixture of free base and silicotungstate at a pH of 7.3 to 7.5.

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A. K. Balls 555

With fairly large quantities of pseudomorphine, the former pro- cedure is satisfactory, but with only a few mg., the protein acts as a protective colloid, and interferes with the precipitation of the base, so that the alternative method must be used.

It is necessary first to remove the bases to be determined from the greater part of the tissue constituents. This is done by taking advantage of the solubility of both morphine and pseudomorphine in benzyl alcohol. At the pH of ordinary tissue fluids, pseudo- morphine is present entirely as the free base.

From 10 to 30 gm. of meat are thoroughly disintegrated in a mortar with a known weight of sand, and then transferred to a weighed flask. About 50 cc. of absolute alcohol are added, and the mixture shaken until thoroughly uniform. It is allowed to stand for a few minutes until dehydration of the shreds of tissue is complete, then 100 to 125 cc. of benzyl alcohol are added. The shaking is repeated, and the contents of the flask heated to about 70” by immersing in warm water. The tissue should now present a rather translucent and uniform appearance and all fat should be dissolved. The flask is allowed to stand until cool, and again weighed, in order to determine the exact amount of material (exclusive of sand) which it contains. An aliquot part of this material is taken by weighing the solvent filtered off through a dry hardened filter paper into a tared flask. This aliquot generally represents about 80 per cent of the total. A correction may be made for the weight of the anhydrous tissue present in the solvent. This method is easier than the corresponding volumetric procedure, when handling a substance as viscid as the benzyl alcohol mixture. The expense and difficulty of handling large amounts of benzyl alcohol make the more accurate method of exhaustive extraction impracticable.

Besides its aliquot of the alkaloids, the solvent contains fat, very small amounts of protein, and coloring matter. The bases are next extracted from the solvent by dilute hydrochloric or acetic acid. Since the specific gravity of benzyl alcohol is near that of water, the solvent is mixed with an equal volume of benzene which decreases its specific gravity and also lessens the solubility of the bases in the non-aqueous phase during extraction. The extraction is made repeatedly in a funnel with 3 per cent hydrochloric acid until the extract gives no opalescence with phosphotungstic acid. One or

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556 Morphine and Pseudomorphine

two extractions are then made with distilled water, all the extracts united, and washed by shaking twice or thrice with benzene. If the water layer is not clear, and it usually is not, it is filtered through hardened paper. This extraction should be thorough, and very dilute acid must be used, because of the insolubility of pseudomorphine salts in acid. No trouble is caused by permanent foam or emulsion.

The acid extract is next evaporated to small volume, never over 15 cc. It should be at the most only slightly yellow. It is brought into a small beaker, cooled, a few crystals of potassium chloride added, and then sufficient silicotungstic acid for com- plete precipitation. After the precipitate, which may become sticky, has collected together, it is filtered, washed with a little 0.2 per cent hydrochloric acid, and then dissolved off the filter with a few drops of warm dilute potassium hydroxide solution. The volume should not be more than 15 or 20 cc., unless large amounts (50 mg. or more) of morphine are present.

The pseudomorphine is next precipitated, by first neutralizing the liquid to phenolphthalein with hydrochloric acid and then adding a solution of KHzPOd drop by drop until the liquid is just slightly but definitely alkaline to litmus paper. The mixture of pseudomorphine and its silicotungstate is allowed to stand until the supernatant fluid is quite clear, and then handled as previously described. If the quantity of precipitate is very small, instead of filtering on a Gooch mat it is better to use a circle of hardened filter paper, wash as usual with 30 per cent alcohol, then redissolve in a little alkali, add a drop of silicotungstic acid solution, and precipitate the pure silicotungstate with hydrochloric acid. This more bulky precipitate is easily filtered in a Gooch crucible, dried, and weighed.

The filtrate from the pseudomorphine contains some peptones, as well as the morphine. It is handled by extraction with chloro- form or amyl alcohol, precisely as described for the separation of morphine and its oxidation products.

Table VII shows the results obtained by this method from a series of consecutive experiments.

The results are, in general, gratifyingly low. There seems to be little chance of impurities leading to high results. In the case of pseudomorphine, the greatest single error apparently lies in

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A. K. Balls 557

incomplete extraction from the benzyl alcohol. With morphine, however, there is danger of loss by oxidation while carrying out the method. It is not wise to prolong the analysis unduly, nor to allow alkaline solutions to stand in the air longer than necessary, as they are more easily oxidized than when acid. The presence of ammonium salts is to be avoided because of the difficulty in maintaining a constant alkalinity during extraction of the mor- phine. As a method for morphine alone, the procedure is not bad, and is rather more rapid than those ordinarily in use.

The author wishes to thank Dr. L. A. Ryan of this faculty for several helpful suggestions, particularly regarding this phase of the work.

TABLE VII.

Recovery of Morphine and Pseudomorphine from Ground Meat.

Sai?J? Meat.

gm. mg.

12 17.3 14 3.3 24 15.9 21 16.6 25 7.2 26 1.4

Pseudomorphine.

Taken. Recovered. Taken. Rewvered.

mg. *

3.7 15.6 15.1 5.1 1.4

ml. ml.

2.6 2.0 17.6 *

10.5 7.9 9.3 6.6 1.7 1.9 2.5 1.2

i- Morphine. -

* Not estimated.

SUMMARY.

Pseudomorphine may be quantitatively determined in several forms, which make possible a method for the chemical separation and analysis of a morphine solution which is undergoing oxidation. The reaction may be abruptly terminated and the composition of such a system determined at any given time with respect to residual morphine, pseudomorphine, and the other morphine- derived substances precipitable by silicotungstic acid, taken to- gether as a class. If desired, five variables can thus be evaluated in the system, when the initial concentration of morphine and the quantity of oxidant used are also known.

A modification of the method enables the estimation of morphine

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Morphine and Pseudomorphine

and pseudomorphine in amounts as small as 2 mg. in 20 to 30 gm. of biological material like ground meat,. with an error of about 30 per cent. In the analysis of tissues for morphine alone, the method is also useful, because rapid.

BIBLIOGRAPHY.

1. Marmk, W., Deutsch. med. Woch., 1883, ix, 197. 2. Donath, J., Arch. Physiol., 1886, xxxviii, 538. ges. 3. Kolthoff, J. M., Biochem. Z., 1925, clxii, 338. 4. Bertrand, G., Comp. rend. Acad., 1899, cxxviii, 743.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. LXXI. PLATE 1.

FIG. 1

RatB8338’. Rat B 7468’. FIG. 2.

(Osborne, Mendel, Park, and Winternits: Diets rich in protein.)

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A. K. BallsAND RELATED SUBSTANCES

OF MORPHINE, PSEUDOMORPHINE, THE SEPARATION AND ESTIMATION

1927, 71:543-558.J. Biol. Chem. 

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