The alkaloid thus far most thoroughly examined is cocain. Crystalline deposits have been obtained with each of the following eleven reagents, viz, palladous chlorid, platinum chlorid, gold chlorid, picric acid, chromic acid and hydrochloric acid, potassium dichromate and hydrochloric acid, potassium permanganate, potassium chromate, sodium carbonate, ferric chlorid, and potassium hydrate or sodium hydrate; noncrystalline deposits were obtained with chlorzine iodid, picralonic acid, Mayer's reagent, phosphomolybdic acid, phosphotungstic acid, Kraut's reagent, Wagner's reagent, barium mercuric iodid, and potassium cyanid.

The following observations were noted concerning the various reactions with cocain in which crystals were produced:

Palladous chlorid.-This is one of the most characteristic tests for cocain, though not quite so sensitive as gold chlorid. The crystals vary in form greatly, according to the conditions of precipitation. There is at first formed, except in very dilute solutions (1:300 and up), an orange-colored amorphous-like or oily precipitate from which, on standing, crystalline forms of golden brown color are produced. One of the most common forms is that obtained with a 1:100 dilution, when feathery crystals are formed which have a strong tendency to twin. With a solution of 1:20 a dense precipitate is thrown down, out of which hexagonal plates are at first formed and frequently followed later by sheaf-like clusters of fine-pointed acicular crystals. A dilution greater than 1:500 gives crystals only with difficulty, crystallization being induced by rubbing the slide with the glass stirring rod. The limit of the test is 0.2 ugr.

Platinum chlorid.-With a 1:20 solution a dense white precipitate is formed and quickly followed by the production of very narrow feathery crystals-many times twinned so as to resemble a bird with outspread wings. Clusters of more than two are also abundant. If the reagents are mixed slowly the crystals are more like those of greater dilution. With a 1:100 dilution the feather type is much more prominent, the secondary branches being well developed into frost-like forms. With 1:1,000 solutions either short thick crystals are formed or else plate crystals twinning in a most characteristic manner are produced. The dilution limit is about 1:4,000, and the limit in 1:1,000 is 0.2 ugr.

Gold chlorid. This is the most sensitive reagent for cocain so far found. At 1:100 feathery frost-like crystals are produced, together with some nearly smooth star-like aggregates. At 1:1,000 the form is much the same, but the branches usually bear a rough outline. Diamond plates are also produced. At 1:4,000 a cross-like form predominates, the cross-bar being short. A few rosette crystals frequently are present. Crystals can be obtained in dilution up to 1:20,000 and the limit of the test for dilutions of about 1:3,000 is 0.033 μgr.

Picric acid. This is a good reagent for dilutions up to 1:800, though the crystals produced are not very characteristic for this alkaloid. They are produced in spherical rosettes (or sheafs) of fine lemon-yellow acicular forms. The reaction takes place quickly, and no difficulty is experienced in producing them nearly to the limit of dilution. At 1:300 the limit is 0.2 μgr.

Potassium permanganate.-With cocain, solutions up to a dilution of 1:700 give purple-colored square plates, or aggregates of this form. Vigorous rubbing of the slide is often necessary to start the crystallization, which then proceeds readily. When they begin to crystallize spontaneously, the plates are sometimes deposited in spherical aggregates. The limit at 1:400 is 2 ugr.

Chromic acid and hydrochloric acid. This test is made by adding a small drop of 5 per cent chromic acid solution to the test drop. A precipitate is formed which on stirring disappears (if too much has not been added).~ A small drop of strong hydrochloric acid is added and a yellowish deposit is produced, which after rubbing of the slide should in a few moments be transformed into loose spherical clusters of an acicular crystal. This test appears to be one of the most uncertain because of the difficulty with which the crystallization is sometimes induced to begin in dilutions greater than 1:60. A concentration of 1:1,000 has produced positive results on standing several minutes. The limit appears to be for 1:100 about 3 μgr.

Potassium bichromate and hydrochloric acid. This test gives the same form of crystals as the chromic acid and the test is conducted in a similar manner. The limit of dilution is about 1:1,000 while at 1:100 the limit is 3 μgr.

Ferric chlorid.-The crystals are spherical aggregates of rather coarse blade-like crystals with chisel-shaped ends. The limit of dilution is about 1:1,000 and in a dilution of 1:100 the limit is 3 ugr.

Potassium hydrate, or sodium hydrate.—This produces a white amorphous precipitate which changes into crystals on standing or by rubbing the slide with a glass rod.

The crystals are rod-like, frequently with more or less chisel-like ends and a V-shaped recess extending backward into the crystal. There is a strong tendency to form coarse clusters up to about 15 branches. In open drops tree-like forms are frequent. For each of these reagents dilutions up to 1:1,000 give the reaction and the limit at 1:100 is 3 ugr.

Sodium carbonate. This gives a precipitate with cocain like that produced by potassium hydrate, both in the amorphous and crystalline forms. Limit of dilution is 1:1,000. In 1:100 solution the limit is 3 μgr.

In order to determine the usefulness of some of the above tests when other alkaloids are present the palladous chlorid test was made on test drops to which had been added solutions of one of the following alkaloids, codein sulphate, atropin sulphate, heroin, dionin, acoin powder, cinchonin sulphate, hydroxylamin hydrochlorid, apomorphin hydrochlorate, narcotin, papaverin, brucin, narcein, morphin, thebain, gujasanol, orthoform (new), cinchonidia sulphate, quinidia sulphate, beta-eucain, holocain, caffein, quinin sulphate, strychnin, and tropacocain. In each case the crystals of the cocain compound were obtained and in the case of brucin, gujasanol, caffein, strychnin, and tropacocain, with which the palladous chlorid regularly gives a crystalline precipitate, it was found that when cocain was also present the cocain product was given in addition to that for the other alkaloid, though occasionally with modified form.

The foregoing serves to give an idea of the scope of the work undertaken, which it is hoped will be carried much further during the coming year.

COOPERATIVE WORK ON HEADACHE MIXTURES.

By W. O. EMERY.

After making investigations of various suggested methods for determining the different constituents present in the many headache mixtures containing acetanilid and similar agents, a method was finally devised which proved quite satisfactory to the members of the Division of Drugs, and it was therefore decided to place this method in the hands of as many chemists interested in this line of work as could assist. A circular letter requesting cooperation was sent out, and a gratifying number responded, signifying their willingness to assist, eleven of whom sent in results. All who expressed a desire to cooperate were supplied with a sample of a mixture containing known amounts of acetanilid, sodium bicarbonate, and caffein, with the following instructions, the U. S. Pharmacopoeia, eighth revision, as amended and corrected May 1 and June 1, 1907, being used as a basis for all calculations and reagents unless otherwise specified:

SEPARATION OF CAFFEIN, ACETANILID, AND SODIUM BICARBONATE.

Caffein.

Weigh out about 0.3 gram of headache powder on a small (5.5 cm) tared filter,a wash with successive small portions of chloroform to the amount of about 30 cc, collecting the solvent in a 100 cc Erlenmeyer. Distil off chloroform by means of a small flame until only a few cubic centimeters remain. Add 10 cc of dilute sulphuric acid, then continue the distillation till all the chloroform has gone over, disconnect from condenser, heat gently, first on wire gauze to complete solution, finally on a steam or hot

a In cases of powder mixtures or tablets containing ground celery seed, much coloring matter, cinchona alkaloids, laxative or extractive principles other than acetanilid or phenacetin, it is our practice to shake out the latter by means of chloroform from dilute sulphuric acid solution.

In case the preparation contains ground celery seed or certain oily principles, it sometimes happens that the acid solution does not become entirely clear at this

water bath until the contents of the flask have evaporated to about 3 to 4 cc. Cool, transfer by washing with water to a separatory funnel, so that the final volume does not greatly exceed 20 cc. Add four times the volume, or about 80 cc of chloroform, shake for some time vigorously, allow to stand until the chloroform clears perfectly, pass through a small dry filter into a dry 100 cc Erlenmeyer, distil off the solvent and use distillate for a second extraction, observing the same method of shaking, clearing, and filtering as above noted. Distil off chloroform to a small volume, transfer residue to a small tared beaker, or crystallizing dish, by means of a few cubic centimeters of chloroform. Allow to evaporate spontaneously, or if desired on a steam or hot-water bath to dryness, in the latter case partially covering the dish toward the end of operation with a watch glass in order to avoid possible loss from "popping." Cool in desiccator and weigh as caffein, dry alkaloid.a

Acetanilid.

First method. The acid solution remaining in the separator and containing anilin sulphate is run into a 100 cc Erlenmeyer, the filter through which the chloroform passed is washed once with a little water, allowing the latter to run into the separator. Rinse the latter thoroughly, adding the aqueous rinsings to the acid solution. Now, run in slowly and with constant agitation a standard solution of potassium bromidbromate to a faint but distinct yellow coloration. The number of cubic centimeters employed, multiplied by the value of 1 ce in terms of acetanilid, will give the amount of acetanilid present.

Second method.-The acid solution aforesaid is treated with successive small portions of sodium bicarbonate until an excess of this reagent is observed in the bottom of the separator. Add 50 cc of chloroform and 15 to 20 drops of acetic anhydrid, shake for some time vigorously, allow the chloroform to clear, then pass through the same filter used for the caffein into a 100 cc Erlenmeyer, and distil off most of the chloroform. Use this distillate for a second shake out, clear, filter, and distil down to a small volume, transferring the residue and the subsequent chloroform washings to a tared beaker or dish precisely as in the case of caffein. Allow the solvent to evaporate spontaneously or by means of a blast or fan, avoiding, however, undue heat. Dry in desiccator over quicklime to constant weight.

Verify the final weight by means of titration with standard potassium bromidbromate solution as in the first method. Heat the residue with 10 cc dilute sulphuric acid a half hour on the steam or vapor bath, cool, add 5 cc of water and titrate as directed above.

Sodium bicarbonate.

The residue left after the first treatment with chloroform is weighed when dry and represents very nearly the amount of sodium bicarbonate present. It may be more accurately estimated by titrating with tenth-normal sulphuric acid, using congo red as indicator, or it may be ignited with dilute sulphuric acid and weighed as sodium sulphate.

Calculate results in parts per 100.

a Should the caffein not be colorless or nearly so, the residue is dissolved in about 10 cc of water, filtered, if necessary (in case oily matters are present), through a wet filter, the filtrate acidified with dilute hydrochloric acid, the caffein precipitated with 15 to 20 cc of Wagner's reagent, allowed to stand a half hour, filtered, and the precipitate washed with a few cubic centimeters of same reagent, the filter, together with precipitate, transferred to separator, decolorized by means of sodium sulphite, and the caffein finally extracted with chloroform.

For this purpose the solution is prepared by adding bromin in slight excess to a concentrated aqueous solution of 50 grams caustic potash, the liquid diluted till the separated salts redissolve, boiled, to expel any excess of bromin, and finally made up to 1 liter. This solution is standardized with weighed amounts of acetanilid, or it may be so adjusted by further dilution that 1 cc is exactly equivalent to 1 centigram of acetanilid. For purposes of titration 1 to 2 decigrams are heated a half hour on the steam or water bath with 10 ce of dilute sulphuric acid.

cAcetanilid suffers appreciable loss when heated above 40°,

The results reported are tabulated as follows:

Results obtained in the cooperative work on an acetanilid mixture.

b In cases where two percentages for volumetic and gravimetic determinations of the same substance were reported, the mean of such percentages has been taken in computing the total percentage.

Owing to an ambiguity in the expression "dilute sulphuric acid" employed in the method under caffein, as also in the footnote a, page 100, for standard bromid-bromate solution, some of the workers quite naturally used the pharmacopoeial strength, with the result that the acetanilid was not completely hydrolyzed. This undoubtedly explains the somewhat high results for caffein and the correspondingly low ones for acetanilid. The strength of acid intended and the one actually employed for this purpose in the Bureau of Chemistry is that ordinarily used in laboratory work and is made by diluting 1 part of concentrated sulphuric acid (whose specific gravity is not less than 1.826 at 25°) with 5 parts of water. From two to three hours' heating on the steam bath is usually required to completely hydrolyze the acetanilid.

Notwithstanding this ambiguity the results obtained are very gratifying, in view of the fact that the method is new and the workers have entered into a comparatively new field. The percentages of variation are so small as to almost warrant the referee in recommending it as a provisional method to the association. He believes, however, that the method should receive additional study, and so recommends. It is also recommended that additional mixtures be tested with this and such other methods as may be found desirable.

President Snyder introduced the Secretary of Agriculture with a few words of appreciation concerning the long-sustained attitude of the Secretary in fostering agricultural chemistry, especially the work of the official chemists, by making possible the close affiliation between the Department of Agriculture and the association. The Secretary then briefly addressed the convention, after which the reading of the drug reports was resumed.

THE NECESSITY FOR ANIMAL EXPERIMENTATION IN DETERMINING THE PURITY AND STRENGTH OF MEDICINAL PREPARATIONS.

By WILLIAM SALANT.

Experiments on animals have long been recognized in medical jurisprudence as a valuable adjunct to chemical and microscopical methods in the detection of poisons in animal tissues and fluid. Notwithstanding the improvements in the methods of analytical chemistry witnessed within recent years, tests on animals, or, as Kobert a terms it, "biological testing," is still resorted to in order to corroborate the findings of the analytical chemist in cases of suspected poisoning with alkaloids and other poisonous substances of plant or animal origin. The French chemist, Boutmy,b who made extensive studies on poisoning with alkaloids, concluded that in all cases in which the presence of an alkaloid in the body is suspected experiments on animals should be made for the purpose of confirming the results of chemical analysis.

The work of some investigators indicates that the biological method is in certain cases much more delicate than the chemical. Rankec reports experiments on dogs which were given 0.1 of a grain of strychnin by mouth. Chemical examination of the organs of these animals failed to show the presence of strychuin, but when extracts of the same organs were injected into frogs tetanus followed. Falckd has shown long ago that one-twentieth of a milligram of strychnin was sufficient to induce tetanus in a medium-sized frog. It might be added that if smaller frogs are used the same effect may be obtained with one-eightieth of a milligram. Atropin is another example of a drug of which small quantities are sufficient to produce a physiological reaction. Only one-twentieth of a milligram is necessary to produce dilation of the pupil.

Likewise cocain, which produces characteristic effects on the mucous membranes, and by its action on the frog's pupil, can be identified, even when very small quantities are present in biological solutions.

Aconitin can be identified in milligram doses by its action on the tongue, eye, heart, and central nervous system. No chemical methods have as yet been devised by which such small quantities of this alkaloid can be detected. A striking illustration of the delicacy of the biological method is afforded by the work of Hunt. e In his investigations on the functions of the thyroid he has shown that mice fed for a few days with the extract of this gland acquire greater resistance to poisoning with acetonitrile. One milligram of the official dried thyroid fed to white mice daily for a few days may enable the animals to recover from double the dose of acetonitrile fatal to the controls. Seidell, working under the direction of Hunt, found that forty to fifty times as much thyroid would be required to give the iodin test.

a Ber. deutsch. pharm. Ges., 1903, 13: 325.

Ann. hyg. publique med. legale, 1880, [3] 4. 193.

c Virchow's Archiv, 1879, 75: 20.

d Vierteljahrschr. gericht. Med., N. F., 1874, 20: 198.
eJ. Amer. Med. Assoc., 1907, 49: 240.

f Ibid.