soluble).

Summary of Results.

The foregoing tables show that market samples of neoarsphenamine may deteriorate in ampule under ordinary conditions. They may change in color, solubility, toxicity, mobility in ampule, and odor after varying periods.

The change in color in some instances consisted of a slight deepening of the original golden-yellow powder, whereas in other cases a change to a brick red occurred, such as takes place when the powder is exposed to the air for a day or more. Prolonged incubation at 37° C. or heating at 100° C. caused deepening in color.

A relatively large number of samples showed a decrease in solubility; in one lot it occurred within two weeks after manufacture; in other lots from four months to slightly over two years had clasped before the condition was noticed. The decrease in solubility varied as shown by the production of a turbid solution or a solution containing a fine flocculent precipitate with some lots, to that of a coarse suspension with other lots, the coarse suspension in some cases being practically insoluble in water within half an hour. Incubation of the ampules at 37° C., as well as at higher temperatures, hastens the development of a decrease in solubility.

Many lots, although showing a change in color and solubility, did not show an increase in toxicity. Some had become more toxic as well as difficultly soluble. An increase in toxicity was noticed in some lots after about one year, even though no apparent changes in their physical properties had occurred, the increase in some cases being slight enough to permit the preparations to pass the official toxicity tests, whereas other lots which had retained their ready solubility failed to meet the official toxicological requirements.

The change in the mobility of the powder in ampule was usually so pronounced that a solid mass was formed which was frequently adherent to the sides of the ampule. A marked deepening in color usually accompanied the change from a freely mobile powder to the solid form.

The odor of the neoarsphenamines varied greatly. Some lots emitted an odor resembling ether, others emitted a garlicky odor on opening the ampule. Incubation at 37° C. may produce a pronounced ethereal or a garlicky odor. It is interesting to note that ampules from the same lot varied as to the odor emitted on opening the ampule. Ampules which emitted a garlicky odor usually produced incomplete solutions which emitted a similar odor.

Discussion of Results in Tables and Additional Investigations.

The fact that the chemical constitution of neoarsphenamine is still in dispute should be borne in mind in a discussion of these results.

Neoarsphenamine, as previously stated, is usually considered to be sodium 3, 3'-diamino-4, 4'-dihydroxy arsenobenzene-Nmethylene sulphinate, and therefore should have an arsenic content of about 30 per cent. In reality, however, the Government regulations require that commercial samples shall have only about 20 per cent arsenic. They must therefore contain either a large amount of by-products or impurities, or diluent, to reduce the arsenic from the theoretical amount to that which exists in commercial samples. Macallum, states that certain neoarsphenamines consist to a great extent of reaction by-products. Raiziss and Falkow, have recently reported that considerable variation was found upon analysis of a number of samples of neoarsphenamine obtained from three different laboratories. They found that the deviation in the arsenic to nitrogen ratio, which they regard as a comparatively good indicator of the purity of the arsenical component, was greater for the neoarsphenamines than for the arsphenamines which they examined, indicating that neoarsphenamine is not as definite a compound as arsphenamine. In addition, the values which they obtained for the oxygen requirements were in excess of those required merely by the arseno group, the excess being attributed to the presence of uncombined sodium formaldehyde sulphoxylate. They found, furthermore, that the amount of combined sulphoxylate was greater than that necessary for one amino group and less than that calculated for a substitution product with both amino groups closed, from which they concluded that neoarsphenamine may be assumed to be a mixture of both the mono- and di- substituted products.

We must, therefore, regard commercial neoarsphenamine as a mixture rather than as a definite chemical compound. It would seem to be more nearly exact to describe neoarsphenamine as a substance prepared from arsphenamine by means of formaldehyde sulphoxylate, as prescribed by the present Treasury Department regulations. The causes for the above deterioration, for the most part, can only be conjectured. The occurrence of physical deterioration in some lots within a relatively short time, about two weeks, for example, whereas it required a year or more for other lots to show a similar change, supports the idea of there being essential chemical differences in the Macallum, A. Douglas, The Examination of Neoarsphenamine: Jour. Amer. Chem. Soc., 1921, vol. 43, p. 643.

Raiziss, George W., and Falkow, M., The Chemistry of Neoarsphenamine and Its Relation to Toxicity: Jour. Biol. Chem., 1921, vol. 16, p. 209.

character of the various lots. But in addition to this cause there are other influences which tend to bring about this condition, chief of which are the influences of age and temperature. Our results indicate that when neoarsphenamine was kept in ampule at ordinary temperature, many of the lots had deteriorated within two years, so that a lot which is over two years old should be looked upon as one requiring careful investigation before being used clinically.

Temperature was found to be a potent factor in hastening the deterioration of neoarsphenamine in ampule. It was found that a lot would remain unchanged longer at a temperature of 18°-20° C. than when kept at a temperature of 37° C. Whereas profound changes may be readily effected in some lots by heating to 100° C. for 20 minutes, other lots may be practically unchanged by such treatment. The gross character of the changes produced by heating in ampule at 100° C. for 20 minutes are the development of a relatively insoluble product emitting an ethereal or garlicky odor, and the production of a relatively immobile powder which may become adherent to the sides of the ampule. These observations suggest that neoarsphenamine should be kept under conditions similar to those required for vaccines and allied biologics, namely, at ice-box temperature rather than at room or higher temperatures, and that some neoarsphenamine is unsuited for use in the Tropics.

The effect of storage (probably heat) is probably further shown by the following observation. During the war our forces in France employed French neoarsphenamine to some extent. Upon return of our forces the stock of French neoarsphenamine, which was forwarded to the medical supply officer at Washington, was referred to the Hygienic Laboratory for examination, and it was found that much of the returned French neoarsphenamine had become insoluble in water and in some cases had deepened in color markedly; also some had become practically immobile in ampule. Inasmuch as a number of lots of this product which had previously been received from other sources were readily soluble in water, it would appear that conditions of storage may have been responsible for the abovementioned changes. Age may have played a part; but it would seem that it was not the most important factor, as some of the more recent lots were more profoundly affected than the older ones.

A very pertinent question in connection with the foregoing findings is, Does the above deterioration occur only in domestic products! We are led to believe that deterioration may occur in all products, inasmuch as we have found that certain samples of Canadian and French products were likewise insoluble. This together with the observation of Verda, that a sample of German neosalvarsan ob

Verda, A., Esam ísico chimico del neosalvaisan: Schweizerische Apoth, Zeit, 1920, Vol. 58, p. 280.

tained from a foreign military store and which was less than two years old had become insoluble, is indeed strong evidence that deterioration is a general phenomenon and may occur in all products. In order to ascertain the relative occurrence of deterioration in neoarsphenamine, as evidenced by a change to a more difficultly soluble product, all of the lots which complied with the official requirements, received during the period February, 1919, to February, 1920, from manufacturers B and C were tested in July, 1921. Out of the 49 lots received from manufacturer B, 13 were found to be difficultly soluble, whereas out of the 25 lots received from manufacturer C, 8 lots had changed from a readily soluble to a difficultly soluble powder; in other words, about 25 per cent of the lots issued by manufacturer B and about 30 per cent of those issued by manufacturer C had deteriorated in less than three years' time.

The behavior of lot 9, manufacturer B, indicates that incomplete drying of the product before ampuling is a factor in causing the deterioration of this lot. This lot was sent to the Hygienic Laboratory in two shipments. The history of these two shipments as obtained from the manufacturer is as follows:

Shipment 1 was sent to the Hygienic Laboratory on April 30, 1920, after 6 days' drying. On June 3, 1920, analysis showed that the remainder of the batch, which was kept in a vacuum chamber, had lost approximately 2 per cent of volatile matter and was cut accordingly. Shipment 2 was made on June 8, 1920, and differed from shipment 1 only in having lost about 2 per cent in moisture.

On reexamination of these two shipments in June, 1921, the samples from shipment 1 were found to have turned a deep orange red, were difficultly soluble in water, and emitted a strong ethereal odor, whereas samples from shipment 2 had remained practically unchanged in color, solubility, and odor.

Both shipments had been kept under identical conditions while at the Hygienic Laboratory; that is, at storeroom temperature. The occurrence of a strong ethereal odor in shipment 1 and none in shipment 2 suggested that the loss due to drying was probably largely due to volatilized ether. Upon analysis for arsenic, shipment 1 was found to contain 17.96 per cent arsenic; shipment 2, 18.32 per cent." It is possible that the heat required to seal the ampules may have been sufficient to induce in some lots changes of so slight a nature as to be undiscoverable at first examination, but which progressed during the ageing of the lots. If such is the case, it could be readily obviated by increasing the length of the ampules.

'Determined by Mr. C. G. Remsburg, of the Division of Chemistry, Hygienic Laboratory.