ANALYTICAL EDITION
JUNE 15, 1939
from extraction 1was treated as described above, recoveries of 99 per cent were obtained. An efficient method was adopted for preparing and keeping dithizone solutions. A chloroform-dithizone solution (6 mg. per liter) prepared from purified dithizone and chloroform redistilled and treated with hydroxylamine (1, 7 ) has been in use intermittently for a period of 6 months without showing signs of deterioration. Positive findings for the blank determinations shown in Tables I1 and I11 are not due to bismuth, but are due partly to errors in density readings and partly to the fact that minute quantities of lead introduced through contamination after extraction 2 have been estimated as bismuth. Although the analytical results shown in Tables I1 and I11 have been obtained with 10-gram samples of blood and 100ml. samples of urine, the method can be applied to larger or smaller samples, depending upon the concentration of bismuth present and the amount of material available.
Summary
A photometric “mixed color” dithizone method for the determination of bismuth, applicable t80biological material, has been devised. Quantitative extractions are made possible by
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isolating the bismuth as the sulfide, interference by other metal sulfides being prevented by complex salt formation with potassium cyanide and specific separation of the bismuth a t pH 2. Although used specifically for the analysis of blood and urine samples, the method is applicable to other materials. It is very sensitive; amounts of bismuth below 5 micrograms can be determined with a high degree of accuracy and 95 per cent recoveries have been obtained for quantities above 50 micrograms.
Literature Cited (1) Assoc. Official Agr. Chem., “Official and Tentative Methods”, 4th ed., p. 378, 1935. (2) Bambach, K., personal communication (data to be published later). (3) Cholak, J., ISD.
[email protected].,Anal. Ed., 9, 26 (1937). (4) Fischer, H., 2. angew. Chern., 50, 919-38 (1937). (5) Fischer, H., and Leopoldi, G., Ibid., 47, 685 (1934). (6) Haddock, L. A., A n a l y s t , 59, 163-8 (1934). (7) Hubbard, D. M., IND.ENG.CHEM.,Anal. Ed., 9, 493 (1937). (8) Tompsett, S. L., Anal@, 63, 250-2 (1938). (9) Willoughby, C. E., Wilkins, E. S., Jr., and Kraemer, E. O . , IXD. ENO.CHEW,Anal. Ed., 7, 285 (1935). ,
PRESENTED before the Division of Microchemistry at the 97th Meeting of t h e Bmerioan Chemical Society, Baltimore, Md.
Microidentification of Metrazole in Mixed Aqueous Solutions VINCENT E. STEWART, Chemical Laboratory of the Florida State Racing Commission, Miami, Fla.
ETRAZOLE (cardiazole) has become recognized as a stimulant, and its identification must be of increasing interest to the toxicologist. The literature contains but little information of this nature. The method of Wollner and Matchett (1, 2 ) is well adapted for the extraction and separation of alkaloids and other drugs which the toxicologist may encounter in body fluids. Because of its pronounced solubility in water, metrazole is not so readily extracted by this method as the alkaloids, unless appreciable amounts are present. The faintly ammoniacal, aqueous solution is extracted with ethyl acetate in a special extraction device (1) and the solvent is evaporated. The residue is taken up with a few milliliters of chloroform in a special microseparatory tube (%). This solution is extracted first with a small portion of 5 per cent potassium hydroxide and then with 0.5 N hydrochloric acid. Amphoteric alkaloids such as morphine are contained in the alkali extract, strongly basic alkaloids such as strychnine appear in the acid extract, and weakly basic alkaloids such as caffeine remain dissolved in the chloroform. When a small amount of metrazole is present it is found entirely in the chloroform fraction. Traces of metrazole occur in the alkali extract when a considerable amount is present in the original solution. Metrazole is recovered by evaporation of the chloroform, and the residue is taken up in a drop of 0.1 N hydrochloric acid on a microscope slide for the microcrystalline test. Metrazole in very dilute solution fails to form microcrystals with the usual alkaloidal reagents. In more concentrated solutions microcrystals and amorphous precipitates are formed with some reagents. Zwikker (3) has suggested a hydrochloric acid solution of cuprous chloride as a reagent for metrazole, claiming a sensitivity of 1 in 40,000. He prepares this reagent from cupric chloride and sodium sulfite im-
mediately before use, stating that the reagent cannot be preserved. The author prepares a satisfactory reagent by dissolving cuprous chloride (Baker’s) in dilute hydrochloric acid. This operation requires considerably less time than the preparation of Zwikker’s reagent. However, this reagent also must be prepared daily, since it is converted rapidly to cupric chloride. For routine use a 5 per cent solution of cupric chloride in 0.5 N hydrochloric acid seems to be much more satisfactory. This reagent is stable and does not have to be prepared daily; furthermore, its sensitivity is comparable to that of cuprous chloride reagent. One drop of a metrazole solution 1. to 1000 (about 50 microgramsj with a drop of the reagent, upon
FIGURE 1. METRAZOLE WITH CUPRICCHLORIDE REAGENT, DILUTION1 TO 1000 ( X 100)
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INDUSTRIAL AND ENGINEERING CHEMISTRY
partial spontaneous evaporation, gives an abundance of crystals which are visible even to the naked eye. Even with a metrazole solution so dilute as 1 to 10,000 (about 5 micrograms), a few microcrystals are formed. In these more dilute solutions it is advisable to allow the drop to evaporate completely before examining it under the microscope. Then the addition of a drop of water will dissolve the readily soluble cupric chloride crystals, leaving the well-formed metrazole crystals plainly visible. The microcrystals are characteristically clusters and rosettes of fine needles, mostly exhibiting arborescent forms resembling a fir tree, and polarizing poorly. Cupric chloride crystals polarize brilliantly and are considerably different in form, so that they scarcely could be confused with metrazole crystals. None of the principal alkaloids or other common drugs which the author has tested with cupric chloride reagent gave microcrystals which might be confused with metrazole. These alkaloids in mixed aqueous solution with metrazole did not appreciably inhibit its separation and identification. For obvious reasons the results of animal experimentation and data on the mode of elimination of metrazole cannot be made public a t the present time.
Modified Method of Wollner and Matchett SEPARATION AND IDENTIFICATION OF METRAZOLE IN BODY FLUIDS.Small amounts (1.0,0.5,0.1, and 0.05 mg.) of metrazole were added to 100-ml. samples of body fluids (and washings). Twenty-five grams of ammonium sulfate, c. P., were dissolved in each sample (d), and the solution was made faintly ammoniacal to litmus paper. The solution was then extracted with ethyl acetate in the special extraction device ( I ) , and the solvent was filtered and evaporated t o dryness on the steam bath. The residue was taken up by washing repeatedly with small portions of chloroform (total volume about 3 ml.) and trans-
ferred to a microse aratory tube (2) by means of a medicine dropper. A 0.25-mf portion of 5 per cent potassium hydroxide was added t o the tube, which then was shaken about 6 minutes in a blood pipet shaker, and centrifuged, and the alkali layer was removed to a second tube by means of a Wright pipet. This alkali extraction was repeated once. Then the chloroform solution was extracted with 0.5 ml. of 0.5 N hydrochloric acid by shaking 2 minutes, centrifuging, and transferring the acid layer t o a third tube. The acid extraction was repeated once with 0.25 ml. of 0.5 N hydrochloric acid, and the chloroform solution was filtered and evaporated t o dryness on the steam bath. The residue was taken up in one drop of 0.1 N hydrochloric acid and transferred to a microscope slide and one drop of the cupric chloride reagent was added to it. Microcrystals were formed in the three samples which contained 1.0, 0.5, and 0.1 mg. of metrazole, but not in the sample which contained 0.05 mg. Therefore, the smallest amount of unchanged metrazole which can be extracted and identified by this method is about 0.1 mg. (100 micrograms) per 100 ml.
Summary A toxicological method for the extraction and separation of metrazole from mixed aqueous solutions with other drugs is described, which is capable of extracting and identifying 0.1 mg. of metrazole in 100 ml. of aqueous solution. Cupric chloride is suggested as a reagent for the detection and identification of metrazole. The sensitivity is 1 part in 10,000.
Literature Cited (1) Wollner and Matchett, IND.E m .CHEM., Anal. Ed., 10,31 (1938). (2) Wollner and Matchett, unpublished work. (3) Zwikker, J. J. L., Pharm. Weekblad, 71,1170-82 (1934).
A Microbiological Assay for Riboflavin E. E. SNELL AND F. M. STRONG University of Wisconsin, Madison, Wis.
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I
N A recent critical article, Ellinger (6) discussed the errors involved in the chemical and physicochemical
methods for the quantitative estimation of riboflavin in biological products, concluding that a t present biological assay provides the most reliable method for the estimation of this vitamin. The disadvantFges of current biological methods for riboflavin are common knowledge. They are expensive, time-consuming, and have limited applicability because of the quantities of material required for assay. In addition, the reliability and accuracy of such assays are open to question (3). For example, the value of the BourquinSherman unit (3') in terms of pure riboflavin as reported in the literature (or as it may be estimated from data in various papers) varies from less than 2 to approximately 5 micrograms per unit ( I , $ , 9,1.4). I n previous papers (16, 17) riboflavin was shown to be essential for the growth of certain lactic acid bacteria, and the amount of growth was found in some cases to be directly proportional to the concentration of riboflavin in the culture medium. The growth-promoting activity of various isomers and homologs of riboflavin was also determined (16). The naturally occurring substance proved more active than any of the synthetic variants, while degradation products produced from riboflavin by light were completely inactive. The present paper describes an assay method for riboflavin in natural materials, which has been developed on the basis of the above information.
Stock Culture and Inoculum The organism used for assay purposes is carried in this laboratory as Lactobacillus casei; it is probably identical with the Bacillus casei E of Freudenreich (8). The requirement of this organism for riboflavin and other growth factors has been previously described (17, 18). Stab cultures of the organism are carried in yeast-water agar containing 1 per cent of glucose. The method of preparing and carrying cultures is indicated in the following diagram: /Stock culture a Stock culture b
4
Culture c 4
\
Cultured, etc.
Inoculum e+Inoculumf+Inoculum
4 Assay tubes h
g, etc.
From the original culture, a, a series of stab transfers (b, c, d, etc.) are made into yeast water-glucose agar. After 24 hours' incubation at 37" C. these are stored in the refrigerator. At least one tube, b, is reserved as the stock culture. The other tubes (c, d, etc.) may be used to prepare inoculum as described below. If assays are t o be made on each of several successive days, one need not grow inoculum from stock cultures (c, d ) each day, but can transfer a drop of inoculum e t o a similar tube, f, which is incubated for use the next day. Inoculum cultures should not be used after they are more than 36 hours old. One should return to a stock culture about every 5 days to minimize chances of contamination and of bacterial variation. New stock cultures corresponding t o b, c, and d are prepared at monthly intervals from a tube such as b of the preceding month.
