Colorimetric Determination of Diethylstilbestrol MANUEL TUBIS, 4860 North 8th St., Philadelphia, Penna. AND
ALBERT BLOOM, Samet Laboratories, Philadelphia, Penna.
T
HE universal interest in diethylstilbestrol as a substitute for the naturally occurring estrogens has resulted in the proposal of several methods for its quantitative estimation. Most of the methods (9,4,6,8) are not applicable in the presence of the usual tablet compositions. Dechene (2) recently applied the “xanthoproteic” reaction to the compound and used this as a basis for a colorimetric method. According t o this author, this reaction is not applicable in the presence of a n y other “phenyl compounds”. In undertaking this investigation, the writers sought a reaction which was specific for one of the functional groups present. As seen from the structural formula, diethylstilbestrol contains two phenolic hydroxyl groups : C&-C C-CzHs
TABLE 11. ANALYSISOF COMMERCIAL TABLETS OF DIETHYLSTILBESTROL Tablet
a
b
I
OH
Labeled Quantity
Difference Found Ma.
Standard set at 20,average of 5 readings. Average of 5 readings.
mately 5 mg. of diethylstilbestrol is transferred to a 100-ml. volumetric flask, 30 ml. of alcohol are added, and refluxed on a water bath for 15 minutes. The solution is then cooled and made to volume with distilled water, mixed, and filtered. A 10-ml. aliquot is treated as described above and, a t the same time, a standard containing 0.5 mg. of diethylstilbestrol per 10 ml. in 30 per cent alcohol is treated similarly. After 1 hour the colors are compared in a colorimeter. This unknown solution may also be examined photometrically and the concentration derived from the graph. COLORED TABLETS.The weighed powdered sample, prepared as above, is transferred to a 100-ml. separatory funnel containing 50 ml. of water and extracted with one 20-ml. and three 10-ml. portions of ether. The ether extracts are combined and evaporated. The residue is dissolved in 30 ml. of alcohol, transferred to a 100-ml. volumetric flask, made to the mark with distilled water, and filtered. A 10-ml. aliquot is used for the analysis.
n n I
Quantity Recovered Photometerb Av. MQ. Mo.
Color-
imetera
I
I
OH
Since colorimetric methods for the determination of phenols are well established, the writers applied several of these reactions t o the compound and found that the reaction of Fslin and Ciocalteu (6, 7‘) gave t h e best results. In this reaction the labile complex phosphomolybdic phosphotungstic acids are reduced b y t h e phenolic hydroxyls t o give a blue solution of tungstic oxides. It was found that the color produced was stable and readily adaptable t o colorimetric or photometric determination and gave a linear relationship over t h e range of concentrations required. The presence of alcohol, sugars, talc, and fatty acids and their salts did not interfere. Watersoluble colors were eliminated b y the technique described below. Figure 1 shows the straight-line graph obtained by plotting t h e concentrations against the photometer readings. Table I gives data on the extraction of diethylstilbestrol from a n artificial mixture. The method was applied t o t h e analysis of commercial tablets and the results are given in Table 11.
Experimental A neutral wedge photometer with a B. & L. EQUIPMENT. Smoke C. glass wedge and filter No. 65 (1) was used with a 2.5-cm. (1-inch) cell. A Hellige Duboscq colorimeter was also used. COLORREACTION.Diethylstilbestrol (0.2 to 0.8 mg.). in 10 ml. of 30 per cent alcohol contained in a 100-ml. volumetric flask is diluted with 50 ml. .of distilled water and 5 ml.f ! reagent (7) are added. After mixing, 15 ml. of 20 per cent sodium carbonate (anhydrous) solution are added and the solution is diluted to the mark with distilled water, mixed, and allowed to stand at room temperature for 1 hour, at which time the color is fully developed. The concentrations are plotted against photometer readings to ve a graph, Figure 1, from which the concentration of an ung o w n solution may be read. ANALYSIS OF COMPRESSED COLORLESS TABLETS.A number of tablets equivalent to 10 mg..of diethylstilbestrol are weighed and ground. An accurately weighed sample equivalent to approld-
GRAPHRELATING WEIGHTOF DIETHYLFIGURE 1. TYPICAL STlLBESTROL TO PHOTOMETER READINGS
Acknowledgment The authors gratefully acknowledge receipt of the materials used in this investigation from the following: Abbott Laboratories, North Chicago, Ill.; Eli Lilly and Company, Indianapolis, Ind.; Merck & Co., Inc., Rahway, N. J.; and Carroll Dunham Smith Pharmacal Company, Orange, S . J.
Literature Cited
OF DIETHYLSTILBESTROL FROM A TABLET TABLE I. EXTRACTIOX MIXTURE
Sample0
Ether Used
1
20 20,lO 20, 10, 10 20. 10. 10, 10
M1. 2 3 4
Diethylstilbestrol Present Recovered Mo. M!? 5 3.4 5 5
5
4.6 4.8
4.6
a 1 gram of mixture of starch, lactose, and stearic acid containing diethylstilbestrol.
309
(1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 5th ed., p. 349 (1940). (2) Dechene, E. B., J. A m . Pharm. Assoc., 30, 208 (1941). (3) Dingemanse, E., Acta Breoia Neerland. Physiol. Pharmacol. Microbial., 10, 118 (1940). (4) Dingemanse, E., Nature, 145, 825 (1940). (5) Elvidge, C., Quart. J . Pharm. PharmacoZ., 12, 347 (1939). (6) Folin, O., and Ciocalteu, V., J. Biol. Chem., 73, 627 (1927). (7) Snell, F. D . , and Snell, C. T., “Colorimetric Methods of Analysis”. Vol. 2, p. 351, New York, D . Van Nostrand Co., 1937. (8) Sondern, C. W., and Burson, C., IND.ENG.CHEM.,ANAL.ED., in press.
