Detection and Quantitative Determination of 4-Amino-2-methyl-1-naphthol A Synthetic Vitamin K AMEL R. MENOTTI Chemical Laboratory, American Medical Association, Chicago, Ill.
T
HE hydrochloride of 4-amino-2-methyl-1-naphthol has
vantage over the more readily prepared sodium pentacyanoammineferroate; consequently, the latter compound was chosen for the quantitative determination of 4-amino-2methyl-1-naphthol. The procedure finally adopted depends on the interaction of sodium pentacyanoammineferroate in alkaline solution with 4-amino-2-methyl-1-naphthol to produce an intense blue color and the comparison of the intensity of this color with that produced by known quantities of the aminonaphthol. The production of a blue color through the interaction of sodium pentacyanoammineferroate, Naa[Fe(C^U)5NH3],and the aminonaphthol may be attributed to a reaction analogous to that which has just been shown-i. e., the replacement of the coordinately bound molecule of ammonia by a molecule of the aminonaphthol. Curve A in Figure 1 represents a n absorption spectrum typical of the colored solutions employed in the quantitative determination of 4-amino-2-methyl-1-naphthol. The solution employed to determine the curve shown was prepared by the addition of 1.0 mg. of 4-amino-2-methyl-1-naphthol to 1 ml. of the sodium pentacyanoammineferroate reagent solution. This mixture was allowed to stand for 15 minutes and was then diluted to a concentration corresponding to 0.004 mg. of aminonaphthol per ml. The solution exhibited but little absorption in the blue region of the spectrum. Curve B illustrates the contribution to the total absorption made by the yellow colored reagent solution. The reagent exhibits no appreciable absorption above 5000 A. For this reason, visual matching of the blue color of the solution is simplified, and more precise spectrophotometric measure-
been introduced to the medical profession as a watersoluble therapeutic agent which possesses vitamin K activity. Increased interest in this compound has necessitated the development of a chemical method for its detection and quantitative determination in preparations now on the market. The procedure described has been found suitable for the assay of preparations containing 4-amino-2-methyl-1-naphthol and yields consistent results with quantities as low as 0.02 mg. Hitherto, the assay of solutions and dry mixtures of 4-amino2-methyl-1-naphthol hydrochloride has been dependent on the curative effect of the compound on vitamin K-depleted chicks (4). It is hoped that the proposed chemical method may supplant, in part, the more complicated biologic assay procedure. Of possible chemical methods to be investigated, it was felt that a colorimetric method should take precedence over volumetric or gravimetric procedures because of the small quantities of material to be dealt with and the relative ease of colorimetric manipulation. Accordingly, a reagent was sought which would Possess a high degree of sensitivity Produce a color which would obey Beer’s law of light absorption and be sufficiently stable to allow time for accurate color comparisons Not be adversely affected by the presence of sodium bisulfite, since this compound is used to stabilize aqueous solutions of
4-amino-2-methyl-1-naphthol Possess sufficient specificity t o permit differentiation of the compound in question from its possible decomposition products The latter characteristic is of importance because in the biologic method of assay such differentiation is not possible. One of the oxidation products of 4-amino-2-methyl-1-naphthol is 2-methyl-1,4naphthoquinone (menadione), which possesses the same antihemorrhagic activity when measured on a molecular basis. Investigation of sodium pentacyanoammineferroate, Na3[Fe(CN)6NH3], as a possible reagent revealed that this compound possessed the desired characteristics. Anger (1) described a colorimetric test for the detection of primary aromatic amines which required sodium pentacyanoaquoferriate, Na2[Fe( C S ) 5 H 2 0 ] as , the reagent. This test provides a basis for the procedure followed herein. In a discussion of Anger’s test, Feigl ( 2 ) attributed the production of color to the substitution of the coordinately bound water molecule, in the pentacyanoaquoferriate complex, by a molecule of the aromatic amine according to the following reaction : [Fe(C S ) jHzO]- - + R-SHz + [Fe(CX)sR--?;H,]-- + H,O During the course of this investigation, FIGURE1. DETERMINATION O F 4-AMINO-Z-METHYL-1-NAPHTHOL sodium pentacyanoaquoferriate as employed by A . Absorption of solution resulting from interaction of 4-amino-2-methyl-1-naphthol Feigl(2) and sodium pentacyanoammineferriate hydrochloride with sodium pentacyanoammineferroate were tested but were found t,o possess no adB . Absorption of sodium pentacyanoarnmineferroate reagent solution 601
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ments of the absorption maximum at 6650 A. would provide a hiahlv - " accurate determination. log 7 to To test the applicability of Beer's law k = ; Io> I this solution, the values of log f were determined at 6500 1. for various dilutions. It is obvious from the values of IC (Table I) that the solution satisfies Beer's law within the limits of experimental error.
(
Accuracy of Method The data reproduced in Table I1 were obtained by visual colorimetric assay of synthetic mixtures of known composition. The greatest error occurred with the solutions of lowest aminonaphthol content because the concentration of the standard solution was maintained a t 1.0 mg. per ml., while the synthetic mixtures varied in aminonaphthol content. The error is due to the difference in color caused by the presence of excess reagent in the solutions of low aminonaphthol content and may be eliminated by the use of a standard of appropriate strength. The effect of bisulfite on the developed color was determined by adding known amounts of sodium bisulfite to solutions of equal aminonaphthol content, then adding the reagent and comparing the resulting colors. From the data obtained i t was apparent that amounts of sodium bisulfite up to 5 mg. per milligram of 4-amino-2-methyl-1-naphthol had no effect on the color; quantities above this amount caused the color to develop more slowly and resulted in a reduction of the final color intensity. TABLE I. APPLICABILITYOF BEER'SLAW TO COLORED SOLUTIOh. OBTAIKED BY INTERACTION O F SODIUM PENTACYANOAYMINEFERROATE WITH 4-AhlIluO-2-hlETHYL-1-NAPHTHOL C.
I log -2 I
Concentration of Aminonaphthol Mg./ml.
1.000 0.608 0.402 0 192 0,098
0.005 0.004 0.002 0.001
0.010
k,
1 log II' 100 101 100 96 98
The presence of 2-methyl-1 ,4-naphthoquinone, a n oxidation product of 4-amin0-2-methyl-l-naphtho1, was found to have no effect on the development and final intensity of the color.
Specificity of Reaction Sodium pentacyanoammineferroate in alkaline solution reacts with other primary aromatic amines to yield blue colors, with aliphatic and aromatic nitroso compounds to yield green colors, and with hydrazine and its derivatives to produce red to yellow derivatives. Such compounds might interfere in the application of this method; however, it is believed that there is little likelihood of their presence in dosage forms of 4-amino-2-methyl-1-naphthol intended for oral or parenteral use. Aliphatic and secondary or tertiary aromatic amines do not interfere.
Determination of 4-Amino-2-methyl1-naphthol The reagent, prepared as described below, may be employed for the detection of this drug in aqueous solution. One drop of the unknon-n solution is placed in the depression of a spot plate and one drop of reagent is added. After a few minutes the appearance of a blue or green color indicates the presence of 4-amino2-methyl-1-naphthol. Used in this manner, the test will detect 0,0005 mg. of the aminonaphthol. (This te,t must be employed with due regard to the interferences mentioned above.) REAGENTS.Standard 4-amino-2-methyl-1-naphthol solution: 60.53 mg. of pure 4-amino-2-methyl-1-naphthol hydrochloride and 50.0 mg. of sodium bisulfite dissolved in 50.0 cc. of distilled
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water contained in a dark glass-stoppered bottle. Gnder conditions of normal usage this solution is stable for from 4 to 6 hours; when allowed to stand overnight it may decrease in strength as much as 10 to 15 per cent. Ssdium pentacyanoammineferroate solution: 250 mg. of sodium pentacyanoammineferroate and 500 mg. of anhydrous sodium carbonate dissolved in 25 cc. of distilled water. This solution is stable for about one ~ e e k . TABLE11.
h A L Y S I S OF
4-8mino-2-methyl-lnaphthol Added
M Q.
1.00 1.00 0.95 0.93 0.90 0.85 0.80
SAMPLES O F KNOWN COMPOSITION Q;;i? Mg.
1.00
1.00 0.97 0.94 0.93 0.87 0.84
Error
M Q.
0.00 0.00 0.02 0.01 0.03 0.02 0.04
The crystalline reagent Kas[Fe(CS)6XHH3] was first described by Hofmann ( 3 ) . I t may be prepared as follows: Ten grams of sodium nitroprusside are ground to a fine powder and added to 30 ml. of concentrated ammonium hydroxide solution. The mixture is shaken to dissolve the salt, and the resulting solution is left overnight at from 0" to IO" C. The crystals which form are removed by filtration, washed several times with 95 per cent ethanol and once with absolute methanol, and dried over sulfuric acid in vacuum. (Additional crystalline material may be obtained in a finely divided state from the mother liquor by adding 95 per cent ethanol, filtering, and washing in the manner described. The material thus obtained is of lower purity than the crystalline substance which forms overnight, but it may be used effectively as a reagent.) PROCEDURE. For the assay of ampouled solutions, appropriate dilutions are made, so that the resulting solution contains approximately 1 mg. of 4-amino-2-methyl-1-naphthol per ml. Powdered mixtures which contain the drug may be extracted with distilled water containing 0.1 per cent of sodium bisulfite, added to inhibit decomposition of the drug. The extract should be diluted to contain about 1 mg. of 4-amino-2-methyl-1-naphthol per ml. of solution, and the final solution may be filtered if necessary. The unknown solution (1 cc., calculated to contain about 1 mg. of 2-methyl-4-amino-1-naphthol) is transferred to a 50-ml. volumetric flask, 1.0 ml. of the standard aminonaphthol solution is placed in a second 50-ml. flask and, if necessary, the contents of the flasks are adjusted to approximately equal volumes with distilled water. Then 1.0 ml. of sodium pentacyanoammineferroate reagent is added t o each flask, mixed, and set aside in the dark for 15 minutes. Finally, distilled water is added to the mark of each flask and mixed, and the solutions are compared in a colorimeter. If the solutions to be compared are found to vary more than 10 per cent in aminonaphthol content, it is best to carry out a second determination after adjusting the concentration of the standard to approximately that of the unknown. Consistently accurate results may be obtained in this manner.
Summary
A method developed for the quantitative determination of 4-amino-2-methyl-1-naphthol depends on the interaction of sodium pentacyanoammineferroate in alkaline solution with 4-amino-2-methyl-1-naphthol to produce a blue color. The absorption spectrum of a typical solution obtained in this determination has been determined. The reagent described will detect the presence of 0.0005 mg. (0.5 microgram) of 4-amino-2-methyl-1-naphthol. Acknowledgment The data for the absorption curves were obtained by means of a Cenco-Sheard spectrophotelometer through the courtesy of Thorfen R. Hogness, in the laboratories of Spectroscopic Biological Investigations, University of Chicago.
Literature Cited (1) Anger, V., Mikrochim. Acta, 2 , 3 (1937). (2) Feigl, F., "Spot Tests", p. 317, New York, Nordemann Publishing Co., 1939. (3) Hofmann, K., Liebigs Ann. Chern., 312,21 (1900). (4) Thayer, S. A,, MoKee, R. W., Binkley, S. B., M a c C o r q u o d a l e , D. W., and Doisy, E. A , , Proc. SOC.Ezptl. B i d . M e d . , 40, 47s (1939); 41, 194 (1939).
