Colorimetric Determination of Alkaloids in Tissues by Means of Methyl Orange ALEXANDER 0. GETTLER AND IRVING SUNSHINE1 .Vew York Unicersity, .Yew York, .Y.Y.
A method was sought that would enable the toxicologist to estimate microgram quantities of alkaloids isolated from human tissues in a simple reliable routine analysis. Research has indicated that a relatively simple extraction method can isolate alkaloids from human tissues. The compound isolated is colored and lends itself to a colorimetric method for a quantitative determination. ldentification can be made with existing physical methods. A group specific reaction for alkaloids requiring minimal amounts of original material gives a quantitative estimation of the drug concurrent with its detection.
I
T i 1940, Prudhomme ( 7 ) reported a colorimetric method foi, the
determination of quinine based upon the reaction of that alkaloid with eosin to form a colored compound which is estra.c& able vit.h chloroform. Lehman and Aitken ( 4 ) in 1942 demonstrated the existence of a similar reaction b e h e e n demerol and bromothymol blue which they used for the colorimetric determination of demerol in urine. In 1943, Oberst (6) slightly modified the method of Lehman and Aitken. hlarshall and Rogers (5)in 1945 applied the bromothymol blue react'ion to the determination of cinchona alkaloids. Brodie and Gdenfriend ( 1 ) in 1945 introduced the sulfoiiic acid, methyl orange, for the colorimetric estimation of cinchona alkaloids and other organic bases in plasma and urine. The authors have modified the methyl orange reaction developed by Brodie and Udenfriend in order to apply it to the quantitative determination of alkaloids in human organs. The main steps in the method are as follows: 1. Extraction of alkaloids from tissue with boiling acidified water 2. Extraction of these alkaloids from the filtered aqueous solution by means of chloroform 3. Formation of a chloroform-soluble colored compound of the alkaloids with methyl orange
Quantitative Determination. Transfer 25 ml. of aqueous extract into a 60-ml. glass-stoppered bottle and add 4 M sodium hydroxide solution until the pH of this aliquot attains a value of 7.5 to 8.2 as indicated by the use of Hydrion indicator paper B. Two to four drops of the sodium hydroxide solution will usually be sufficient to attain the proper p H value as indicated by the dcvelopment of a green color on the indicator paper, Other experiments performed in this research project indicate that, a t this pH, alkaloids are completely extracted by the chloroform and a t the same time the extraction of organic bases normally present in tissues is kept' a t a minimum. After the adjustment of the pH, 25 ml. of chloroform are added and the mixture is shaken vigorously for 20 minutes, preferably on a mechanical shaking machine. The bott,le and contents are then centrifuged a t high speed for 3 to 4 minutes. If an emulsion is evident a t the interface, the mixture should be stirred vigorously with a glass rod and again centrifuged. The aqueous layer and any remaining emulsion a t the interface are then removed k~y aspiration. The remaining chloroform phase is transferred to a test tube and again centrifuged. Any remaining aqueous layer and residual emulsion are removed by aspiration. The chloroform layer is transferred to a glass-stoppered bottle and mixed with 0.7 ml. of the met,hyl orange reagent, and the resultant mixture is shaken mechanically for about 10 minutes. After shaking, the mixture is transferred to a test tube and the aqueous methyl orange layer is removed by aspiration. The content of the tefit tube is centrifuged, and all remaining aqueous methyl orange layer is removed by aspiration. It is imperative to remove all of the aqueous methyl orange solution to ensure that subsequent readings Kill he valid. Ten milliliters of the ehlorcform solution are pipet,ted into a cuvette and 1.0 ml. of the acidified alcohol reagent is added. In the present investigation a Coleman Junior spectrophotometer, Model 6 A.S., was employed to determine the optical densities of the solutions a t 520 nip using reagent blank to set the instrument a t 10070transmiwion.
REAGENTS
Tartaric acid, saturated solution. Sodium hydroxide solution, 4 M . Chloroform, analytical grade essential. Hydrion indicator paper B. Methyl orange solution. Saturated solution essential. May be prepared in the following manner: Add 500 mg. of methyl orange to 100 ml. of water and maintain the mixture a t a temperature of approximately 40" C. for about 20 minutes with occasional stirring. Allow the solution to cool to room temperature and filter. Boric acid, saturated solution. To ensure a saturated solution, a small amount of solid boric acid should be maintained in contact with the solution. Methyl orange reagent. Should be prepared just prior to use by mixing equal volumes of boric acid and methyl orange solutions. Phosphate buffer, p H 8.0. Mix 25 ml. of 0.2 1M potassium dihydrogen phosphate and 46.85 ml. of 0.1 M sodium hydroxide and dilute to 100 ml. Ethyl alcohol, acidified by adding 2 ml. of concentrated sulfuric arid to 100 ml. of absolute ethyl alcohol. PROCEDURE
Extraction of Alkaloid. To 500 grams of finely macerated tissue, preferably brain or liver, contained in a 2-liter Florence flask, are added 500 ml. of water and 2 ml. of the tartaric acid 1
solution. The mixture is steam distilled until about, 150 nil. of distillate have been collected. During the st.eani distillatioil process, the flask and its contents itre immersed in a boiling water bath. This operation serves to coagulate prot.eins and to facilitate the extraction of alkaloids from the tissue. If desired, the distillate may be examined for volatile poisons, but in the present investigation, inasmuch as these substances were not pertinent to an evaluation of the colorimetric method for alkaloids, the distillate was discarded. The total volume of niat,erial in the flask is measured and t,hen filtered while still hot. The volume of insoluble matter is estimated as 30Y0 of the original brain or liver tissue, in the present case 150 ml. (0.30 X 500). If other tissues are used, a correct'ion should be applied depending on the per cent of solids present in t'hat tissue. This amount is subtracted from t,he total volume in order to estimate the volume of the aqueous extract. An aliquot of the aqueous extract is employed for subsequent steps of the analysis.
In cases where the alkaloid content is extremely small, the sensitivity may be increased t,went,ytimes by extracting the 10
Prebent address, City of Kingston Laboratory, Kingston, S . Y.
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ANALYTICAL CHEMISTRY
nil. of chloroform solution containing the alkaloid-methyl orange compound with 0.5 ml. of 1 M hydrochloric acid and reading this aqueous layer in a suitable cuvette a t the same wave length. Thus the colored compound has been concentrated from a volume of 10 nil. to 0.5 ml. The analysis of plasma for alkaloids may be conducted by the original method of Brodie and Udenfriend ( 1 ) .
Table I. Recovery of Alkaloids and Other Organic Bases from Tissue Using Methyl Orange Method (Recovery, mg.) Alkaloid Added t o 500 Grama of Tissue, M g . 1.00 2.00 3.00 4.00 Atropine
1.18
Benzedrine
1.12 0.91 0.92
Cocaine Codeine Demerol Dilaudid Heroin Neohetramine Nicotine Nupercaine Pontocaine Quinine Strychnine
...
...
0.97 0.91 1.05 1.08 1.10 1.09 1.05 1.03 0.99 0.97 0.96 0.93 0.76 1.02 0.93 0.99 0.93 0.96
2.16 2.08 2.02 2.07 1.34 1.42 2.06 1. a 5 1.84 1.93 2.03 2.06 2.09 2.00 1.92 2.13 1.98 0.93 1.00 1 91 1.91 1.91 2.10 1.88
3.13 3.00 3.10 3.06 2.63 2.36 2.71 2.88 3.06 2.96 3.10 3.00 3.05 2.98 2.74 2.95 3.03 1.18 1.22 1.45 2.94 2.98 2.85 2.74
.
4.20 4.12 3.96 4.04 3.12 2.93 3.95 3.70 4.01
concentrations were plotted against their corresponding optical densities thus obtaining the standard optical density curve shown in Figure 1. The rectilinear nature of the curve indicates that the compounds obey Beer’s law within the range from 1 to 6 microgranls per ml. ANALYSIS OF TISSUES CONTAINING NO ALKALOIDS
Upon application of the above method to 70 human brains containing no alkaloids, the optical density of the chloroform solution of methyl orange, the tissue blank, was 0.013 A 0.003. Inasmuch as the reagent blank gives an optical density reading of 0.008, it is evident that a correction for the normal brain tissue blank is not necessary. The liver tissue blanks were somewhat higher with an average optical density of 0.028. With liver’this value should be subtracted from the optical density of solutions containing alkaloids, especially if the alkaloidal content is very small. It was also found that the tissue blank does not increase if the aqueous extract of the tissues is kept in the refrigerator from 4 to 6 days. Putrefied tissue blanks, however, are so high as to invalidate the method.
...
3.97 3.70 4.10 4.15 3.82 3.96 3.90 1.58 1.84 2.38 3.96
1.00
1
/ /’
/
...
3.61
...
Isolation and Identification. The aqueous extract of the tissue (100 ml.) is made alkaline and shaken with 100 ml. of chloroform in a 250-ml. bottle by means of a mechanical shaker. The mixture is then centrifuged to break any emulsion and the aqueous phase is removed by aspiration. The chloroform layer is evaporated to about 5 ml. and transferred to a sublimation tube, and all the remaining chloroform is then evaporated. The residue is subjected to a vacuum sublimation procedure as described by Gettler, Umberger, and Goldbaum (2). The resultant sublimate will contain the pure alkaloid which may then be identified by crystalline structure, micro melting point, and eutectic point using Kofler’s technique (S), and also by color reactions. A n excellent English presentation of Kofler’s technique is given by Reimers (8). EXPERIMENTAL WORK
Standard Optical Density Curves. A sample of the pure alkaloidal salt, equivalent to 50 mg. of the free base, is weighed and dissolved in 250 ml. of the phosphate buffer solution. Five milliliters of this solution are diluted to 100 ml. with distilled water to yield a working standard alkaloid solution equivalent to 10 micrograms per ml. of solution. Into a series of 60-ml. glass-stoppered bottles there are pipetted 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 ml. of the working standard alkaloid solution, Containing 25, 50, 75, 100, 125, and 150 micrograms of thealkaloid, respectively. Volumes of the buffer solution are added to each of these standard samples in order to bring the volume to a total of 25 ml. These solutions were processed in the manner d e scribed above for the aqueous extract. The samples prepared represented an alkaloid content of 1, 2, 3, 4, 5, and 6 micrograms per ml. of chloroform solvent. These alkaloidal
P O N T O C A I NL-
ATROPINE---------
----
CODLIN EI
I
I
t
I
t
3
4
I 6
I 6
GAMMA/ML,
Figure 1. Effect of Concentration on Optical Density
Because the reaction involved is a general one for alkaloids and many synthetic organic bases, a reading equivalent to the tissue blank (0.013) indicates that none of these substances is present. The method, therefore, also serves as a general qualitative test. If the optical density of the processed aqueous extract is definitely higher than the optical density of the tissue blank, some basic organic substance is present. The next step is the isolation and identification of the substance, followed by the preparation of a standard optical density curve for that substance. ANALYSIS OF TISSUES CONTAINING ALKALOIDS
Known quantities of alkaloids were added to tissues, These were then processed in the manner described. From the optical density of the colored chloroform extract, the micrograms of alkaloid present per milliliter of chloroform solvent are obtained from the standard curve. This value multiplied by the corrected
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V O L U M E 23, NO. 5, M A Y 1 9 5 1 volume of t h e original filtrate will give the quantity of alkaloid present in the 500 grams of tissue used for the analysis. The results are given in Table I. Each value is given as the average of two concurrent analyses of the same sample. Duplicate values checked within an average of 3%. Should the chloroform solvent contain much more than 6 niicrogrems of the alkaloid per ml., the analysis should be repeated, using a smaller volume of the aqueous extract. Similarly, should the solvent contain less than 1 microgram of alkaloid, a larger volume of the aqueous extract should be processed. h direct comparison of the color of the final chloroform extract of tissue filtrate with that of a standard alkaloid solution processed in the same manner, may also be used. iln analysis of the data in Table I leads to the following convlusions: 1. The recoveries of most of the alkaloids and other organic ompounds are good. 2. Some antihistamines-for example, Keohetramine-can also be determined by this method with good results. 3. The low recoveries in the case of cocaine are probably due to decomposition during the steam distillation, because aqueous solutions of cocaine that were not heated gave good recoveries. 4. The paminobenzoic acid derivative pontocaine, and nupercaine, gave good recoveries from aqueous solutions under the itlentical conditions of the method. When added to tissues, howw e r , the recoveries were low. This x a s probably due to decomposition by enzymatic action. pAminobenzoic acid is not determinable by this method and the aliphatic base obtained from the decomposition is extractable only a t high alkalinity and with another sdvent. a. Morphine, not included in Table I, gave poor results under the conditions of this method. Further studies involving the re(
covery of morphine by altering experimental conditions % ,conthe templated. The poor results are probably attributgfffe amphoteric nature of morphine. ‘aw
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IIor ACKNOWLEDGMENT
)nu
The authors are deeply indebted to Milton Levy of New York University Medical School and Bernard €3. Brodie of the Research Service, Third (New York University) Medical Division, Goldwater Memorial Hospital, who placed their laboratory facilities a t the authors’ disposal. Sidney Udenfriend of New York University Medical School merits thanks for his many helpful suggestions. LITERATURE CITED
(1)
Biodie, B. B., and Udenfriend, S.,J . Bid. Chern., 158, 705-14
(2)
Gettler, A. O., Umberger, C. J., and Goldbaum, L., AX\IZL. CHmi.,
(1945).
22, 600-3 (1950).
Kofler, A., “Mikromethoden Bur Kennzeichnung organischer Stoffe und Stoffegemische,”Vols. I and 11,Philadelphia, hrthur H. Thomas Co., 1947. (4) Lehman, R. A., and ditken, T., J . Lab. Clin. Med., 28, 787-93
(3)
(1943).
( 5 ) llarshall. P. B., and Rogers, E. W., Biochen. J . , 39, 258-60 (1945). (6) Oberst, F. W., J . Pharmucol. Esptl. Therap., 79, 10-5 (1943). (7) Prudhomme, R. O., J. pharm. chim., 9,8-17 (1940). ( 8 ) Reimers, F., Anal. Chim. Acta, 2, 1-16 (1948).
RECEIVEO September 13, 1950. Presented before the Division of Analytical Chemistry a t the 119th Meeting of the AMERICAX CHEMICAL Boston, Mass. From a thesis submitted in partial fulfillment SOCKETS. of the requirements for the degree of doctor of philosophy a t Xew York Universitv.
Colorimetric Determination of Tungsten Study of Variables Involved in Stannous Chloride-Thiocyanate Method H I R R Y FREUND, Oregon State College, Corvallis, Ore. AND
MARK L. WRIGHT
ROBERT K. BROOKSHIER Northwest Electrodevelopment Laboratory, Bureau of Mines, Albany, Ore. AND
Erratic results in the determination of tungsten led to a study of the variables influencing the stannous chloride thiocyanate method. Free acid and chloride concentrations determine the degree of reduction of the tungsten. With 7.00, 5.95, and 3.63 moles of chloride per liter, the lower limits of free acid to achieve complete reduction are 9.5,11.2, and 13.9 moles per liter, respectively. The thiocyanate concentration should be maintained about 0.2 molar for best results. The reliability of the method is thereby improved by selection of optimum operating conditions.
T
HERE are only a few chemical methods for determining
tra’ce amounts of tungsten. Current interest is directed primarily toward the colorimetric method, based on the reduction of tungstate and subsequent formation of the yellow tungsten thiocyanate complex. A bibliography relating to the development of the method is given by Geld and Carroll ( 2 ) . The changes in procedure and reagents suggested in the literature stress the need for studying the reactions involved. This paper discusses some of the variables involved in the stannous chloride thiocyanate method, and makes possible selection of optimum operating conditions, thereby improving the reliability of the method. Experiments on the nature and rate of formation of the color when the reduction and color development
are carried out simultaneously indicate the limitations of this method. A modificd procedure, separating the reduction and complexation steps, is considered in detail. The influence of free acid, chloride, and stannous ion concentrations on the reduction and the choice of proper conditions for color development comprise the major parts of the investigation. The addition of stannous chloride and then potassium thiocyanate t o an acid tungstate solution results in a greenish color, whereas the same additions to an initially alkaline solution yield a yellow color. Two tungstate samples treated according to the general procedure of Sandell ( 4 ) were identical in all respects, except that one was initially acid and the other initially alkaline. The two absorption curves, plotted in Figure 1, show a maximum
