Colorimetric Determination of Desoxycholic Acid in Ox Bile C. R . SZALKOWSKI
AND
PC'. J. MADER, Merck & Co., Znc., Rahway, N.J .
Adequate chemical methods were needed for determining desoxycholic acid in ox bile in the presence of cholic acid, cholesterol, and fatty acids. Desoxycholic and cholic acids occur in combination, partly with glycine and partly with taurine, as glyco- and, taurocholic and glyco- and taurodesoxycholic acids. The linkage between the amino acids and bile acids is of an amide nature. On alkaline hydrolysis the nitrogenous constituents are split off. The liberated desoxycholic acid is extracted from an acid solution with ether and determined colorimetrically with salicylaldehyde.
D
ESOXYCHOLIC acid, which occurs in ox biles and is made
from cholic acid, is uaed in the synthesis of many important hormones. With the advent of the commercial production of cortisone, it became necessary to have available chemical means of determining desoxycholic acid in various biles and also to control the manufactured and isolated material. The principal bile acids in ox bile are cholic (3,7,12-trihydroxycholanic acid) and desoxycholic acid (3,12-dihydroxycholanic acid) (2). Lithocholic and chenodesoxycholic acids are present in minor amounts. The bile acids are present as the sodium salts conjugated with taurine and glycine, the linkage being of an amide nature. On alkaline hydrolysis the nitrogenous constituents are split off and the desoxycholic acid and cholic acid are liberated ( 6 , 7 ) . Because of the close similarity of the bile acids, especially cholic and desoxycholic acids, such standard methods as selective precipitation and physicochemical methods proved of little value. Methods using benzaldehyde (4,9,11),vanillin (1,6), and furfural (S,8,IO)have been recommended for the determination of the bile acids. However, these reagents were found to lack specificity. I n an attempt to find a reagent showing specificity, a series of aldehydes was investigated and salicylaldehyde was found to be sufficiently specific for the purpose of the investigation, when carefully controlled conditions were used as outlined.
minutes in an oven to ensure complete removal of the alcohol. Cool to room temperature. Pipet exactly 1.0 ml. of salicylaldehyde into a graduated cylinder, and dilute to 35 ml. with the dilute sulfuric acid. RIis by inverting the cylinder several times. Transfer to a fastdraining buret and use immediately. This solution may be slightly turbid. Add 3.0 ml. of this solution to each tube. swirl the tubes to moisten the residue completely. Place the stoppered tubes in a water bath a t 40' C. for 15 minutes, Remove the tubes from the water bath and allow to stand a t room temperature for 5 minutes. Put exactly 20 ml. of glacial acetic acid from a fastdraining 100-ml. buret into each of the tubes and shake vigorously
REAGENTS AND APPARATUS
MICROGRAMS OF OESOXYCHOLIC AGIO
Unless otherwise indicated, all reagents were C.P. or reagent grade. Desoxycholic Acid. Desoxycholic acid was recrystallized from methyl ethyl ketone to a constant melting point after vacuum drying and the purity was determined by phase solubility analysis (1.3). Various samples prepared by this procedure show slopes of less than 0.2'30. Standard Desoxycholic Acid Solution. Weigh 80.0 mg. of pure desoxycholic acid into a 100-nil. glass-stoppered volumetric flask, dissolve in Qj", ethyl alcohol, dilute to 100 ml., and mi.;. Pipet exactly 10 ml., of this alcoholic solution into a 100-ml. glassstoppered volumetric flask, dilute to 100 ml. with 95% ethyl alcohol, and mix. Each 5 ml. of the latter solution contains 0.40 mg. of desoxycholic acid. Salicylaldehyde, C.P. Dilute Sulfuric Acid. AIix 100 ml. of water and 200 ml. of concentrated sulfuric acid. Glacial Acetic Acid. Hydrochloric Acid. Ethyl Ether. Spectrophotometer. For the measurement of color a fastreading spectrophotometer such as a Becknian 1Iodel B is used.
Figure 1. Absorbancy of Desoxycholic .4cid
D
-'".
D DCSOXYCHOLIC ACID Y-METHYL DESOXYCHOLATE Ca-S-e- CHOLIC ACID L LITHOCHOLIC ACID A ----APOCHOLIC ACID C -a-*CHOLESTEROL
-.-._
1
EXPERIMENTAL PROCEDURE FOR DESOXYCHOLIC ACID
Weigh exactly 80.0 mg. of the sample into a 100-ml. lassstoppered volumetric flask, dissolve in 95% ethyl alcohol, &lute to 100 ml., and mix. Pipet exactly 10 ml. of this alcoholic solution into a 100-ml. glass-stoppered volumetric flask, dilute to 100 ml. with 95% ethyl alcohol, and mix. Accurately measure 5.0-ml. aliquots of the sample solution into -s oppered tubes, Evaporate the alcohol by heating k?:u$E?inta boiling water bath and drying a t 110" C. for 15
400
600
SO0
700
WAVELENGTH
Figure 2.
1602
Absorption Curves
1603
V O L U M E 2 4 , N O . 10, O C T O B E R 1 9 5 2 Table I.
Effect of Lithocholic Acid, Apocholic Acid, and Cholesterol
Desoxycholic Acid, Mg.
Lithocholic .kcid, J I g .
-4pocholic Acid, M g .
0 30 0 03
.... .... .... ....
Cholesterol, hIg.
0.30
....
0.30 0.30 0.30 0.30
0 030 l o
....
...
0.30 0.30 0.30 0.30
.... .... .... ....
....
0.30 0.30 0.30
0.025
0.443 0.448 0.460 0 475 0.010 0.443 0.445 0.450 0.458
....
....
0.30 0.03 0.10 1.0 3.0
.... .... ....
0.30
0.010 0.440 0.450 0.450 0.460
....
.... .... .... ....
0.30 0.03 0.30 1.0 3.0
....
Absorbancy 0.440
Table 11. Effect of Temperature and Time on Absorbancy of 200 Micrograms of Desoxycholic Acid Room Temperature
Time, Min.
40' C.
60' C.
0.210 0.240 0.240 0.230
0.120 0.100 0.085
for 10 seconds, making sure that a clear solution and a uniform color are produced. Read the color exactly 5 minutes after the addition of glacial acetic acid to the first tube. Use a suitable fast-reading instrument such as a Beckman B spectrophotometer set a t 680 mp with a red phototube and a sensitivity setting of 1
0.7
for 100% transmittance with a blank prepared simultaneously with the sample using all reagents. Treat 5.0-ml. aliquots of the standard desoxycholic acid solution in exactly the same manner and a t the same time as the sample. Prepare no more than a total of five tubes (blank, standards, and samples) a t one time, as the time limit must be carefully observed.
(X) (lO000) - 'j&desosycholic acid on dry basis ( 8 ) (100 = yoloss on drying) where X = optical density of sample and S = optical density of standard. The absorbancy of desouycholic acid and its adherence to Beer's law is shown in Figure 1. PROCEDURE FOR DESOXYCHOLIC ACID IN BILE
A4ccuratelyweigh 0.5 to 1.0 gram of bile sample into a 250-nil. flask and add 25 nil. of xater and 25 nil. of 3070 sodium hydrouide. Attach a reflux condenser and place the apparatus on a hot plate. Gently reflux for 6 hours, cool, and remove the condenser. Carefully make the solution acid to Congo red paper with concentrated hydrochloric acid. Cool, carefully transfer the solution to a 25-ml. Squibb-ty e separator funnel, wash the flask with 50 ml. of ether, add t f e ether to txe funnel, and shake for about 5 minutes. Allow the liquids to separate, draw off the aqueous layer into another funnel, and again extract with 50 ml. of ether. Repeat this procedure four times. using 50 ml. of ether each time. Combine the ether extracts and mash with 50 ml. of water. Carefully concentrate the combined ether extracts t o about 20 ml. on a steam bath. Dissolve the ether residue in alcohol and carefully transfer to a 200-1n1. volumetric flask with the aid of a stream of alcohol. Dilute to the mark with alcohol and mix thoroughly. Pipet exactly 20 ml. of this solution into a 100-ml. volumetric flask, dilute to 100 nil. nith 95% alcohol, and mix. Accurately measure 4.0-ml. aliquots o! this sample solution into large glass-stoppered tubes and proceed as for pure desoxycholic acid.
/-
EFFECT O F VARIABLES
Other Bile Acids. I n Figure 2 are given the absorption curves
0 3 t 0.2 0.1
1
t
2
I
I
I
I
I
I
I
1
4
6
8
IO
12
14
16
18
20
TIME I N MINUTES
Figure 3.
as measured on a Carey recording spectrophotometer of the color produced by desoxycholic acid, lithocholic acid, apocholic acid, and cholesterol using the technique given. Only the desoxycholic acid and the methyl desoxycholate shon a peak a t 680 mp. The other materials show some color formation and if present in large quantities will produce a background that ill cause high results. The quantitative aspects of this effect are shown in Tables I and 11. Cholic acid is present to the extent of three to four times the concentration of desoxycholic acid in the average ox bile; however, because the usual ox bile contains only 10 to 12% desoxycholic acid, the interference of cholic acid TI o d d amount to about 0.5% actual (Table 111).
Stability of Color Table 111. Effect of Cholic Acid on Absorbancy Produced by Desoxycholic Acid
I
Desoxycholic Acid, Mg. 0.30
Absorbancy 0.440 0.015 0.442 0.445 0.450 0.460 0.475
..../-+I .... 0.30 0.30 0.30 0.30 0.30
MG. OF SALICYLALDEHYDE
Figure 4.
Cholic Acid,
Effect of Salicylaldehyde on Color Formation
Mg.
0.30 0.03 0.10 0.30 1.00 3.00
Temperature and Time. The effect of temperature and time on the color formation was determined by treating 200 micrograms of desoxycholic acid with 3.0 ml. of diluted sulfuric acid containing 100 mg. of salicylaldehyde. The best results were obtained a t 40" C. and a 15-minute period of heating. This is illustrated in Figure 3 and Table 11. Salicylaldehyde. The concentration of the salicylaldehyde affects the reaction. Clear solutions were obtained when desoxycholic acid was treated with 3.0 ml. of dilute sulfuric acid containing 10 to 200 mg. of salicylaldehyde. Larger quantities of salicylaldehyde produced turbid solutions in the glacial acetic acid (Figure 4). The quantity of salicylaldehyde used in the procedure is 98 mg.
ANALYTICAL CHEMISTRY
1604
Sample 1 2 3 4
5 6
7 8 9
10
Table IV. Reproducibility of Method % Desoxycholic Acid Average 90.5-90.0-90.4 91.5-94,1-89.4 90.0-92.7-91.5 98.6-98.8-99.5 95,1-96.2-94.3 98.7-97.0-98.7 81.2-80.4-80.0 84.1-83.9-84.1 96.1-95.9-96.6 92.1-94.2-92.0
90.3 f0.3 9 1 . 7 12 . 2 9 1 . 4 10 . 9 98.9 1 0 . 3 9 5 , 2 =t0 . 7 98.1 + 0 . 8 80.5 1 0 . 4 84.0 1 0 . 1 96.2 * 0 . 3 9 2 . 8 11 . 0
Table V. Desoxycholic Acid in Typical Biles Sample
DISCUSSION OF RESULTS
The results given in Table IV indicate the reproducibility of this method when applied to commercially produced desoxycholic acid, and Table V presents assays of typical biles collected from various countries. LITERATURE CITED
(1) Abe, Yoshimi, J . Biochem. ( J a p a n ) , 25, 181-9 (1937). (2) deHaen, P., J . Am. P h a r m . Assoc., 33, 161 (1944). (3) Gregroy, R., and Pascoe, T. A , J . Biol. Chem., 83,35-42 (1929). (4) Kajiro, K., and Shimada, T., 2. physiol. Chem., 254, 57-60 (1938). (5) Kawaguti, S., J . Biochem. ( J a p a n ) , 28, 445-9 (1938). Biochem. J., 11,ll-13 (1917). (6) Sair, W.,
Desoxycholic Acid Found, % 10.3-10.9-10.6 12.0-12.4-11.9 12.5-12.1-12.3 10.8-10.9-1 1 . 3 9.5-9.9 14.2-14.8 12.7-12.8 10.9-10.6-11.0 0.5- 0 . 5 4.8- 4 . 6 9.6- 9 . 6 12.3-11.8-12.2 1.4- 1.5 1.1- 1 . 1
Average, % 10.6 12.1 12.3 11.0 9.7 14.5 12.8 10.9 0.5 4.7
9.6 12.1 1.5 1.1
(7) Pregl, F., and Buchtala, H., 2. physiol. Chem., 74, 198-211 (1911). (8) Reinhold, J. G., and Wilson, D. W.,J . Biol. Chem., 96, 637-46 (1932). (9) Scherrer, I., Helv. Chim. A c t a , 22, 1329-40 (1939). (10) Shimada, T., J . Biochem. ( J a p a n ) , 28, 149-60 (1938). (11) Ibid., 29,41-50 (1939). (12) TTebb, T. J., ASAL. CHEM.,20, 100-3 (1948). RECEIVED for review April 16, 1952. Accepted July 31, 1952. Presented a t the Meeting-in-3Iiniature, of the North Jersey Section, AXERICASCHEMICAL SOCIETY, January 28,1952.
Determination of Barbiturates Ultraviolet Spectrophotometric Method with Diflerentiation of Several Barbiturates LEO R. GOLDBAUM Department of Biochemistry, Army Medical Service Graduate School, Washington, D. This study was made to perfect a n ultraviolet absorption procedure for the determination and differentiation of barbiturates in biological materials. A method was developed which is based on the change in the absorption spectra of barbiturates i n strong alkali and i n solution a t pH 10.5. When the optical densities in pH 10.5 solution are subtracted from those in strong alkali, differences appear that are highly characteristic of all barbiturates except the n-methyl and thio derivatives. By comparingthe differences a t various wave lengths with t h a t a t 260 mp, ratios are obtained t h a t can differentiate among many of the commonly used barbitu-
R
ECESTLY a number of ultraviolet spectrophotometric
procedures have been developed for the determination of barbiturates (f-4), but none has been able to combine specificity, accuracy, sensitivity, and simplicity, nor have any attempted a differentiation of the various barbiturates. Such a procedure is needed in toxicological investigations of barbiturate poisonings and in pharmacological studies of these sedatives. This report described a simple, rapid, ultraviolet spectrophotometric procedure for the specific identification and quantitative determination of micro quantities of barbiturates and for the differentiation of many barbiturates. EXPERIMENTAL
This method is based on the observation that substituted barbiturates have an absorption band in strong alkali characteristic
C.
rates. Absorbing substances appearing in the extracts of drug-free biological materials as well as other drugs show no significant differences a t these pH’s and do not interfere in this procedure. The difference i n optical density a t 260 mp is related to the barbiturate concentration. The method can determine barbiturate concentrations as low as 0.1 mg. per 100 ml. of blood and 0.3 mg. per 100 grams of tissue. This rapid, simple, highly specific procedure is invaluable in the clinical diagnosis of barbiturate poisoning, in toxicologicalinvestigation of barbiturate deaths, and in pharmacological investigation of barbiturate metabolism.
of one resonance form, while in solution of approximately pH 10 there is a different absorption band characteristic of another resonance form. Thus, the absorption spectra of barbiturates in strong alkali show characteristic maxima a t 255 mp and minima a t about 235 mp. I n a buffer solution a t pH 9.8 to 10.5 there occur higher maxima a t 240 mp with no minima. When the optical densities of the buffered solution a t the various n-ave lengths are subtracted from the optical densities of the strong alkali, differences result which provide a highly specific identification of barbiturates. The differences are greatest a t 260 mp, decreasing through zero a t about 280 mp (isosbestic point) to a maximum negative a t around 240 or 235 mp, and increasing through zero a t about 227 mp (isosbestic point) to positive differences a t the lower wave lengths. The differences a t 260 mp for alkaline solutions containing 2 to 30 micrograms of bar-
