Colorimetric Method for Determination of Desoxycholic Acid in the Presence of Certain Other Bile Acids E. L. PR4TT 4 Y D H. R . COKBTTT Winthrop-Stearns l n r . , Rensselaer, \. I .
hR1OI;S methods of estimating desoxycholic acid have been sorbancy of the standard desoxycholic acid and of the unknown solution containing desoxycholic acid and probably cholic acid a t b'sugge&d. Thesse are exemplified by the Use of vanillin and 545 and 465 m ~ . phosphoric acid (1-4), benzaldehyde ( 5 ) ,and salicylaldehyde with Calculate the desoxycholic acid content, and the cholic acid sulfuric acid ( 6 ) . content from the folloxing rqu:itions, the derivation of which is Abe ( I , 2 ) recognized that vanillin and strong (88%)phosphoric c3alculatioIls: deacrihec{ arid gave a chromophore with desoxycholic and cholic acids upon heating, whereas only cholic mp) _ X 0 ,$I4 ab. of unknois n a t 545 mp) - (ab.: of unknown a t 465 _ Desoxycholic acid, mg. = ( A(1) acid gave a color when a (ab.. of standit]rd a t 545 mp) - (ah% of standard a t 465 mp) X 0 '314 more dilute (78%) acid was uwd. IVhile the author recognized there was some difference in The cholic acid content ii eitimated on the same sample as the colors produced, the wave length at which the maxima for follo~x.s: the\? chromophores occurred wab not determined; the colors were estimated with but one filter (abs. of unknown a t 465 mp) (abs. of 1 mg. standard a t 545 mp)in the inbtrument described. (abs. of unknown a t 515 mp)(abs. of 1 mg. standard a t 465 mp) Prior to this work of . 4 k , Cholic :icid, mg. = (2) (ahs. of standard a t 545 m p ) - (abi. of standard a t 465 mp) X 0.311 Chabrol, Charonnat, et al. ( 3 ) ,described the use of vanillin and The abyorbancy values for the bile acid chromophores obtained phosphoric acid as a test for bile salts. Charonnnt 2nd Gnuthier with this vanillin-phosphoric acid reagent were determined with ( 4 ) 1:tter described the action of vanillin and phosphoric arid as a Model DU Beckman spectrophotometer. Routine tests are a test, for cholic acid (3,7,12-trihydroxycholanicacid). The negrun with n lIode1 R Beckm:in qmtrophotometer. ative reaction with desoxycholic acid (3,12-dihydroxycholanic acid) and the positive reaction wit.h cholic acid are due t.o their C4LCUL4TIONS use of a dilute aqueous vanillin solution. In the method described in this paper, vanillin dissolved in The calculation of the content of desoxycholic acid and of cholic> 85% phosphoric acid gives with desoxycholic acid a chromoacid is based on the usual procedure for the estimation of the inphore having a maximum a t 545 mp. Cholic acid, and likewise dividual contributions to a multiple component system in coloriapocholic acid (~8~'4-3,12-dihydroxycholanicarid), react with metric analysis. The maxima of cholic acid and desoxycholic the proposed reagent giving maxima at 465 mp. acid, as previouslv mentioned, are 465 and 545 mp, respectively. Sulfuric acid may not be substituted for phosphoric acid in this For a mixture of t,he chromophores dependent on the concentratest, since the former reacts with lithocholic acid and cholic acid tion of these two compounds, the equality may be writttm: as well as with desoxycholic acid, with maxima a t approximately Total xbsorbancy, 545 mp = K1 concn. of the same wave length for the three hile arids. desoxyc.holic ncid R?co~icn.of cholic acid ( 3 ) ~~
+
RE4C7EYTS
To prepare vanillin reagent dissolve 100 mg. of U.S.P. grade vanillin in 100 ml. of 85% (sirupy) phosphoric acid. Filter the solution through a Selas crucible of coarse porosity. (The snlution when storrd in the dark iQstable for several chvs.) PROCEDURE
Weigh 100 nig. of standard desoujcholic acid and diwolve in acetone. Make u p t o 100 ml. in a volumetric flask F i t h acetone IYeigh 100 mg. of sample, dissolve, and make up t o 100 nil with acetone as for the standard desoxycholic acid. Have on hand a quantitv of borosilicate glass-stopprred teit tubes (16 X 150 mm., T 16/15) uhich have been thoroughl) cleaned and dried. To one test tube add 1 ml. of the standard desoxycholic acid solution. To the second tube add 1 nil. of the unknown solution Place the tubes, unstoppered, in a water bath regulated to 70" to 80" C., and allow the acetone to evaporate completely. Transfei the tubes to a 100" C. dry oven, and heat for 10 minutes. Placrj stoppers in tubes, and cool in an ice bath. Five minutes later add rapidly 10 ml. of vanillin reagent. Replace stoppers immediately after each addition. Prepare a blank of the reagent by adding 10 ml. of vanillin reagent t o a clean dry tube. Withdraw simultaneously from the ice bath, and place in a :onstant temperature water bath regulated t o a temperature of (0" 5 1' C. Allow the tubes to remain in this bath for 20 minutes. l l i x the contents of each tube during the heatingperiod by inverting a t the end of 5 minutes and a t the end of 15 minutes. At the end of this 20-minute period remove tubes and allow to stand a t room temperature for 30 minutes. Using the blank as 1 0 0 ~ otransmittancv determine the ab-
and Total absorbancy, 465 mp = K ; concn. of desoxycholic acid S A concn. of cholic acid
+
(4)
Solving for the desoxycholic acid and cholic: arid conc.entration+, respectively, follows: Concrntration of desoxycholic acid
=
Concentration of cholic acid =
The K values may be given as follows: K , = absorbancy a t 515 mp per mg. of desoxycholic acid reference standard taken KI. = absorbancy a t 545 mp per m g . of cholic acid reference standard taken k'; = absorbancy a t 465 mp prr mg. of desoxycholic acid reference standard taken S A = absorbancy a t 465 mp per mg. of cholic acid referenrr standard taken T h r values of K2 and K: have been determined (Figure 2 ) : I *
k', 0.474 h7(2= 1.509
These values are subst,ituted as constants in Equations 5 and 6. The values K1and K ; are the values determined from the desoxycholic acid reference standard during the course of any particular
1665
A N A L Y T 1,CA L C H E M I S T R Y
1666 analysis. They are actually the observed absorbancy values of the standard for the 1.0-mg. sample taken. Final simplification leads t o the equations written under Procedure (Equations 1 and 2).
030
c
DISCUSSION
Data. Figures 3 and 4 represent the spectra of desoxycholic acid and cholic acid, respectively. Figure 1, the absorbancy us. concentration curve for desoxycholic acid, shows the adherence of the chromophore formation to Beer’s law and also the concentration range over which this procedure may be applied.
I
0.4
1
>. 0.15 n
p 0.10
t
/
x)
0.0
0.05 0.10 0.1 5 0.20 CONCENTRATION, MG. PER 10 ML.
Figure 2. Absorbancy of Cholic Acid at 545 and 465 mp
0.1
0.35
ILL_
0.P
0.4 0.6 0.8 1.0 CONCENTRATION, MG. PER 10 ML.
0.0
Figure 1.
-4bsorbancy of Desoxycholic Acid at 545 and 465 m p
Typical data obtained in the routine analysis of various crude lots of desoxycholic acid-cholic acid mixtures by the foregoing procedure are given in Table I. These results are presented to demonstrate the reproducibility within individual group analyees. Table I1 represents data obtained when varying known amounts of cholic acid are added to a k n o m amount of desoxycholic acid. General. Desoxycholic acid may be obtained as a natural product in bile. When obtained from this source, cholic acid may be expected as an impurity, the amount of which depends on the extent of purification.
0.30
s2
20
035
o.Pol 0.1 5
\ 450
500 550 WAVE LENGTH, rns
600
Figure 3. Spectra of Desoxycholic Acid Table I. Sample NO.
42G
38A
48C
48E
48D
Desoxycholic Acid and Cholic Acid Content,, (Each sample consisted of 1 mg. of substance) Desoxycholic Cholic Sample Desoxycholic Cholic Acid, % Acid, % No. Acid. % Acid, 70 81.9 48B 82.8 81.0 82.8 0.5 1.8 80.1 83.5 Av. 8 1 . 0 Av. 8 3 . 0 89.6 83.6 89.8 1.6 184 83.6 6.0 89.8 84.4 Av. 8 9 , 7 Av. 8 3 . 9 88.7 XI-54 94.3 1.3 88 7 2.2 93.9 88.7 94.3 Av. 8 8 . 7 Av. 9 4 . 2 86.8 56 86.8 87.8 0.5 86.3 3.4 87.3 86.7 Av. 8 7 . 3 Av. 8 6 . 6 84.2 56.4 87.7 8 4 . 2 Negligible 87.7 3.3 87.7 84.6 Av. 8 4 . 3 Av. 8 7 . 7
Synthetic desoxycholic acid derived from cholic acid might possibly contain mono- , di- , and triketocholanic acids to some extent. llore probable is the contamination of the synthetic product with the parent substance, cholic acid, and the product resulting from overoxidation, lithocholic acid (3-hydroxycholanic acid).
Table 11.
Recovery of Desoxycholic and Cholic Acids from Known Mixtures
Desoxycholic ricid Taken, Mg.
Cholic Acid Addition. y
545mp
I 00 1 00 1.00
10
0.325 0.325 0.333 0.339 0,334
465mr 0.262 0.276 0.283 0.300 0.306
0.316
0.250
1.00 1.00 Standard 1.00
20 30 40 50
Absorbancy
Desoxycholio .4cid Found, Mg. 1 02 1.00 1.02 1.03 1.00
Cholic Acid Found, Y
F,
25 25 42 58
V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 The foregoing assay procedure can be used to evaluate the content of desoxycholic acid in the presence of any of the above hy-
1667 The proposal of a reaction mechanism for the preceding chromophore formation would, a t this time, be speculative. Hoaever, observations made during the course of this study suggest the involvement of oxygen units with two unshared electron pairs. In the case of desoxycholic acid (3,12-dihydroxycholanic acid) and cholic acid (3,7,12-trihydroxycholanicacid) specific chromophores are realized with the prescribed vanillin reagent. Certain other sterols form specific chromophores, and for such sterols esterification does not change the quality of spectrum. Short-chain alcohols, low molecular weight acids, and water, when present in appreciable concentration, inhibit the chromophore formation described herein. If added subsequent to the color development they quench the chromophore formation, and a negative rate curve will be observed. For this reason these substances are minimized in the reaction mixture. Lithocholic acid (3-hydroxycholanic acid) does not form a colored complex with the vanillin reagent Whether it combines in any way with the reagent is not known. Its failure to form a chromophore makes possible highly specific assay procedure for desoxycholic acid especially when this acid is derived synthetically from cholic acid. LITERATURE CITED
450 500 550 WAVE LENGTH, rnr
Figure 4.
600
Spectra of Cholic Acid
droxy- and ketocholanic acids. The quantities of cholic and desoxycholic acids are evaluated simultaneously; no color is given with the ketocholanic acids and lithocholic acid.
Abe, Y., J . Bzochem. ( J a p a n ) ,25, 181-9 (1937). I b i d . , 26, 323-6 (1937). Chabrol, E., Charonnat, R., Cottet, J., and Blonde, P., Compt. rend. soc. b i d . , 115, 834-5 (1934). (4) Charonnat, R., and Gauthier, B., Compt. rend., 223, 1009-11 (1946). ( 5 ) Kaairo, K., and Shimada, T., 2. physiol. Ciiem., 254, 57 66 (1938). (6) Saalkowski, C. R., and hIader, TT’. J., d a a ~ .CHEM., 24, 1602 (1952). RECEIVED for review March 18, 1952.
Accepted July 2, 1952.
Application of line-Width Method of Spectrogram Evaluation to Spectrochemical Analysis of Plant Products ROBERT T. O’CONNOR AND D. C . HEINZEL3IAN Southern Regional Research Laboratory, New Orleans, La.
S E of the main obstacles to the extended use of spectrochemical methods for the quantitative determination of metallic cations in plant products has been the requirement of a large number of working curves. Plants and their products are submitted for analysis in an almost endless variety of widely diff went physical forms. The preliminary problem of reducing these samples to a form suitable for spectrochemical analysis has retarded widespread acceptance of this method in the analysis of plant products, in contrast to its extensive use in the analysis of metals and alloys where often a rod of predescribed diameter and length can be tooled and compared directly with readily available rods of standard composition. Generally applicable procedures for preparation of samples and a reduction in the requirement of working curves are essential if advantage of the simplicity and speed of spectrochemical methods for the analysis of plant products for metallic cations is to be realized. A generally applicable method has been devised and evaluated for the ready analysis of plant products. It involves the preparation of ashes sufficiently buffered to permit the evaluation of metallic cations in most plant products from a single set of working curves. A modification ( I d ) of the line-width method is used for the evaluation of the spectrograms. The line-width method was first thoroughly investigated by Coheur ( 4 , 6 ) , following simultaneous suggestions regarding its use by Eisenlohr and .4lexy (7) and by Gerlach and RollRTagen (9). The advantages of the method, arising from the fact that it is in-
sensitive to time of exposure variations and t o changes in dcveloping conditions, and that it can be used ovsr wide ranges of concentrations, as it is independent of plate density and even not affected by self-absorption, have been pointed out by Ahrens ( I ) , Harvey ( 8 ) , and Nachtrieb ( I O ) . In spite of these advantages the line-width method does not seem to have been generally adopted. A special application of the method to the determination of relatively high concentrations of zinc (up to 10%) by Eastmond and JVilliams ( 6 )appears to be the only description of its use except in a paper from this laboratory (fa). This nonacceptance has been due, in part a t least, to difficulties in obtaining accurate measurements of line width. Coheur ( 5 )measured the width of the line of the internal standard a t a galvanometer reading equal to the densest portion of the analysis line with a direct-reading Zeiss photometer or a modified direct-reading ?Jo11 photometer. Eastmond and Williams (6) describe their technique as follows: “For width measurements, line contours were recorded on photographic plates with the 16 mm. microscope objectives, a 0.5 mm. slit and a 6 4 X magnification on the recorder. The widths of the zinc line a t maximum density of the same barium and cadmium lines were measured from the photographic recording by means of a small scalemagnifier.” Use of an automatic recording microphotometer, Euch as the Leeds and Northrup instrument employed by the authors, and measurement of the line width directly from the profiles on the recorder strip, does not seem to have been de-
