-Table II.
Foreign Ion, P.P 11. Ca, 500 Alg, 100 l l n + T , 10 Fe+-, 10
Interferences
F Present, P.P.AI. 0 00 1 00 F Found, P.P.M.
*
5 Ce-+-: 5 Cr (as Clo,), 5 Cd, 5 h-i+-. 5 Pb++: 5 Zn, 5 AI, 5 Al, 10 Cl? (as hypochlorite), 10
0.10 0.05 0.05
0.10
0.00 0.00 0.05 0.00 0.05 0.00
1.00 1.00 0.95 1.10
Location Woodboro. l l d . Rocky Ridge, l l d . Antietam, Md. Sumter, S.C. Chico, Calif. Colusa, Calif.
Dunsmuii, Calif. Tuscan Spring, Calif. Azores Islands
Dissolved Solids 42 306 464 31 20, 700 15,600
4,420 21,500 617
1.00 0.94 0.75 0 98
HCOaj 12,000 sO3, 2000
1 .oo
C1, 10,000 C1, 20,000 Si&, io0 B (ae tetraboi,ate), 10 POA. 5 PO;; 10 PO,, 25 P207, 10 POI, 10 CX, 10
1.05 1.00 0.97 1.00 1.00 0.97 1.oo
1.05 1.oo
Table 111.
Foreign Ion, P P AI.
pensates for the absorbance of the sumended matter in the same way that F present, p,p,i\~. it conipensates for color. 0 00 1 00 APPLICATION F Found, P.P.11.
i.00
0 00 0.05 0.30 0.52 0.60
1.05 1.00 1.00 1 00 1.00 1.10 1.40 1, 4 3
1.00
A4pproximately 500 natural surface and ground water samples of varying types have been analj zed by the method since its adoption by one U. S. Geological Survey laboratory. Recovery tests applied to randomly selected samples have been satisfactory. I n Table 111 the results of nine such tests on widely varied watm samples are giyen. I n each case 1.00 p.p.m. of fluoride was added to the water sample. ACKNOWLEDGMENT
Recovery Tests
Significant Constituents HCOI, 389 Fe, 1.9 SO,,3050 HCOI, 5680 SiO,, 109 Fe, -6.9 NO,. 254 Fe,"9.4 H2S, 172
Samples of swamp water with Hellige color yalues as high as 150 have been successfully analyzed b y use of the correction solution. Natural turbidity from suspended matter seldom causes
Fluoride, P.P.hI. 5 Initial Recoveled Recovery 0 15 1 15 100 105 0.05 1 10 9i 1 27 0 30 98 0 17 1 15 100 2 80 1 80 2 75 95 1 so
0.15 2.50 0.55
1.07 3 60 1.55
92
110 100
trouble because the precipitation of barium sulfate brings down the suspended matter with it. I n the rare case where the suspended matter refuses to flocculate, the correction solution coni-
The author wishes to acknowledge the effective assistance of Gerald Gordon who performed much of the experimental work. LITERATURE CITED
(1) Lamar, TV. L., IND.ENG. CHEiI., AUL. ED. 17,4S (1945).
(2) XIegregian, S., AKAL.CHEW26, 1161 (1954). (3) Megregian, S., hlaier, F. S., J . A m . W a t e r Works Assoc. 44. 239 (1952). (4) Revinson, D., Hartley, J. H., ' h A 1 . . CHERI. 25, 794 (1953). ( 5 ) Sanchis, J. AI., IND.ENG. CHEX, .%NAL. ED.6 , 134 (1934). 16) Thrun, w.E., h . 4 ~ .CHEar. 22, 918 ' (1950). (7) Willard, H. H., Winter, 0. B., IND. ESG. CHE~I., iix.4~.ED. 5 , 7 (1933). RECEIVEDfor review October 13, 1956. iiccepted July 3, 1957.
Microdetermination of Hippuric Acid in Urine HOWARD C. ELLIOTT, Jr.' Biochemistry Deparfmenf, University o f Alabama Medical College, Birmingham, Ala.
A new spectrophotometric micromethod for the analysis of hippuric acid in urine was developed in order to study the kinetics of the excretion of hippuric acid by man. The method requires a sample of only 0.1 to 1.0 ml. and is based on a partial separation of hippuric acid from other urinary components by ion exchange chromatography, Hippuric acid in the aqueous eluate is quantitatively determined by ultraviolet spectrophotometry. Recovery studies show a range of 98.8 to 101.3% recovery of hippuric acid added to urine. The method is rapid and precise, so that experiments, in which 30 to 40 urine samples are collected in several hours, can be completed the same d a y they are started. Kinetic data obtained following small
1712
ANALYTICAL CHEMISTRY
doses of benzoate or hippurate are of value in the study of liver and kidney function.
T
he primary drawback t o expansion of studies of benzoic acid metnbolism has been the lack of a rapid, sensitive method for the determination of hippuric acid in small samples of urine. Several micromethods have appeared in the literature in recent years. Dickens and Pearson (3) reported a technique which was applicable t o samples containing 0.05 to 10.0 mg. of hippuric acid which lacked the sensitivity required for the experiments contemplated by the author. Gaffney and coworkers (6) reported a method n-hich utilized paper chromatographic separation of hippuric acid. As 20 to 40 samples were to be
analyzed in each experiment, paper chromatography was eliminated as too time-consuming. R u (14) had reported a differential extraction method for the determination of hippuric acid ivhich had good precision, but n hich required a sample of a t least 10 ml. for 3 single determination. Evaluation of techniques available in the literature led to the choice of X u ' s method for comparison with the method presented in this paper. Spectrophotometric studies indicated that microgram quantities of hippuric acid could be quantitatively determined, provided the hippuric acid could Present address, University of Alabama, Birmingham Center, Birmingham 3, Ala.
06 05
W
2
aK
04
O3
0
2
0.2
a
01 6
210
220
230
240 250
260
270
280-290
WAVE LENGTH - m l Figure 1.
Absorbance spectra
be separated from the ot'lier components of urine, which would also have a strong absorption in the ultraviolet. Anion exchange resins were first considered for this purpose. Several esperimenta,l types lvere tried, but they all had a high and variable ultraviolet absorption blank. The use of cation exchange resins to achieve a partial separation n-as then studied, and it was shown t,hat Dowex 50-X-%hydrogen form would allow hippuric acid to pass unchanged through the colunin, ix-hile creatinine and amino acids were adsorbed on the column. The only other major urinarJ- component appearing in the eluate from the cationic column was uric acid. -4s uric acid has a \vel1 defined ultraviolet absorption ( 2 , @, it, was posqible to measure hippuric acid in the presence of uric acid using the principle cii additive absorbancy. METHOD
Apparatus. All absorbance measrnents IT-ere made with a 11odel DU Beckman spectrophotometer. Potentiometric balance was obtained b y v:iij-ing t h e setting of t h e sensitivity control. T h e wave length scale of t h e instrunient was calibrated against a h j drogen discharge lamp. After consideration of the absorption data, a constant slit width of 0.4 nim. was chosen as representing the best choice with regard t o sensitivity and the possibility of stray light interference. The photometric accuracy of the instrument n-as checked by the method of Ewing and Parsons (5) and found to be n ithin the limits described by these authors. -411 measurements mere made ill matched 1-em. silica cells against a water blank. Reagents. T h e hippuric acid used as t h e priniaiy standard for t h e spectrophotometric study n-as obtained commercially. It melted a t 188" C. after recrystallization from water. Xitrogen analysis, in triplicate. by t h e Kjeldahl method gave an average
value of 7 3 0 % compared with the theoretical nitrogen content of 7.81%. Benzoylglucuronic acid was isolated from dog urine according to the methods of Pryde (9) and Quick (IO). ,4 mixed melting point determination with a known sample indicated that the two compounds were the same. The uric acid used for the spectrophotometric study was obtained commercially. Sitrogen analysis, in triplicate, gave a n average value of 33.1y0 compared with the theoretical of 33.34%. PROCEDURE
The cation Pvchange resin Do~veu 50-X-8, 50 to 100 mesh, is prepared as follows: The resin is alternately stirred in 11vhydrochloric acid and sodium hydroxide for 1 hour. The resin is treated in this manner three times and then washed with distilled water by decantation until chloride free, as indicated by a negative reaction with 1% silver nitrate solution. Twenty milliliters of moist resin are poured into a standard type chromatographic column, 12 mm. in diameter, and distilled water is allowed to flow over the column until the p H of the wash water is approximately 7 . There may be a very slight attraction of the resin for hippuric acid when the resin is treated in this manner. A feiv batches of the resin required treatment with a solution of hippuric acid in water (1 mg. per ml.) before pouring into the column. The existence of this attraction may be determined b y setting up a column, adding a known amount of hippuric acid, and determining the recovery, which should be 100%. If recovery is less than loo%, 50 ml. of moist resin are stirred for 1 hour with 100 ml. of hippuric acid solution. The resin is then poured into the column and washed free of excess acid solution with distilled nater. It usually requires about 200 ml. of mater to conipletely wash the column. One milliliter of urine is added to the column of resin. Then about 5 nil. of distilled n-ater are added slonly to the column. This step allows hippuric and uric acid to pass unchanged through the column while creatinine ( I S ) and
free amino acids ( 7 ) are quantitatively adsorbed on the resin. After 5 minutes to allow the solution to equilibrate in tlic column, the hippuric acid, along with uric acid, is eluted into a 200-ml. volumetric flask by allowing distilled water to flow through the column until tlic solution fills the flask to the mark. The flask is stoppered with a rubber stopper and the solution mixed well by inversion and sn-irling. The absorbance of the solution is determined a t 232 and 287 mp against distilled water from a blank column and the hippuric acid concentration determined as described in detail under spectrophotometry. Spectrophotometry. A search of t h e literature did not reveal a previous study of t h e ultraviolet absorption spectrum of hippuric acid. Hippuric acid, in a concentration of 0.01 mg. pel nil., vias found t o have t h e absorption spectrum indicated in Figure 1 n i t h a single broad B-band maximum a t 230 mp and a weak secondary or C-band a t 272 mp. Ungnade and Lamb (12) reported similar findings for the esters of benzoic acid. The molar extinction coefficient for hippuric acid a t 232 mp was determined to be 10,300. d series of dilutions of a stock solution of hippuric acid showed a linear relationship between absorbance and concentration a t 232 mp within the limits of 0.001 to 0.025 mg. per ml. The equation for this line as determined by the method of least squares ( I ) is: Y = 0..003
+ 57.38X
where X is concentration and I' is absorbance. I n order to check the new method for hippuric acid analysis with a standard method, a number of urine samples were analyzed in duplicate by both the ion exchangespectrophotometric technique and the Wu (14) technique. The results of these simultaneous determinations are indicated in Table I. Recovery experiments recorded in Table I1 indicated that hippuric acid added to urine samples was recovered in the range of 98.8 to 101.3%. Known concentrations of hippuric acid in pure aqueous solution nere analyzed in order to indicate the precision
Table I.
Recovery of Hippuric Acid Added to Urine
Actual
Concn.
Concn. in
b x-
Urine Sam le. alg.f;ll.
VOL. 29,
2 1 1 1
80 84
78 20 2 2b 2 55 1.85
3.10
NO. 1 1 ,
SiW
hIethod, Mg./hIl. 2 77 1 85 1 78 1 19 2 28 2 58 1.84 3.08
NOVEMBER 1957
Concn. h\\l=U
Method, Mg./lIl. 2 80 1 82 1 80 1 15 2 30 2 50 1.81
3.14
1713
Table II. Recovery of Hippuric Acid from Urine b y Ion ExchangeSpectrophotometric Technique (Concentration x 103)
Total Hippuric Acid, Mg / n u 2 80 1 201 1 846 0 846 1 713 2 040 1 309 2 628 2 285 1 470 1 681 1 538 0 764 1 224
Hippuric Acid Found, Mg /Ml. 2 Ti 1 192 1 850 0 845 1 723 2 015 1 322 2 664 2 2i0 1 460 1 686 1 5$2
0 764 1 210
Recovei y, 99 0 99 2 100 3
100 100 ')8 101 101
0
00
')
6
8 0 3 99 3
100 3 99 0 100 0 98 8
Table 111. Determination of Hippuric Acid Concentration in Aqueous Solution b y New Method
Hippuric Acid in Sample, Mg./Ml. 0.00311 0.00543 O.OOi15
0 00997 0.01740
Hippuric Acid Found, ?.Ig./Ml. 0,00313 0.00308 0.00540 0.00544 O.OOi10 0.00719 0,00994 0 00996 0 01004 0.01730 0.01i5i
Recovery, c-
100.6 99.0 99.8 100 5 99.2 100.5 99 i 99.8 100.7 99.4 100.9
of the method. The data in Table 111 show that the values range from 99.0 to 100.9~oof the amount present.
Calculation of Hippuric Acid Concentration. 1. Determine the cell constants absorbance a t 232 and 287 mp of the silica cells, using distilled water which has been run over a control ion exchange column treated exactly like the columns used for analysis. Set the slit width of the spectrophotometer at 0.4 mm., and balance the potentiometer by varying the sensitivity control. 2. Determine the average absorbance readings of the unknowns a t 232 mp and 287 mp. Readings are made in triplicate. 3. Correct each average reading a t the two wave lengths by algebraically adding the cell constant determined in Step 1. 4. Convert the corrected 287-mp reading due to uric acid to the corresponding value at 232 mp, and subtract from the total 232-mp reading. J. Determine the hippuric acid concentration from the calibration curve or from the equation determined by the method of least squares. DISCUSSION
Sources of Error. I t is recognized t h a t there are many compounds which may be present in urine lyhich will absorb ultraviolet light a t t h e wave 1714
ANALYTICAL CHEMISTRY
length of interest. While creatinine is quantitatively removed from solution by Dowex 50 (H+), uric acid, which also absorbs ultraviolet light a t 232 mp, is quantitatively washed through the column into the effluent. At first an attempt was made to separate the hippuric acid from the uric acid in the effluent by means of anion exchange resins. The strongly acidic solutions v\-hich were necessary t o elute these compounds also carried a large and variable blank absorption a t the wave length of interest, and it seemed doubtful that complete separation of the two compounds could be achieved. It was therefore decided to determine hippuric acid in the presence of uric acid. The absorption spectrum of uric acid in the ultraviolet range found in these experiments agrees well with those reported by previous investigators (2, 8 ) . The uric acid absorption peak a t 232 mp makes it possible to correct for the contribution of uric acid to the total absorbance of the eluate a t this n-ave length. The fact that hippuric acid in dilute solution does not absorb a t 287 mp makes it possible to correct for the absorption of uric acid a t 232 mp. This has been done by measuring the absorbance of known amounts of uric acid in nater a t 232 and 287 nip and plotting absorbance vs, concentration curves. The equations of the two lines have also been determined by the method of least squares ( 1 ) and are as follows:
V i t h these curves it is a simple matter to convert the total absorbance due to uric acid a t 287 mp to its corresponding value a t 232 mp, and to subtract this value from the total absorbance a t 232 mp. It was determined that the experimental curve resulting from a niixture of 0.005 mg. per nil. of hippuric acid plus 0.005 mg. per nil. of uric acid closely followed that of a curve prepared by adding the absorbances of the pure compounds in equivalent amounts and plotting the points. At the nave lengths of interest the curyes coincided, indicating that the absorbance measure-
Table IV. Enzymatic Uric .4cid, hIg./hIl. 0.27
Uric Acid in Urine
Ion ExchangeSpectrophotometric Uric Acid, Mg./hIl. 0.26
0 36
0.38
0.15 0.42 0.52 0.48 0.15
0.80 0.16 0.44 0.52 0.53 0.15
0.6s
nients of the two compounds are additive. This relationship makes possible the determination of hippuric acid in the presence of uric acid. I n order to determine hov much of the absorption a t 287 nip was actually due to uric acid under the experimental conditions, several other methods of analysis ( 6 , 8)of uric acid were studied. S o n e of the methods available gave complete recovery, even with aqueous solutions of uric acid, as compared with the spectrophotometric method except the enzymatic method of Praetorius ( 8 ) . I n this method the absorption a t 287 mp can be measured before and after action of the specific uricase. Table I V shows the comparison of the enzymatic method u-ith the ion exchange-spectrophotometric method. The presence of benzoic acid in the urine sample would be a source of error in the determination of hippuric acid, since it is not affected by passage over the Dowex 50 column, and it has a strong absorption (12) in the ultraviolet a t the n-ave length of interest, The logarithm of the molar extinction coefficient of benzoic acid a t 228 mp in 0.01.\- sodium hydroxide 11as determined to be 3.965 compared with 3.97 reported by Ungnade (12). The presence of 0.002 mg. per ml. of benzoic acid can be detected in urine qualitatively by extracting 1.0 ml. of urine a t p H 2 with 5.0 ml. of petroleum ether, and measuring the absorption against a petroleum ether blank a t 232 mp. I n the experimental procedure which was followed in these studies, benzoic acid was never detected qualitatively. The problem of quantitative estimation of the absorption a t 232 mp due to benzoic acid was not encountered. A search of the literature did not reveal a spectrophotometric study of benzo! lglucuronic acid. As one would predict from the n-ork of Ungnade and Lanib (12) that this compound should show a strong absorption a t the wave length of interest, it was studied further. The absorption spectrum is shown in Figure 1. Ultraviolet absorption measurenients of dilutions of a stock aqueous solution of this compound indicated a linear relationship between absorbance and concentration in the range of 0.001 to 0.014 mg. per ml. I n order t o determine if glucuronate were present in the urine sample in increased amounts, the naphthoresorcinol reaction as modified by Teague ( 1 1 ) was used for analysis. As it was shown (4) that glucuronates were excreted at a constant rate under normal fasting conditions, it was reasoned that a n increase above this rate on ingestion of benzoate should be due to benzoylglucuronic acid. If extra glucuronate was detected, honever, the correction for the effect of the extra glucuronate on the absorption a t 232 mp could be made b y
conversion of the benzoylglucuronic acid concentration determined b y the naphthoresorcinol reaction t o its corresponding ultraviolet absorption at 232 mp. The problem of interferences in the method is greatly simplified in studies in which hippurate is used rather than benzoate, as this eliminates biological conjugation. This makes the technique extremely useful for clearance studies of kidney function. The nieasurement of the absorption a t 287 mp makes it possible to follolv the effect of small doses of benzoate on the urinari- excretion of uric acid by the kidney. These results vi11 be reported elsewhere along with the liver and kidney function studies. The effects of other components in the urine n-hich might absorb ultraviolet light a t 232 mp h a r e been considered to be negligible, as dilutions of
1 to 200 through 1 to 5000 were used for the spectrophotometric analysis. ACKNOWLEDGMENT
The author is indebted to A. J. Quick, AIarquette University, Alilwaukee, Kis., for a sample of benzoylglucuronic acid. Discussions u-ith Hsien Wu, E. B. Carmichael, and J. TT’. Woods are acknon ledged ii-ith gratitude. LITERATURE CITED
(1) .irkin, H., Colton, R. R., “An Outline of Statistical Methods,” Barnes and Noble, S e w York, 1950.
(2) Bergmann, F., Dikstein, S., J . B d . Chenz. 211, 691-6 (1‘354). (3) Dickens, F., Pearson, J., Biochon. J . 48, 216-21(1951). (4) Elliott. H. C., Ph.D. thesis, Cniveieity of Alabama Medical Center. 1956. (5) Ewing, G., Parsons, T., ANAL.CHEN 20, 423-5 (1948).
(6) Gaffney, G. W.,Schreur, K., Ferrante, N., Altman, K., J . BioE. Chem. 206, 695-8 (1954). ( 7 ) JIoore, S., Stein, W. H., Ibid., 192, 663-81 (1951). (8) Praetorius, E., Poulsen, H., Scand. J . Clin. Lab. Invest. 5 , 2’73-80 (1953). (9) Pri.de, J., Williams, R. T., Biochem. J . 27, 1210-15 (1933). J . B i d . Chem. 69, 549-
(11) Teague, R. S., Dept. Pharmacology, Universitr of Alabama Medical Center, personal communication. (12) Cngnade, H. E., Lamb, R., J . Am. Chem. SOC.74, 3789-94 (1952). (13’1 Kall, J. S., ASAL. CHEJI. 25, 950-3 (1953). (14) K u , H., JT-u. D., Federatzon Proc. 11, 314 (1!)52). RECEIVED for review March 30, 1957. .iccepted July 1, l%i. Extracted from the thesis of H. C. Elliott, Jr., presented in partial fulfillment of the requirements for the Ph.D. degree in biochemistry to the 1-niversity of AAlabamahIedical Center.
Spot Tests Based on Nencki Synthesis of Rhodanine (Rhodanic Acid) FRITZ FEIGL and VICENTE GENTIL laboraforio da Producao Mineral, Ministerio da Agriculfura, Rio de laneiro, B r a d Translated b y RALPH E. OESPER, University of Cincinnati, Cincinnati, Ohio
b The color reaction of rhodanine (rhodanic acid) with 1,2-naphthoquinone-4-sulfonic acid i s used as the basis of indirect tests for compounds which can participate in the Nencki synthesis of rhodanine. These include rnonochloro(bromo)acetic acid, thiocyanates, thiourea and its N-monoalkylated or -arylated derivatives. Cyanamide and its salts also can be indirectly detected in this manner by virtue of their ready conversion into thiourea. All of these tests can be rapidly accomplished within the framework of spot test analysis. The limits of identification are within the rnicroanalytical bounds.
R
hodanine (I), as well as the numerous other compounds with a reactive methylene group, forms colored quinoidal compounds in alkaline solution on treatment with 1,2-naphthoquinone-4sulfonic acid (11) which is known as the Ehrlich-Herter reagent ( 2 ) . The practically instantaneous condensation 0
HN-CO
I
I
SC
CHz
‘d (1)
+~
I1 a O , S - ~ = o
c_> (11)
HS-CO
ONa
U
(111)
+ Sa2S03 + 2H20
(1)
yields the water-soluble blue-violet p-quinoidal compound (111) (7’) and permits the detection b y means of a spot test (3) of rhodanine with an identification limit of 0.6 y. During studies of the analytical usefulness of syntheses and methods of formation of various organic compounds (4) it was found that the color reaction cited above permits the sensitive and selective detection of those compounds which can participate in a synthesis of rhodanine. According to h’encki (e), rhodanine results if an aqueous solution of monochloroacetic acid is warmed with ammonium thiocyanate:
+ 2SHdCNS + HzO + 1 + NHiCl + COn + 2KH3 CHQ
CHzClCOOH HK-CO
I
SC
Although this reaction gives only about a 30% yield of rhodanine, even
a brief warming of the reaction mixture is sufficient t o cause color Reaction 1 to occur to a satisfactory extent after the addition of alkali and 1,2naphthoquinone-4-sulfonic acid. Either a slight quantity of monochloroacetic acid or ammonium thiocyanate can be employed in the starting mixture, provided a n excess of the other reacta n t is present. Rhodanine can be formed by Reaction 2 either in aqueous solution or in molten monochloroacetic acid (melting point 63” C.) Alkali thiocyanates can be substituted for the ammonium salt, and monobromoacetic acid for the chloro acid. Thiourea, which is isomeric and tautomeric with ammonium thiocyanate, shows a remarkable behavior. Volhard (8) observed that the socalled mustard oil-acetic acid is produced in aqueous solution by reaction with monochloroacetic acid: CHzClCOOH HK-CO
I
OC
I
CH,
+ CS(NHz)z + iu”dC1
(3)
‘S/ This product, analogous to rhodanine, contains a reactivemethylene group and consequently is capable of reacting with 1,2 - naphthoquinone - 4 - sulVOL. 29, NO. 1 1 , NOVEMBER1957
1715
