(7) G. F. Kirkbright, A. F. Ward, and T. S . West, Anal. Chim. Acta, 82, 241 (1971). (8) G.F. Kirkbright and M. Sargent, “Atomic Absorption and Fluorescence Spectroscopy”, Academic Press, London, 1974,p 516. (9) S. Greenfield, I. L. Jones, and C. T. Berry, AnalVst(London), 89, 713 (1964). (IO) G.F. Larson, V. A. Fassel, R. H. Scott, and R. N. Knlseley, Anal. Chem., 47,238 (1975).
(11) M. H.Abdallah, J. M. Mermet, and C. Trassy, Anal. Chim. Acta, 87, 329 (1976).
RECEIVED for review April 5, 1977. Accepted June 3, 1977. We thank the British Council for the award of a Postdoctoral Scholarship to one of us (L.N.O.).
Colorimetric Determination of Hippuric Acid in Urine and Liver Homogenate Shinji Ohmorl” and Mikiko Ikeda Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Tsushima-Naka-I , Japan
Shohel Kira and Masana Ogata Department of Public Health, Okayama University Medical School, Okayama 700, Shikata 2-5, Japan
To a dried extract Containing hippuric acid (HA), 1.0 mL of acetic anhydride and 2.0 mL of 0.5% p-dlmethyiamlnobenzaldehyde (DAB) solution In pyridine were added, and the solution was kept at 40 O C for 1 h after thorough mlxing. The absorbance was then determlned at 458 nm agalnst a blank containing acetic anhydride, DAB, and pyridine. This method was in good agreement with Beer’s law within 1 to 100 pg and the mean f SD absorbance for 20 pg HA was 0.939 k 0.013 ( n = 5). The apparent molar absorptivity of 2.5 X 104/mol/cm, and the relative standard deviation of 1.4% was calculated.
The determination of hippuric acid (HA) in urine is of great significance, mainly for testing of liver function ( I ) , diagnosis (2-4), and estimation of the detoxication of alkylbenzenes and drugs (5-7). In this paper a convenient and reproducible method based on colorimetry is presented. About 40 papers dealing with methods of determination of HA have been published. These methods depend on column chromatography (8), extraction (9), colorimetry (10-13), gas chromatography (14-1 7)) thinlayer Chromatography (18), paper chromatography (19), fluorimetry (20), titration (21),and determination of radioactivity (22). The method most widely used at present is based on gas chromatography. Applying this method to the determination of HA in liver homogenates of rat and eel (instead of urine), we had difficulties with overlapping peaks. The method to be described here is applicable to urine as well as liver homogenates. Two colorimetric methods have been presented for the determination of HA. With the method of Umberger (10) which was later modified by Ogata, the color is produced in a mixture of the HA-containing sample with benzene-sulfonylchloride and pyridine. With the method reported by Gaffney et al. (19)and Ogata et al. (23),DAB is used. Gaffney e t al. employed paper chromatography. They detected the HA spot by spraying a 4% solution of DAB in acetic anhydride which contained a few crystals of sodium acetate with subsequent heating of the chromatogram at 130-150 “C for 1-2 min. The absorbance of the color was determined after elution of the spot. Ogata et al. improved this method. Their reaction 1494
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
mixture additionally contained silica gel. The colorimetric procedure reported in this paper is based on the DAB method.
EXPERIMENTAL Chemicals and Instruments. Analytical grade chemicals from Wako Pure Chemical Industries Ltd (Osaka, Japan) were used. A Hitachi-139 Spectrophotometer was used for measuring the absorbance at 458 nm and a Hitachi-124 Spectrophotometer for recording the absorption spectra. Determination of HA. Extraction of HA from Urine. Urine (0.1 mL), 10 pL 6 N HC1, ca. 20 mg NaC1, and 1 mL ethyl acetate in a 10-mL test tube were mixed for 30 s with a Vortex mixer (Thermonics Inc., Japan). After 5 min, a 0.1-mL aliquot of the ethyl acetate layer was transferred to a 20-mL test tube and evaporated to dryness under reduced pressure. Extraction of HA from Liver Homogenates. Method A: for homogenates containing more than 20 pg HA per 0.5 mL. Rat liver (10 g) was homogenized in 90 mL of 1.15% KC1 in a Potter-Elvejhem Teflon pestle homogenizer and then centrifuged at 1000 X g for 10 min. To a 10-mL glass stoppered centrifuge tube, 0.5 mL of the supernatant and 20 pL 6 N HC1,20 gL glacial acetic acid, ca. 150 mg NaC1, and 2.5 mL ethyl acetate were added. The mixture was agitated with a Vortex mixer for 30 s and centrifuged at 1500 X g for 5 min. The ethyl acetate layer was dried over anhydrous sodium sulfate. An aliquot (0.5 mL) was then evaporated under reduced pressure. Method B: for homogenates containing less than 20 pg HA per 0.5 mL. To a 10-mL glass stoppered centrifuge tube 0.5 mL of the supernatant of the homogenate and 50 pL 6 N NaOH, ca. 150 mg NaC1, and 5 mL ethyl acetate were added. The mixture was agitated with a Vortex mixer. It was then centrifuged at 1500 x g for 10 min and the ethyl acetate layer was removed as thoroughly as possible. The aqueous layer was acidified with 0.1 mL 6 N HCl and extracted with 5 mL n-hexane by vortexing and centrifuging. After the hexane layer was removed as thoroughly as possible, the aqueous layer was extracted with 5 mL ethyl acetate by vortexing and centrifuging. The extract was dried with anhydrous sodium sulfate and an aliquot (3.5 mL) was evaporated under reduced pressure. Development of the Color and Its Measurement. To the dried HA-containing residue, 1.0 mL of acetic anhydride and 2.0 mL of 0.5% DAB solution in pyridine were added in turn. After thorough mixing, the solution was kept at 40 O C for 1 h. The absorbance was then determined at 458 nm against a blank containing acetic anhydride, DAB, and pyridine.
0
A a
at 4 0 ' ~ at 30' C
20 ug hippuric acid IO u g hippuric acid
p---:
I
LO[
05
b/
..O
-
D
O
Absorbance at at
458 nm 489 nm
n 4
-
L 025
0 0 . 1
Time in h o u r s
Flgure 1. Time course of color development. HA (10 and 20 pg) was allowed to react with 1.0 mL of acetic anhydride and 2.0 mL of 0.5% DAB solution in pyridine at 30 or 40 O C
-
IO
05 % DAB i n pyridine
Figure 3. Influence of the concentration of DAB on color development. HA (15 pg) was allowed to react with 1.0 mL acetic anhydrlde and 2.0 mL of 0.5% DAB in pyrldine at 40 O C for 1 h
Ratio of A A to pyridine 2.5 : 0.5 . . 2.0 : 1.0 1.5 : 1.5 1.0 : 2.0
-
I
.
0 IO
,
30
50
-
100
200 Water(u1 )
Flgure 4. Influence of water on color development. HA (15 pg) was allowed to react with 1.0 mL acetic anhydride and 2.0 mL of 0.5% DAB in pyridine at 40 O C for 1 h, in the presence of different amounts of water
0
"
"
"
-
450
"
"
500 Wavelength ( nm )
Figure 2. Absorption spectra under varying ratios of acetic anhydride and pyridine. HA (15 pg) was reacted in a final concentration of 0.33% DAB at 30 OC for 2 h RESULTS Time Course of Color Development. As is shown in Figure 1, it takes about 1h at 40 OC or up to 2 h at 30 "C to develop the maximum absorbance. The color is stable for at least one day. Absorption Spectra under Varying Ratios of Acetic Anhydride a n d Pyridine. In a total volume of 3 mL with constant amounts of DAB and HA, the color was developed in the presence of different ratios of acetic anhydride and pyridine. As shown in Figure 2, there was a minute red shift with increasing pyridine concentration. The maximum absorbance at 458 nm was obtained with a pyridine/acetic anhydride ratio of 2. Influence of Concentration of DAB on Color Development. HA (15 pg), 1.0 mL acetic anhydride and 2.0 mL of pyridine containing different concentrations of DAB were kept at 30 "C for 2 h. As shown in Figure 3, almost the same absorbance was found in the range of 0.375 to 1.0% DAB in pyridine. A final concentration of 0.33% DAB was, however, chosen as standard concentration to avoid HA-independent color development. Figure 3 also shows that the color is
Table I. Reliability of Color Development under the Standard Conditiona Mean t SD Re1 std HA, pg N absorbance at 458 nm dev, % 10 20 30
5
0.466
5 5
0.939
f +_
1.444 t
0.015
3.2
0.013 0.017
1.4
1.2
a Using a standard ethyl acetate solution of HA, the color was developed according to the procedure in Exper-
imental after ethyl acetate was evaporated. developed in the absence of DAB.It took, however, more than 3 h until the absorbance reached a constant value. Influence of Water on Color Development. The color was developed under standard conditions except that varying amounts of water were added to the reaction mixture. As shown in Figure 4, up to 30 pL of water do not influence the color development significantly. Calibration Curves. ( a ) Calibration Curve without Extraction. The calibration curves which were prepared using a standard ethyl acetate solution of HA showed fine linearity within 1 to 100 pg. Moreover as shown in Table I, this method is precise to 1.4% relative standard deviation for 20 pg HA. The apparent molar absorptivity was 2.5 X 104/mol/cm at 458 nm. ( b ) Calibration Curves with Extraction. The calibration curves, which were prepared using an HA aqueous standard solution according to the extraction procedures for urine or ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
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0
a)
Rf 0 70
b)
400
450
500
Table 11. Recovery Studies of Hippuric Acid Added t o Urine and Liver Homogenate by the Present Method HA HA HA(pg)/O.5 add- HA(pg)/O.l Re- add- mL of liver Reed, mL of urine covery, ed, homogenate covery, pg ( n = 3) % pg ( n = 3) % 0 15.9 ?: 0.2 0 N.D. 5 3.62 i 0.08 72.4O 10 25.4 ?: 1.5 95.0 7.43 i 0.28 74.3' 15 30.5 i 0.7 97.3 10 20 35.9 + 1.2 100.0 1 5 11.22 i 0.39 74.8' 50 65.3 i 4.1 98.7 20 17.9 f 1.2 89.5b 100 121.2 i 1.0 105.3 50 46.4 ?r 3.0 92.86 93.6b 150 169.8 + 3.3 102.6 70 65.5 i 4.6 95.0b 200 218.9 i 2.8 101.5 100 95.0 r 7.2 100.1 Mean 73.8a Mean 92.7b 1.30 SD 3.4 SD 2.3b ' HA was extracted according to Method B. HA was extracted according to Method A.
Buchet et al. (14),and from data a correlation coefficient of 0.977 and the regression line of Y = 0.896X 0.090 was calculated (Y = the present method). With the present method the absorbance can also be measured at 489 nm in good agreement with Lambert-Beer's law. Compared with measurements at 458 nm, the sensitivity is about 70%.
+
-
Wavelength( nm )
Figure 5. Absorption spectra of two yellow spots separated on a thin-layer chromatogram. The solid lines represent the absorption spectra in methanol and the dotted lines in the mixed solution of acetic anhydride and pyridine (1:2)
liver homogenate, also showed good agreement with Beer's law and about 94% of absorbance by comparing with (a). Recovery Tests. As shown in Table 11, recoveries of HA added to urine are 100.1 f 3.4%, and in the case of liver homogenates, they are 73.8 f 1.3% for Method B and 92.7 f 2.3% for Method A. When Method A is applied to the sample containing less than 20 pg HA per 0.5 mL, an HAindependent color development attributable to the contents of liver homogenate prevents accurate determination. This is why two methods are applicable respectively.
DISCUSSION The colorimetric method for determination of HA, presented in this paper, is simpler, more reproducible, and about 5 times more sensitive than the previous colorimetric methods. With thin-layer chromatography of the colored substance on silica gel developing with toluene/acetic acid 9:1, three yellow spots (Rf0.70,0.48, and 0.42) could be separated. The absorption spectra of the eluted spots with methanol indicated that the colored compound with the Rf value of 0.42 mainly contributed to the absorbance at 458 nm (see Figure 5). This substance may be 2-phenyl-4-(1-hydroxyethy1idene)oxazol&one which was synthesized by Attenburrow et al. (24). The substance with the Rf value of 0.70 may be 2-phenyl-4-(pdimethylaminobenzal)oxazol-5-one, which was synthesized by Schueler et al. (25) and Gaffney et al. (19). The spot with the Rf value of 0.48 was paler than the other two. The color faded away after elution with methanol. Work is in progress to identify the colored substances. Seventeen urine samples were used for comparing the present method with the gas chromatograph method by
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
ACKNOWLEDGMENT We are grateful to Y. Yoshida for help in the laboratory. LITERATURE CITED (1) A. J. Quick, Am. J. Clin. fafhol., I O , 222 (1940). (2) A. Hili, G.N. Hoag, and W. A. Zaleski, Clin. Chim. Acta, 37, 455 (1972). (3) C. F. van Sumere, H.PE,R. Verbeke, and J. Bekaett, Clin. Chim. Acta, 26. 85 (1969). (4) T. D. Beardmore and W. N. Keiiey, Clin. Chem. ( Winston-Salem,N.C), 17, 795 (1971). (5) M. Ikeda and H. Ohtsuji, Br. J. Ind. Med., 26, 244 (1969). (6) S.P. James and D. A. White, Biochem. J., 104, 914 (1967). (7) Y. Kato, Yakugaku Zasshi, 92, 1140 (1972). (8) L. A. Eamess, G. Morrow 111, R. E. Nocho,and R. A. Maresca, Clin. C h m . ( Winston-Salem, N.C.), 16, 20 (1970). (9) H. Chantrenne, J. Biol. Chem., 189,227 (1951). (10) C. J. Umberaer and F. F. Fiorese, Clin. Chem. (Winston-Salem. N.C.). 9, 91 (1963j. (11) K. Tomokuni and M. Ogata, Clin. Chem. (Winston-Salem, N . C . ) , 16, 349 11972). -
\
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I
(12) H. Kehi, Clin. Chem. (Winston-Salem, N.C.), 13, 475 (1967). (13) A. M. EL Masry, J. N. Smith, and R . T. Williams, Blochem. J., 64, 50 (1956). (14) J. P. Buchet and R. R. Lauwerys, Br. J. Ind. Med., 30, 125 (1973). (15) 0.A. Mamer, J. C. Crawhail, and S.S.Tjoa, Clin. Chlm. Acta, 32, 171 (1971). (16) U. Langenbeck and J. E. Seegmiiier, J. Chromatogr., 80, 81 (1973). (17) R . F. Coward and P. Smith, J. Chromatogr., 45, 230 (1969). (18) H. Teuchy and C. F. van Sumere, Clin. Chim. Acfa, 25, 79 (1969). (19) G.W.Gaffnev, K. Schreier, N. Diferrante. and K. 1. Altmn. J. Bbl. Chem.. 208, 695 (1954). (20) G.L. Eiiman, A. Burkhaiter, and J. La Don, J. Lab. Ciin. Med., 57, 813 119611 -- , (21) M. L. Verma and R. K. Srivastava, Microchem. J., 14, 396 (1969). (22) J. W. Bridges, M. R. French, R. L. Smith, and R. T. Williams, Biochem. J., 118, 47 (1970). (23) M. Ogata, K. Tomokuni, and Y. Takatsuka, Br. J. Ind. Med., 26, 330 (1969). (24) J. Attenburrow, D. F. Elliot, and G. F. Penny, J. Chem. SOC.,310 (1948). (25) F. W. Schueier and S.C. Wang, J. Am. Chem. SOC.,72, 2220 (1950). I
RECEIVED for review November 1, 1976. Accepted June 21, 1977. Work supported in part by a grant (No. 111315) from the Ministery of Education of Japan.
