1107
Anal. Chem. 1985, 57, 1107-1109
Spectrophotometric Determination of Hydrogen Peroxide with Titanium 2-((5-Bromopyridyl)azo)-5-(N-propylN-sulfopropy1amino)phenol Reagent and Its Application to the Determination of Serum Glucose Using Glucose Oxidase Chiyo Matsubara, Kiyoji Kudo,l Takahisa Kawashita? and Kiyoko Takamura*
Tokyo College of Pharmacy,
1432-1, Horinouchi, Hachioji,
A mixture of titanium( I V ) and 2 4 (5-bromopyridyi)azo)5-(Npropyl-N-sulfopropylamino)phenol (denoted as TI-PAPS reagent) was found very useful for the spectrophotometrlc determination of traces of hydrogen peroxlde. The addition of hydrogen peroxlde to the Ti-PAPS reagent resulted In 539 nm) due to the formation of red-purple coloration (A, the tltanlum( IV) complex with hydrogen peroxlde and PAPS. Absorbance at 539 nm was proporllonai to the concentratlon of hydrogen peroxide, when [PAPS]/[TI(IV)] = 1 In a pH reglon from 6.3 to 8.0. Molar absorptivlty was found to be 5.7 X lo4 M-' cm-' The reagent was successfully applied to the assay of serum glucose when used In combinatlon with glucose oxidase. With 10 pL of serum, glucose within a concentration range from 3 to 270 mg/dL could be determined. The data were reproduclble with a coefflclent of variatlon of less than 2.0%. The advantage of the present method Is that the results obtained are influenced very llttle by reduclble substances such as ascorbic acld In the serum.
.
Methods widely used for determining trace amounts of biological substances are based on the detection of hydrogen peroxide produced through the specifically inherent enzymatic oxidation of these substances. In recent years the development of enzymatic techniques has made it possible for hydrogen peroxide to be produced quantitatively through the enzymatic oxidation of various biological substances a t trace levels. Consequently, high sensitivity and selectivity are required for determining the trace amounts of hydrogen peroxide. For clinical and biochemical studies, spectrophotometry using a peroxidasechromogen such as the 4-aminoantipyrine-phenol system is widely used. Present methods, however, do not permit an accurate estimation of trace amounts of biological substances owing to the inadequate sensitivity and selectivity of peroxidase (1, 2). In our previous paper, a mixture of titanium(1V) and 4(2-pyridy1azo)resorcinol (denoted as Ti-PAR reagent) was proposed as a colorimetric reagent having the required sensititivy and selectivity (3). The addition of hydrogen peroxide to the Ti-PAR reagent caused red coloration (A, 508 nm) due to the formation of titanium(1V) complex with hydrogen peroxide and PAR (4). The reagent was thus successfully applied to the determination of trace amounts of biological substances in serum, using an appropriate combination of enzymes to produce hydrogen peroxide through enzymatic oxidation ( 5 , 6 ) . In the course of this study, for the detection of extremely small amounts of hydrogen peroxide, it was Present address: Wako Pure Chemical Industries, Ltd. Present address: Kyushin Seiyaku, Ltd. 0003-2700/85/0357-1107$01.50/0
Tokyo, 192-03, J a p a n
necessary to use a reagent that would form a complex with molecular absorptivity (e) and bathocromic shift greater than that of the titanium(1V) complex with PAR and hydrogen peroxide. In the present paper, an attempt was made to get the TiPAPS reagent using 2-( (5-bromopyridyl)azo)-5-(N-propyl-Nsulfopropy1amino)phenol disodium (abbreviated as PAPS) instead of PAR to improvement of the Ti-PAR reagent. The Ti-PAPS reagent was also used for the determination of serum glucose. EXPERIMENTAL SECTION Reagent. All chemicals were of analytical reagent grade and used without further purification. The stock solution of Ti(1V) (1mM) was prepared as follows: 24 mL of titanium(1V) tetrachloride was dissolved in 500 mL of 4 M hydrochloric acid. The solution thus obtained was standardized by titrating with ethylenediaminetetraacetic acid, followed by dilution with water to 1 mM concentration of Ti(1V). The stock solution of PAPS (1mM) was prepared by dissolving 0.0617 g of PAPS (Dojin Co., Ltd.,Kumamoto Japan)in 100 mL of water. The Ti-PAPS reagent was prepared by mixing 30 mL of the stock solution of titanium(1V) with 30 mL of the stock solution of PAPS. This solution was diluted with water to 100 mL and had a pH of below 3. The hydrogen peroxide solution (0.1 M) was prepared as follows: 5.5 mL of 30% hydrogen peroxide was diluted with water to 500 mL and the solution was standardized by titration with potassium permanganate. The glucose solution (5 X M) was prepared by dissolving 0.0908 g in 100 mL of water. Glucose oxidase (EC 1.1.3.4) from Aspergillus niger was purchased from Sigma Chemical Co. Procedure for Hydrogen Peroxide Determination Following the addition of 2.5 mL of the Ti-PAPS reagent to 10 mL of sample solution containing an aliquot amount of hydrogen peroxide, 2.0 mL of 0.2 M phosphate buffer (pH 7.0) was added to the solution. The solution was diluted to 25 mL with water and incubated at 37 "C for 5 min. The solution was cooled to room temperature and the absorbance was measured at 539 nm using a reagent blank as a reference. Procedure for Serum Glucose Determination. To 0.5 mL of a solution containing 0.01 M phosphate buffer (pH 5.6), 2 U/mL glucose oxidase, and 3.3 X lo4 M NaN3,10 WLof serum was added and incubated at 37 "C for 15 min. A 0.5-mL portion of the Ti-PAPS reagent, 3.5 mL of water, and 0.5 mL of 0.2 M phosphate buffer (pH 7.0) were then added, and the solution was incubated at 37 "C for 10 min. The solution was cooled to room temperature and the absorbance was measured at 539 nm. For the control assay, a solution containing the same constituents as the above was prepared, except that the glucose solution, instead of serum, was added.
.
RESULTS AND DISCUSSION Determination of Hydrogen Peroxide. When hydrogen peroxide was added to the Ti-PAPS reagent at pH ca. 1.0, a 0 1985 American Chemical Society
1108
0
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
1 . 500
80
550
600
Wavelength, n m
Figure 1. Absorption spectra of (1) the Ti-PAPS-H O2system and, (2) PAPS at pH 7.0: [Ti(IV)]and [PAPS], 2 X 10-P M; [H202],1 X
-
Os
8
7
9
10
11
PH
Figure 2. Effects of pH on absorbance of the Ti-PAPS-H,O, system using (1) Na2HP04-KH,P04, (2) trls(hydroxymethyl)aminomethane, (3) NH,-NH,CI, and (4) Na2C03-NaOH buffers: [Ti(IV)]and [PAPS], 2 X M; [H202],2 X
M.
10-5 M.
sharp peak appeared at ,A, 539 nm due to the formation of the Ti-PAPS-H202 complex, as shown by curve 1 in Figure 1. With the addition of hydrogen peroxide, constant absorbance values were obtained within a few minutes at 37 O C and remained virtually unchanged over a period of 24 h at room temperature. The absorbance at 539 nm was proportional to the concentration of hydrogen peroxide. The composition of the complex was confirmed by Job's method: only a 1:l:l complex was found to form in the solution containing Ti(IV), PAPS, and hydrogen peroxide. Though the structure of the Ti(IV)-PAPS-H202 is still uncertain, the most probable one which can be presumed at present is shown below. m
dH 'OH
The structure is postulated on the basis of our previous findings about the titanium(1V) 4-(2-pyridylazo)resorcinol (PAR)-H202 complex, in which PAR, like PAPS, contains a hydroxoazo group (4).Then it can be considered that the Ti(IV)-PAPS-H202complex is stabilized by bridging of the pyridine N atom and Ti(1V) through hydrogen bonding involving hydrogen peroxide and that the coloration is derived from the conjugated double bond system of PAPS coordinated. The absorption spectrum of the intact Ti-PAPS reagent at pH 2 showed a peak a t A,, 515 nm, perhaps due to the Ti(IV)-PAPS complex. However, by incubation of the solution at 37 "C after raising pH to 7.0,this peak rapidly diminished and disappeared completely within a few minutes. At the same time, a peak appeared anew at 4.47 nm, and the spectrum was essentially the same as that obtained for PAPS alone at pH 7.0. Thus no Ti-PAPScomplex was present in the reagent blank since the formation of the stable hydroxotitanyl polymer eliminated Ti(1V) from the Ti-PAPS complex in the Ti-PAPS reagent. When hydrogen peroxide is determined, interference from an excess of the Ti-PAPS complex can be avoided by incubating the solution at 37 O C for 5 min following the addition of the Ti-PAPS reagent. To decide what conditions would be appropriate for using the reagent to carry out spectrophotometry, the effects of pH and constituents of the pH buffer solution on absorbance were examined. As shown in Figure 2, a constant absorbance was obtained in the pH range from 6.3 to 8.0 when using the phosphate buffer. Taking the composition of the complex stated above into consideration, the [Ti(IV)]/ [PAPS] ratio was fixed at unity in the Ti-PAPS reagent. On the basis of the experimental results, the conditions for the spectrophotometric determination of hydrogen peroxide using the TiPAPS reagent were decided upon as indicated above.
0o+o+ GOD, UI rnl Time, rnin Figure 3. Effects of glucose oxidase concentration (A) and incubation time (B) on absorbance: (A) incubation of 20 rnin at 37 O C , (B) activity M in the final solution. of 1 U glucose oxidase; [glucose], 1 X
The absorbance determined with the standard hydrogen peroxide solution was plotted against concentration to give a linear relation in the concentration range from 0.5 to 30 pM. The coefficient of variation was less than 2.0 and 1.8% at concentrations of 5 pM and 15 pM,respectively. Assay of Serum Glucose. The above results indicate that the Ti-PAPS reagent can be used in the determination of hydrogen peroxide in biological fluids. The following experiments were performed to establish optimum conditions for determining serum glucose using this reagent along with glucose oxidase. pH values in the vicinity of 5.6 were optimum for the enzymatic reaction of glucose oxidase (7), whereas optimum coloration by the Ti-PAPS reagent was possible within a pH range from 6.3 to 8.0. The solution was first adjusted to pH 5.6 for incubation with glucose oxidase and then readjusted to pH 7.0 for coloration. An ideal temperature for the enzymatic reaction of glucose oxidase to proceed to completion was around 37 "C. During incubation at 37 "C,constant absorbance values were obtained within 5 min following the addition of the Ti-PAPS reagent. The temperature of the solution was maintained at 37 "C throughout both the enzymatic and chromogenic reactions. The effects of glucose oxidase activity and incubation time on absorbance were examined to determine the optimum concentration of glucose oxidase. As shown in Figure 3,constant absorbance values were obtained in the range of 1-2 U of glucose oxidase during a 15-min incubation period using 10 pL of the 5 X M standard glucose solution. The absorbance obtained using the standard glucose solution was plotted against the concentration of glucose. The linear relationship in the concentration range from 3 to 270 mg/dL in serum was obtained. The coefficient of variation was less than 2.0%. The effects of certain foreign substances usually present in serum or added to the test solution were examined and the results are summarized in Table I. Inorganic ions such as Na+, K+,C1-, and P04s-hardly had any effect even when present at several thousands times the concentration of glucose. Absorbance was hardly affected with addition of anticoagulants such as F- and heparin at their usual concentations in the sample solution. But sodium citrate caused a
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1965
1
Table I Effects of Inorganic and Organic Substances on the Determination of Glucose Using the Ti-PAPS Reagent substance KC1 MgCb NazHPOl NaF ascorbic acid citric acid pyruvic acid uric acid heparin cysteine bilirubin
concn added,” M 1.0 x 10-1 1.1 x 10-2
2.0 x lo-’
2.4 x 5.6 X 3.4 x 3.4 x 1.3 x 1.3 x 0.3c 2.0 x 4.2 x 1.0 x
10-3 lo4 10-2 10-3 10-3 10-3 10-2 10-3 10-6
glucose found: %
4.58
r=o.ssa
/
97.1 98.9 96.0 100.0 100.0 52.0
97.1 98.5 98.4 97.5 84.9 98.7 99.5
a Concentration in the final solution for the test. *Concentration M in the final solution for the test. CUnits of glucose, 1.00 X of concentration g/L.
decrease in absorbance since it interfered with coloration by the Ti-PAPS reagent. The presence of cysteine resulted in minor error in measurement. But at its final concentration in the test solution prepared from human serum, cysteine exerted practically no effect on absorbance. Examined reducible compounds, such as ascorbic acid, glutathione, and uric acid at concentrations equal to or even higher than those present in normal serum, had no significant effect on the glucose assay. For example, the addition of ascorbic acid (10 mg/dL in serum) caused only a slight decrease in coloration. But on using the peroxidaseaminoantipyrinephenolmethod, ascorbic acid (6 mg/dL in serum) gave rise to a large negative error in the glucose assay (I). The presence of catalase in the serum sample decreased the absorbance by catalyzing the decomposition of hydrogen peroxide generated in the enzymatic oxidation with glucose oxidase. However, the addition of sodium azide (final concentration 1 x M) hindered the catalytic action and prevented the decrease in absorbance. The presence of glucose oxidase had no effect on the coloration by the Ti-PAPS reagent. Further experiments were carried out to examine the usefulness of the Ti-PAPS reagent for determining glucose in human sera. The recovery of 25 and 50 mg/dL glucose added (n = 10) a t concentrations from 72.0 to 120 mg/dL serum glucose was 99.02 and 99.30%, respectively. Glucose in the serum sample (n = 50) was assayed by the present method, and the results were compared with those obtained by the glucose oxidase-peroxidase-aminoantipyrinephenol method (commercial kit glucose B-Test Wako, by Wako Pure Chemical Industries, Ltd., Japan) as shown in Figure 4. A correlation coefficient (r) of 0.988 and a linear regression equation of Y = 0.920~ 4.58 (mg/dL) were found by these two methods 0, refers to data obtained by the new met hod). The present data show that the Ti-PAPS reagent can be used to assay trace amounts of hydrogen peroxide and serum glucose, using glucose oxidase. The new method is preferable to the usual methods because of its simplicity, specificity, and accuracy. In the usual methods using peroxidase, color development of chromogen by hydrogen peroxide is inhibited by the presence of certain reducible substances, such &s ascorbic acid, for the incomplete selectivity of peroxidase (I). In contrast, coloration by the Ti-PAPS reagent requires no peroxidase,
+
.-
v=o.sz x
1lOB
a
Peroxidase method, mgldl serum
Figure 4. Correlation of results obtained by peroxidase-aminoanti-
pyrine-phenol and presented methods. because of the formation of a Ti(1V) complex containing hydrogen peroxide and PAPS. Thus, by the present method, the presence of reducible substances has hardly any or no effect on absorbance. The colorimetric method based on the formation of a mixed ligand complex in a ternary Ti(1V)xylenol orange-hydrogen peroxide system has been reported by Richmond for determining trace amounts of hydrogen peroxide (8). However, because of the considerable absorbance of the reagent blank due to the remaining Ti(1V)-xylenol orange complex, the Ti(IV)-xylenol orange-hydrogen peroxide system is not adequate for practical application. On the present method, the effect of the reagent blank by presence of the Ti(1V)-PAPS complex on the absorbance measurement is readily eliminated by incubating the solution at 37 “C. The small absorbance of the reagent blank was effective for high accuracy measurement. The red-purple coloration by the Ti-PAPS reagent is also preferable over red coloration by the Ti-PAR reagent, since it prevents interference from bilirubin present in the serum. Following formation of the complex at a molar absorptiviity of 5.7 X lo4M-’ cm-l, the sensitivity of the Ti-PAPS reagent was found to be better than that of the Ti-PAR reagent at 3.6 X 104 M-l cm-l. The molar absorptivity of colored species in the Ti(IV)-PAPS-H202 system was about 9 times that observed in the peroxidase-aminoantipyrine-phenol system (9).
The present method should prove quite useful for the spectrophotometry of other substances such as uric acid and activity determination of enzyme such as uricase. Registry No. Ti-PAPS-H2O2,95069-72-0; HzOz,7722-84-1. LITERATURE CITED (1) Sharp, P. Clin. Chim. Acta 1972, 4 0 , 115-120. (2) White Stevens, R. H. Clh. Chem. (Winston-Salem, N . C . ) 1982, 2 8 ,
578-589. (3) Matsubara, C.; Takarnura, K. Bunsekl Kagaku 1980, 2 9 , 759-764. (4) Matsubara, C.; Iwarnoto, T.; Nishikawa, Y.; Takarnura, K.; Yano, S.; Yoshlkawa, S. J . Chem. SOC. Dakon Trans. 1985, 1 , 81-84. (5) Matsubara, C.; Nishlkawa, Y.; Yoshlda. Y.; Takamura, K . Anal. Blochem. 1983, 730, 128-133. (8) Matsubara. C.;Nishlkawa. Y.; Takamura, K. Yakugaku Zasshl 1983,
103, 884-888. (7) Bentley, R. “Methods In Enzymology”;Academic Press: New York, 1955; Vol. 1 , p 340. (8) Richmond W. Clln. Chem. (Wlnston-Salem, N . C . ) 1976, 2 2 , (9)
1579-1588. Pesee, M. A.; Bondourian, S. H. Clin. Chem. (Winston-Salem, N . C . ) 1977, 2 3 , 757.
RECEIVED for review August 9,1984. Accepted November 9, 1984.
