ANALYTICAL SCIENCES APRIL 2004, VOL.

20 707
2004 The Japan Society for Analytical Chemistry

Spectrofluorometric Determination of Hydrogen Peroxide Based

on Oxidative Catalytic Reactions of p-Hydroxyphenyl Derivatives
with Metal Complexes of Thiacalix[4]arenetetrasulfonate on a
Modified Anion-Exchanger
Junichi ODO, Kozue MATSUMOTO (ne MIEDA), Eri SHINMOTO, Yoko HATAE, and Atsushi SHIOZAKI

Department of Biological Chemistry, Faculty of Science, Okayama University of Science,

Ridai-cho, Okayama 7000005, Japan

The peroxidase-like catalytic activity of metal complexes of thiacalix[4]arenetetrasulfonate (TCAS[4]) on a modified anion-
exchanger (Men+-TCAS[4]A-500; Men+ = H2, Fe3+, Fe2+, Mn3+, Co3+, Co2+, Cu2+, Zn2+, Ni2+) for the oxidation of p-
hydroxyphenyl derivatives to produce fluorescent substances in the presence of hydrogen peroxide has been investigated.
Among the Men+-TCAS[4]A-500 tested, Fe3+-TCAS[4]A-500 exhibited the highest level of catalytic activity for the oxidation
of p-acetoamidophenol in a carbonate buffer solution of pH 10. The catalytic activity of Fe 3+-TCAS[4]A-500 was then used
for the spectrofluorometric determination of hydrogen peroxide. The calibration curve for the Fe3+-TCAS[4]A-500 method
was linear over a range spanning from 0.1 to 5.0 g of hydrogen peroxide in a 1.0 ml sample solution.

(Received December 1, 2003; Accepted February 4, 2004)

In clinical analyses, vital compounds such as glucose and uric
acid are routinely measured using enzymatic methods.1 With
such methods, the amount of hydrogen peroxide produced by
the oxidase corresponding to the compound (for example,
glucoseoxidase and uricase for glucose and uric acid,
respectively) is indirectly measured based on a color reaction
with the aid of peroxidase.1 Thus, hydrogen peroxide is an
important target in the determination of vital compounds in
clinical analyses. Accordingly, it is necessary to develop a
more sensitive method for measuring hydrogen peroxide. In
order to determine a trace amount of hydrogen peroxide, various
fluorometric methods in which peroxidase was used as an
enzyme have been developed.26 However, even if peroxidase
was immobilized on a carrier, such as chitosan and glass
beads,7,8 peroxidase in these methods has still some
disadvantages regarding stability, handling and storage. Thus,
the development of an effective artificial mimesis with high
peroxidase-like activity has been desired to overcome these
advantages.
Recently, thiacalix[n]arene derivatives have been developed.9
Thiacalix[n]arenes have a specific structure in which the
methylene units of the parent calix[n]arenes have been replaced
by S atom linkages, as shown in Fig. 1. Much attention has
been focused on the characteristics of thiacalix[n]arenes for
forming stable metal complexes without modifying their upper-
and/or lower-rims with functional groups.1013 We have recently
found that Fe3+-thiacalix[4]arenetetrasulfonate on the modified
anion-exchanger (Fe3+-TCAS[4]A-500) exhibited very strong
peroxidase-like catalytic activity, and was useful as a except for Mn3+-TCAS[4]A-500, were prepared according to
peroxidase mimetics.14 Moreover, we have developed new
methods for the spectrophotometric determination of hydrogen
peroxide using the peroxidase-like catalytic activity of Fe3+-
TCAS[4]A-500, and applied them to measurements of glucose and
uric acid.14,15 It has been demonstrated that methods employing
Fe3+-TCAS[4]A-500 are effective for determining trace amounts
of vital compounds in clinical analyses.
However, in clinical analyses the amount of vital samples,
especially blood, should be minimized as much as possible,
making it necessary to develop more sensitive methods.
Accordingly, the peroxidase-like catalytic activity of Men+-
TCAS[4]A-500 for the oxidation of p-hydroxyphenyl derivatives
in the presence of hydrogen peroxide was investigated in this
study.
Experimental
Reagents and apparatus
Sodium thiacalix[4]arenetetrasulfonate (TCAS[4], Fig. 1) was
kindly provided by Cosmo Oil Co. DEAE cellulofine A-500
(an anion-exchanger of cellulose-type with diethylaminoethyl
groups), purchased from Seikagaku Kogyo Co., was washed
with water several times and dried over P 2O5 under reduced
pressure. p-Acetoamidophenol from Nacalai Tesque Co. was
recrystallized from hot water several times. Peroxidase (POD,
from horseradish) was purchased from Wako Pure Chemical
Industries Co. All other chemicals were of analytical or reagent
grade and used without further purification.
The anion-exchangers modified with Men+-TCAS[4] (Men+-
TCAS[4]A-500, Men+: H2, Fe3+, Fe2+, Co3+, Co2+, Cu2+, Zn2+, Ni2+),
method A in the literature.14 H2- and Co3+-TCAS[4]A-500 were
prepared by using TCAS[4] and K3[Co(CN)6], respectively.
The modified anion-exchanger (Men+-TCAS[4]A-500) contained
100 mol of Men+-TCAS[4] per gram of dry anion-exchanger.

To whom correspondence should be addressed. The proposed structure of Fe3+-TCAS[4]A-500 is shown in Fig. 1.
708 ANALYTICAL SCIENCES APRIL 2004, VOL. 20

Fig. 1 Proposed structure of Fe3+-TCAS[4]A-500.

Table 1 Ex and Em of each oxidized product of p-hydroxyphenyl derivatives

Exa Ema
HO R R
(maximum, nm) (maximum, nm)

p-Acetoamidophenol NHCOCH3 330 430

p-Cresol CH3 292 417
p-Ethylphenol CH2CH3 292 419
p-Hydroxyphenylacetic acid CH2COOH 291 404
p-Hydroxyphenethylalcohol CH2CH2OH 292 406
3-(4-Hydroxyphenyl)propionic acid CH2CH2COOH 293 402
4-(2-Aminoethyl)phenolHCl CH2CH2NH2HCl 291 404
p-Hydroxyphenyl-1-propanol CH2CH2CH2OH 293 406

a. Excitation (Ex) and emission (Em) spectra were observed for the mixture after the treatment with peroxidase for p-hydroxyphenyl
derivatives in the presence of H2O2 at pH 10.

Mn3+-TCAS[4]A-500 was prepared as follows. First, 50 ml of a
1 mM ethanol solution of Mn(CH3COO)32H2O was added to
TCAS[4] (100 mol) suspended in 50 ml of ethanol while
stirring; this mixture was then refluxed at 70 75C for an
additional 2 h. The produced Mn3+-TCAS[4] was filtered off,
washed with ethanol and dried over P 2O5 under reduced
pressure. Next, 50 ml of a 1 mM Mn3+-TCAS[4] solution was
added to DEAE cellulofine A-500 (500 mg) suspended in 20 ml
of water while stirring; this mixture was then stirred at room
temperature for an additional 1 h. The Mn 3+-TCAS[4]A-500
produced was filtered off, washed with water and dried over
P2O5 under reduced pressure. Mn3+-TCAS[4] was completely
adsorbed on the anion-exchanger because it was not found in
the filtrate.
The fluorescence spectra and fluorescence intensities were
recorded with a quartz cell (1 cm 1 cm cross-section) on a
Shimadzu RF-5300 spectrofluorometer equipped with a xenon
lamp and a dual monochromator. The bandwidths for both
excitation and emission were set at 3 nm.
Procedures for determining H2O2
Fe3+-TCAS[4]A-500 method. The determination of H2O2 using
this method was carried out by measuring the fluorescence
intensity of a fluorescent substance produced through an
oxidative reaction (1). The fluorescence intensity was measured
with excitation and emission wavelengths of 330 and 430 nm,
respectively. Fe3+-TCAS[4]A-500 (20 mg, 100 mol/g) was
added to a mixture containing the sample solution (1.0 ml, 0.1
6.0 g of H2O2), 40 mM p-acetoamidophenol solution (2.0 ml)
and buffer solution (3.0 ml); the mixture was incubated at 40C
for 20 min. After Fe3+-TCAS[4]A-500 was filtered off, the
fluorescence intensity of the supernatant was measured. A
blank in the absence of H2O2 was also measured under the same
conditions.
Oxidative reaction (1)
POD method as a reference. A mixture of a sample solution
(1.0 ml), a POD solution (1.0 ml, 10 U/ml), a 40 mM p-
acetoamidophenol solution (2.0 ml) and a buffer solution (2.0
ml) was incubated at room temperature for 20 min. The
fluorescence intensity of this reaction mixture was measured
under the same conditions as with the Fe3+-TCAS[4]A-500
method.
The buffer solutions used were 0.1 M KH2PO40.05 M
Na2B4O7 or 0.05 M Na2B4O7(0.2 M H3BO3 + 0.05 M NaCl) for
pH 6 9, 0.1 M NH4Cl0.1 N NH4OH for pH 8 11, 0.05 M
Na2B4O70.05 M Na2CO3 for pH 10 12.4, and 0.05 M Na2CO3
0.1 M NaHCO3 for pH 9 11.
ANALYTICAL SCIENCES APRIL 2004, VOL. 20 709

Table 2 Fluorescence intensities of products of the reaction by

Fe3+-TCAS[4]A-500 for each p-hydroxyphenyl derivative at pH 10
in the presence of 4.0 g of H2O2

HO R Fluorescence
intensitya

p-Acetoamidophenol 195
p-Cresol 10
p-Ethylphenol 12
p-Hydroxyphenylacetic acid ~0
p-Hydroxyphenethylalcohol ~0
3-(4-Hydroxyphenyl)propionic acid ~0
4-(2-Aminoethyl)phenolHCl ~0
p-Hydroxyphenyl-1-propanol 15

Fig. 2 Excitation and emission spectra of a fluorescence substance
produced by the oxidation of p-AP catalyzed by POD at pH 10. The
spectra (A and B) show the respective blank fluorescence.
a. Observed at the corresponding Ex and Em indicated in Table 1.
Table 3 Effects of Men+-TCAS[4]A-500 on the oxidative activity
for p-AP at pH 10 in the presence of 4.0 g of H2O2

Fluorescence
Results and Discussion Men+-TCAS[4]A-500 intensity

Peroxidase-like catalytic activity of Fe3+-TCASA-500 for the Fe3+ 187

oxidation of p-hydroxyphenyl derivatives
Table 1 gives the observed Ex and Em of each fluorescence
substance produced through an oxidative reaction (1) catalyzed
by a peroxidase for p-hydroxyphenyl derivatives. As an
example, the excitation and emission spectra of the reaction
mixture by p-acetoamidophenol at pH 10 are shown in Fig. 2.
We have demonstrated that Fe3+-TCAS[4]A-500 exhibited the
strongest peroxidase-like catalytic activity. Thus, the
peroxidase-like catalytic activity of Fe3+-TCAS[4]A-500 for an
oxidative reaction (1) was investigated by using the Fe 3+-
TCAS[4]A-500 method in a carbonate buffer solution of pH 10.
The fluorescence intensity of the reaction mixture was measured
at the corresponding Ex and Em for each p-hydroxyphenyl
derivative given in Table 1. As shown in Table 2, Fe3+-
TCAS[4]A-500 exhibited the highest fluorescence intensity for the
oxidation of p-acetoamidophenol among the p-hydroxyphenyl
derivatives. In the case of 3-(4-hydroxyphenyl)propionic acid
and p-hydroxyphenylacetic acid, their adsorption by the mother
ion-exchanger may result in a decrease of fluorescent intensity.
Accordingly, it is clear that p-acetoamidophenol is the most
suitable fluorogenic reagent for the spectrofluorometric
determination of H2O2 using Men+-TCAS[4]A-500.
Although three calix[n]arene derivatives (Fig. 1, X = CH2, R =
SO3Na, n = 1, 3, and 5) were also examined for reaction (1),
their activity could not be evaluated, because their metal
complexes were not prepared under the same conditions in this
study.
Catalytic activity of Men+-TCAS[4] A-500 for the oxidation of p-
acetoamidophenol
The fluorescence intensities of each reaction mixture after a
treatment with Men+-TCAS[4]A-500 in the presence of H2O2 are
given in Table 3. H2-, Mn3+-, Co3+-, Cu2+-, Zn2+-, and Ni2+-
TCAS[4]A-500 showed no activity, while Fe2+- and Co2+-
TCAS[4]A-500 showed very weak activity. Only Fe3+-TCAS[4]A-500
exhibited strong activity, indicating that it was the most
effective catalyst for the oxidation of p-acetoamidophenol in the
presence of H2O2.
To establish the optimal conditions for determining H2O2
using Fe3+-TCAS[4]A-500, the factors that influence the catalytic
activity of Fe3+-TCAS[4]A-500 for the oxidation of p-
Fe2+ 35
Mn3+ ~0
Co3+ ~0
Co2+ 20
Cu2+ ~0
Zn2+ ~0
Ni2+ ~0
H2 ~0
acetoamidophenol in the presence of H2O2 were investigated. In
these experiments, the activity of Fe3+-TCAS[4]A-500 was
evaluated for a sample solution containing 4.0 g of H2O2 in a
carbonate buffer solution.
Effect of the pH on the determination of H2O2
The peroxidase-like catalytic activity of Fe3+-TCAS[4]A-500 for
the oxidation of p-acetoamidophenol in the presence of H2O2
was investigated in various buffer solutions. Figure 3 shows the
fluorescence intensities of the reaction mixtures obtained
through reaction (1) after a treatment with Fe3+-TCAS[4]A-500 in
each buffer solution. As shown in Fig. 3, Fe3+-TCAS[4]A-500
exhibited strong activity in alkaline buffer solutions, especially
in a carbonate buffer solution of about pH 10. Accordingly, a
carbonate buffer solution of pH 10 was selected for the
determination of H2O2 using the Fe3+-TCAS[4]A-500 method.
Effects of the concentration of p-acetoamidophenol, the
incubation time and the temperature in the determination of
H2O2
The concentration of p-acetoamidophenol, the incubation time
and the temperature affected the catalytic activity of Fe3+-
TCAS[4]A-500. Since the fluorescence intensity reached a
maximum at 30 mM and remained almost constant at up to 50
mM, 40 mM of p-acetoamidophenol was the optimal
concentration as a fluorogenic reagent. The fluorescence
intensity was almost maximum and constant after 20 min of
incubation. Thus, the incubation time was set at 20 min. The
higher was the incubation temperature, the higher was the
fluorescence intensity of the reaction mixture. Since the
fluorescence intensity of the reagent blank was high above
710 ANALYTICAL SCIENCES APRIL 2004, VOL. 20

Table 4 Effect of foreign substances on the Fe3+-TCAS[4]A-500

method for the determination of H2O2

Added/ Added/
Substance g Error, %a Substance g Error, %a

Heparin 40 0.7 Fe3+ 40 9.8

NaF 40 4.8 K+ 40 1.6
EDTA 40 2.3 Ca2+ 40 9.8
Glycine 40 5.3 PO43 40 0.3
Ascorbate 40 89.7 CO
NH3
2
40 2.9
4
Citrate 40 24.9 + 40 1.9
Albumin (HSA) 104 1.3 Br 40 1.6
Fig. 3 Effects of the pH and buffer on the activity of Fe3+- TCAS[4]A- I 40 1.2
500. Na2CO3NaHCO3; Na2B4O7NaOH; NH4OH
NH4Cl; Na2B4O7KH2PO4; Na2B4O7(H3BO3 + NaCl). H2O2 added: 4.0 g.
a. Error (%) = 100 (H2O2 (found) H2O2 (added))/H2O2 (added).

40C, and the calibration curve was a straight line at 40C the
incubation temperature was set at 40C. amount of H2O2.

Effect of repeated use

The effect of the repeated use of Fe3+-TCAS[4]A-500 on the
catalytic activity was investigated in order to elucidate whether
Fe3+-TCAS[4]A-500 shows catalytic activity for the oxidation of p-
acetoamidophenol in the presence of H2O2. Fe3+-TCAS[4]A-500 was
applied repeatedly after being separated from the reaction
mixture, washed with water and dried. The catalytic activity of
Fe3+-TCAS[4]A-500 was maintained after repeated use, 5 times.
Thus, Fe3+-TCAS[4]A-500 has catalytic activity for the oxidation
of p-acetoamidophenol, and can be used repeatedly.
Acknowledgements
The authors thank Cosmo Oil Co. for supplying a sample of
sodium thiacalix[4]arenetetrasulfonate for the study. They are
especially indebted to Drs. H. Kumagai and S. Miyanari of
Cosmo Oil Co. for supporting this work.
References and Notes

Calibration curve for the determination of H2O2
Under the optimal conditions described above, a linear
calibration curve for the Fe3+-TCAS[4]A-500 method was
obtained between 0.1 and 6.0 g of H2O2 in the sample solution
(1.0 ml). The fluorescence intensity using the Fe3+-TCAS[4]A-500
method was about 70% of that for the corresponding reaction
using the POD method as a reference. The correlation
coefficient and the relative standard deviation (N = 4) were
greater than 0.989 and 5.5% for 4.0 g of H2O2, respectively.
For the determination of H2O2, the Fe3+-TCAS[4]A-500 method is
more sensitive by a factor of 10 100 than the previous
methods, in which 4-AAP-phenol and MBTH-HALPS systems
were employed as a chromogen.14,15
Effect of foreign substances on the determination of H2O2
The influence on the Fe3+-TCAS[4]A-500 method by the
presence of various foreign substances was investigated. As
shown in Table 4, human serum albumin (HSA) and other
substances caused almost no interference, while ascorbic acid
had an appreciable effect. With the previous method using the
MBTH-HALPS system,8 the trend of interference by HSA and
ascorbic acid was the same as that for the present method. If the
sample solution contains ascorbic acid, the addition of ascorbic
acid oxidase should minimize the interference from ascorbic
acid.1
In conclusion, Fe3+-TCAS[4]A-500 showed the strongest
peroxidase-like catalytic activity for the oxidation of p-
acetoamidophenol in a carbonate buffer solution in the presence
of H2O2 at pH 10. The sensitivity of the present method for the
determination of H2O2 was about 10 100 times higher than that
of previous methods using 4-AAP-phenol and MBTH-HALPS
systems. The catalytic activity of Fe3+-TCAS[4]A-500 was
effective for the spectrofluorometric determination of a trace
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