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22 2006 The Japan Society for Analytical Chemistry

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Application of Crude Extract of Kohlrabi (Brassica oleracea gongylodes) as a Rich Source of Peroxidase in the Spectrofluorometric Determination of Hydrogen Peroxide in Honey Samples
Jamshid L. MANZOORI, Mohammad AMJADI, and Maghsood OROOJI
Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran

Crude extract of kohlrabi (Brassica oleracea gongylodes) was prepared by a simple procedure and its enzymatic activity and total protein concentration were determined. It was found that this crude extract is a rich source of peroxidase (POx) and has high specific activity. Cross-linked polyvinylpyrrolidone was used as a stabilizer in the preparation of the crude extract. The POx activity of kohlrabi crude extract did not vary for at least 2 months when deoxygenated and stored at 4C. This extract was applied for the spectrofluorometric determination of hydrogen peroxide using homovanillic acid as a fluorogenic substrate. POx catalyzes the hydrogen peroxide oxidation of homovanillic acid to produce a dimer which shows strong fluorescence at 420 nm with excitation at 312 nm. In the optimum conditions, the calibration graph for hydrogen peroxide was linear up to 190 ng mL1, with a detection limit of 4.4 ng mL1. The relative standard deviation (RSD) was 1.48% for 50 ng mL1 hydrogen peroxide. The proposed method was successfully applied to the determination of hydrogen peroxide in honey. The concentrationtime profile of H2O2 produced upon dilution of honey was studied and H2O2 contents of some different honeys from various areas of Iran were determined. (Received September 5, 2005; Accepted May 15, 2006)

Hydrogen peroxide is a product of many biological reactions catalyzed by several oxidase enzymes. It has been shown that enzymically produced hydrogen peroxide is a major source of the antibacterial activity of honey. It is produced by the action of small amounts of glucose oxidase present in honey on glucose. The enzyme has been found to be practically inactive in fullstrength honey and gives rise to hydrogen peroxide only when the honey is diluted. Thus honey can act as a continuous generator of hydrogen peroxide at a level, which is antibacterial but not tissue-damaging. It should also be mentioned that the hydrogen peroxide content of honey and consequently its antibacterial activity varies with origin and processing.14 Therefore the determination of hydrogen peroxide is important in characterization and selection of honey samples for use as an antimicrobial agent. Several analytical methods have been proposed for the determination of H2O2, including spectrophotometry,58 spectrofluorometry,913 chemiluminescence1417 and electrochemical techniques.1821 Most of these methods are enzymatic methods that are of interest because of their high sensitivity and selectivity. Peroxidase (POx, EC 1.11.1.7) is the most commonly used enzyme for the determination of H2O2. It is a hem-containing enzyme that is extensively distributed in plants, animals and microorganisms but its main source for commercial production is the roots of horseradish. This enzyme utilizes hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds.22 Although enzymatic determinations are widely used in biochemistry and clinical chemistry, natural enzymes are expensive and their solutions are not stable. Therefore, the
To whom correspondence should be addressed. E-mail: manzoori@tabrizu.ac.ir

study of potential alternatives is one of the interesting trends in analytical biochemistry. Several metal-porphyrin2325 and metalphthalocyanine12,26 complexes, hemin27 and -CD-hemin28,29 have been used as mimetic enzymes instead of POx for the determination of H2O2. In recent years the use of plant tissue materials and crude extracts of various vegetables as novel biocatalysts has also received considerable interest for replacing isolated enzymes.3037 The use of such biological materials is very attractive because of their simplicity, high stability, very low cost and fewer cofactor requirements in comparison with the pure enzymes. Tissues of several plants such as asparagus,38,39 grape,40 pineapple,41 kohlrabi,42 tobacco callus,43 horseradish roots,44,45 turnip46 and sweet potato47 have been used as POx sources. Vieira and Fatibello-Filho48 have chosen the crude extract of zucchini as a POx source of very high specific activity and have used it for the spectrophotometric determination of H2O2. Nevertheless, there is no report on the use of crude extracts for the spectrofluorometric determination of H2O2. However, spectrofluorometry has been the method of choice for many organic and inorganic materials because it has the advantages of both sensitivity and simplicity. Furthermore, it is more selective than other spectrophotometric methods. In the present paper, crude extract of kohlrabi (Brassica oleracea gongylodes) was prepared and its activity was compared with that of zucchini. It was found that the crude extract of kohlrabi is a rich source of POx and has higher specific activity than zucchini. This extract was applied for the spectrofluorometric determination of H2O2 in several honey samples using homovanillic acid (HVA) as a fluorogenic substrate.

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Table 1 Activity, total protein and specific activity of kohlrabi and zucchini crude extracts Vegetable Kohlrabi Zucchini Activity/ unit mL1 9610 1420 Total protein/ mg mL1 1.50 0.84 Specific activity/ unit mg1 6400 1690

Experimental
Apparatus Fluorescence spectra and intensity measurements were made on a Shimadzu RF-540 spectrofluorometer equipped with a 150 W xenon lamp, using a 1.0 cm quartz cell. Slit widths of both monochromators were set at 5 nm. All measurements were performed at 20C by use of a thermostated cell holder and thermostatically controlled water bath. Absorbance measurements were carried out on a Shimadzu UV-265FW spectrophotometer. A Metrohm Model 654 pH meter was used for pH measurements. A Hettich Model EBA centrifuge was used in preparation of crude extract of kohlrabi. Reagents All reagents used were of analytical reagent grade. Doubly distilled water was used throughout. Homovanillic acid (HVA) was purchased from Acros Chemical Company and its stock standard solution of 0.01 mol L1 was prepared in water. Working standard solutions were prepared daily by proper dilution with water. An H2O2 stock standard solution (0.1 mol L1) was prepared from 30% H2O2 solution and standardized by titration with KMnO4. A working standard solution was prepared daily by proper dilution with water. Cross-linked polyvinylpyrrolidone was purchased from Acros Chemical Company and purified as described in the literature.34 4Aminoantipyrine was purchased from Merck and its stock solution was prepared in water. Varieties of healthy kohlrabi were obtained from a local producer. They were washed, hand-peeled and frozen in a refrigerator. Preparation of kohlrabi crude extract A piece of the frozen peeled kohlrabi was finely chopped and 25 g of it was mixed with 100 mL of 0.1 mol L1 phosphate buffer (pH 6.5) and 7.5 g of polyvinylpyrrolidone. The mixture was stirred for 3 5 min at 4 7C, rapidly filtered through a layer of cheesecloth, and centrifuged at 3000 rpm for 15 min at this temperature. The supernatant was deoxygenated by Ar bubbling and was stored at 4C in a refrigerator. In this condition the solution was stable for at least two months. This solution was utilized as our enzymatic source. Peroxidase activity and total protein determinations Peroxidase activity present in the crude extract of kohlrabi was determined by measuring an increase in absorbance at 510 nm resulting from the reaction of 4-aminoantipyrine and phenol with hydrogen peroxide.49 The crude extract was diluted 10 times and 0.1 mL of the resulting solution was mixed with 1.4 mL of a mixture solution containing 0.0025 mol L1 4aminoantipyrine and 0.17 mol L1 phenol, and 1.5 mL of 0.0017 mol L1 H2O2 solution. The increase in absorbance at 510 nm and 25C was recorded for 5 min. One activity unit is defined as the amount of enzyme that causes an increase of 0.001 absorbance per minute under the conditions described above. Total protein concentration was determined by the Lowry method50 using bovine serum albumin as a standard. General procedure To a set of 10 mL calibrated flasks containing various volumes of H2O2 standard solution, we added 0.8 mL 1 103 mol L1 HVA, 2 mL 0.25 mol L1 phosphate buffer (pH 8.5) and 50 L crude extract (with activity of 9610 unit). The samples were diluted to the volume with water. The final concentrations

of H2O2 in these solutions should be in the range of 10 190 ng mL1. The fluorescence intensities of the solutions were measured at an excitation wavelength of 312 nm and an emission wavelength of 420 nm after 15 min. Determination of hydrogen peroxide in honey samples Four types of honey from various regions of Iran were purchased from local sources. A 10.0 g portion of each honey sample was weighed and mixed with about 30 mL distilled water and diluted to 50.0 mL in a calibrated flask. After 1 h standing, 0.5 mL of the solution was taken for the determination of hydrogen peroxide according to the proposed procedure and by using the calibration graph. The recovery assays were carried out using the same procedure but adding known amounts of H2O2 in honey solution after 1 h.

Results and Discussion

Preparation of crude extract So far the crude extract of some vegetables has been prepared; among them zucchini has been selected as a best source of POx.48 In this work, POx activity and total protein concentration of kohlrabi was determined and the results compared with those obtained from zucchini (Table 1). As can be seen, the kohlrabi crude extract shows higher specific activity (more than 5 times the activity of zucchini). Therefore, this extract was selected as a source of enzyme. Some important parameters affecting the activity of crude extract were optimized. The buffer to tissue ratio was studied in the range 2:1 to 6:1 (v/m) and the highest specific activity was obtained at 4:1 ratio. The effect of buffer pH was also investigated in the pH range 5 8.0. The results are shown in Fig. 1. As can be seen, the highest enzymatic activity was reached at pH 6.5. The effect of stirring time, under the Ar bubbling and at 4 7C, was also studied. It was found that stirring for 3 5 min is sufficient for achieving the maximum extraction yield and that stirring for more than 5 min causes a decrease in activity. The enzymatic activity of kohlrabi crude extract is decreased rapidly in time. This is known to be due to the effects of natural phenolic compounds present in the extract47 and of oxidation by atmospheric oxygen. To minimize these effects, cross-linked polyvinylpyrrolidone (PVP) was used as a stabilizer in preparation of the crude extract together with deoxygenating of solution by Ar bubbling. The effect of amount of PVP was investigated and the PVP to tissue ratio of 3:10 (m/m) was selected as the optimum amount. Under these conditions, the enzymatic activity of kohlrabi crude extract did not vary for at least 2 months when stored at 4C in a refrigerator. The crude extract of six different kohlrabi samples was prepared and variation of enzymatic activity from sample to sample was investigated. According to the results, the average activity and relative standard deviation were found to be 9672 and 8.6%, respectively.

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Fig. 1 Effect of 0.1 mol L1 phosphate buffer pH on the enzymatic activity of kohlrabi crude extract. Buffer to tissue ratio, 4:1; stirring time, 5 min.

Fig. 3 Effect of the pH on the fluorescence intensity of HVAH2O2kohlrabi crude extract (s) and HVAkohlrabi crude extract (a) systems. [HVA], 8 105 mol L1; [crude extract], 100 unit mL1; [H2O2], 100 ng mL1. Other conditions are as in Fig. 1.

Fig. 4 Effect of HVA concentration on the fluorescence intensity of HVAH2O2kohlrabi crude extract (s) and HVAkohlrabi crude extract (a) systems. pH, 8.5. Other conditions are as in Fig. 3.

Fig. 2 Fluorescence excitation (Ex) and emission (Em) spectra of a) HVAH2O2-crude extract; b) HVAcrude extract and c) HVAH2O2 systems. pH, 8.5; [HVA], 8 105 mol L1; [H2O2], 150 ng mL1; [crude extract], 50 unit mL1; T, 20C; incubation time, 15 min; ex, 312 nm; em, 420 nm.

fluorescence signal that was studied by using phosphate buffers. As can be seen, the fluorescence intensity reaches a maximum in the pH range 8.0 8.5. The effect of buffer concentration was also studied. The fluorescence increased by increasing the concentration and reaches a maximum at 0.04 mol L1. Therefore, pH 8.5 phosphate buffer with concentration of 0.05 mol L1 was chosen for pH adjustment. Effect of homovanillic acid concentration and crude extract activity Figure 4 shows the effect of HVA concentration on the fluorescence signals of HVAH2O2crude extract and HVAcrude extract systems. As can be seen, the fluorescence intensity of both systems is increased with HVA concentration. Then, 8 105 mol L1 was chosen as the optimum concentration, because after this point the difference between the fluorescence intensity values of the two systems remained constant. The effect of POx activity was also investigated in the range The results show that, of 1 200 unit mL1 (Fig. 5). fluorescence intensity is increased by increasing POx activity until about 20 units mL1 and then remained constant. The activity of about 50 units mL1 was selected as a suitable amount of the enzyme. This activity can be achieved by adding 50 L of the crude extract to a 10 mL calibrated flask. Effect of time and temperature Figure 6 shows the kinetic curves for HVAH2O2crude extract system at various temperatures. As the temperature is raised, the fluorescence intensity is decreased; this can be

Reaction of HVA with hydrogen peroxide in the presence of peroxidase Homovanillic acid is a well-known fluorogenic substrate for the determination of hydrogen peroxide. It reacts with H2O2 in the presence of POx to give a fluorescent dimer. Figure 2 shows the excitation and emission spectra of the reaction product of HVA with H2O2 in the presence of the kohlrabi crude extract. As can be seen, the fluorescence spectra of HVAH2O2crude extract system is similar in shape to that of HVAH2O2 system, but the fluorescence intensity of the former system is greatly enhanced due to the catalysis by POx present in the crude extract. The excitation and emission maximum wavelengths are at 312 and 420 nm, respectively. Effect of pH and buffer The oxidation of HVA by H2O2, using the crude extract of kohlrabi as a source of POx, is dependent on the pH and also on the specific buffer used. Four kinds of buffer system, phosphate, carbonate, borate and glycine, were tested. In the presence of phosphate buffer solution the highest fluorescence signal was obtained. Figure 3 shows the effect of pH on the

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Table 2 Comparison of the detection limits of the proposed method with those of other methods for the determination of hydrogen peroxide Method Spectrofluorometry by using Pure enzyme Fe-TSPC Mn-TMPyP Mn-TSPC Immobilized hemin Spectrophotometry by using the crude extract of zucchini Amperometric biosensors by using asparagus tissue Amperometric biosensors by using tobacco callus tissue This method Detection limit/ ng ml1 17 5.8 0.44 4.4 2.1 0.51 71.4 13.6 25.5 4.4 Reference 13 24 12 24 26 27 48 38 43

Fig. 5 Effect of the crude extract activity on the fluorescence intensity of HVAH2O2kohlrabi crude extract (s) and HVAkohlrabi crude extract (a) systems. pH, 8.5. Other conditions are as in Fig. 3.

Table 3 Tolerance limits of interfering species on the determination of 100 ng mL1 H2O2 Substance Na+, K+, Mg2+, Ca2+, Cl, NO3, SO42, CO32, Zn2+, glucose, sucrose, galactose, maltose, glutamic acid, glycine Cu2+ Ba2+ Ni2+, vitamin B6 Cd2+, vitamin B1 Al3+, vitamin B2 Co2+ Cr3+ Fe3+ Mn2+ Ascorbic acid Tolerance limit (mass ratio to H2O2) 1000

Fig. 6 Kinetic curves for HVAH2O2kohlrabi crude extract system at various temperatures (C). [HVA], 5 105 mol L1. pH, 8.5. Other conditions are as in Fig. 3.

attributed to a gradual decline in POx activity due to heat inactivation. Based on these results, a temperature of 20C was chosen for our experiments. At this temperature the fluorescence reaches a maximum within 12 min. Therefore, in all experiments the fluorescence intensities were measured after 15 min. Analytical characteristics In the optimum conditions described above, a spectrofluorometric method was developed for the determination of H2O2. The calibration graph (n = 14) was found to be linear in the range 10 190 ng mL1; its equation was F = 0.382(0.005)C + 0.377(0.55), where F is fluorescence intensity and C is the H2O2 concentration in ng mL1. The correlation coefficient (r) was 0.9990. The detection and quantification limits (calculated as 3/m[sb2 + sh2 + (h/m)2sm2]1/2 and 10/m[sb2 + sh2 + (h/m)2sm2]1/2 where sb, sh and sm are the standard deviation of the blank, intercept and slope, respectively)51 were found to be 4.4 and 14.4 ng mL1, respectively. The slope of the calibration graph (m) is the calibration sensitivity according to IUPAC definition. In order to study the precision of the method, we measured a series of nine standard solutions of 50 ng mL1 H2O2 on the same day. The relative standard deviation (RSD) was 1.48%. Table 2 compares the detection limit of the proposed method with some published methods for the determination of H2O2. As can be seen, our proposed method has comparable or better detection limits than the methods studied except for some of those methods that used synthetic catalyst as mimetic enzymes. But the preparation of crude extract is much simpler than the synthesis of most of these mimetic enzymes.

900 800 500 200 100 80 60 40 25 0.5

Study of interferences Several inorganic ions and some organic compounds are investigated for their interferences in the determination of H2O2 by using the proposed method. A foreign species was considered to interfere when it gave an error of more than 5% in fluorescence signal. The results are shown in Table 3. According to these results, most inorganic ions and organic compounds do not affect the determination of H2O2 when they are present in 1000-fold excess. The severe interference of ascorbic acid is due to its reducing effect on H2O2 and is independent of the method used. In the case of honey samples, our goal is the determination of net amount of H2O2 produced in sample under our conditions. Determination of hydrogen peroxide in honey As mentioned before, H2O2 in honey is produced by the action of glucose oxidase on glucose when honey is diluted. Dilution is needed for decreasing the acidity of the medium and for adjusting the pH for proper action of glucose oxidase. To the best of our knowledge, there is no report on the quantitation of H2O2 in honey by using chemical analysis methods. Therefore, the releasing of H2O2 upon dilution was studied. Figure 7 shows the profiles of H2O2 concentration with time when honey is diluted with distilled water or phosphate buffer of pH 7.4.

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Table 4 Determination of hydrogen peroxide produced upon dilution of several honey samples with water Hydrogen peroxide Sample Maragheh Found/g g1 6.9 0.3 8.9 0.7 8.5 0.2 10.5 0.2 Addeda/g 20.0 40.0 60.0 20.0 40.0 60.0 20.0 40.0 60.0 20.0 40.0 60.0 Recovery, % 99.3 1. 7 98.8 1.5 99.4 0.2 104.4 4.6 97.8 1.6 99.8 1.5 101.0 1.3 99.1 1 101.3 0.7 98.0 0.7 99.5 0.8 99.4 0.2

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Hashtrood

Orumieh

Tarom

Fig. 7 Profiles of H2O2 concentration vs. time when 10 g of honey is diluted to 50 mL with (s) water and (a) phosphate buffer (pH 7.4).

a. Amount added to 50 mL honey solutions.

Upon dilution with water, the concentration of H2O2 is increased with time and a maximum is reached in 50 min. After about 80 min, the concentration of H2O2 decreased, probably due to the fact that the rate of degradation of H2O2 by some substances present in honey, such as ascorbic acid or some cations, becomes greater than the rate of its production. When honey is diluted with phosphate buffer of pH 7.4, the maximum concentration is reached sooner (after about 20 min); after about 30 min, the concentration decreases rapidly. These results clearly show that dilution of honey lead to the continuous release of H2O2, which can act as a source of antibacterial agent. Hydrogen peroxide concentrations of 4 honey samples from various regions of Iran, after dilution with water, were determined as described in the Experimental section. The results are shown in Table 4. Recovery experiments on honey solutions spiked with different amounts of H2O2 were also carried out. As can be seen from Table 4, the obtained recoveries are between 99 and 104%; such values confirm the accuracy of the proposed method.

Conclusions
It was shown that the crude extract of kohlrabi is a rich source of peroxidase and has higher specific activity than previously used zucchini crude extract. The kohlrabi crude extract was applied as an inexpensive and stable source of POx for the spectrofluorometric determination of H2O2. The use of this extract gave a detection limit for H2O2 which is comparable with that obtained by using the pure enzyme. The hydrogen peroxide contents of several honey samples were successfully determined by using the proposed method. The results confirmed that dilution of honey lead to a continuous production of H2O2.

Acknowledgements
We would like to thank Prof. O. Fatibello-Filho for helpful advice about preparation of the crude extract.

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