SHORT COMMUNICATION Spectrofluorimetric determination of uric

Chemical Papers 62 (3) 318–322 (2008) DOI: 10.2478/s11696-008-0029-8

SHORT COMMUNICATION

Spectrofluorimetric determination of uric acid based on its activation of catalytic oxidation of pyronine Y Suling Feng*, Xueping Liu College of Chemistry and Environmental Science, Henan Normal University, Xinxiang, 453007, China Received 11 April 2007; Revised 15 July 2007; Accepted 17 July 2007

A novel kinetic spectrofluorimetric method for the determination of uric acid based on the activation effect of uric acid on the Cu(II) ion catalyzed oxidation of pyronine Y by hydrogen peroxide was developed. The influence of different buffer solutions was tested and the Britton-Robinson buffer solution with pH 2.2 was found to be the optimum. The detection limit and the linear range for uric acid are 0.09 µg mL−1 and 0.3–3.0 µg mL−1 , respectively. The RSD for eleven determinations of 1.6 µg mL−1 uric acid was 1.6 %. Satisfactory results were obtained when using this method of uric acid determination in human urine. c 2008 Institute of Chemistry, Slovak Academy of Sciences Keywords: kinetic spectrofluorimetric method, activation, uric acid, pyronine Y

Uric acid [7,9-dihydro-1H-purine-2,6,8(3H )-trione], as a primary end-product of purine metabolism, is a constituent of human body fluids. It is very slightly soluble in water and forms soluble salts with alkali. Therefore, when there is a local rise of [H+ ] in tissue, urate depositions in kidneys may ultimately lead to renal failure. Gout is caused by an excess of uric acid in the body (Akyilmaz et al., 2003). For these reasons, the assay of uric acid is of great importance in biochemical and clinical diagnoses. Various methods for uric acid determination, such as electroanalytical techniques using enzyme based electrodes and modified electrodes (Akyilmaz et al., 2003; Dobay et al., 1999; Frebel et al., 1997; Gilmartin et al., 1992; Hoshi et al., 2003; Matos et al., 2000; Roy et al., 2004; Sun et al., 2003; Wang et al., 2002; Zen et al., 1997; Zhang et al., 1998), high performance liquid chromatography (Chen et al., 1997; Czauderna & Kowalczyk, 1997; Jen et al., 2002), and chemiluminescence (Hong & Huang, 2003; Li et al., 1998; Yao et al., 2003), have been developed. In some of these methods, the electrodes lack stability (Akyilmaz et al., 2003; Frebel et al., 1997; Hoshi et al., 2003), or need to be renewed after each measurement (Gilmartin et al., 1992; Sun et al., 2003; Wang et al., 2002). Some

methods demand an enzyme immobilization technology (Akyilmaz et al., 2003; Dobay et al., 1999; Zhang et al., 1998), provide a narrow linear range (Matos et al., 2000), need uric acid preconcentration before each measurement (Sun et al., 2003), or require a long analytical time (Chen et al., 1997; Czauderna & Kowalczyk, 1997). Up to now, there have been few reports on the spectrofluorimetric determination of uric acid (Galban et al., 2001). Galban et al. (2001) described a fluorimetric method for the measurement of uric acid in blood serum based on its reaction with uricase. The linear range for uric acid was 3 × 10−5 –6 × 10−4 mol L−1 . To the best of our knowledge, no kinetic spectrofluorimetry based on the activation effect of uric acid was reported for the determination of uric acid. The development of a sensitive spectrofluorimetry detection procedure for the uric acid analysis should be of interest. The activation effect of uric acid on the reaction of pyronine Y (PRY) with H2 O2 in the presence of Cu(II) was confirmed, therefore, PRY was used as a new reagent in the present work. The purpose of the work described here was to develop a sensitive kinetic spectrofluorimetric method for the determination of the uric acid concentration. Since the variables are

*Corresponding author, e-mail: [email protected]

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S. Feng and X. Liu/Chemical Papers 62 (3) 318–322 (2008)

usually not fully independent in the experiment, a controlled and weighted centroid simplex method was used to optimize some variables (Zhang et al., 1993). The presented method avoids the use of enzymatic reagents. Satisfactory results were achieved in the determination of the uric acid concentration in human urine without pretreatment. Uric acid (UA) standard stock solution of 1 mg mL−1 was prepared by dissolving 0.2500 g of uric acid in 250 mL of 0.01 mol L−1 sodium hydroxide solution, stored at 4 ◦C and protected from light. Working solutions were prepared by appropriate dilution of the stock solution with water before their use. The Britton–Robinson buffer solution of pH 2.2 was prepared by mixing 100 mL of 0.04 M acid (3.92 g of H3 PO4 + 2.40 g of acetic acid + 2.47 g of H3 BO3 ) with appropriate volumes of 0.2 mol L−1 NaOH, and adjusting the solution to pH 2.2. The hydrogen peroxide solution of 0.33 mol L−1 was obtained by appropriate dilution of 30 % H2 O2 standardized by titration with KMnO4 as the secondary standard. The Cu(II) standard solution of 10 mg mL−1 was prepared by dissolving 9.8226 g of CuSO4 · 5H2 O in 250 mL of water. The pyronine Y (PRY) solution of 1.0 × 10−3 mol L−1 was prepared by dissolving 0.0670 g of pyronine Y in 250 mL of water. Working solutions were prepared by appropriate dilution before their use. All the reagents were of analytical or guaranteed grade, and doubly distilled water was used throughout the experiments. An FP-6200 spectrofluorometer (Jasco, Japan) was used to obtain the fluorescence spectra and to measure the fluorescence intensity. A 501 thermostatted bath (Chongqing Experimental Instrument Factory, China) was used for heating. A pH S-3C digital acidimeter (Shanghai Weiye Instrument Factory, China) was employed in the pH measurements. To a series of 25 mL measuring flasks containing uric acid in final concentrations between 0.3 µg mL−1 and 3.0 µg mL−1 , 2.5 mL of the pH 2.2 BrittonRobinson buffer solution, 1.6 mL of 0.33 mol L−1 hydrogen peroxide, 0.5 mL of 1.0 × 10−4 mol L−1 pyronine Y solution, and 0.5 mL of 10 mg mL−1 Cu(II) ion solution were added. They were diluted with water to the mark, heated in a thermostatted bath at (75 ± 0.2) ◦C for 11 min, and to stop the reaction, the flasks were cooled to room temperature by running water. Fluorescence measurements for both the reagent blank (F0 ) and the sample solutions (F ) containing uric acid were done at 552 nm with the excitation at 522 nm. Decreased fluorescence intensity, ∆F = F0 − F , was the result. Pyronine Y belongs to xanthene dyes. It emits very strong red-yellow fluorescence in aqueous solutions with maximum excitation and emission wavelength at 522 nm and 552 nm, respectively (Fig. 1 a, a’). Its fluorescence intensity decreases slightly when oxidized by H2 O2 (Fig. 1 c, c’) or co-existing with Cu(II) (Fig. 1 b, b’). However, the reaction of pyro-

A

B

Fig. 1. Excitation (A) and emission (B ) spectra of pyronine Y in the presence of different reagents. Conditions: 2.5 mL of pH 2.2 B-R buffer solution; H2 O2 , 2.11 × 10−2 mol L−1 ; PRY, 1.8 × 10−6 mol L−1 ; Cu(II), 0.18 mg mL−1 ; uric acid, 2.0 µg mL−1 ; temperature, 75 ◦C; time, 11 min; a,a’ B-R + PRY; b,b’ B-R + PRY + Cu(II); c,c’ B-R + H2 O2 + PRY; B-R + H2 O2 + PRY + UA; d,d’ B-R + H2 O2 + PRY + Cu(II); e,e’ B-R + H2 O2 + PRY + Cu(II) + UA.

nine Y with hydrogen peroxide speeds up with the presence of Cu(II) (Fig. 1 d, d’) indicating that Cu(II) catalyzes the oxidation of pyronine Y with hydrogen peroxide. Furthermore, with the addition of uric acid, the catalytic oxidation reaction is faster and the fluorescence intensity is lower (Fig. 1 e, e’). This demonstrates the ability of uric acid to activate the oxidation of pyronine Y by hydrogen peroxide in the presence of Cu(II). There is also a linear relation between the decreased fluorescence intensity and the concentration of uric acid. However, a reasonable mechanism of the reaction is unclear. Several buffer solutions of potassium biphthalatehydrochloric acid, sodium citrate-hydrochloric acid, sodium acetate-hydrochloric acid, potassium chloridehydrochloric acid, and Britton-Robinson with pH regulated in the range of 1.85–2.25 in each case were compared. It was found that the solution became brown-yellow in the potassium biphthalatehydrochloric acid medium and the maximum emission wavelength of pyronine Y shifted. In the potassium chloride-hydrochloric acid buffer solution, the reagent blank faded very quickly which resulted in low sensitivity. Uric acid could hardly activate the Cu(II)catalyzed oxidation reaction in the sodium acetatehydrochloric acid and sodium citrate-hydrochloric acid media. When the Britton-Robinson buffer solution was present in the system, PRY + H2 O2 + Cu(II) + UA, the sensitivity of the method improved. Therefore, the effect of pH was tested in the range of 1.85–2.88. The results are shown in Fig. 2. ∆F was kept nearly constant in this pH range. Therefore, the Britton-Robinson buffer solution of pH 2.2 was chosen for the subsequent experiments. A univariate method was conducted over a wide


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Table 1. Simplex shifting process with uric acid concentration of 2.0 µg mL−1 Reagent solution volume/mL Point number

1 2 3 4 5 6 7 8 9 10 11 12

Retention point

2, 1, 6, 4, 5, 3 2, 1, 6, 7, 4, 5 2, 1, 6, 8, 7, 4 2, 1, 6, 8, 7, 4 10, 2, 1, 6, 8, 7 10, 2, 1, 11, 6, 8

B-R

H2 O2

Cu(II)

PRY

3.09 3.09 3.09 3.09 3.09 1.91 2.53 2.40 2.28 1.86 2.38 2.46

1.85 1.85 1.85 1.85 1.50 1.50 1.47 1.78 1.49 1.29 1.67 1.60

0.45 0.45 0.45 0.40 0.35 0.35 0.38 0.48 0.46 0.48 0.53 0.46

0.45 0.40 0.35 0.35 0.35 0.35 0.42 0.44 0.47 0.54 0.49 0.45

Time/min

∆F

10.18 10.18 9.00 7.82 7.82 7.82 9.32 10.99 11.80 13.80 13.26 11.04

29.39 30.47 21.85 25.26 24.02 28.59 27.17 28.50 31.61 31.69 29.18 32.54

36

32

32

∆F

∆F

28

28 24

24 20

20

16

1.8

2.0

2.2

2.4

2.6

2.8

50

3.0

60

70

80

90

Temperature/°C

pH Fig. 2. Influence of pH on ∆F. 2.5 mL B-R buffer solution; H2 O2 , 2.11 × 10−2 mol L−1 ; PRY, 1.8 × 10−6 mol L−1 ; Cu(II), 0.18 mg mL−1 ; uric acid, 2.0 µg mL−1 ; temperature, 75 ◦C, time, 11 min.

Fig. 3. Influence of temperature on ∆F. 2.5 mL of pH 2.2 B-R buffer solution; H2 O2 , 2.11 × 10−2 mol L−1 ; PRY, 1.8 × 10−6 mol L−1 ; Cu(II), 0.18 µg mL−1 ; uric acid, 2.0 µg mL−1 ; time, 11 min.

range of variables which included the concentration of reagents and the reaction time. On basis of the univariate method results, the controlled and weighted centroid simplex method was employed to optimize these interrelated variables to achieve high sensitivity. The results are shown in Table 1. The simplex shifting would meet the expected requirement reaching the value of 11, the relative deviation of the experiment response value of the six retention points was R = 3.88–5 %, and the process stopped with 12 being the centroid of the six retention points. The temperature was optimized by the univariate method. The results are shown in Fig. 3. The maximum ∆F was obtained at 75 ◦C. Thus, the temperature of 75 ◦C was selected for subsequent experiments. In order to stop the reaction, the solution was cooled to room temperature by running water. The fluorescence intensity of the system remained stable for at least 5 h at room temperature. Under the optimum conditions, a linear calibration graph was obtained over the uric acid concentration

range of 0.3–3.0 µg mL−1 . The regression equation was ∆F = 4.83 + 14.12C with a regression coefficient of 0.9993 (n = 7), where C stands for the concentration of uric acid in µg mL−1 . The detection limit, calculated as 3s0 /S, where s0 is the standard deviation of the blank measurements (n = 11) and S the slope of the calibration graph, was 0.09 µg mL−1 . The limit of quantitation, calculated as 10s0 /S, was 0.3 µg mL−1 . The relative standard deviation for eleven determinations of 1.6 µg mL−1 uric acid was 1.6 %. The effect of various ions and compounds on ∆F was examined in the presence of 1.2 µg mL−1 uric acid. The tolerance limit is defined as the concentration which does not result in more than ± 5 % deviation from the standard value in the absence of the disturbing species. The results are shown in Table 2. It was found that ascorbic acid in concentrations higher than 1.2 µg mL−1 caused errors in the determination of 1.2 µg mL−1 uric acid. Fortunately, the concentration of uric acid in body fluids is much higher than that of ascorbic acid. The interference of ascorbic acid


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raphy. Journal of Chromatography A, 763, 187–192. DOI: 10.1016/S0021-9673(96)00740-6. Czauderna, M., & Kowalczyk, J. (1997). Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography. Journal of Chromatography B, 704, 89–98. DOI: 10.1016/S03784347(97)00459-3. Dobay, R., Harsanyi, G., & Visy, C. (1999). Detection of uric acid with a new type of conducting polymer-based enzymatic sensor by bipotentiostatic technique. Analytica Chimica Acta, 385, 187–194. DOI: 10.1016/S0003-2670(98)00662X. Frebel, H., Chemnitius, G. C., Cammann, K., Kakerow, R., Rospert, M., & Mokwa, W. (1997). Multianalyte sensor for the simultaneous determination of glucose, L-lactate and uric acid based on a microelectrode array. Sensors and Actuators B: Chemical, 43, 87–93. DOI: 10.1016/S0925-4005(97)00133-0. Galban, J., Andreu, Y., Almenara, M. J., Marcos, S, & Castillo, J. R. (2001). Direct determination of uric acid in serum by a fluorometric-enzymatic method based on uricase. Talanta, 54, 847–854. DOI: 10.1016/S0039-9140(01)00335-6. Gilmartin, M. A. T., Hart, J. P., & Birch, B. (1992). Voltammetric and amperometric behavior of uric-acid at bare and surface-modified screen-printed electrodes: studies towards a disposable uric-acid sensor. The Analyst, 117, 1299–1303. DOI: 10.1039/AN9921701299. Hong, H. C., & Huang, H. J. (2003). Flow injection analysis of uric acid with a uricase- and horseradish peroxidase-coupled Sepharose column based luminol chemiluminescence system. Analytica Chimica Acta, 499, 41–46. DOI: 10.1016/S00032670(03)00950-4. Hoshi, T., Saiki, H., & Anzai, J. I. (2003). Amperometric uric acid sensors based on polyelectrolyte multilayer films. Talanta, 61, 363–368. DOI: 10.1016/S0039-9140(03)00303-5. Jen, J. F., Hsiao, S. L., & Liu, K. H. (2002). Simultaneous determination of uric acid and creatinine in urine by an eco-friendly solvent-free high performance liquid chromatographic method. Talanta, 58, 711–717. DOI: 10.1016/S00399140(02)00377-6. Li, Z., Feng, M., & Lu, J. (1998). KMnO4 –octylphenyl polygylcol, ether chemiluminescence system for flow injection analysis of uric acid in urine. Microchemical Journal, 59, 278–283. DOI: 10.1006/mchj.1997.1537. Matos, R. C., Augelli, M. A., Lago, C. L., & Angnes, L. (2000). Flow injection analysis - amperometric determination of ascorbic and uric acids in urine using arrays of gold microelectrodes modified by electrodeposition of palladium. Analytica Chimica Acta, 404, 151–157. DOI: 10.1016/S00032670(99)00674-1. Roy, P. R., Okajima, T., & Ohsaka, T. (2004). Simultaneous electrochemical detection of uric acid and ascorbic acid at a poly(N,N-dimethylaniline) film-coated GC electrode.

Table 2. Study of interferences with the determination of 1.2 µg mL−1 uric acid in the proposed method Tolerance limit Interfering substance

µg mL−1 8.04 × 104 3.96 × 103 300 81.6 60.0

Urea Zn2+ Allantoin Glucose, K+ Mg2+ , Na+ , C2 O2− 4 , sucrose

Lactic acid, HCO− 3 Creatinine CH3 OH Ca2+ Fe3+ , caffeine, C2 H5 OH Inosine Ascorbic acid L-Cysteine

32.4 25.9 19.2 12.0 3.22 2.0 1.2 0.24

in body fluids can be eliminated by appropriate dilution of the body fluids sample (Hong & Huang, 2003). In order to examine the applicability of the presented method, five human urine samples from volunteers were appropriately diluted and analyzed without pretreatment according to the procedure described above. The results were compared with those obtained by the phosphotungstic acid method, as shown in Table 3. It is evident from the calculated t-test values that the results for uric acid in human urine were in good agreement with those obtained by the classical spectrophtometric method. The recovery of 98– 104 % was determined assuming the standard addition method. This confirms the validity of the method proposed in this work. References Akyilmaz, E., Sezginturk, M. K., & Dinckaya, E. (2003). A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine. Talanta, 61, 73– 79. DOI: 10.1016/S0039-9140(03)00239-X. Chen, Y. M., Pietrzyk, R. A., & Whitson, P. A. (1997). Quantification of urinary uric acid in the presence of thymol and thimerosal by high-performance liquid chromatog-

Table 3. Determination of uric acid in human urine Urine concentrationa /(102 µg mL−1 )

Recovery/%

Sample Proposed method n = 4 1 2 3 4 5

6.9 3.4 4.1 5.8 3.5

± ± ± ± ±

0.2 0.1 0.2 0.08 0.09

Reference method n = 3

n=2

± ± ± ± ±

104 103 104 102 98

6.6 3.3 4.0 5.6 3.4

0.2 0.03 0.06 0.07 0.07

a) Mean value ± standard deviation. b) Theoretical value t = 2.57, n = 5 for 95 % confidence interval.


tb

1.25 1.10 0.55 1.53 1.20

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