CHEM. RES. CHINESE UNIVERSITIES 2011, 27(6), 929—933

Flow-injection Spectrophotometric Determination of Uric Acid in Urine via Prussian Blue Reaction WASEEM Amir1*, YAQOOB Mohammad2, NABI Abdul2, MURTAZA Ghulam3 and HUSSAIN Izhar3 1. Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan; 2. Department of Chemistry, University of Balochistan, Quetta 87300, Pakistan; 3. Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Abstract A simple and sensitive flow injection(FI) spectrophotometric method was reported for the determination of uric acid based on the reduction of Fe(III)/ferricyanide in the presence of uric acid. The in situ reduced ions reacted with unreduced portion of ferricyanide/Fe(III) to form soluble Prussian blue, which was monitored at an absorption wavelength of 735 nm. The optimized conditions allow a linear calibration graph in a concentration range of 1―100 µmol/L. The relative standard deviation was in a range of 0.5%―2.5%, with a detection limit(3σ blank) of 0.3 µmol/L and a sampling frequency of 60 injection/h was obtained. The effect of common substances present in human physiological fluids on the determination of uric acid was examined. The method was applied to determining uric acid in human urine samples with the recoveries in a range of 96%―105%. The results agree well with those by spectrophotometric reference method at a confidence level of 95%. Spectrophotometric procedures for uric acid determination in clinical samples were reviewed briefly. Keywords Flow injection analysis; Spectrophotometry; Uric acid; Prussian blue; Urine Article ID 1005-9040(2011)-06-929-05

1

Introduction

Growing demand for accuracy and rapid determination of analytes in human physiological fluids has always been observed. It is mainly focused on the devices that allow one to make simple, reliable and cheaper analysis performed close to the patients. Uric acid[7,9-dihydro-1H-purine-2,6,8(3H)trione] is the final metabolite of a purine base among the nucleic acids. That is, an inosine or guanosine is produced from the purine base in the biochemical reaction process and the product is converted to a xanthine which is oxidized to uric acid by the action of xanthine oxidase. In humans and higher primates, uric acid is the final oxidation(breakdown) product of purine metabolism, present in blood and excreted in urine[1,2]. The improvement of dietary life by an economical development causes the over nutrient of high protein for people so that the uric acid is increasing in blood serum by physiological reaction, which is further increased by over drinking of alcoholic beverages, under exercise and heavy stress. An elevated level of uric acid in blood can be associated with gout[3], Lesch-Nyhan Syndrome[4] and urolithiasis[5]. Several analytical methods have been reported in the literature for the determination of uric acid in human physiological fluids, some of them include chromatographic methods[6―12], electrochemical[13,14], capillary electrophoresis[15,16], fluorimetric[17,18] and spectrophotometric methods[19―27].

Enzymatic procedures are based on the oxidation of uric acid in the presence of uricase, generating allantoin, CO2 and H2O2. The analyte consumption can be directly monitored by ultraviolet(UV) spectrophotometry[19], but the procedure requires complex sample treatment to eliminate other absorbing species. Alternatively, the concentration of uric acid can be related to the generation of H2O2, which reacts with 3,5dichlorohidroxibenzenesulphonic acid and 4-aminophenazone, yielding a red product with absorption maximum at 512 nm[21]. This is the usual method for uric acid determination in clinical analysis, in spite of reagent instability and the need for sample heating before measurement. Uric acid can also be quantified by the reduction of phosphotungstate ion to tungsten blue, monitored by spectrophotometry at 700 nm[20]. The method lacks selectivity in view of the precipitation of proteins by phosphotungstic acid. Derivative spectrophotometric method has been reported for uric acid and allopurinol in urine samples[22]. Uric acid is determined through first derivative signal measurement at 284 nm and allopurinol with second derivative signal measurement at 293 nm. Flow injection(FI) methods for the individual and simultaneous determination of ascorbic acid and uric acid have also been reported by means of spectrophotometer and miniamperometric detectors connected in sequence[23]. The simultaneous determination of uric acid and ascorbic acid is based on the measurement of the absorbance of uric acid at 393 nm and amperometric determination of both the analytes at

——————————— *Corresponding author. E-mail: [email protected] Received January 19, 2011; accepted April 25, 2011. Supported by the CIIT-Project Funded by COMSATS Institute of Information Technology, Pakistan.

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+0.6 V. Another FI-spectrophotometric procedure reported for uric acid determination in the presence of ascorbic acid took into account the difference between signals obtained in two residence time[24]. The sample is mixed with a solution containing Fe(III) and the reduced metal ion forms a colored complex with tripyridiltriazine. The first signal corresponds to the complex formed due to the fast reaction between Fe(III) and ascorbic acid. Then, the sample zone passes through another coil, being redirected to the detector for obtaining a signal corresponding to the sum of ascorbic and uric acid concentrations. Another spectrophotometric method reported for the determination of uric acid is based on fading of o-hydroxyhydroquinonephthalein-palladium(II)-hexadecyltrimethylammonium [QPpalladium(II)-HTA] complex[25]. The method is applied to uric acid in urine samples. Recently, a spectrophotometric multicommuted flow system has been described for the determination of uric acid in urine samples[27]. The method is based on the reduction of Cu(II) ions by uric acid to Cu(I) that reacts with 2,2-biquinoline 4,4-dicarboxylic acid(BCA) to form a purple complex with absorption maximum at 562 nm via solenoid micro-pumps. The method is fast, but has a complex operation. Flow analysis techniques are well-established tools for the automation and miniaturization of analytical methodologies, which has been reviewed excellently[28], showing advantages such as: (a) increased sampling frequency, (b) high versatility, (c) high robustness, (d) new analytical improvements based on operating modes under non-stationary conditions, (e) decrease of the human exposure under hazardous chemical/physical sample pretreatments, (f) more environmentally friendly procedures obtained due to process downscaling and (g) use of alternative detection systems with the concomitant simplification of the operating conditions(viz. chemiluminescent detection). Prussian blue or iron(III) hexacyanoferrate(II) is a coordination compound with different application since its discovery[29]. At first, this compound was only used as blue pigment for paints, lacquers or dyes due to the great intensity and stability of its color. Nowadays, its application range covers very different research areas such as clinical diagnostic or treatment[30]. Formation of intensely colored Prussian blue by the reduction of Fe(III) with ferricyanide can be easily detected spectrophotometrically and is the basis of detection of compounds having reducing properties[31―34]. This paper presents a simple FI method for the determination of uric acid via soluble Prussian blue formation and detection by spectrophotometry in urine samples. Experimental results show that Fe(III)/ferricyanide is reduced to Fe(II)/ ferrocyanide by uric acid in water at neutral pH. Soluble Prussian blue was formed by the reaction between the in situ formed Fe(II) and potassium ferricyanide and vice verse. The maximum absorption of this product was found around 735 nm[31,32]. Absorbance of soluble Prussian blue is the indicative of the amount of uric acid and the Beer’s law is obeyed in a concentration range of 1―100 µmol/L. The proposed method has high sensitivity, simple operation as well as low cost and is rapid. The method has been found to be significant in clinical

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2

Experimental

2.1 Reagents and Solutions All the reagents used were of analytical grade and the solutions were prepared in ultra-high purity(UHP) deionized water(