ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
A FIBER OPTIC SPECTROPHOTOMETRIC DETERMINATION OF URINARY INDOXYL SULFATE (INDICAN) AFTER CLOUD POINT EXTRACTION
© 2014 Hayati Filik, Dilek Kilcan
Istanbul University, Faculty of Engineering, Department of Chemistry 34320AvcilarIstanbul, Turkey Received 09.02.2012; in final form 28.11.2012
This work describes a new, simple, and sensitive colorimetric determination method for indoxyl sulfate (indican) by fiber optic UV-Vis spectrophotometry coupled to cloud point extraction as the separation-precon-centration method. This method is based on the diazotization of sulphanilic acid in acidic medium followed by its coupling with indoxyl sulfate (indican), which gives an azo product and extraction of the colored product using the cloud point extraction (CPE) technique. The optimal extraction and reaction conditions (e.g., acid and reagent concentrations, effect of time) were studied, and the analytical characteristics of the method (e.g., limit of detection, linear range, preconcentration factor) were obtained. Linear response was achieved within 0.9—44 p.g/mL and the detection limit was estimated as 0.6 p.g/mL. The inter-day and intra-day relative standard deviations were in the ranges 1.2—1.3% and 1.6—1.8% for indican. The method was applied to the determination of indican in human spiked urine samples; Recoveries within 96—99% were obtained.
Keywords: indoxyl sulfate, indican, cloud point extraction, spectrophotometry, urine analysis.
DOI: 10.7868/S0044450214030050
In biochemistry indican refers to indoxyl sulfate (IS) found in urine due to bacteria action on tryptophan. The essential amino acid tryptophan is converted to indole by intestinal bacterial cleavage of the tryptophan side chain. Some indole is absorbed into the blood stream and converted in the liver first to indoxyl and then to the sulfate ester, which is termed indican a colourless, water-soluble compound which is excreted in the urine [1, 2]. Normally, only a small amount of indican is found in the urine. Conversion of IS in the urine to indoxyl is catalyzed by a sulfa-tase/phosphatase [3]. An alkaline environment favors conversion of indoxyl to indigo, and a low oxygen environment favors the production of indirubin [3]. IS is one of the uremic toxins circulating at elevated concentration in the serum of patients with uremia. Because IS is mainly excreted via the kidney, the toxin cannot be efficiently removed from uremic patients with renal failure [4—8]. It is increased with high protein diets or inefficient protein digestion [1, 2]. Its Increased amounts result from putrefactive reactions and intestinal obstruction, gastric cancer, hypochlo-rhydria, biliary obstruction, and malabsorptive syndromes. The quantitation method of indoxyl in urine has been established and validated in this study.
Various analytical methods have been applied to identify and quantitate the indoxyl derivatives. Methods involving spectrophotometry, thin-layer chromatography (TLC) [9, 10], high performance thin-layer
chromatography (HPTLC) [11], high performance liquid chromatography with ultraviolet detection (HPLC/UV) [12], HPLC coupled with evaporative light scattering detection (ELSD) [13, 14] and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of indican, isatin, indirubin and indigotin in the roots and leaves of I. indigotica [15, 16] have been developed. HPLC with UV detection was applied to determine indican in human urine and plasma [17]. HPLC/ECD (electrochemical detection) method with a palladium hexacyanoferrate chemically modified electrode (PdHCF CME) was also developed to determine isatin in rat brain, while HPLC/UV methods were also used to determine indirubin and indigotin in the leaves and roots of I. indigotica [18]. Among these methods, spectrophotometry continues to gain wide popularity because of common availability of the instrumentation and simplicity of procedures as well as low cost.
Few methods have been described for the determination of indican in urine till now. The urine indican test is also called Obermeyer test, which is an indicator of intestinal toxemia and overgrowth of anaerobic bacteria. Direct UV-Vis spectrophotometric detection of indican depends upon its decomposition to indoxyl and subsequent oxidation to indigo blue which is then concentrated into a layer of chloroform for easier measurement [19]. In 1913 Jolles introduced alcohol-
ic thymol as a reagent for the detection of indican in urine [20]. Various modifications of this method has been proposed [21, 22]. A spectrophotometric procedure for the determination of indican in urine based in Jolles' reaction, modified by Bermejo-Martinez and Barrera-Ramallo, was described and applied for the determination of urine in adulterated cow milk, addition of 2% human urine was estimated [22]. Curzon and Walsh [23] reported on the spectrophotometric determination of urinary IS. For this purpose, indoxyl sulphate was treated with ^-dimethyl-aminobenzalde-hyde-HCl and after making alkaline, the red Gessner compound was extracted into petroleum spirit and determined colorimetrically. Bryan described a procedure to determine the indican in urine samples [24]. The description of the 24-h urine collections, the reagents used in the analytical procedure for indican and the determination of urinary IS by diazotization and coupling have been presented [24]. This method is based upon preliminary column chromatography on a cation-exchange resin followed by diazotization and coupling.
In this work, a cloud point extraction (CPE) methodology has been developed and optimized for the extraction and determination of indican. The quantitation method of indican or indoxyl sulfate in urine was established and validated. The reaction involves the transformation of nitrite into nitrous acid in acid medium, subsequent diazotization of sulphanilic acid and formation of a diazonium salt. This diazonium salt couples with indican to produce 4-(sulphophenyla-zo)-1-indoxyl. This reaction called coupling and is an electrophilic substitution reaction. The CPE technique was used to extract the color product, and spectrophotometry was used to determine it.
EXPERIMENTAL
Apparatus. Experiments were carried out using a commercially available miniature fibre-optic based spectrometer (Ocean Optics Inc., HR4000CG-UV-NIR) which utilises a small tungsten halogen lamp (Ocean Optics Inc.) as the light source and a charge-coupled device (CCD)-based detector for absorbance measurements. The spectral resolution declared by the manufacturer was 0.02 nm. A thermostated bath maintained at the desired temperature was used for reaction (Hettich, Universal). The pH values of the solutions were measured by a Hanna HI 221 pH-meter using the full range of 0—14. Conical bottom disposable plastic centrifuge tubes (10 mL) made of clear and autoclavable polypropylene were used for phase separations. All glassware was rinsed carefully with 1 : 3 diluted HCl.
Chemicals. All chemicals used were of analytical reagent, or better yet, grade without any additional purification. Distilled water was used throughout this study. Indoxyl-P-D-glucoside (indican), indoxyl sulfate (IS) and TX-114 were obtained from Sigma-Ald-rich Chemical Co. (St. Louis, MO, USA). NaCl and
HCl were purchased from Mallinckrodt Baker, Inc. (Philllipsburg, NJ, USA). Stock standard solution of 1.0 x 10-3 M was prepared by dissolving standard indican sample in 1.0 mL ethanol and diluting to the mark in a 100 mL volumetric flask with distilled water. The resulting solution was kept in refrigerator before use. Working solutions of indican were prepared from the stock solution by appropriate dilutions. Sulphanilic acid (Merck) 1% (w/v) solution was prepared in 1.0% (w/v) Na2CO3. Sodium nitrite (1.0 x 10-4 M ) solution was prepared in distilled water.
Sample preparation. Urine samples were collected during 24 h from two normal healthy volunteers and and stored in polyethylene bottles at 4°C. The urine samples were initially diluted in a ratio of 1 : 10. Ali-quots (5.0 mL) of each urine sample were analyzed directly without any pretreatment.
Procedure. An aliquot of the solution containing 1.0 mL of 1.0 x 10-4 M NaNO2, 1.0 mL of 3.0 M HCl solution, and 1.0 mL of % 1.0 (w/v) sulphanilic acid (SA) solution was transferred into a 10-mL centrifuge tube. The solution was diluted to approximately 7 mL with water and allowed to stand for 5 min. Then 1.0 mL of indican solution and 1.0 mL of Triton X-100 solution were added and made up to the mark with water. Subsequently, the mixture was shaken and left to stand in a thermostated water bath for 20 min at 100°C before centrifugation. The phase separation was accelerated by centrifuging for 5 min at 4000 rpm. The mixture was cooled in an ice bath to increase the viscosity of the surfactant-rich phase, and the aqueous phase was easily decanted by simply inverting the tube. The mi-cellar extract of this procedure was diluted with 0.3 mL of tetrahydrofuran (THF), transferred into a quartz cell, and the absorbance of the colored azo dye was measured at 572 nm against the reagent blank.
RESULTS AND DISCUSSION
Absorption spectra. Free indoxyl is released by hydrolysis from indican, which subsequently dimerizes to indigotin (also known as indigo) (X = 607 nm). Figure 1b shows the UV-Vis absorption spectrum of indigo dye (dimerized indoxyl) after CPE. The CPE technique was used to extract the color product, and spec-trophotometry was used to determine it. The absorbance intensity of the solution at 572 nm, the Xmax of the product, is proportional to the indican concentration. Therefore, the new system is suitable for spectrophotometric determination of indican. Figure 1a shows the absorption spectrum of diazo coupling reaction product. As can be seen in Fig. 1, the absorption signal of diazo coupling product was higher than indigotin (i.e. dimeriz
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