Influence and role of metal ions in enzymatic catalysis with e.c. 1.2.3.2. xanthine oxidase Application to trace analysis

The effect of metal ions on the reductive half-reaction of xanthine oxidase (XOD) in the catalytic conversion of xanthine into uric acid has been studied spectrophotometrically in Tris-HCl buffer at pH 7.4, 37 +/- 0.1 degrees and ionic strength 0.04M. Some metal ions display inhibitor properties, the sequence of inhibiting efficiency being Ag(I) > Hg(II) > Cu(II) > Cr(VI) > V(V) > Au(III) > Tl(I) and for these the I(50) values were determined. Only Tl(I), V(V) and Cu(II) showed reversible inhibition and therefore for these the mechanisms were assessed [competitive for V(V) and Tl(I); uncompetitive for Cu(II)]. The conditional inhibition constants (K(i)) were also determined. The effect of EDTA for protection of the enzyme against metal inhibition, and for its reactivation after inhibition, was also investigated. Utilization of the linear relationship between relative enzyme activity and inhibitor concentration allowed sensitive and selective (though not specific) determination of Ag(I) and Hg(II) (10(-9)-10(-8)M), and of Cu(II) and Cr(VI) (10(-7)-10(-6)M), the maximum relative error being +/- 4%. For a few metal ions, e.g., Ag(I) and Cr(VI), in the presence of EDTA, a certain specificity is observed.     

Application of spectral beta-correction method and partial least squares for simultaneous determination of V(IV) and V(V) in surfactant media

Simultaneous determination of V(IV) and V(V) was performed by application of partial least squares when the calibration matrix was obtained using beta-correction spectra. Two reaction between V(IV) and V(V) and Pyrogallol Red as a ligand in presence of cethyltrimethylammoniumbromide (CTAB) media has been investigated and applied to the simultaneous spectrophotometric determination of these species. The parameters controlling behavior of the system were investigated and optimum conditions selected. Determinations were made over the concentration range 0.6-4.50 microg ml-1 of V(IV) and 0.3-5.50 microg ml-1 of V(V). Applying this method to simultaneous determination of these metal ions in several real samples with total relative standard error less than 5% validated the proposed method.     

Separation of V(V)-4-(2-pyridylazo)resorcinolato complex from a large excess reagent using an ODS cartridge for high-performance liquid chromatography.

A selective off-line preconcentration technique for the V(V) complex with 4-(2-pyridylazo)resorcinol has been developed and successfully applied to the determination of V(V) in an air-borne sample. The target complex was separated from excess reagent using an ODS cartridge and water as the eluent. The complex was then concentrated on another ODS cartridge using tetrabutylammonium bromide and eluted with methanol; the eluate was applied to a one-drop concentration/HPLC. A detection limit as low as (6.05 +/- 0.82)x 10(-11) M (5 ppt) was achieved.     

Gas chromatographic determination of volatile anaesthetic agents in blood. Part 2. Clinical studies

A method is described for the direct determination of the volatile anaesthetics halothane and isoflurane in blood by gas chromatography with flame-ionisation detection. The method is accurate and precise and allows rapid measurements of blood levels of anaesthetic agents. Headspace concentrations of anaesthetic agents in the concentration range 0-3% V/V are determined with an accuracy of +/- 0.01% V/V. The relative standard deviation of these results is less than 4.0%. A relatively small volume of blood is required for each determination, a factor of great significance in the treatment of children. The need for separate blood calibration graphs for each patient is discussed, further emphasising the need for a rapid calibration procedure. The results from the clinical application of this method show conclusively its suitability for the management of anaesthetised subjects.     

Application of the spectral correction method and "neural networks" for simultaneous determination of V(V) and Al(III)

Simultaneous determination of V(V) and Al(III) was performed by application of neural networks when the calibration matrix was performed using β-correction spectra. Two reactions between V(V) and Al(III) and Alizarin Red S (ARS) as a ligand have been investigated and applied for the simultaneous spectrophotometric determination of these metal ions. The parameters controlling behavior of the system were investigated and optimum conditions selected. Feed-forward neural networks have been trained to quantify considered metal ions in mixtures under optimum conditions. Sigmoidal functions were used in the hidden and output layers. Determinations were made over the concentration range 0.10-7.80 μg ml⁻¹ of V(V) and 0.11-4.20 μg ml⁻¹ of Al(III). Applying this method satisfactorily to simultaneous determination of these metal ions in several synthetic solutions with total relative standard error less than 4.02% validated the proposed method.     

Optical fibre chemical sensor for trace vanadium(V) determination based on newly synthesized palm based fatty hydroxamic acid immobilized in polyvinyl chloride membrane

Fatty hydroxamic acid (FHA) immobilized in polyvinyl chloride (PVC) has been studied as a sensor element of an optical fibre chemical sensor for V(V). By using this instrument, V(V) in solution has been determined in the log concentration range of 0-2.5 (i.e. 1.0-300 mg/L). The detection limit was 1.0 mg/L. The relative standard deviation (R.S.D.) of the method for the reproducibility study at V(V) concentration of 200 mg/L and 300 mg/L were calculated to be 2.9% and 2.0%, respectively. Interference from foreign ions was also studied at 1:1 mole ratio of V(V):foreign ions. It was found that, Fe(III) ion interfered most in the determination of vanadium(V). Excellent agreement with ICP-AES method was achieved when the proposed method was applied towards determination of V(V).     

Chemometrics-assisted spectrophotometric methods for simultaneous determination and complexation study of Fe(III), Al(III) and V(V) with morin in micellar media

Evolutionary factor analysis (EFA) and rank annihilation factor analysis (RAFA) were applied to resolve the two-way equilibrium spectrophotometric data belonging to the complexes of Fe(III), Al(III) and V(V) with morin (3,5,7,20,40-penta hydroxy flavone) as chelating agent in triton X-100 micellar media. Then, partial least square regression combined with genetic algorithm for wavelength selection (GA-PLS) was used for simultaneous determination of the metal ions. The parameters controlling behavior of the system were investigated and optimum conditions were selected. The predictive abilities of partial least squares regression (PLS) and genetic algorithm-partial least squares regression (GA-PLS) were examined in simultaneous determination of ternary mixtures of metal ions over the concentration range of 17.0-170.0ngml(-1), 25.0-180.0ngml(-1) and 40.0-325.0ngml(-1) for Fe(III), Al(III) and V(V), respectively. The relative standard errors for prediction of the ions in synthetic mixtures were lower than 5% and the mean recoveries in the tap water spiked samples were 104.2 and 101.7% for PLS and GA-PLS, respectively.     

黄嘌呤在离子液体-纳米金-碳纳米管复合物修饰电极上的电化学行为

研究了黄嘌呤在离子液体-纳米金-碳纳米管修饰玻碳电极上的电化学行为。结果表明,在0.1mol/L磷酸盐(pH=4.4)介质中,修饰电极对黄嘌呤氧化具有强的电催化作用,黄嘌呤在0.9V(vs.SCE)左右产生一灵敏的氧化峰。在优化的实验条件下,用此峰测定黄嘌呤的线性范围为1.5×10-7～1.0×10-5mol/L,检出限为3.5×10-8mol/L。该修饰电极具有良好的重现性和稳定性。     

Flow-injection spectrophotometric determination of paraoxon by its inhibitory effect on the enzyme acetylcholinesterase.

A spectrophotometric enzymatic flow injection (FI) system for the determination of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method was based on the determination of the acetic acid formed by the enzymatic reaction of the acetylcholinesterase, immobilized on glass beads, with the substrate acetylcholine. The acetic acid formed permeates through a PTFE membrane and is received by a solution (pH 7.0) containing the acid-base indicator Bromocresol Purple (B.C.P.), leading to a pH change and therefore to a color change. The variation of the absorbance of the solution is detected spectrophotometrically at 400 nm. The determination of paraoxon is related to its inhibitory action on the enzyme. Therefore the analytical signal is the difference between the signal that corresponds to the free and the one that corresponds to the inhibited enzyme, considering a fixed acetylcholine concentration. The correlation between the peak height and paraoxon concentration at a given acetylcholine concentration is linear in the range from 5.0 x 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 (r = 0.998) of paraoxon, with a relative estimated standard deviation (R.S.D.) of +/- 1.7% (n = 10) considering a solution containing 5.0 x 10(-6) mol L-1 of paraoxon and a solution containing 5.0 x 10(-3) mol L-1 of acetylcholine. Therefore, the quantitative limit detection is about 2.5 x 10(-7) of paraoxon (3 sigma). A 1,1'-trimethylene-bis(4-formylpyridinium bromide)dioxime (TMB-4) solution was used to reactivate the enzyme.     

Analytical application of the complexation of Nb(V) with bromopyrogallol red in micellar media

A spectrophotometric method for the détermination of trace amounts of Nb(V) based on the formation of a ternary complex with Bromopyrogallol Red (L) and cetylpyridinium bromide (CPB) in 1M hydrochloric acid/15% dimethylformamide medium has been developed. The ternary 1:2:2 Nb-L-CPB complex is formed. The absorbance maximum is at 645 nm, the molar absorptivity being (4.00 +/- 0.04) x 10(4) l.mole(-1).cm(-1). The relative standard deviation is 1.9% and Beer's law is obeyed up to 1.4 mug of Nb(V) per ml. The application of the method to the determination of Nb in pyrochlore-bearing rocks is described. A possible mechanism of interaction of the surfactant with the Nb-L complex is discussed.     

Influence of some metal ions on oxidation of nadh and on formation of the superoxide anion radical (O(2)(-)), during enzymatic catalysis by E.C. 1.2.3.2 xanthine oxidase

The inhibitory effect of selected metal ions [Ag(I), Hg(II), Cu(II), Cr(VI), V(V), Au(III), T1(I) and Zn(II)], on the xanthine oxidase (XOD) catalysis of xanthine oxidation, has been investigated with reference to the XOD catalysis of oxidation of NADH. Hg(II), Ag(I), Zn(II) and Au(III) act as inhibitors, T1(I) has no effect and Cu(II), Cr(VI) and V(V) act as activators. The formation of O(2)(-) during XOD catalysis of oxidation of either xanthine or NADH has also been studied. All the metal ions considered act as inhibitors with respect to O(2)(-) production when the reducing substrate is xanthine, but only a few of them when the substrate is NADH, the others showing no effect whatsoever whether or not they activate NADH oxidation in the course of the same reaction. Vanadium (V) has an anomalous effect: it inhibits xanthine oxidation but considerably increases NADH oxidation, and thus appears to modify the catalytic properties of the enzyme. This behaviour appears promising as the basis for a kinetic method for determination of V(V).     

Simultaneous kinetic spectrophotometric determination of vanadium(V) and iron(III)

Vanadium(V) and iron(III) can be determined simultaneously at pH 2 and 25 degrees C by a single experiment using their kinetic effect on the oxidation of indigo carmine by bromate which goes through an induction period and then decreases in absorbance, at lambda(max), 612 nm. The rate of the color-fading of indigo carmine is proportional to the concentration of vanadium and is independent of the concentration of iron. The length of the induction period of the reaction is related to the concentration of iron and is independent of the concentration of vanadium. Concentrations of 0.3-2 (mug ml(-1)) vanadium(V) and 6-12 (mug ml(-1)) iron(III) were determined with mean relative errors of 2.7 and 1.6%, respectively. The interference effects of various cations and anions on determination of mixtures of vanadium and iron is reported. Application of the method to real samples and several mixtures of standard solutions are performed which gave acceptable results.     

Applications of catalytic reactions. Kinetic microdetermination of W(VI) and V(V)

Summary Applications of Catalytic Reactions. Kinetic Microdetermination of W(VI) and V(V) A kinetic method is described for the microdetermination of tungsten and vanadium. The method is based on the catalytic action of tungstate and vanadate ions on the oxidation of 2,4-diaminophenol with hydrogen peroxide. The effect of reagent concentration is studied and the maximum tolerable amounts of interfering ions are determined. Procedures for the determination of 0.46 to 73.5g/ml tungsten are given with a relative error of about 1.5%. Quantities of 2.02·10^–2 to 2.5g/ml vanadium could be determined with a relative error of about 2%.Furthermore a kinetic determination of tungstate and vanadate ions in their mixtures is proposed.Part of PhD Thesis-University of Thessaloniki, 1975.     

Direct measurements of xanthine in 2000-fold diluted xanthinuric urine with a nanoporous carbon fiber sensor

High selectivity and sensitivity is reported in the measurements of xanthine in urine by fast scan cyclic voltammetry (FSV) with a nanostructured carbon fiber sensor of 3.5 +/- 0.4 mum radius. Fabrication of the sensors for the measurements is described. Fabrication of the nanostructure at the carbon fiber sensor surface exposes surface pores. SEM images confirm the formation of the nanostructure. The results indicate that the nanostructure improves the sensitivity and limit of detection (LOD) in the measurements of xanthine and uric acid. The sensors allow rapid direct measurements of xanthine in 2000-fold diluted xanthinuric urine and of uric acid in 2000-fold diluted normal urine. The sensitivity and the LOD of xanthine is 0.40 +/- 0.02 nA microM(-1) (0.995) and 1 microM, respectively, and 0.99 +/- 0.01 nA microM(-1) (0.998) and 500 nM for uric acid. The concentration of xanthine in 2000-fold diluted xanthinuric urine is 1.6 +/- 0.2 muM from FSV and from HPLC. The concentration of xanthine and uric acid in urine can be determined by pre- or post-calibration of the sensor in buffer or by the method of standard addition.     

Indirect spectrophotometric determination of small amounts of selenium(IV) and arsenic(V) by simple extraction using flotation columns.

A simple, rapid and selective procedure for the indirect spectrophotometric determination of Se(IV) and As(V) has been developed. It is based on the reduction of Se(IV) to Se(0) and As(V) to As(III) with hydroiodic acid (KI + HCl). The liberated iodine, equivalent to each analyte, is quantitatively extracted with oleic acid (HOL) surfactant. The iodine-HOL system exhibits its maximum absorbance at 435 nm. The different analytical parameters affecting the extraction and determination processes have been examined. The calibration graphs were found to be linear over the ranges 5-120 and 0.25-20 ppm of Se(IV) and As(V), with lower detection limits of 2.5 and 0.15 ppm and molar absorptivities of 1 x 10(4) and 0.5 x 10(4) dm3 mol(-1) cm(-1), respectively. Sandell's sensitivity was calculated to be 0.0078 and 0.0149 microg/cm2 in the same order. The relative standard deviation for five replicate analyses of 40 ppm Se(IV) and 4 ppm As(V) were 1.0 and 0.9%, respectively. The proposed procedure in the presence of EDTA as a masking agent for foreign ions has been successfully applied to the determination of Se(IV) in a reference sample and As(V) in copper metal, in addition to their determination in spiked and polluted water samples.     

Monitoring of V(IV) and V(V) in Etnean drinking-water distribution systems by solid phase extraction and electrothermal atomic absorption spectrometry

Monitoring activities carried out since 1994 showed the presence of significant levels of vanadium in drinking waters delivered in a lot of Etnean towns. The highest vanadium concentration was found in ground waters collected in the drainage gallery Ciapparazzo located on the Northwestern flank of Mt. Etna in Bronte's area (Catania, Italy). This drainage gallery, with a flow rate of near 500 l s^− 1, is an important water source for several towns of the Etnean province. On account of different toxicological behaviours of V(IV) and V(V), which are the only possible oxidation states in aqueous media, a research project was set up to evaluate the ratio between their concentrations before and after disinfection treatments (chlorination or UV irradiation). Data were acquired in the most representative sites of the drainage gallery and the distribution network to evaluate the effect of residence times and disinfection treatments on possible species interconversion. The average total concentration of vanadium was 165 μg l^− 1. Speciation analyses performed by solid phase extraction of both species followed by furnace atomic absorption spectrometric determination of V(IV) eluates revealed that the latter was the predominant species (90-100%) in untreated waters. Moreover, among the two disinfecting treatments applied by the water supplier, only sodium hypochlorite altered the species ratio and determined an instant increase of near 20% in V(V) relative concentration. No significant effect was observed as residence time varied in the drainage gallery or in the distribution systems. Other physico-chemical and chemical parameters (i.e. pH, E_H, water temperature, electrical conductance, dissolved oxygen as well as major and minor inorganic cations and anions) were determined in the collected water samples to evaluate if they are proper or not for interconversion of the two V species. Redox potential of the water was also correlated to the percentage of V(IV).     

Kinetic-spectrophotometric determination of trace amounts of vanadium(V) based on its catalytic effect on the reaction of DBM-arsenazo and potassium bromate

A simple and sensitive kinetic-spectrophotometric method is developed for the determination of trace vanadium(V), based on the catalytic effect of vanadium(V) on the oxidation of DBM-arsenazo by potassium bromate in 0.0138 moll(-1) phosphoric acid medium and at 100 degrees C in the presence of citric acid as activator. The absorbance is measured at 528 nm with the fixed-time method. The optimization of the operating conditions regarding concentrations of the reagents, temperature and interferences are also investigated. The working curve is linear over the concentration range 0-20 ngml(-1) of vanadium(V) with good precision and accuracy and the detection limit was down to 3.44 ngl(-1). The relative standard deviation for a standard solution of 14 ngml(-1) is 0.28% (n=11). The apparent activity energies of the catalytic reaction and the non-catalytic reaction are 73.48, 113.5 kJ/mol, respectively. The proposed method proved highly sensitive, selective and relatively rapid for the assay of vanadium at low-level range of 0-20 ngml(-1) without any pre-concentration step. Thw method was applied to the determination of vanadium(V) in steels, rice, flour, cabbage, potato, fish, shrimp and tea samples with satisfactory results. The obtained results for the steel samples were excellent agreement with the standard reference values. The analytical results of the rice, flour, cabbage, potato, fish, shrimp and tea samples were excellent agreement with those of atomic absorption spectrometry. The recovery experiments have been made for the rice, flour, cabbage, potato, fish, shrimp and tea samples except the steels; excellent results were obtained. The relative standard deviations were over the range of 0.18-2.60% and the recoveries were over the range of 98.00-102.4%, respectively. The analytical results obtained were satisfactory.     

Determination of V(V), Nb(V), and Ta(V) with 2-(2-Thiazolylazo)-5-diethylaminophenol by Reversed-Phase High Performance Liquid Chromatography

The simultaneous determination of vanadium, niobium, and tantalum by reversed-phase high performance liquid chromatography (HPLC) with 2-(2-thiazolylazo)-5-diethylaminophenol (TADAP) as the precolumn chelating reagent has been established. Optimum conditions for the separation, such as the methanol content, the addition of tartaric acid, pH, and the concentration of acetate buffer, were investigated. The metal chelates were eluted in 8 min with a mobile phase of methanol–water (55/45, v/v) containing tartaric acid (0.1%, m/v) and acetate buffer (pH 3.5, 10 mmol/liter) on an ODS column at 568 nm. The detection limits (signal-to-noise RATIO = 3) for V(V), Nb(V), and Ta(V) were 0.16, 0.32, and 1.77 ng/ml, respectively. The proposed method was applied to the analyses of a reference of mineral and synthetic samples. The result was in accord with the certified value, and the recoveries were 98.9–101.8%.     

High-performance liquid chromatographic method with fluorescence detection for the determination of total homocyst(e)ine in plasma.

A high-performance liquid chromatographic method for the determination of total plasma homocyst(e)ine [H(e)] after reduction with sodium tetrahydroborate and precolumn derivatization with o-phthaldialdehyde is described. The analyses, carried out on a reversed-phase C18 column, were based on spectrofluorimetric detection. The sensitivity was 1 pmol per injection and the intra- and inter-assay relative standard deviations were 1.8% and 5%, respectively. The plasma H(e) concentration determined in 40 healthy volunteers (20-60 years old) was 12.4 +/- 2.9 microM (mean +/- S.D.), in good agreement with reference values.