Determination of Uric Acid in Human Serum: Reversed-Phase Liquid Chromatography with Electrochemical Detection

Abstract

A method for the simultaneous determination of uric acid in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection has been developed. Human serum (0.5 ml) was mixed with 0.5 ml of 0.2 N perchloric acid solution and the mixture was centrifuged at 3,000 g for 20 min. An aliquot (10 μl) of the supernatant (deproteinized human serum) was injected into the chromatographic system employed in this study. The assay limit for quantitation was about 10 pg for uric acid. Complete separation of uric acid was achieved in about 8 min under the present chromatographic conditions.     

Electrochemical Detection of Ascorbic and Uric Acids in the Eluate from Reversed-Phase High-Performance Liquid Chromatography with Newly Developed Rigid-Type Porous Polymer Packing (Polymetacrylate Gels)

Abstract

It has been found that the combination of ethylenediaminetetraacetate and metaphosphoric acid is useful for the stabilization of ascorbic acid (AA) and precipitation of proteins in body fluids and also that the complete separation of catecholamines, AA, and uric acid (UA) on a column packed with a newly developed rigid-type porous polymer packing (polymetacrtlate gels) can be achieved. On the basis of the above findings, we have developed a method for the determination of endogenous AA and UA in body fluids such as human serum, urine, and cerebrospinal fluid by reversed-phase high-performance liquid chromatography with electrochemical detection. Determination of AA and UA in each body fluid has been carried out by injecting 10 μl of each simply deproteinized body fluid into a chromatographic system.     

Determination of Plasma Norepinephrine and Epinephrine by High Performance Liquid Chromatography Using a Two-Column System and an Electrochemical Detector

Abstract

A method for the simultaneous plasma norepinephrine (NE) and epinephrine (E) determination by reversed-phase ion-pair liquid chromatography with electrochemical detection has been developed. Catecholamines were extracted from a 4 ml plasma sample using an alumina adsorption procedure. A two-pump, two-injection valve, two-column system allowed both to detect plasma NE and E with a good sensitivity due to large injected volumes of extract without any electrochemical detector disturbance and to eliminate uric acid and dopa the low k' of which would prevent the NE detection. Using this method, NE and E would be detected in respective injected amounts down to 30 and 50 picograms. Plasma NE and E determinations were found to be linear in the range of 288 to 788 pg/ml and 24 to 274 pg/ml respectively. The reproducibility, expressed as the coefficients of variation, varied from 2.1% for NE to 10.8% for E.     

Non-enzymatic sensing of uric acid using a carbon nanotube ionic-liquid paste electrode modified with poly(β-cyclodextrin)

We describe a nonenzymatic electrochemical sensor for uric acid. It is based on a carbon nanotube ionic-liquid paste electrode modified with poly(β-cyclodextrin) that was prepared in-situ by electropolymerization. The functionalized multi-walled carbon nanotubes and the surface morphology of the modified electrodes were characterized by transmission electronic microscopy and scanning electron microscopy. The electrochemical response of uric acid was studied by cyclic voltammetry and linear sweep voltammetry. The effects of scan rate, pH value, electropolymerization cycles and accumulation time were also studied. Under optimized experimental conditions and at a working voltage of 500 mV vs. Ag/AgCl (3 M KCl), response to uric acid is linear in the 0.6 to 400 μΜ and in the 0.4 to 1 mΜ concentration ranges, and the detection limit is 0.3 μΜ (at an S/N of 3). The electrode was successfully applied to the detection of uric acid in (spiked) human urine samples.

SEM images of (a) carbon ionic liquid electrode (CILE) (b) MWNT-CILE (c) β-CD/CILE (d) β-CD/ MWNT-CILE. The surfaces of carbon ionic liquid electrode (CILE) (a) and MWNT-CILE (b) were homogenous and no separated carbon layers can be observed; After β- cyclodextrin (CD) was modified on CILE and MWNT-CILE, the surfaces of β-CD modified electrodes (c and d) exhibited loose and porous morphologies.

     

A Uricase Method for the Peak Identification of Uric Acid Appeared in a Liquid Chromatogram Amperometrically Monitored

Abstract

A uricase method for the peak identification of uric acid appeared in a liquid chromatogram monitored by aid of an electrochemical detector has been developed. Uricase (EC 1.7.3.3, from Candida utilis) catalyzes the conversion of uric acid to allantoin. We have found that uric acid can be oxidized under the chromatographic conditions employed in this study, whereas allantoin cannot be oxidized. The complete disappearance of a uric acid peak in a chromatogram of a biological sample after the uricase treatment indicates that the uric acid peak does not contain any other electroactive components. We observed the complete disappearance of the uric acid peaks in the chromatograms of human serum and gastric body.     

Determination of Ascorbic Acid and Dehydroascorbic Acid in Blood Plasma Samples

Abstract

Following the stabilization of the plasma samples with HClO₄ and EDTA, the samples could be directly analyzed by HPLC using electrochemical detection and reversed-phase columns. The accuracy and precision of the method was evaluated using plasma samples spiked with ascorbic acid (10 μg/ml) and the results were also compared to the classical colorimetric procedure. Dehydroascorbic (5 μg/ml) was determined in plasma samples using UV detection following derivatization at room temperature for 45 minutes with o-phenylenediamine.     

Electrochemical Enhanced Detection of Uric Acid Based on Peroxidase-like Activity of Fe3O4@Au

Fe₃O₄@Au nanomaterials were prepared by the self-assembly method. An enzyme-free, ultrasensitive electrochemical detection of uric acid was achieved based on the peroxidase-like activity of Fe₃O₄@Au. The proposed procedure has exhibited excellent catalytic activities and achieved significant enhancements of the current responses to uric acid. The detection range was from 0.1 to 10 mmol/L, and the limit of detection was 0.087 μmol/L. Under the action of external magnetic field, the magnetic particles can be easily separated from the bottom liquid, which has the advantages of simple operation and high separation efficiency. Moreover, this detection method combining a simulated enzyme and electrochemical can enhance the effective output of the overall electrochemical signal without modifying the electrode, and excellent reproducibility can be achieved. Compared to colorimetric assay, the electrochemical one has higher sensitivity and selectivity, and was further applied in ultrasensitive detection of uric acid in food samples. In short, the proposed electrochemical assay has great potential in the fields of food quality control and biomedical analysis.     

Electrocatalytic oxidation and determination of norepinephrine in the presence of ascorbic and uric acids at a poly (Evans Blue)—modified glassy carbon electrode

A sensitive and selective electrochemical method for the determination of norepinephrine using a poly (Evans Blue) film-modified glassy carbon electrode was developed. The polymer film-modified electrode shows excellent electrocatalytic activity toward the oxidation of norepinephrine (NE) in phosphate buffer solution (pH 5.0). The linear range of 5.0 × 10⁻⁷–1.8 × 10⁻⁵ M and detection limit of 3.5 × 10⁻⁸ M were observed for the determination of NE in pH 5.0 phosphate buffer solutions. The interference studies showed that the modified electrode had excellent selectivity for the determination of NE in the presence of large excess of ascorbic acid (AA) and uric acid (UA). The differences of the oxidation peak potentials for NE-AA and NE-UA were about 175 and 172 mV, respectively. The resolution is large enough to determine AA, NE and UA individually. This work provides a simple and easy approach to selective detection of NE in the presence of AA and UA in physiological samples. The article is published in the original.     

A Sensitive Electrochemical Approach for Melamine Detection Using a Disposable Screen Printed Carbon Electrode

We report here a simple and easy electrochemical approach for sensitive detection of non‐electroactive melamine using a disposable screen printed carbon electrode (SPCE) with uric acid as the recognition element. It is based on the competitive adsorptive behavior of melamine at the preanodized SPCE causing suppression in the oxidation current of uric acid. A linear range up to 126 ppb with a detection limit of 1.6 ppb (S/N=3) is achieved at the preanodized SPCE by differential pulse voltammetry. The electrochemical method is successfully applied to detect the melamine content in tainted milk powder and dog food.     

A Method for the Determination of Methionine in Human Serum by High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

We have found remarkable enhancement of electrochemical response for the analysis of methionine by use of a glassy carbon electrode preanodized in a 0.2 M phosphate buffer (KH₂PO₄?KOH, pH 6.5) at +1.9 V vs. Ag/AgCl for 2 min. On the basis of this finding, we have developed a method for the determination of methionine in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection using the electrochemically pretreated glassy carbon electrode. The minimum detectable quantity of methionine has been found to be about 1 ng.     

Ferrocene Derivatization Reagents for Optical Resolution of Carboxylic Acids by High-performance Liquid Chromatography with Electrochemical Detection

Abstract

New derivatization methods using chiral ferrocene reagents have been developed for the optical resolution of carboxylic acids by high-performance liquid chromatography with electrochemical detection. Two chiral derivatization reagents, 1-ferrocenylethylamine and 1-ferrocenylpropylamine, were readily prepared from acetylferrocene and propionylferrocene in two steps, respectively. Condensation of carboxylic acids with the chiral reagent was effected in the presence of water-soluble carbodiimide and 1-hydroxybenzotriazole. The diastereomeric amides formed from N-acetylamino acid and α-arylpropionic acid enantiomers were efficiently resolved by reversed-phase chromatography and showed the satisfactory sensitivity at +0.45 V vs. an Ag/AgCl reference electrode with a detection limit of 0.5 pmole (S/N=5).     

Electrochemical behavior of small biological molecules at organic donor—acceptor electrodes in aqueous media

The electrochemical behavior of NADH, xanthine, uric acid, 6-mercaptopurine, 6-thioxanthine, dopamine and ascorbic acid was investigated at the organic metal-like polymer paste electrodes, tetrathiafulvalene- and N-methylphenazene-tetracyanoquinodimethane. The reactivity of small biological molecules at polymer paste electrodes is generally lower than at most active graphite electrodes. Typical detection limits of about 10^?5 M and the typical upper limit of linearity of 10^?3 M are observed for the compounds studied.     

Simultaneous Determination of Fourteen Catecholamines and Their Metabolites by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A simple method has been developed for simultaneous determination of 14 catecholamines and their metabolites in cerebrospinal fluid and brain tissue by reversed-phase, ion-pair high-performance liquid chromatography with electrochemical detection. The time required for complete separation and analysis of all compounds was less than 35 min. Quantitation was based on the use of an internal standard isoproterenol. The mobile phase consisted of a 91:9 (v/v) mixture of 0.1 M formic acid and acetonitrile containing sodium-1-octane sulfonic acid. Using this method, analysis of neurotransmitters in brain tissue can be accomplished without a clean-up procedure.     

基于纳米结构的电化学传感器用于伏安法测定苄丝肼、尿酸和叶酸(英文)

A carbon paste electrode modified with carbon nanotubes and ferrocene was fabricated.An electrochemical study of the modified electrode and an investigation into its efficiency for the electrocatalytic oxidation of benserazide,uric acid and folic acid were undertaken.The electrode was also used to study the electrocatalytic oxidation of benserazide using cyclic voltammetry,chronoamperometry,and square wave voltammetry(SWV).We found that the oxidation of benserazide at the surface of the modified electrode occurs at a potential about 285 mV lower than that of unmodified carbon paste electrode.SWV gave a linear dynamic range from 8.0×10-7 to 7.0×10 4 mol/L.The detection limit was 1.0×10-7 mol/L for benserazide.This modified electrode was used for the determination of benserazide,uric acid,and folic acid in an urine sample.     

Electrochemical Behaviors of Ascorbic Acid and Uric Acid in Ionic Liquid

Cyclic voltammetric measurements at platinum electrode have been carried out to investigate the electrochemical oxidation of ascorbic acid and uric acid in ionic liquid, [bmim][BF₄]. It is important that a typical redox couple of ascorbic acid was obtained and it is oxidized to dehydroascorbic acid in [bmim][BF₄]. However, there is no electron-transfer for uric acid and no electrochemical oxidation carried out in the same ionic liquid. It provides a new way to eliminate the interfering between ascorbic acid and uric acid in the study of the electrochemical behaviors for them.     

Study on the Electrochemical Behavior of Dopamine and Uric Acid at a 2‐Amino‐5‐mercapto‐[1,3,4] Triazole Self‐Assembled Monolayers Electrode

Selected from a series of structurally related heteroaromatic thiols, a newly synthesized reagent 2‐amino‐5‐mercapto‐[1,3,4] triazole (MATZ) was used to fabricate self‐assembled monolayers (SAMs) on gold electrode for the first time. The MATZ/Au SAMs was characterized by electrochemical methods and scanning electronic microscopy (SEM). In 0.04 mol/L Britton–Robinson buffer solution (pH 5), the electrochemical behavior of dopamine showed a quasireversible process at the MATZ/Au SAMs with an electrode kinetic constant 0.1049 cm/s. However, the electrochemical reaction of uric acid at the SAMs electrode showed an irreversible oxidation process, the charge‐transfer kinetics of uric acid was promoted by the SAMs. By Osteryoung square‐wave voltammetry (OSWV), the simultaneous determination of dopamine and uric acid can be accomplished with an oxidation peak separation of 0.24 V, the peak current of dopamine and uric acid were linearly to its concentration in the range of 2.5×10^?6–5.0×10^?4 mol/L for dopamine and 1×10^?6–1×10^?4 mol/L for uric acid with a detection limit of 8.0×10^?7 mol/L for dopamine and 7.0×10^?7 mol/L for uric acid. The MATZ/Au SAMs electrode was used to detect the content of uric acid in real urine and serum sample with satisfactory results.     

Chromatographic Properties of Ethanol/Water Mobile Phases on Silica Based Monolithic C18

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L⁻¹ and 0.86 μmol L⁻¹, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.

     

CE Determination of Creatinine and Uric Acid in Saliva and Urine During Exercise

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L^?1 and 0.86 μmol L^?1, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.     

Electrochemical Determination of Xanthine Oxidase-Catalyzed Oxidation of Xanthine

Abstract

A rapid electrochemical (chronoamperometric) method for the determination of xanthine oxidase catalyzed oxidation of xanthine and hypoxanthine is described. The assay is based on the anodic oxidation of the product, uric acid, at a stationary carbon paste electrode. Metabolism was monitored as reaction proceeded by direct insertion of a three-electrode assembly into incubation mixtures, applying a potential and measuring current after a 7 sec controlled electrolysis. The method requires no sample preparation, nor utilization of external reagents, and is compared with the on-line spectrophotonetric analysis based on monitoring the appearance of uric acid detected as an increase in absorbance at 290 nm.     

High Performance Liquid Chromatography-Electrochemical Assay for Monitoring the Formation of 8-Oxo-7,8-dihydroadenine and its Related 2′-Deoxyribonucleoside

Abstract

A sensitive method involving the combined use of reversed-phase high-performance liquid chromatography and electrochemical detection has been developed for monitoring the formation of 8-oxo-7,8-dihydroadenine and 8-oxo-7,8-dihydro-2′-deoxyadenosine. The limit of detection for both compounds is close to 0.2 ng under optimised conditions of detection at an oxidation potential of 850 mV. This assay allows the quantitative measurement of the hydroxylated purine components in aqueous solutions of adenine and 2′-deoxyadenosine exposed to a dose of ionizing radiation as low as 2 Gray (Gy).