The analysis of uric acid in urine using microchip capillary electrophoresis with electrochemical detection

Clinical studies have linked irregular concentrations of uric acid in urine to several diseases. Conventional methods for the measurement of uric acid are however temperature-dependent, expensive, and require labile reagents. The miniaturization of analytical techniques, specifically capillary electrophoresis, offers an ideal alternative for clinical analyses such as uric acid determination. The added benefits include reduced reagent and analyte consumption, decreased maintenance costs, and increased throughput and portability. A microchip capillary electrophoresis-electrochemical system for the analysis of uric acid in urine is described. The poly(dimethylsiloxane) (PDMS)/glass microchip utilizes amperometric detection via an off-chip platinum working electrode. Linear responses from 1 to 165 microM and 15 to 110 microM were found for dopamine and uric acid, respectively. The limit of detection for both compounds was 1 microM. Once characterized, the system was used to measure the concentration of uric acid in a dilute urine sample in less than 30 s. The measured uric acid concentration was verified with the uricase reaction and found to be acceptable. Six additional urine samples were evaluated with the microchip device and the uric acid concentration for each sample was found to be in the expected clinical concentration range.     

Microchip capillary electrophoresis with a boron-doped diamond electrode for rapid separation and detection of purines

Microchip capillary electrophoresis (CE) coupled with a boron-doped diamond (BDD) electrode has been employed for the separation and detection of several purines and purine-containing compounds. The BDD end-channel amperometric detector offers favorable signal-to-noise (S/N) characteristics at the high detection potential (+ 1.3 V) essential for detecting purine-related compounds. Factors influencing the separation and detection processes were examined and optimized. Five purines (guanine, hypoxanthine, guanosine, xanthine, and uric acid) have been separated within 6 min at a separation voltage of 1000V using a borate/phosphate run buffer (pH 8.2). Linear calibration plots are observed for micromolar concentrations of the purine compounds. Good stability and reproducibility (R.S.D. < 5%) are obtained reflecting the minimal adsorption of purines at the BDD surface. Applicability for the detection of nucleosides, nucleotides, and oligonucleotides is illustrated. The new microchip protocol offers great promise for a wide range of bioanalytical applications involving assays of purines and purine-containing compounds.     

Determination of phytosiderophores by anion-exchange chromatography with pulsed amperometric detection

Phytosiderophores of the mugineic acid family are separated by anion-exchange HPLC using NaOH gradient elution. Separation of mugineic acid (MA), 2'-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA) and 3-epi-hydroxymugineic acid (epi-HMA) is obtained within 15 min. Detection of the underivatised phytosiderophores is performed using pulsed amperometric detection (PAD) at pH 13. The sensitivity of the detection increases in the order DMA < MA < HMA < epi-HMA and respective detection limits of 5 microM (DMA), 1 microM (MA) and < 0.5 microM (HMA, epi-HMA) are achieved. PAD is discussed in comparison with the well-established fluorimetric detection method after post-column derivatisation with ortho-phthaldialdehyde. The main advantage of PAD is the simplicity of the method (no derivatisation) and the high sensitivity for hydroxylated mugineic acids. The method is used for the determination of phytosiderophores in root washings of iron-deficient and non-deficient wheat and barley plants.     

Pulsed amperometric detection of carbohydrates on an electrophoretic microchip

The first report of pulsed amperometric detection (PAD) on an electrophoretic microchip is presented. A hybrid poly-(dimethylsiloxane)/glass device was coupled with a platinum working electrode for the electrochemical detection of glucose, maltose, and xylose. Under optimized detection conditions, glucose was found to respond linearly from 20 to 500 microM with a measured detection limit of 20 microM. The coupling of PAD with a microchip provides a straightforward approach to the analysis of a wide range of carbohydrates using microfluidics.     

Comparison of pulsed amperometric detection and integrated voltammetric detection for inorganic sulfur compounds in liquid chromatography

A Variety of potential–time waveforms are useful in pulsed electrochemical detection (PED) when applied for the amperometric detection of numerous polar organic compounds following their separation by liquid chromatography (LC). Here, we compare the waveforms for pulsed amperometric detection (PAD) and integrated voltammetric detection (IVD) applied for detection of organosulfur compounds at Au electrodes in acidic media. In PAD waveforms, electrodes response is measured at a constant detection potentials. In IVD waveforms, electrodes current is integrated throughout a fast cyclic scan of the detection potential. As a consequence of this difference in detection strategy, the background signal for IVD is significantly smaller for PAD in the detection of organosulfur compounds whose response mechanisms require the concomitant formation of surface oxides on Au electrodes. Furthermore, in comparison to Pad, IVD has a larger sensitivity and a diminished system peak from 0₂ dissolved in the sample. Use of a preadsorption step increases detection sensitivity in both PAD and IVD. The limit of detection (S/N=3)for cysteine in LC-IVD is ca. 6 nM for a 50-μl injection (i.e., 300 fmol) using a detection waveform that includes a 1000-ms preadsorption period.     

Application of microchip-CE electrophoresis to follow the degradation of phenolic acids by aquatic plants

In this paper, we describe the separation and detection of six phenolic acids using an electrophoretic microchip with pulsed amperometric detection (PAD). The selected phenolic acids are particularly important because of their biological activity. The analysis of these compounds is typically performed by chromatography or standard CE coupled with a wide variety of detection modes. However, these methods are slow, labor intensive, involve a multistep solvent extraction, require skilled personnel, or use bulky and expensive instrumentation. In contrast, microchip CE offers the possibility of performing simpler, less expensive, and faster analysis. In addition, integrated devices can be custom-fabricated and incorporated with portable computers to perform on-site analysis. In the present report, the effect of the separation potential, buffer pH and composition, injection time and PAD parameters were studied in an effort to optimize both the separation and detection of these phenolic acids. Using the optimized conditions, the analysis can be performed in less than 3 min, with detection limits ranging from 0.73 microM (0.10 microg/mL) for 4-hydroxyphenylacetic acid to 2.12 microM (0.29 microg/mL) for salicylic acid. In order to demonstrate the capabilities of the device, the degradation of a mixture of these acids by two aquatic plants was followed using the optimized conditions.     

糖的高效阴离子交换色谱-脉冲安培检测法分析

高效阴离子交换色谱-脉冲安培检测法是新近发展的一种分析糖的有效方法。本文讨论了糖的高效阴离子交换色谱分离和脉冲安培法检测的原理,色谱条件的选择,包括分离糖的固定相、流动相以及检测糖的脉冲电位波形,并对方法的应用进行了较为详细的叙述。     

A comparison of pulsed amperometric detection and conductivity detection for carbohydrates

Pulsed amperometric detection (PAD) at a gold electrode and conductivity detection are compared for glucose, as a representative of the general challenge of the chromatographic detection of carbohydrates. In alkaline solutions of barium hydroxide, which are useful for anion-exchange separation of carbohydrates, PAD is significantly more sensitive than the conductivity detector but the latter provides linear response to higher concentrations than those observed for PAD. The combined technique of conductivity in series with PAD is linear for glucose over the range 6×10^?7?1×10^?2 M (ca. 6 ng-10 mg per 50-μl sample injection).     

Analysis of etimicin sulfate by liquid chromatography with pulsed amperometric detection

A new method for determination of etimicin's (ETM) purity and content is developed by liquid chromatography (LC) and pulsed amperometric detection (PAD). A reversed-phase ion-pair LC method with pulsed amperometric detection on a gold electrode after post-added NaOH is described. The mobile phase consisted of an aqueous solution containing 0.033 mol L(-1) oxalic acid, 0.012 mol L(-1) heptafluorobutyric acid, and 210 mL L(-1) acetonitrile with pH 3.40 adjusting by dilute NaOH solution. The total analysis time was not more than 30 min. The effects of the different chromatographic parameters on the separation were also investigated. A number of commercial samples of etimicin sulfate were analyzed using this method.     

Separation of urea, uric acid, creatine, and creatinine by micellar electrokinetic capillary chromatography with sodium cholate

The capillary electrophoretic separation of the four nonprotein nitrogenous compounds (NPNs; urea, uric acid, creatine, and creatinine) typically employed in clinical and medical settings for the monitoring of renal function is described. Successful resolution of these compounds is achieved with the use of a bile salt micelle system composed of sodium cholate at phosphate buffer pH 7.4. The elution patterns of four NPNs are obtained within 30 min with a voltage of 30 kV. The effect of varying the applied voltage, temperature, and the mole ratio of phosphate buffer with bile salt surfactant on the migration behavior is also examined.     

Simultaneous determination of creatinine, uric, L(+)-ascorbic and orotic acids in milk by reversed-phase ion-interaction HPLC chromatography

Summary Separation methods have been developed by means of reversed-phase ion-interaction HPLC chromatography, using octylamine ortho-phosphate as the interaction reagent. In particular the separation of some antioxidants of relevant interest (known also as radical scavengers) such as L(+)-ascorbic acid, uric acid, orotic acid and cysteine, as well as the separation of creatine and creatinine have been performed. The results have been applied to the analysis of different kinds of milk, namely a dairy farm milk, a commercial whole fresh milk and a UHT-sterilized one: creatinine, uric, L(+)-ascorbic and orotic acids were identified and quantified.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Characterization of low-molecular-weight metal species in plant extracts by using HPLC with pulsed amperometric detection and cyclic voltammetry

An analytical separation scheme is presented for the isolation of low-molecular-weight metal species (< 10 kDa) in plants. After ultrafiltration of the aqueous plant extracts isotachophoresis or gel chromatography is used for pre-separation and HPLC at a cyclodextrin-phase for further separation of metal containing fractions. Trace metals are detected off-line by using either adsorptive stripping voltammetry for platinum or AAS for zinc and magnesium. It is shown that platinum and zinc detection closely correlate with pulsed amperometric detection (PAD), while magnesium behaves differently. PAD is carried out after post-column addition of sodium hydroxide, thus enabling a sensitive and selective detection of carbohydrates. It is demonstrated that cyclic voltammetry is a useful tool for further characterization of the PAD-detectable metal species. By comparison of the cyclic voltammograms of carbohydrate standards, including carbohydrate-metal species, with the respective voltammograms obtained from HPLC fractions, carbohydrate species isolated from the samples can be considered as being either partly oxidized sugars (sugar alcohols or sugar acids) or glycosidically bound sugars.     

Qualitative and quantitative analysis of uric acid,creatine and creatinine together with carbohydrates in biological material by HPTLC

Summary In clinical diagnosis creatine, creatinine and uric acid are important parameters for the evaluation of renal diseases, and are partially responsible for gout and the formation of renal calculi. The determination of various carbohydrates, especially glucose, in urine and serum serves as an indicator of diabetes and other carbohydrate anomalies. A simple, rapid and economic method for the simultaneous screening and quantitation of these compounds is presented. No sample preparation is necessary for the determination in urine or in serum. The proposed method consists of separations on an amino modified HPTLC precoated plate. The detection of all relevant substances is reproducibly performed by simply heating the chromatogram to give stable fluorescent derivatives. The detection limits in all cases lie significantly below the physiological range.     

HPLC-Methode zur simultanen Bestimmung von Harnstoff, Kreatinin und Harns?ure in Serum und Urin

Summary A simple, rapid, and reproducible method for the determination of urea, creatinine, and uric acid in human serum and urine by ion-pair reversed-phase high-performance liquid chromatography with UV detection (196 nm) has been developed. The method involves the pretreatment of serum samples with trichloroacetic acid and centrifugation followed by the isocratic separation of compounds on a -Bondapak C18 column using a mobile phase consisting of 1.25 mmol/l tetrabutylammonium phosphate. For urine samples no special pretreatment is necessary. In addition, this method allows, with some limitations, the determination of creatine in serum.     

Characterization of low-molecular-weight metal species in plant extracts by using HPLC with pulsed amperometric detection and cyclic voltammetry

An analytical separation scheme is presented for the isolation of low-molecular-weight metal species (< 10 kDa) in plants. After ultrafiltration of the aqueous plant extracts isotachophoresis or gel chromatography is used for pre-separation and HPLC at a cyclodextrin-phase for further separation of metal containing fractions. Trace metals are detected off-line by using either adsorptive stripping voltammetry for platinum or AAS for zinc and magnesium. It is shown that platinum and zinc detection closely correlate with pulsed amperometric detection (PAD), while magnesium behaves differently. PAD is carried out after post-column addition of sodium hydroxide, thus enabling a sensitive and selective detection of carbohydrates. It is demonstrated that cyclic voltammetry is a useful tool for further characterization of the PAD-detectable metal species. By comparison of the cyclic voltammograms of carbohydrate standards, including carbohydrate-metal species, with the respective voltammograms obtained from HPLC fractions, carbohydrate species isolated from the samples can be considered as being either partly oxidized sugars (sugar alcohols or sugar acids) or glycosidically bound sugars. Received: 26 January 1998 / Revised: 19 June 1998 / Accepted: 24 June 1998     

High-performance capillary zone electrophoretic assay for markers of diabetic nephropathy in plasma and urine

A new high-performance capillary zone electrophoretic assay for creatine (Cr), creatinine (Cn), urea (U) and uric acid (Ua), markers of human diabetic nephropathy, both in plasma and urine has been developed with UV detection at 200 nm. The plasma sample was deproteinized with trichloroacetic acid and centrifuged at 10 000 rpm for 10 min. The urine sample was diluted 20-fold with buffer before analysis. The optimum separation conditions for the markers was investigated with respect to the concentration of the buffer, the pH, the voltage and the capillary temperature. Baseline separation was achieved in 25 mmol/L phosphate buffer (pH 3.45) using a 21 cm x 75 microm I.D. fused-silica capillary at 40 degrees C with an electric field of 1190 V/cm. The calibration curves showed good linearity in the range 3.5-1000, 0.18-700, 500-5000 and 2-800 microM (r2 min > 0.998) for Cr, Cn, U and Ua, respectively. The proposed method also has a high reproducibility (peak area RSD max < 3%) and has been successfully applied to the determination of clinical samples.     

Direct amperometric detection of glucose on a multiple-branching carbon nanotube forest

We show here that a re-structured carbon nanotube (CNT) forest can be used 'as-received' for high selectivity, non-enzymatic glucose detection without the need to incorporate catalytic metal nanoparticles or an exclusion polymeric membrane. Direct amperometric sensing of glucose has been achieved by using multiply-branched CNTs prepared by growing secondary or tertiary 'offshoot' CNTs on top of the existing CNT layer. The increased roughness factor on these re-structured CNTs enhances the amperometric detection sensitivity for glucose by a factor of 20x over that of interfering species like ascorbic acid or uric acid. A sensitivity of 60 microA mM(-1) cm(-2) and a detection limit of 1 microM, with a linear detection range of 1-11 mM (R(2) = 0.999), could be attained on the re-grown CNTs. When the re-grown CNTs were subjected to further anodization in acid to introduce a highly-charged interface, the sensitivity to glucose was enhanced to 157 microA mM(-1) cm(-2).     

Internal standard in flow injection analysis with amperometric detection

In this work, we describe for the first time the use of the internal standard method in flow injection analysis (FIA) with amperometric detection. The method is based on the application of sequential potential pulses to the working electrode in an electrochemical flow cell. The sequence of potential pulses is selected in such a way that the analyte and internal standard compound are detected and monitored individually and independently at the same working electrode. This approach compensates for random errors associated with variations of flow rate, injection volume, ionic strength difference between standards and samples, and accidental insertion or formation of air bubbles in the carrier stream. In addition, this method can overcome the major drawback of amperometric detection using solid electrodes, which is gradual electrode passivation. To illustrate the potential of this method, the flow-injection amperometric detection of uric acid using [Fe(CN)₆]^3? as an internal standard (IS) is presented as an example.     

Extension of the application of the thermal in-situ reaction on NH2 layers to the detection of catecholamines in biological materials

Summary This article describes a thin-layer chromatographic method for the determination of catecholamines in human urine. Chromatographic separation on NH₂-modified silica gel layers is followed by in-situ visualization brought about by heating the plate. The use of this derivatization technique for other urine components, such as creatine, creatinine, uric acid and glucose, is also discussed.