高效毛细管区带电泳测定人尿液中尿酸、肌酐和伪尿核苷的含量

报道了采用高效毛细管区带电泳技术直接将人尿液注入毛细管进行尿液中肌酐、尿酸及伪尿核苷含量测定的新方法。试验表明,以磷酸盐（ｐＨ６．１）作缓冲液,对人体尿液中肌酐、尿酸及伪尿核苷进行直接分析具有较高的灵敏度和较好的重复性。     

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.     

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.

     

Direct detection of renal function markers using microchip CE with pulsed electrochemical detection

Creatinine, creatine, and uric acid are three important compounds that are measured in a variety of clinical assays, most notably for renal function. Traditional clinical assays for these compounds have focused on the use of enzymes or chemical reactions. Electrophoretic microchips have the potential to integrate separation power of capillary electrophoresis with devices that are small, portable, and have the speed of conventional sensors. The development of a microchip CE system for the direct detection of creatinine, creatine, and uric acid is presented. The device uses pulsed amperometric detection (PAD) to detect the nitrogen-containing compounds as well as the easily oxidizable uric acid. Baseline separation of creatinine, creatine and uric acid was achieved using 30 mM borate buffer (pH = 9.4) in less than 200 s. Linear calibration curves were obtained with limits of detection of 80 microM, 250 microM and 270 microM for creatinine, creatine and uric acid respectively. An optimization of the separation conditions and a comparison of PAD with other amperometric detection modes is also shown. Finally, analysis of a real urine sample is presented with validation of creatinine concentrations using a clinical assay kit based on the Jaffé reaction.     

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.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

Determination of uric acid and creatinine in human urine using hydrophilic interaction chromatography

Uric acid is the end-product of purine metabolism and a major antioxidant in humans. The concentrations of uric acid in plasma and urine are associated with various diseases and routinely measured in clinical and biomedical laboratories using enzymatic conversion and colorimetric measurement. In this study a hydrophilic interaction chromatographic (HILIC) method was developed for simultaneous determination of uric acid and creatinine, a biomarker of urine dilution and renal function, in human urine. Urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration. Uric acid and creatinine were separated from other components in urine samples and quantified using HILIC chromatography. A linear relationship between the ratio of the peak area of the standards to that of the internal standard and the concentration of the standards was obtained for both uric acid and creatinine with the square of correlation coefficients >0.999 for both analytes. The detection limits were 0.04 μg/mL for creatinine and 0.06 μg/mL for uric acid. The described HILIC method has proved to be simple, accurate, robust and reliable.     

神经递质类相关物质的毛细管电泳分离及实际尿液的检测应用

建立了单胺类神经递质（5-羟色胺、多巴胺和肾上腺素）、神经递质类代谢产物（高香草酸、5-羟吲哚乙酸和香草扁桃酸）及神经递质类前体（精氨酸和酪氨酸）混合物的毛细管电泳（CE）分离方法. 利用标准试剂混合样考察了缓冲体系的组成、pH值及添加剂对分离的影响,并探讨了尿液中基体成分如肌酸酐、尿酸和乙酰乙酸对分离的干扰. 在Na₂B₄O₇-NaOH缓冲体系（pH=9.90）及紫外（UV）检测（波长200 nm）条件下对8种神经递质类相关物质的分析获得了良好的定量线性关系,检出限（LOD）为0.04~0.60 μmol/L,迁移时间和峰面积的相对标准偏差（RSD,n=5）范围分别为0.09% ~0.48%和0.47% ~3.34%. 利用该方法对实际尿液中的精氨酸和香草扁桃酸进行了定性和定量分析,其定量结果分别为（95.8±3.8）和（44.6±3.5） μmol/L,加标回收率为96.65%~104.5%.     

Determination of unconjugated aromatic acids in urine by capillary electrophoresis with dual electrochemical detection – Potential application in fast diagnosis of phenylketonuria

A novel method of CE coupled with dual electrochemical detection has been developed for the determination of pathological metabolites of phenylalanine in urine samples. Factors influencing the separation and detection were examined and optimized. Five aromatic acid metabolites and a major coexisting interfering compound uric acid could be well separated within 23 min at a separation voltage of 16 kV using a 35 mmol/L SDS/60 mmol/L H₃BO₃‐Na₂B₄O₇ running buffer (pH 8.2). Highly linear response was obtained for these five biomarker compounds over three orders of magnitude with detection limits ranging from 6.6 to 0.064 μg/mL (S/N=3). The average recovery and RSD were within the range of 92.6–121.0 and 1.0–12.0%, respectively. The proposed method has been used to detect the unconjugated aromatic acids simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost, thus could find potential applications involving assays of biomarker compounds for the purpose of fast diagnose of some metabolic diseases including phenylketonuria.     

基于纳米结构BDD电极的人体尿酸检测

利用纳米结构硼掺杂金刚石(nBDD)电极的优点对人体尿液中的UA含量进行检测,并与常规玻碳(GC)电极做了比较。检测不同尿样的结果可知,nBDD电极检测的回收率为95.3%～98.4%,GC电极79.6%～87.1%;检测同一尿样的重复性实验得出,nBDD电极上峰电流的相对标准偏差(RSD)为8.0%,GC电极上峰电流的RSD为39%。     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

Determination of creatinine and purine derivatives in ruminants' urine by reversed-phase high-performance liquid chromatography.

A procedure is described for the rapid and simultaneous determination of allantoin, creatinine, uric acid, hypoxanthine and xanthine in sheep urine. Separation was achieved on a Novapak C18 column under isocratic conditions. The mobile phase was potassium phosphate buffer (10 mM, pH 4.0). A flow-rate of 0.5 ml/min, detection at 218 nm and a column temperature of 25 degrees C were employed with a total analysis time of less than 15 min. Detection limits for allantoin, creatinine, uric acid, hypoxanthine and xanthine were 1.0, 0.5, 0.5, 0.5 and 0.2 micrograms/ml, respectively, at a signal-to-noise ratio of 3 in a 20-microliters injection volume of tenfold-diluted urine. This sensitivity permits the precise determination of these compounds in ruminants' urine.     

Separation and simultaneous determination of uric acid and ascorbic acid on a dynamically modified poly(dimethylsiloxane) microchip.

Poly(dimethylsiloxane) microfluidic channels alternately modified by poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) were successfully used to separate uric acid and ascorbic acid. Results show that uric acid and ascorbic acid can be well separated and detected simultaneously in modified microchips coupled with in-channel electrochemical detection. Under the optimal conditions, the linear ranges of uric acid and ascorbic acid were both from 25 to 600 microM, with the correlation coefficients of 0.997 and 0.996, respectively. The detection limits were 8 microM for uric acid and 5 microM for ascorbic acid. Factors influencing separation and detection, including buffer solution, detection potential and separation voltage, were investigated and optimized. In addition, the dependences of the current response on sensitivity and reproducibility were studied, and the stability of the device was also evaluated in detail. This method was successfully used to determine uric acid and ascorbic acid in human urine.     

Simultaneous determination of creatinine and uric acid in human plasma and urine by micellar electrokinetic chromatography

High performance capillary electrophoresis using a buffer solution containing micelles of ionic surfactant (e.g. sodium dodecyl sulfate), called micellar electrokinetic chromatography, has been applied to the separation and simultaneous determination of creatinine and uric acid in human plasma and urine. The sample was introduced into the capillary by siphoning an appropriate volume of untreated plasma or urine spiked with an internal standard (antipyrine). Creatinine, uric acid, and antipyrine were separated mutually, and from other endogeneous components within 18 min. The calibration plots showed good linearity (correlation coefficient > 0.999) over the concentration range needed for clinical analysis. Standard addition tests indicated that the recoveries of creatinine and uric acid from urine samples ranged, respectively, from 97 % to 106 % and 97.4 % to 108 % with a coefficient of variation (C.V.) of 3.3 % (n = 5), and that those from plasma samples ranged, respectively, from 100 % to 112 % and 101 % to 107 % with a C.V. of 4.7 % (n = 5). The results were in agreement with those obtained by conventional methods.     

A Novel Method for Uric Acid Determination Using CdS Quantum Dots as Fluorescence Probes

A novel method has been developed for uric acid analysis based on the quenching of fluorescence emission from CdS quantum dots by uric acid. Also, the effect of the presence of different surfactant agents, in order to improve the fluorescent signals of the CdS QDs, has been investigated, and the cetyltrimethyl ammonium bromide (CTAB) was selected. Under optimum conditions, the calibration graph was linear over the range of 0.1 ng/mL to 12.0 ng/mL (r = 0.9950). The limit of detection (S/N = 3) was 0.1 ng/mL. The RSD for ten determinations of 5.0 ng/mL uric acid was 3.5%. The method was applied to determine uric acid in human serum and urine sample with satisfactory results.     

On-line automatic SPE-CE coupling for the determination of biological markers in urine

Automatic SPE has been coupled on-line to CE by a transfer tube and the replenishment system of the CE instrument. The approach allows the target analytes (viz. creatinine, creatine, xanthine, hypoxanthine, uric acid, p-aminohippuric acid and ascorbic acid in urine samples) to be removed from the sample matrix, cleaned up, preconcentrated and injected into the capillary. The detection limits range between 0.14 and 4.50 microg/mL, the quantification limits between 0.45 and 15.0 microg/mL, and linear dynamic ranges - which include the reference healthy human values - from the quantification limits to 1332 microg/mL. The precision, expressed as RSD, ranges between 0.38 and 2.22% for repeatability and between 1.79 and 7.61% for within-laboratory reproducibility. The errors, expressed as RSD for all compounds, range between 0.20 and 6.90%. The time for automatic SPE and that necessary for the individual separation-detection of the target analytes are 13 and 12 min, respectively; the analysis frequency is 5 h(-1). The accuracy of the method and potential matrix effects were studied by using spiked samples and recoveries between 96.00 and 103.07 % were obtained. The proposed method was applied to samples from healthy young students.     

Spectrofluorimetric determination of uric acid based on its activation of catalytic oxidation of pyronine Y

A novel kinetic spectrofluorimetric method for the determination of uric acid based on the activation effect of uric acid on the Cu(II) ion catalyzed oxidation of pyronine Y by hydrogen peroxide was developed. The influence of different buffer solutions was tested and the Britton-Robinson buffer solution with pH 2.2 was found to be the optimum. The detection limit and the linear range for uric acid are 0.09 μg mL⁻¹ and 0.3–3.0 μg mL⁻¹, respectively. The RSD for eleven determinations of 1.6 μg mL⁻¹ uric acid was 1.6 %. Satisfactory results were obtained when using this method of uric acid determination in human urine.     

Microchip capillary electrophoresis with electrochemical detector for precolumn enzymatic analysis of glucose, creatinine, uric acid and ascorbic acid in urine and serum

An integrated multiple-enzymatic assay was performed on a (microchip capillary electrophoresis) μCE-EC chip capable of precise intake of sample or reagents in nanoliters. Incorporating multiple-enzyme assay into the μCE chip is relatively new—rendering simultaneous analysis of creatinine and uric acid a snap.Added to the list of merits in this study are the enhanced sensitivity down to 1 μM and a broader spectrum of analytes—inclusive of glucose for the long-time sufferers of diabetes. The performance was orchestrated to attain the claimed level: employing the end-channel electrode mode to tame the noises and the precolumn enzymatic reaction to stabilize the baseline. The 10 μm embedded Pt electrode, deposited at the end of the 30 μm wide separation channel, benefited chip fabrication besides noise reduction. The optimized conditions were 20 mM phosphate buffer (pH 7.5), +1.5 kV separation voltage and +1.0 V detection potential (versus Ag/AgCl). The migration time was repeatable within the deviation of 0.5% R.S.D. (n=7), but the peak currents ranged from 1.5 to 2.2% R.S.D. The detection limits (S/N=3) ranged from 0.71 μM for ascorbic acid to 10 μM for glucose. The calibration curve was linear from 10 to 800 μM (R²>0.995). Glucose, creatinine, uric acid and ascorbic acid as model analytes, in pure form or in serum and urine samples, were tested to verify its feasibility.     

UPLC-MS/MS法测定高尿酸血症大鼠血清及尿液中的尿酸和肌酐

建立了一种测定血清和尿液中尿酸和肌酐含量的超高效液相色谱-串联质谱联用（UPLC-MS/MS）方法. 样品经自动进样器引入液相色谱通过预柱富集和去除杂质后,直接引入质谱中进行分析. 采用正离子电喷雾电离模式下的多反应监控模式对肌酐进行定量分析,纯溶剂标准曲线的肌酐线性范围为0.03~400 μmol/L;基质标准曲线的肌酐线性范围为0.2~400 μmol/L;它们的最低定量限分别为0.03和0.2 μmol/L. 采用负离子电喷雾电离模式下的多反应监控模式对尿酸进行定量分析,纯溶剂标准曲线的尿酸线性范围为0.1~350 μmol/L;基质标准曲线的尿酸线性范围为0.5~300 μmol/L;它们的最低定量限分别为0.1和0.5 μmol/L. 该方法的提取回收率在91.8%~103.7%之间,日内和日间RSD分别小于5.9%和6.8%,满足生物分析的要求. 利用该方法进行抗痛风中药筛选及其作用机制研究,结果表明,与阳性对照药物别嘌呤醇和苯溴马隆相比,中药二妙丸、黄柏和苍术均能在一定程度上降低血尿酸水平,并可显著逆转肾功能损伤,对肾功能具有一定的保护作用.     

流动注射化学发光法测定尿酸

在碱性条件下,铁氰化钾氧化鲁米诺,产生化学发光,尿酸对该体系的化学发光有显著的增强作用（亚铁氰化钾存在时）。基于此,建立了一种直接测定尿酸的流动注射化学发光分析法。方法的线性范围为２．０×１０－８～５．０×１０－６　ｇ／ｍＬ;检测限（３σ）为６．７×１０－９　ｇ／ｍＬ;相对标准偏差为１．１％（尿酸１．０×１０－７　ｇ／ｍＬ,ｎ＝１１）。用于血清及尿样中尿酸的分析,结果令人满意。