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.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

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.     

高效液相色谱法准确测定血清中的尿酸

建立了准确测定血清中尿酸含量的高效液相色谱方法,血清样品利用乙腈沉淀蛋白,过滤后直接进样测定。所采用的色谱柱为Inertsil ODS-SP(4.6mm i.d.×250 mm,5μm),柱温25℃,流动相体系为10 mmol/L乙酸铵(pH 4.5),流速为1.0 mL/min,紫外检测波长为280 nm。线性范围12.5~150μg/g,回收率为99.79%~100.5%。本文采用乙酸铵缓冲体系,对环境的污染小,同时也为建立液相色谱-质谱法测定尿酸奠定了良好的基础。     

Simultaneous determination of pseudouridine and creatinine in untreated urine by ion-pair liquid chromatography with diode-array ultraviolet detection

A simple procedure for the simultaneous determination of pseudouridine and creatinine in urine using ion-pair high-performance liquid chromatography with ultraviolet detection is described. It consists of simply diluting the filtered urine with mobile phase (1:20) followed by direct chromatographic injection. A single analysis takes only 10 min. This method has been applied to the analysis of urine samples from normal donors and patients with different types of cancer. The mean values, means, of the peak-area ratio of pseudouridine to creatinine were 61.79.10(-3) and 81.92.10(-3) for male and female normal donors, respectively. Out of twenty-five urine samples of patients with cancer examined, nineteen (all the forteen males included) had values higher than means + 2 sigma.     

Quantitation of p-aminohippuric acid in biological fluids by high-performance liquid chromatography and dual-wavelength ultraviolet detection

A modification of the method of Verbeke [J. Chromatogr., 177 (1979) 69] is presented. The fatty tissue is dissolved in hexane, partitioned against methanol-sodium acetate buffer (pH 5.2) and extracted with dichloromethane. The crude extract is then purified on a disposable C18 column. The final extract together with a set of reference compounds is spotted on two opposite sides of a high-performance thin-layer chromatographic plate (10 X 10 cm), which is developed with two different eluents in two opposite directions. This mode of operation has the advantage that more reference compounds can be spotted and that the identification is based on two independent chromatographic runs. Also the total analysis time is decreased.     

Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been employed for the separation and determination of homocysteine, cysteine, reduced glutathione, ascorbic acid and uric acid. Effects of several important factors such as the acidity and concentration of the running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 500 microm diameter platinum disk electrode at a working potential of +1.05 V (vs saturated calomel electrode). The five analytes were well separated within 10 min in a 50 cm long fused silica capillary at a separation voltage of 18 kV in a 100 mm phosphate buffer (pH 7.8). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) ranging from 0.83 to 2.58 microm. The proposed method was successfully applied to determine cysteine, reduced glutathione, ascorbic acid and uric acid in human whole blood and rat brain tissues with satisfactory assay results and should find a wide range of bioanalytical applications.     

Simultaneous determination of azelaic and benzoic acids in topical preparations by liquid chromatography

Summary An isocratic, reversed-phase liquid chromatographic (LC) method has been developed for the simultaneous determination of azelaic and benzoic acids in pharmaceutical creams. The compounds were separated on a C₁₈ column (4 μm particles); the mobile phase was methanolwater, 40∶60, containing 10mm ammonium acetate and with the pH adjusted to 5.0. Detection was performed at 220 nm. The method was validated for accuracy, linearity, precision, and selectivity. Recoveries at levels corresponding to 80% to 120% of the declared content of the creams ranged from 99.5 to 101.8% and from 100.4 to 102.1% for azelaic and benzoic acids, respectively. The calibration graphs were linear in the ranges 20–1400 μg mL⁻¹ for azelaic acid (correlation coefficient,r ₁>0.99999), and 0.1–7.0 μg mL⁻¹ for benzoic acid (r>0.99998).     

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

Full automation of serotonin determination by column-switching and HPLC

Summary A simple, fast, fully automated method for plasma serotonin determination is described. Full automation is obtained by coupling two devices: a sample processing station and a solid-phase autosampler. The sample processing station dilutes the plasma sample and is then connected, on-stream, with the solid-phase autosampler. It firstly fills a loop with all the solvents necessary for the sample clean-up, then, inverting the flow, pumps these solvents through the silica-bonded cation-exchange disposable extraction cartridge positioned on the autosampler. For the elution, the cartridge is switched on-stream with the HPLC analytical column and serotonin is eluted by the HPLC mobile-phase. The HPLC separation is performed by ion-pairing reversed-phase liquid chromatography. The column effluent is completely reduced by an electrochemical reactor and serotonin is detected in an oxidation-mode by a dual-cell electrochemical detector. The plasma sample is 50 l, the plasma sensitivity is 40 ng/l, the retention time is 6 min and the recovery is 95%. The repeatibility, the normal ranges for platelet-poor and for platelet-rich plasma have been established and correlation with manual HPLC calculated.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Simultaneous determination of ascorbic acid,dopamine and uric acid based on tryptophan functionalized graphene

A new type of tryptophan-functionalized graphene nanocomposite (Trp-GR) was synthesized by utilizing a facile ultrasonic method via π–π conjugate action between graphene (GR) and tryptophan (Trp) molecule. The material as prepared had well dispersivity in water and better conductivity than pure GR. The surface morphology of Trp-GR was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. The electrochemical behaviors of ascorbic acid (AA), dopamine (DA), and uric acid (UA) were investigated by cyclic voltammetry (CV) on the surface of Trp-GR. The separation of the oxidation peak potentials for AA–DA, DA–UA and UA–AA was about 182 mV, 125 mV and 307 mV, which allowed simultaneously determining AA, DA, and UA. Differential pulse voltammetery (DPV) was used for the determination of AA, DA, and UA in their mixture. Under optimum conditions, the linear response ranges for the determination of AA, DA, and UA were 0.2–12.9 mM, 0.5–110 μM, and 10–1000 μM, with the detection limits (S/N = 3) of 10.09 μM, 0.29 μM and 1.24 μM, respectively. Furthermore, the modified electrode was investigated for real sample analysis.     

Simultaneous determination of tiopronin and d-penicillamine in human urine by liquid chromatography with ultraviolet detection

d-Penicillamine and tiopronin are drugs widely used for the treatment of many diseases. Because of the relatively high frequency of side effects to these compounds, some of which are dose-related, drug monitoring in urine samples during treatment is advisable. In this paper, we describe a simple method for the determination of tiopronin and d-penicillamine in human urine. The method was based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection. 2-S-quinolinium derivatives of thiols were detected at 355 nm. The derivatization was optimized in terms of pH and time of the reaction. Baseline separation was achieved on an analytical Zorbax SB C-18 (5 μm, 150 mm × 4.6 mm) column with a mobile phase consisting of pH 2.0 0.09 mol L⁻¹ trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.0 mL min⁻¹. Gradient elution was used: 0-4 min, 12% B; 4-8 min, 12-40% B; 8-12 min, 40-12% B. The d-penicillamine and tiopronin standards added to the urine show that the response of the detector is linear within the range studied, from 1 to 200 μmol L⁻¹ urine. The imprecision ranges for tiopronin and d-penicillamine were within 1.61-8.24% and 2.92-10.60%, respectively. The analytical accuracy for determined compounds was from 97.24 to 109.39%. The lower limits of detection and quantitation were 0.5 μmol L⁻¹ and 1.0 μmol L⁻¹ urine, respectively. This method can be used for routine clinical monitoring of the title thiol-drugs. Cysteine can be measured concurrently, if needed.     

Simultaneous determination of twelve water- and fat-soluble vitamins by high-performance liquid chromatography with diode array detection

Summary A novel method for the simultaneous determination of twelve water- and fat-soluble vitamins has been established by high-performance liquid chromatography with diode array detection. The vitamins were analyzed on a μBondapak C₁₈ column (300 × 3.9 mm, 10 μm) with methanol-KH₂PO₄ buffer (0.1 M, pH 7.0)-water as mobile phase in a gradient. The linearity of calibration graphs was compound-dependent and the detection limits ranged from 0.02 μg mL⁻¹ to 0.5 μg mL⁻¹. The method was successfully applied to determine vitamins in pharmaceutical preparations. The recoveries were from 95.1% to 103% and the relative standard deviations were in the range of 0.9% to 4.5%.     

Determination of uric acid in human urine by ion chromatography with conductivity detector

A simple,fast,precise and eco-friendly analytical method for the determination of uric acid(UA) in human urine by ion chromatography(IC) was established.The sample pretreatment was not required,only needed centrifugation and filtration.The separation was carried out on a cation exchange column with 2.0 mmol/L nitric acid as mobile phase at the flow-rate 1.0 mL/min.A non-suppressed conductivity detector was used.The IC analysis time for one run was within 10 min under the optimized IC condition.The detection limits were 0.5μg/L(S/N = 3) for uric acid.The recovery was 100.1%while the relative standard deviation (RSD) was 1.8%from 10 measurements.     

Simultaneous detection of ascorbate and uric acid using a selectively catalytic surface

The direct and selective detection of ascorbate at conventional carbon or metal electrodes is difficult due to its large overpotential and fouling by oxidation products. Electrode modification by electrochemical reduction of diazonium salts of different aryl derivatives is useful for catalytic, analytical and biotechnological applications. A monolayer of o-aminophenol (o-AP) was grafted on a glassy carbon electrode (GCE) via the electrochemical reduction of its in situ prepared diazonium salts in aqueous solution. The o-aminophenol confined surface was characterized by cyclic voltammetry. The grafted film demonstrated an excellent electrocatalytic activity towards the oxidation of ascorbate in phosphate buffer of pH 7.0 shifting the overpotential from +462 to +263 mV versus Ag/AgCl. Cyclic voltammetry and d.c. amperometric measurements were carried out for the quantitative determination of ascorbate and uric acid. The catalytic oxidation peak current was linearly dependent on the ascorbate concentration and a linear calibration curve was obtained using d.c. amperometry in the range of 2-20 μM of ascorbate with a correlation coefficient 0.9998, and limit of detection 0.3 μM. The effect of H₂O₂ on the electrocatalytic oxidation of ascorbate at o-aminophenol modified GC electrode has been studied, the half-life time and rate constant was estimated as 270 s, and 2.57 × 10⁻³ s⁻¹, respectively. The catalytically selective electrode was applied to the simultaneous detection of ascorbate and uric acid, and used for their determination in real urine samples. This o-AP/GCE showed high stability with time, and was used as a simple and precise amperometric sensor for the selective determination of ascorbate.     

Simultaneous determination of ascorbic acid, uric acid and neurotransmitters with a carbon ceramic electrode prepared by sol-gel technique

A sol-gel carbon composite electrode (CCE) has been prepared by mixing a sol-gel precursor (e.g. methyltrimethoxysilane) and carbon powder without adding any electron transfer mediator or specific reagents. It was demonstrated that this sensor can be used for simultaneous determination ascorbic acid, neurotransmitters (dopamine and adrenaline) and uric acid. Direct electrochemical oxidation of ascorbic acid, uric acid and catecholamines at a carbon composite electrode was investigated. The experimental results were compared with other common carbon based electrodes, specifically, boron doped diamond, glassy carbon, graphite and carbon paste electrodes. It was found that the CCE shows a significantly higher of reversibility for dopamine. In addition, in comparison to the other electrodes used, for CCE the oxidation peaks of uric acid, ascorbic acid and catecholamines in cyclic and square wave voltammetry were well resolved at the low positive potential with good sensitivity. The advantages of this sensor were high sensitivity, inherent stability and simplicity and ability for simultaneous determination of uric acid, catecholamines and ascorbic acid without using any chromatography or separation systems. The analytical performance of this sensor has been evaluated for detection of biological molecules in urine and serum as real samples.     

Simultaneous determination of ursodeoxycholic acid and its glycine-conjugate in serum as phenacyl esters using multidimensional liquid chromatography

Summary A narrowbore high-performance liquid chromatographic (HPLC) method using column switching is described for the simultaneous determination of ursodeoxycholic acid (UDCA) and glyco-UDCA (GUDCA) from serum samples as their phenacyl esters. Serum samples were subjected to a preliminary clean-up using octadecylsilane reversed-phase extraction and derivatized with phenacylbromide. The purification, fractionation and concentration of UDCA and GUDCA from the esterified serum sample were performed on-line by appropriate switching of columns. Limit of detection (LOD) of UDCA and GUDCA were 5 ng and the absolute mean recoveries averaged 84.4±8.2% and 85.2±8.4%, respectively. This method was successfully applied to the pharmacokinetic study of UDCA in rats and human.