Development of a high-throughput method for preparing human urine for two-dimensional electrophoresis

There is an increasing interest in analysing the human urinary proteome in the search for biomarkers. However, despite the ease of its collection, urine is a difficult fluid to analyse by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) because of its dilute protein content and high salt levels. Here, we describe a method for high-throughput processing of urine for 2-D PAGE. Urine is filtered and applied to solid phase extraction columns. After washing, the urinary proteins are eluted and freeze dried. The lyophilised powder can then be resuspended in an appropriate buffer for downstream proteomic analysis.     

Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.     

Stereoselective determination of clenbuterol in human urine by capillary electrophoresis

The effectiveness of capillary electrophoresis (CE) in the field of stereoselective determination of drugs in biological matrices is demonstrated by analyzing clenbuterol in human urine. Due to the very low therapeutical doses of 20–40 μg per day the total concentrations in urine are 1–10 ng/ml. The sample was extracted with hexane–tert.-butyl methyl ether (99.5:0.5). The reconstituted sample was injected electrokinetically (50 s, 10 kV). Using phosphate buffer, pH 3.3 and hydroxyethyl-β-cyclodextrin as chiral selector the total analysis time was below 15 min. The limit of determination was estimated as 0.5 ng/ml per enantiomer. S-(−)-Bupranolol was used as internal standard. Both precision and accuracy of the method were within the limits for biological samples. The application to human urine from patients having received therapeutical doses showed a slightly predominant excretion of the (+)-enantiomer to the (−)-enantiomer.     

Development and validation of a capillary zone electrophoresis method for the determination of ephedrine and related compounds in urine without extraction

A capillary zone electrophoresis (CZE) method, with UV detection and in the presence of dimethyl-beta-CD, was optimized by means of an experimental design for the separation and the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine) and norpseudoephedrine (cathine) in urine without any extraction. In this application, the optimization of the analytical conditions with an experimental design was preferred to a univariate study. Therefore, a central composite design was used and the following factors were investigated and varied simultaneously: buffer concentration, buffer pH and dimethyl-beta-CD concentration. In order to evaluate the influence of each experimental parameter on the analytical separation, the resolutions between the four compounds, as well as the separation time and generated current were observed and established as responses of the experimental design. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. After acceptance of the mathematical models, the most favorable conditions were determined by maximizing the resolutions between the four compounds and by setting the other responses at threshold values. Successful results were obtained with a 260 mM Tris-phosphate buffer at pH 3.5 in the presence of 13.3 mM dimethyl-beta-CD at 25 degrees C and with an applied voltage of 30 kV. Under these optimized conditions, a baseline separation of the four compounds was achieved in less than 6 min. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of these compounds in urine samples without any extraction as well as in nutritional supplements.     

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection

In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.     

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.     

Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection

Four diuretic drugs banned in sport (amiloride, triamterene, bendroflumethiazide and bumetanide) have been separated by capillary zone electrophoresis (CZE) and detected using conventional fluorescence spectrometry. The effect of pH on electrophoretic parameters such as migration time, peak efficiency and peak height is discussed. Complete separation of the four drugs is achieved in less than 8 min at pH 8. No interference due to endogenous urine components is observed and thus direct urine analysis is feasible. Analytical figures of merit including precision and limits of detection are presented. Limits of detection range between 0.5 fmol for triamterene and 21.6 fmol for bumetanide.     

Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs

Pharmaceutical counterfeiting is becoming a serious problem in the world, especially in developing countries including China. Herein an isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for screening counterfeit medicines and adulterated dietary supplement products. The developed method could be employed to separate and determine simultaneously six anti-diabetic drugs (glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, repaglinide) on an isocratic solvent system using an Alltima C18 column (5 microm, 150 mmx4.6 mm) with an isocratic mobile phase of methanol-phosphate buffer (pH 3.0; 0.01 mol/L) (70:30, v/v), at a flow rate of 1.0 mL/min and at a wavelength of 230 nm. The proposed method was successfully applied to the analysis of medicinal and dietary supplement samples purchased from the local market in China.     

Affinity capillary electrophoresis for the screening of novel antimicrobial targets

The increasing number of multiantibiotic-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), requires the development of novel chemotherapies that are structurally distinct and exempt from current resistance mechanisms. Bioinformatics data mining of microbial genomes has revealed numerous previously unexploited essential open reading frames (ORFs) of unknown biochemical function. The potential of these proteins as screening targets is not readily apparent because most screening technologies rely on knowledge of biological function. To address this problem, the authors employed affinity capillary electrophoresis (ACE) to identify antimicrobial compounds that bound the novel target YihA. Screening a small-molecule library of 44,000 compounds initially identified 115 binders, of which 76% were confirmed. Furthermore, the ACE assay distinguished diverse compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates. These data validate ACE as a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity.     

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.     

Determination of opiates in urine by capillary electrophoresis

A method for the separation of a mixture of opiates comprising pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine and dihydrocodeine by capillary electrophoresis using a running buffer of 100 mM disodium hydrogenphosphate at pH 6 is described. The characteristics of an analytical method based on this separation for the determination of these drugs following extraction from urine and using levallorphan as internal standard are reported. Detection limits in the region of 10 ng cm⁻³ are achieved when using electrokinetic injection. A comparison is made of the sensitivity and reproducibility of electrokinetic and hydrodynamic injection for these drugs. Data are presented to show the results obtained when the proposed method is applied to urine spiked with all the above opiates and also to urine from a subject following consumption of dihydrocodeine and pholcodine. The concentrations found are compared with those obtained by LC.     

Application of single drop liquid-liquid-liquid microextraction for the determination of fluoroquinolones in human urine by capillary electrophoresis

A simple and novel method of single drop liquid–liquid–liquid microextraction (SD-LLLME) coupled with capillary electrophoresis (CE) for the determination of six fluoroquinolones (FQs) was developed. The method was eventually applied to extraction and preconcentration of FQs in human urine samples. Good linear relationships were obtained for all analytes in a range of 40–1000 μg L^?1 with the correlation coefficients from 0.9913 to 0.9995. The limit of detections (LODs) varied from 7.4 to 31.5 μg L^?1 at a signal-to-noise (S/N) of 3. The recoveries at two spiking levels were 81.8–104.9% with relative standard deviations <8.3%.     

Enantioselective determination of metoprolol and major metabolites in human urine by capillary electrophoresis

The enantiomeric separation of metoprolol and its metabolites in human urine was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using carboxymethyl-β-cyclodextrin (CM-β-CD) as the chiral selector. A 100-mM acetate buffer (pH 4.0) containing 5% 2-propanol and 10 mM CM-β-CD resulted in the optimum separation of the metoprolol enantiomers and its acidic metabolite in human urine. Following a single metoprolol oral administration of 100 mg racemic metoprolol tartrate, stereoselective pharmacokinetic analysis showed that urinary acidic metabolite 3 of metoprolol accounted for 62.3% of the dose with an R/S ratio of 1.23 and urinary unchanged metoprolol 1 accounted for 6.3% of the dose with an R/S ratio of 0.72.     

Quantification of pralidoxime methylsulfate (Contrathion) in human urine by capillary zone electrophoresis

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. For the first time, we developed a specific assay for urinary pralidoxime using capillary zone electrophoresis (CZE) in the following conditions: fused-silica capillary (length: 47 cm, internal diameter: 75 microm), electrolyte solution: 25 mM sodium borate (pH 9.1), voltage: 15 kV, temperature: 25 degrees C, injection time: 1 or 2s, on-line UV detection: 280 nm. Sample preparation did not require a deproteinization step (1:5 dilution in water). The method was linear between 0.125 and 2 mg mL-1 of pralidoxime (quantification limit: 0.10 mg mL-1). Coefficients of variation for intra- and inter-assay precision were below 10% for all three control levels (0.15-1.15 mg mL-1). This assay was successfully applied to urine specimens from organophosphate poisoned patients treated by Contrathion (n=10). This CZE method allows the measure of pralidoxime in urine within 15 min with excellent precision, selectivity, and sensitivity. It is simple (no pretreatment) and convenient, thus suitable for the monitoring of Contrathion therapy in organophosphate poisoned patients.     

尿样中三种蛋白质的毛细管电泳分离检测方法研究

目的:建立毛细管电泳分离测定人尿样中转铁蛋白、白蛋白和血红蛋白的新方法.方法:通过选择运行缓冲溶液种类及浓度、pH、表面活性荆种类及浓度、分离电压、进样时间对蛋白质分离效果的影响,优化了毛细管电泳法分离转铁蛋白、白蛋白和血红蛋白的条件.结果:利用此方法测定三种蛋白质的含量,浓度在0.01到1.00 g L-1范围内与峰电流呈良好的线性关系,检出限均为10-4 g L-1.结论:所建立的方法用于人尿样中转铁蛋白、白蛋白和血红蛋白的测定,结果满意.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM): A novel method for biomolecular screening

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a new separation-based affinity method. It has kinetic capabilities exceeding those of surface plasmon resonance (SPR) and does not require immobilization of molecules on the surface. Another distinctive feature of NECEEM is that--if it is combined with an advanced method for the mixing solutions inside a capillary, termed transverse diffusion of laminar flow profiles (TDLFP)--it requires only nanoliter volumes of solutions. The proven applications of NECEEM to biomolecular screening include 1) measuring kinetic and thermodynamic parameters of protein-ligand interactions, 2) quantitative affinity analyses of proteins and hybridization analyses of DNA and RNA, and 3) selection of binding ligands from combinatorial libraries. NECEEM is easy to automate and parallelize. Because of its simplicity and analytical power, NECEEM has the potential to become a workhorse in studies of biomolecular interactions. The author reviews theoretical bases of NECEEM and its applications to biomolecular screening.     

Clinical evaluation and use of urine screening for drug abuse.

Urine drug screening is indicated to evaluate patients who show mental status or behavioral changes and to monitor the abstinence of drug abusers. The appropriate timing for collecting urine specimens may vary depending on the suspected drug of abuse and on laboratory factors. Laboratories use a variety of techniques to do urine screens, and these must be understood by clinicians ordering the screens to interpret results correctly. In treating drug-abusing patients, clinicians must apply structured reinforcement in conjunction with urine screen results to aid patients in achieving abstinence.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Determination of three major catecholamines in human urine by capillary zone electrophoresis with chemiluminescence detection

A sensitive simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detection. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of 20.0mM sodium borate and 1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than 8 min. Detection limits of 7.9 × 10(-8)M, 1.0 × 10(-7)M, and 6.9 × 10(-8)M were observed for EP, NE, and DA, respectively. Relative standard deviation (RSD) values for the peak height were 4.7% to 5.4% (n = 5). Our proposed method was applied to the determinations of the catecholamines in urine samples from 12 healthy individuals and 26 pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.     

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-β-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2μg/ml. The calibration curves are linear from 0.4 to 16 μg/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.