Determination of 8-oxoguanine and 8-hydroxy-2'-deoxyguanosine in the rat cerebral cortex using microdialysis sampling and capillary electrophoresis with electrochemical detection

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values 相似文献   

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomnnose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B withA. saitoi (1–2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.     

Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis

We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na⁺ and Mg²⁺) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.     

Determination of alkaloids in Sinomenium acutum by field‐amplified sample stacking in capillary electrophoresis with chemiluminescene detection

A simple and rapid capillary electrophoresis (CE) with an acidic potassium permanganate chemiluminescence (CL) detection method was developed to determine three alkaloids (curine, sinomenine and magnoflorine) simultaneously. A laboratory‐built CE–CL detection interface was used. The field‐amplified sample stacking technique was applied to the online concentration of alkaloids. Experimental conditions for CE separation and CL detection were investigated in detail to acquire optimum conditions. Under optimal conditions, the three alkaloids were baseline separated within 6 min, and the detection limits (S/N = 3) ranged from 0.03 µg/mL to 0.49 µg/mL. This method was successfully applied to determine the above three alkaloids in Sinomenium acutum, and the result of the determination of sinomenine was in good agreement with those given by high‐performance liquid chromatography and CE methods. In addition, a possible CL reaction mechanism of sinomenine–KMnO₄–H₂SO₄ was proposed. Copyright © 2013 John Wiley & Sons, Ltd.     

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT_8–13), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise = 3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT_8–13, NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT_8–13 in human cerebrospinal fluid sample.     

The method of single-nucleotide variations detection using capillary electrophoresis and molecular beacons

We demonstrate that single-nucleotide variations in a DNA sequence can be detected using capillary electrophoresis (CE) and molecular beacons (MBs). In this method, the region surrounding the site of a nucleotide variation was amplified in a polymerase chain reaction, then hybridize PCR products with each of MBs. The sequences of the PCR products are different at the site of 2,044 in exon of interleukin (IL)-13 which to be identified. Through denaturation, the PCR product became single strand and hybridized with the completely complementary MB. The MB-target duplexes were separated using CE and solution-based fluorescence techniques. The results show that in each reaction a fluorescent response was elicited from the molecular beacon which was perfectly complementary to the amplified DNA, but not from the other MB whose probe sequence mismatched the target sequence. The method of CE based on MBs is able to identify single-nucleotide variations in a DNA sequence and can discriminate the genotyping of the SNP between the homo- and heteroduplexes of DNA fragments.     

On-line drug-metabolism system using microsomes encapsulated in a capillary by the sol-gel method and integrated into capillary electrophoresis

A novel microsome-encapsulation technique using the sol-gel method was developed for the on-line drug-metabolism analytical system integrated into capillary electrophoresis. This analytical system allows both the metabolism of drugs and the determination of the metabolites in a single capillary simultaneously. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane-based silica matrices within a capillary in a single step under mild conditions. The availability of this system was evaluated using UDP-glucuronyltransferase, which is one of the most important microsomal enzymes. 4-Nitrophenol and testosterone, which were metabolized by the different isoforms of UDP-glucuronyltransferase, were used as substrates. The resultant monolithic reactor showed enzymatic activity at the same level as that of the soluble form. The following separation of the unreacted substrates and metabolites in the same capillary also showed high selectivity. Furthermore, the sample amount required for one analysis decreased more than 3 orders of magnitude from conventional reaction schemes in free solution. This on-line system could largely simplify the laborious procedures which were needed in conventional analytical schemes.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.     

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection

DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process, we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products.     

Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis

The aim of this study was to validate, by capillary electrophoresis, the use of synthesized methyl malondialdehyde as the internal standard for the direct quantification of free and total (free+bound) malondialdehyde in biological samples. All analyses were performed in 20 cm x 50 microm uncoated capillaries at 20 degrees C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was -4kV (about 8 microA), the detector being set at 260 nm for a total run time of 8 min per sample. Free malondialdehyde was evaluated after acetonitrile extraction, while the samples evaluated for total malondialdehyde were, before extraction, hydrolyzed for 1h at 60 degrees C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 micromol/L in microsomes and 0.4 micromol/L in plasma. As an application of the method, three pools of rat liver microsomes were quantified before (0.35+/-0.1 and 1.1+/-0.5 nmol/mg protein, free and total malondialdehyde, respectively, mean+/-SD) and after lipoperoxidation induction using systems able to generate oxygen free radicals (18.4+/-3.2 and 19.7+/-2.0 nmol/mg protein). The results were confirmed by isotopic dilution gas chromatography-mass spectrometry, used as the reference method. The feasibility of capillary electrophoresis for malondialdehyde determination in normal and pathological human plasma was also investigated.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

Optimization by factorial design of a capillary zone electrophoresis method for the simultaneous separation of antihistamines

A 3(2) full factorial design was used to optimize the experimental conditions of a capillary zone electrophoresis method aimed at achieving simultaneous separation and quantification of the antihistamines brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine according to their therapeutic group. A statistical program, SPSS, was used to calculate the mathematical model with which to obtain the response surface. Critical parameters such as pH and applied voltage were studied to evaluate their effect on resolution and on efficiency. Optimum separation conditions were phosphate buffer pH 2.0, 5kV, and 2psis(-1) at 214nm. The analysis time was below 9min and the theoretical plates were between 6000 and 63,000N. Calibration curves were prepared for the antihistamines. The limits of detection were 4-14ngmL(-1), which allow their quantification in pharmaceuticals. The RSD% of each antihistamine was fairly good. Up to seven antihistamines belonging to the antihistaminic H(1)-receptor group were separated in the same electropherogram. The proposed method was then applied to the determination of antihistamines in pharmaceutical, urine, and serum samples with recoveries in agreement with the stated contents.     

Quantification of penicillin G during labor and delivery by capillary electrophoresis

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3 cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.