Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM     

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.     

Steroid analysis in single cells by capillary electrophoresis with collinear laser-induced fluorescence detection.

Capillary electrophoresis with collinear laser-induced fluorescence detection was used for the analysis of steroids in single R2C cells. Progesterone secretion was monitored from cultured cells and subsequently detected in single cells. Mass detection limit of 10(-18) mol for dansylated steroids was achieved with the 325-nm line of a helium-cadmium laser. Dansylhydrazine proved to be an effective fluorescent tag for derivatization of steroids outside and inside the biological cell. Fluorescence microscopy indicates that a dimethyl sulfoxide-containing physiological buffer was sufficient to incorporate the tag inside the cell for subsequent steroid derivatization.     

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of

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-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate–methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (λ_ex 325 nm, λ_em 435 nm), a limit of detection of 0.1–0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4–0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86–110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.     

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.     

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.     

Determination of glycosaminoglycan monosaccharides by capillary electrophoresis using laser-induced fluorescence detection

A newly developed capillary electrophoretic method using laser-induced fluorescence detection (CE-LIF) for the analysis of monosaccharides released from acid hydrolysis of glycosaminoglycans was studied. The method was compared with a previously published method using indirect LIF detection (CE-ILIF). For the CE-LIF method, electrophoretic conditions for the separation of the monosaccharides derivatised with 8-aminopyrene-1,3,6-trisulfonate (APTS) were optimised. The best separations were obtained using 100 mM acetate at pH 4.5 as running buffer. The influence of the injection vial volume on the precision and stability of the sample in different conditions was studied. The detection limits of the CE-LIF method were found to be 0.4-0.6 nM, while those obtained by CE-ILIF ranged from 11.4 to 14.3 microM. Other quality parameters of the method, such as run-to-run precision, day-to-day precision, and linearity were also determined. Finally, the new method was applied to the analysis of the acid hydrolysis products from a glucosaminoglycan (heparin) and a galactosaminoglycan (dermatan sulfate) and cross-contamination between the two solutions was determined. The high sensitivity of the new method allows the determination of dermatan sulfate contaminations in a heparin raw sample down to 0.04% (w/w) and broadens the practical applicability of CE-LIF for the quantitation of the endogenous levels of glycosaminoglycans in animal samples and for pharmacokinetic control after therapeutical heparin administration.     

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides that relies on capillary electrophoresis (CE) with laser-induced fluorescence detection is presented. This method enables complete separation of 17 GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone to improve sensitivity. The limit of detection was at the attomole level and approximately 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The relative standard deviations of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7 and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate resulting from the separate analyses of a single sample.     

Determination of duloxetine in human plasma by capillary electrophoresis with laser-induced fluorescence detection

A method based on capillary electrophoresis has been developed for the analysis of the novel antidepressant drug duloxetine in human plasma. The method makes use of laser-induced fluorescence detection after derivatisation of the analyte with 5-(4,6-dichlorotriazinyl)aminofluorescein at pH 11. A single step liquid/liquid extraction procedure with a mixture of hexane/2-propanol allows the sample clean-up with extraction yields always ≥84% and interference removal. The electrophoretic separation is achieved using uncoated fused silica capillaries (60.0 cm effective length, 75.0 cm total length, 50 μm internal diameter) and a background electrolyte composed of borate buffer (40 mM, pH 10.3), tetrabutylammonium bromide (10 mM), and acetone (10%, v/v). The applied voltage is 20 kV; the samples are injected by pressure (50 mbar × 8 s). The method has been fully validated in terms of linearity range (2.5–150 ng mL^?1), LOD and LOQ (1.0 and 2.5 ng mL^?1, respectively), precision (R.S.D. < 6.7%) and accuracy (recovery >78%). Application to samples obtained from patients under treatment with duloxetine gave good results. The method represents the first application of capillary electrophoresis to the analysis of duloxetine in human plasma.     

Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent label molecule. To minimize the amount of additional sample handling and time associated with such labeling procedures, not to mention the sometimes-stringent pH and temperature controls they require, noncovalent labeling is presented as a viable alternative. This review article considers established methods for noncovalent labeling of proteins for their subsequent analysis by CE-LIF. Label molecules suitable for excitation and emission in the ultraviolet, visible, and near-infrared regions of the spectrum are enumerated for a variety of protein analytes.     

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.     

A novel clean-up method for urine analysis of low-molecular mass aldehydes by capillary electrophoresis with laser-induced fluorescence detection

A rapid clean-up method using ultra-filtration was developed to remove sample matrix in the determination of low-molecular mass aldehydes in human urine. The ensuing filtrate was derivatized with fluorescein 5-thiosemicarbazide and the labelled aldehydes determined by capillary zone electrophoresis with laser-induced fluorescence detection. Practical aspects related to the effect of the urine sample matrix on the label chemistry and the electrophoretic separation showed that the urine samples must be diluted 20-fold after their ultra-filtration. By using synthetic urine, linear ranges were established in the range of 15-5000 μg/l with limits of detection between 4.5 and 9 μg/l. The intra- and inter-assay precision (relative standard deviation, %) of the aldehydes ranged from 4.1% to 8.4% and 6.1%-9.6%, respectively, and the average specific uncertainty was 149±12 ng. The average recoveries performed on two levels by enriching synthetic urine samples ranged between 94% and 100%. Finally, the proposed method was applied to check low-molecular mass aldehydes in the human urine of a female volunteer to obtain information about the risk in her exposure to these chemicals in the workplace.     

Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection

A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP's or N4-substituted dCTP's were determined. The method has proven to be very useful in obtaining quantitative analytical data from the small quantities of complex molecules produced in nick translations. Various polymerization conditions using DNA polymerase I were examined to determine optimal labeling density. Simultaneous copolymerization of green fluorescing dCTP and dUTP nucleotides were undertaken in an attempt to maximize labeling density.     

Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.     

Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced fluorescence detection

A capillary electrophoresis method for the simultaneous separation and enantioseparation of the antibacterial drug ofloxacin and its metabolites desmethyl ofloxacin and ofloxacin N-oxide in human urine has been developed and validated. Enantioseparation was achieved by adding sulfobutyl β-cyclodextrin to the running buffer. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd-laser with an excitation wavelength of 325 nm. In comparison with conventional UV detection, LIF detection provides higher sensitivity and selectivity. The separation can be performed after direct injection of urine into the capillary without any sample preparation, because no matrix compounds interfere with the assay. Additionally, the high sensitivity of this method allows the quantification of the very low concentrations of enantiomers of both metabolites. The limit of quantification was 250 ng/ml for ofloxacin enantiomers and 100 ng/ml for each metabolites’ enantiomers. This method was applied to the analysis of human urine samples collected from a volunteer after oral administration of 200 mg of (±)-ofloxacin to elucidate stereoselective differences in the formation and excretion of the metabolites. It could be demonstrated that the renal excretion of the S-configured metabolites, especially S-desmethyl ofloxacin, within the first 20 h after dosage, is significantly lower than that of the R-enantiomers.     

Assay of tramadol in urine by capillary electrophoresis using laser-induced native fluorescence detection

Capillary electrophoresis (CE) with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the direct determination of tramadol in human urine without extraction or preconcentration. The main problem in CE is the small inner diameter of the capillary which causes a low sensitivity with instruments equipped with a UV detector. Laser-induced native fluorescence with a frequency doubled argon ion laser at an excitation wavelength of 257 nm was used for the direct assay of tramadol in urine to enhance the limit of detection about 1000-fold compared to UV absorption detection. The detection system consists of an imaging spectrograph and an intensified CCD camera, which views an illuminated 1.5 mm section of the capillary. This set-up is able to record the whole emission spectra of the analytes to achieve additionally wavelength-resolved electropherograms. In the concentration range of 20 ng/ml–5 μg/ml in human urine coefficients of correlation were better than 0.998. Within-day variation determined on four different concentrations showed accuracies ranging from 90.2 to 108.4%. The relative standard deviation (RSD) was determined to be less than 10%. Day-to-day variation presented accuracies ranging from 90.9 to 103.1% with an RSD less than 8%.     

Evaluation of capillary electrophoresis with post-column derivatization and laser-induced fluorescence detection for the determination of substance P and its metabolites

A method for the detection of substance P and its metabolites using capillary electrophoresis with post-capillary derivatization and laser-induced fluorescence detection is described. The post-capillary chemical derivatization system employs naphthalene-2,3-dicarboxaldehyde and β-mercaptoethanol. Two reactor designs were evaluated for the determination of substance P and its metabolites. The fluorescent spectroscopic properties of the derivatives under optimal separation conditions were also examined. The final system was evaluated for the investigation of substance P metabolism in brain following perfusion of the striatum with substance P using microdialysis sampling.     

Sensitive analysis of [ -Pen]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [ -Pen^2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C₁₈ solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm×50 μm I.D. capillary column with borate buffer (25 mM, pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.     

Determination of dimethylamine and other low-molecular-mass amines using capillary electrophoresis with laser-induced fluorescence detection

The potential of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the separation and determination of dimethylamine (DMA) and other low-molecular-mass amines involving precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) was investigated. Different variables that affect derivatization (pH, FITC concentration, reaction time and temperature) and separation (buffer concentration, addition of various organic modifiers, applied voltage and length of capillary) were studied. The linearity, reproducibility and reliability of the method were evaluated. The estimated instrumental detection limit for a 2-s pressure injection of the FITC-DMA derivative was 50 pg/ml (10⁻⁹ M), using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. However, for practical reasons, a minimum of 5 ng/ml DMA should be subjected to the derivatization. The applicability of the described method to the extract of atmospheric aerosol samples was demonstrated.