Determination of tryptamine derivatives in illicit synthetic drugs by capillary electrophoresis and ultraviolet laser-induced fluorescence detection

A method based on separation by capillary electrophoresis combined with UV-laser-induced fluorescence detection (Lambdaex = 266 nm) was developed for the determination of nine tryptamine derivatives of forensic interest and potential matrix constituents. The composition of the separation electrolyte was optimized with respect to the resolution of solutes of interest and to the sensitivity of fluorescence detection. Native alpha-cyclodextrin was employed as a complex forming modifier of the electrophoretic separation and fluorescence-enhancing agent. With the help of a stacking procedure, limits of detection of 0.1-6 microg/L for all analytes were obtained. The repeatability for the peak area (at a concentration of the analyte about 100 times the LOD) was less than 2.3% RSD. A second HPLC method was developed, and its analytical parameters were evaluated for an estimation of the accuracy of the CE-LIF method and for method comparison. The results of the determination of tryptamine derivatives in the samples of forensic interest obtained with the two independent methods are in good agreement.     

Determination of aluminum in serum by capillary zone electrophoresis with laser-induced fluorescence detection

Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.     

毛细管电泳-激光诱导荧光检测法测定抗癫痫药加巴喷丁

建立了毛细管电泳-激光诱导荧光(CE-LIF)测定抗癫痫药加巴喷丁的方法。加巴喷丁经4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)衍生化后,采用10 mmol/L硼砂-10 mmol/L十二烷基硫酸钠(pH 9.75)的缓冲体系,加巴喷丁在6 min内实现高效基线分离。方法的线性范围为0.01～10 mg/L(r=0.9997),检出限为2 μg/L,定量限为10 μg/L。方法的平均回收率为100.2%～103.1%,相对标准偏差为0.15%～1.00%(n=3)。该方法灵敏、快速、准确和可靠,已用于加巴喷丁药物制剂的质量控制。     

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

The industrial application of nitrocellulose depends on its nitrogen content. When nitrocellulose presents high nitrogen content is used in the manufacture of explosives whereas nitrocellulose with low nitrogen content is used to make a wide range of daily and non-explosive products (e.g. cigarettes, paints, lacquers). This fact makes really important to develop a method for the determination and discrimination of nitrocellulose samples. This work reports, for the first time, the qualitative determination of nitrocellulose previously derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by capillary electrophoresis (CE-LIF) with laser-induced fluorescence detection (CE-LIF). APTS-labeled nitrocellulose was determined in lowly and highly nitrated nitrocellulose samples present in collodions and smokeless gunpowders, respectively, after their pulverization in liquid nitrogen. The method described enables the visual discrimination of different nitrocelluloses on the basis of the different electrophoretic profiles obtained, and provides a useful tool to determine nitrocellulose. Additionally, the use of field-amplified sample injection (FASI) enabled enhanced sample detection, which made it possible to determine nitrocellulose contained in ∼15 μg of gunpowder.     

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

Phycobiliproteins are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae. They are composed of a protein backbone to which linear tetrapyrrole chromophores are covalently bound. Furthermore, they are water-soluble highly fluorescent, and relatively stable at room temperature and neutral pH. For this reason, capillary electrophoresis-laser induced fluorescence (CE-LIF) seems the idea method for determination of these important proteins. The effects of buffer additives such as sodium dodecyl sulfate (SDS)and putrescine on the separation of the three major phycobiliprotein types, namely allophycocyanin, phycocyanin, and phycoerythrin, with excitation and emission maxima at 652/660, 615/647, and 565(494)/575 nm, respectively, are considered. Detection limits for these proteins by CE-LIF are some 60-500 times better than by absorbance detection. The development of a fast and sensitive CE-LIF assay such as this is of potential significance to our understand ing of chemical and biological oceanographic processes.     

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

An immunoassay based on CE–LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL⁻¹) of DNA under a low UVB fluence of 65 J m⁻² for CPDs or 195 J m⁻² for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.     

Determination of abscisic acid by capillary electrophoresis with laser-induced fluorescence detection

A novel method based on capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF) was developed for the determination of abscisic acid (ABA), which is an essential phytohormone during plant growth and development. ABA was labeled with 8-aminopyrene-1,3,6-trisulfonate via reductive amination in presence of acetic acid and sodium cyanoborohydride. The derivatization yield was maximized by optimizing several derivatization parameters including derivatization reagent concentration, reaction temperature and time. The conjugate was separated and quantitated by CE-LIF. The linearity of ABA was determined in the range from 0.1 to 10 micromol l(-1) with a correlation of 0.9979. The derivatization limit of detection for ABA was found to be 56 fmol (corresponding to the concentration of 2.8 x 10(-8) mol l(-1)). The detection limit for ABA was 5.5 amol for an injection volume of 5 nl. As a preliminary application, the proposed method was successfully applied to determining trace amount of ABA in the crude extracts of tobacco without extra purification and enrichment procedure and showed a better selectivity and sensitivity than those conventional methods used in determination of ABA.     

Some factors affecting enantiomeric impurity determination by capillary electrophoresis using ultraviolet and laser-induced fluorescence detection.

The key factors influencing enantiomer trace determination were investigated; these include resolution capillary diameter, limit of detection, linear range and type of detection. Chiral reagents, (+)- and (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC), were employed as probes to demonstrate the influence of the variables. In order to find the best resolution, separation variables were optimized in both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) modes by the application of factorial design experiments. A highly efficient chiral separation of the (+/-)-FLEC, derivatized with nonchiral amino acids, was achieved when using gamma-cyclodextrin as the chiral selector. The benefits of using a small diameter capillary for direct determination of both (+) and (-)-FLEC impurity (0.05-0.1% area/area) were demonstrated using UV detection and applying a sample stacking condition. A frequency-doubled argon ion laser (244 nm) was used as light source for laser-induced fluorescence (LIF) detection. Excitation light was provided by means of an optical fiber directed into the Hewlett Packard 3D capillary cartridge. The signals from UV and LIF were monitored simultaneously. The application of LIF detection greatly improved sensitivity and linear range. Further, as a consequence of the increased sensitivity, sample loading could be decreased, which led to an improvement of separation efficiency. Direct determination of 0.005% impurity could be achieved within the linear range.     

beta-galactosidase assay using capillary electrophoresis laser-induced fluorescence detection and resorufin-beta-D-galactopyranoside as substrate

beta-Galactosidase was incubated for 60 min with the fluorogenic substrate resorufin-beta-D-galactopyranoside, which is converted by the action of the enzyme into resorufin and galactose. A 160 pL aliquot of reaction mixture was analyzed by capillary electrophoresis utilizing laser-induced fluorescence detection. Based on the detection of the resorufin formed, the limit of detection of beta-galactosidase was 1.5 x 10(-15) M or 900 molecules of enzyme in a 1 microL sample.     

Determination of uric acid in human urine by capillary zone electrophoresis with indirect laser-induced fluorescence detection

A CZE with indirect LIF detection method was used for the determination of uric acid (UA) in human urine. UA and its coexisting analytes (i.e. hypoxanthine, xanthine and ascorbic acid) could be well separated within 4.5?min at a voltage of 25?kV with 25°C cartridge temperature in a running buffer composed of 5?mM sodium borate, 10% methanol (v/v) and 0.3?μM fluorescein sodium (apparent pH of the final mixed hydro-organic solution of sodium borate, methanol and fluorescein is 9.5). Under the optimum condition, the method has good linearity relationships (correlation coefficients: 0.9973-0.9987) with ranges of 25-500, 25-350, 25-250 and 25-300?μg/mL for hypoxanthine, ascorbic acid, xanthine and UA, respectively. The detection limits for the analytes were in the range of 0.29-0.90?μg/mL. The intra-day RSD values for migration times and peak areas were less than 0.43 and 3.27%, respectively. This method was applied to the quantitation of UA in human urine with recoveries in the range of 93.1-107.3%.     

Determination of metoprolol in rabbit blood using capillary electrophoresis with laser-induced fluorescence detection

This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis(CE) coupled with laser-induced fluorescence(LIF) detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range(2.0-500 ng/mL) of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation(RSD) less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.     

Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with postcolumn laser-induced fluorescence detection was used to individually detect 6.0, 1.0, 0.5, and 0.2 num diameter polystyrene microspheres and individually measure their electrophoretic mobility. The analysis of a nanoliter-size volume from a microsphere suspension results in an electropherogram characterized by several narrow spikes in a well-defined migration time window. Each spike is associated with one microsphere because, when one single microsphere is introduced into the capillary by micromanipulation, the electropherogram has only one spike in the same migration time window. The distributions of individual measurements resulting from an electropherogram were used to evaluate the reproducibility from run to run, observe the effect of sodium dodecyl sulfate (SDS) added to the running buffer, and to investigate the origin of electrophoretic dispersion. As expected from the interactions between microspheres and SDS, the addition of this surfactant to the running buffer narrowed the range and shifted the average electrophoretic mobility to more negative values. After evaluating common sources of broadening in capillary electrophoresis, electrophoretic dispersion was attributed to microsphere heterogeneity. Unlike electropherograms displaying Gaussian-like profiles, the two-dimensional representations of the individual measurements provide a new alternative to evaluate and study electrophoretic-related properties of microspheres.     

Determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, using capillary zone electrophoresis and laser-induced fluorescence detection

High-dosage methotrexate therapy requires careful monitoring of the drug in serum to ensure minimal toxic effects. A simple, rapid and sensitive method for the separation and quantitation of methotrexate and its major metabolite, 7-hydroxymethotrexate, using high-voltage capillary zone electrophoresis combined with laser-induced fluorescence detection is described. The detection limit for methotrexate is as low as 5.10(-10) M (signal-to-noise ratio = 3), while that for 7-hydroxymethotrexate is 2.10(-9) M. The linearity of the system extends over nearly four orders of magnitude for both methotrexate and 7-hydroxymethotrexate. The extraction efficiency for the drug and its metabolite from serum is 80-85% using a Sep-Pak C18 cartridge. Quantitation of methotrexate in serum was possible in the 10(-10) M range, nearly two orders of magnitude lower than that currently obtainable by existing methods. Good correlation (r = 0.99) for serum methotrexate concentrations was obtained with an enzyme-multiplied immunoassay technique. Comparison with an enzyme inhibition assay also provided similar results.     

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

Two methods for monosaccharide analysis by capillary electrophoresis (CE) using counterelectroosmotic and coelectroosmotic modes with indirect laser-induced fluorescence detection were optimised and compared. A mixture of seven glycosaminoglycan-derived hexoses was separated in alkaline fluorescein-based electrolytes and detected in both counterelectroosmotic and coelectroosmotic conditions. The fluorescein concentration and pH of the background electrolyte, and the influence of the reversal of electroosmotic flow by addition of hexadimethrine bromide on the separation were studied. Coelectroosmotic CE conditions provided better resolution and limits of detection. A 10(-6) M fluorescein solution at pH 12.25 containing 0.0005% (w/v) hexadimethrine bromide was used as background electrolyte. Quality parameters such as run-to-run, day-to-day precision and limits of detection were calculated, and better figures of merit were obtained for the coelectrooosmotic conditions than for the counterelectroosmotic mode. The coelectroosmotic method was applied to the quantitation of the hexosamine contents in glycosaminoglycans after acid hydrolysis. The method proved to be suitable for the determination of dermatan sulfate in heparin down to 2% (w/w).     

Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection

A rapid method using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed to determine free and protein-bound glutathione (GSH) in human HepG2 hepatocarcinoma cells. The samples were derivatized with 5-iodoacetamidofluorescein (5-IAF), and analyzed at 22 kV using sodium phosphate buffer (10mM, pH 11.4) and an uncoated 58 cm x 75 microm I.D. fused silica capillary. The analysis time was less than 10 min and N-acetylcysteine was used as internal standard. The derivatization conditions, such as reaction time, 5-IAF concentration, running buffer and cartridge temperature were optimized. Argon gas was used in the study to prevent the oxidization of GSH during sample preparation. The optimized method required only 30-40 nl sample per analysis and was fast and sensitive. The method was applied to the analyses of HepG2 cells treated with the small metal chelating agent, pyrrolidine dithiocarbamate (PDTC). The results demonstrate that the amount of protein-bound GSH, which reflects the amount of protein S-glutathionylation, increased in a time-dependent manner upon cell treatment with PDTC, reaching a maximum of over 50% increase 2h post-PDTC.     

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.     

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde 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 (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.