Capillary electrophoresis of baclofen with argon-ion laser-induced fluorescence detection

A capillary electrophoretic method with laser-induced fluorescence detection for baclofen (4-amino-3-p-chlorophenylbutyric acid) has been developed. 6-Carboxyfluorescein 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 (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the lowest derivatizable concentration limit for baclofen in aqueous solution was 10 nM (2 ng baclofen/ml). Coupled with a simple clean up procedure, the method can be applied to the analysis of baclofen in human plasma at micromolar level. Recovery of spiked baclofen in plasma was 95%. The relative standard deviation values on peak size (0.5 microM level) and migration time were 8.2 and 1.0% (n=7), respectively. The limit of detection of baclofen in plasma was 0.1 microM (21 ng/ml).     

Capillary electrophoresis of aminoglycosides with argon-ion laser-induced fluorescence detection

A new analytical method for aminoglycosides (kanamycin, bekanamycin, paromomycin and tobramycin) based on capillary electrophoretic separation and argon-ion laser-induced fluorescence detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent aminoglycosides. Optimal separation and detection were obtained with an electrophoretic buffer of 30 mM sodium borate (pH 9.0) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The concentration limits of detection in aqueous solution were 3.9-8.2 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of aminoglycosides in human plasma. A calibration curve ranging from 0.15 to 30 microM was shown to be linear. The limits of detection of aminoglycosides in human plasma were between 14.4 and 24.0 nM. Recoveries of spiked aminoglycosides in human plasma were between 92 and 105%.     

Capillary electrophoresis of catecholamines with laser-induced fluorescence intensified charge-coupled device detection

A capillary electrophoresis-laser-induced fluorescence-intensified charge-coupled device system was used for the separation and determination of catecholamines. Optimization of derivatization and separation conditions was investigated in order to reach a high separation efficiency and sensitivity. All fluorecein isothiocyanate derivatives of catecholamines were satisfactorily separated within 12 min. The detection limits were in attomole ranges. This method allows determination of catecholamines with high separation efficiencies and sensitivity.     

Capillary electrophoresis of FITC labeled amino acids with laser-induced fluorescence detection

FITC labeled amino acids have been separated using a home-huilt capillary electrophoresis with a laserinduced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.     

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5′-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 μm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV?=?4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 μmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5′-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.     

毛细管电泳-激光诱导荧光法测定草甘膦、草胺膦及氨甲基膦酸

建立了一种快速、有效的毛细管电泳分离-激光诱导荧光检测有机磷除草剂草甘膦、草胺膦和草甘膦的代谢物氨甲基膦酸的方法。将荧光衍生试剂5-(4, 6-二氯三嗪基)氨基荧光素(DTAF)成功用于衍生上述3种化合物。最佳衍生条件: DTAF的浓度为1.0 μmol/L,以50 mmol/L硼酸(pH 9.5)作为缓冲溶液,在30 ℃下反应40 min。以pH 9.5的30 mmol/L硼酸缓冲溶液(含15 mmol/L Brij-35)作为电泳背景电解质,3种衍生物得到基线分离。在优化的条件下,草甘膦、草胺膦、氨甲基膦酸的检出限分别为3.21、6.14和1.99 ng/kg。将该方法应用于环境水样和土壤中除草剂及代谢物的测定,回收率为91.3%～106.0%。该方法准确、灵敏,可满足环境样品中有机磷农药及其代谢物残留的检测要求。     

Capillary electrophoresis with laser-induced fluorescence detection of main polyamines and precursor amino acids in saliva

A hollow-fiber liquid-phase microextraction （HF-LPME） method has been developed for the purification and preconcentration of biogenic polyamines and their precursor amino acids in human saliva. Putrescine （Put）, cadaverine （Cad）, spermidine （Spe）, ornithine （Orn）, lysine （Lys）, and arginine （Arg） were determined by the CE-LIF detection after microextraction. Several factors that affect extraction efficiency, separation, and detection were investigated. Under the optimum conditions, six analytes could achieve baseline separation within 30 min, exhibiting a linear calibration at three orders of magnitude （r2 〉 0.998）; the obtained enrichment factors of HF-LPME were between 19 （for Orn） and 2] 8 （for Cad）, and the LODs were in the range of 0.0072-0.26 nmol/L. The proposed HF-LPME/CE-LIF method has been successfully applied for the sensitive analyses of the real-world saliva samples collected from healthy volunteers and different patients with oral diseases, providing a potential method for primary non-invasive diagnosis of some oral diseases.     

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.     

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.     

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.     

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.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

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.     

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.     

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.     

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。     

Microfluidic chip-based cell electrophoresis with multipoint laser-induced fluorescence detection system

In this work, the electrophoretic mobility (EPM) measurement of individual cells was investigated by a simple on-chip electrophoresis system with LIF multipoint detection. The system enabled the characterization of cell electrophoresis behavior as well as the fluorescence signal from individual cells simultaneously. The measurement yielded the electropherograms of a large number of cells labeled with dye, in which the migration time and migration distance could be obtained easily. The EPM has been demonstrated to be different between the K562 cells and K562 cells treated with anticancer drug arsenic trioxide (As2O3). The K562 cells were found to exhibit a lower EPM compared to the cells after drug addition with different concentration. In this preliminary study, over 300 cells could be analyzed within 2 h, demonstrated a much higher analysis throughput compared with traditional methods. The established system is simple and fast, which is expected to be a promising method for evaluating cell surface properties and to be useful in clinical and pharmaceutical applications.     

Solid-state UV laser-induced fluorescence detection in capillary electrophoresis

Two solid-state UV lasers were applied to the laser-induced fluorescence (LIF) detection of various groups of compounds after separation by capillary electrophoresis. These lasers are thermoelectric-cooled, highly compact, and inexpensive. Such lasers provide few mW of quasi-continuous wave (CW) power which are sufficient and stable for LIF detection. Native fluorescence detection of tryptophan-containing proteins and peptides and related indoles was achieved at the nM level with the laser operating at 266 nm. Detection of fluorescamine-labeled amino acids and peptides was also possible at the nM level with the laser operating at 355 nm. Amino acids at a concentration as low as 10 ng/mL could be labeled with fluorescamine. Solid-state UV-LIF detection of the tryptic digest of cytochrome c after fluorescamine derivatization was demonstrated.     

Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies

Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-aminobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency.     

Capillary electrophoresis of urinary porphyrins with absorbance and fluorescence detection

Urinary porphyrins are separated in a 72 cm x 50 microns I.D. fused-silica capillary by micellar electrokinetic capillary chromatography with 100 mM sodium dodecyl sulfate and 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid at pH 11. Detection is accomplished by absorbance at 400 nm or fluorescence with excitation at 400 nm and emission at wavelengths above 550 nm. Substantial trace enrichment is found for porphyrins in urine samples or for porphyrin standards prepared without surfactant in the injection buffer. Limits of detection are in the 100 pmol/ml concentration range with an optimized fluorescence system. The method is shown suitable for the determination of porphyrins in clinical urine specimens. Comparisons are made between electrophoretic and chromatographic methods for the separation and detection of urinary porphyrins.