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%.     

Determination of biogenic amines in lake water by micellar electrokinetic chromatography with fluorescence detection after derivatization with fluorescamine

A simple and rapid method has been developed for the determination of biogenic amines in lake water using micellar electrokinetic chromatography with fluorescence detection. Separation of fluorescamine derivatized biogenic amines was accomplished by using borate buffer of pH 9.5 containing 40 mM of sodium dodecyl sulphate. The method has been optimized with respect to fluorescamine concentration, reaction pH, reaction time, separation voltage and injection time. Detection was performed by using UG-11 excitation filter and 495 nm emission filter. The proposed method for histamine, tyramine and dopamine allowed their separation within 2 min with detection limits in nM range. The interday and intraday reproducibility of peak areas were less than 6.5%. Recovery of spiked samples was 95.76–116.31%.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Determination of vigabatrin in human plasma by means of CE with LIF detection

A method has been developed for the quantitation of the antiepileptic drug vigabatrin (VGB) in human plasma. It is based on CE with LIF detection. The effect of the pH of the buffer and of N-methylglucamine (GLC) as BGE constituent was investigated. The final BGE consisted of 50 mM borate buffer, pH 9.0, with 100 mM GLC and enabled separation within 12 min at 20 kV voltage. An SPE procedure was used for the pretreatment of biological samples, based on mixed-mode lipophilic-cation exchange cartridges, followed by a derivatization step with 6-carboxyfluorescein-N-succinimidyl ester (CFSE). Fluorescence was excited by an Ar-ion laser (lambda(exc) = 488 nm). Linearity was observed in the 10-120 microg/mL plasma concentration range. Extraction yield was >96%, precision (expressed as RSD) <6.7% and accuracy (recovery) was between 97.0 and 101.6%. The method has been successfully applied to the analysis of VGB in plasma of epileptic patients undergoing therapy with the drug.     

Determination of synthesized alpha-vitamin E by micellar electrokinetic chromatography

We developed a micellar electrokinetic chromatography method (MEKC) for the direct determination of the content of synthesized alpha-vitamin E. It was found that under the optimum separation conditions 7 mM borate + 14 mM phosphate + 15 mM sodium dodecyl sulfate (SDS) + 10 mM sodium cholate (NaCh) + 8% acetonitrile (pH 9.2) with UV detection wavelength at 214 nm, 16 kV constant voltage, and 26 degrees C constant temperature, alpha-vitamin E and its isomers can be baseline separated and alpha-vitamin E was quantitatively analyzed. In addition, the sample recovery, the limit of detection and the repeatability of the method were investigated. The influence of various parameters on the separation such as SDS concentration, NaCh concentration, buffer pH and acetonitrile percentage were also discussed.     

Fourier transform infrared spectroscopy with a sample deposition interface as a quantitative detector in size-exclusion chromatography

Micellar electrokinetic capillary chromatography was developed to analyze plant hormones including gibberellic acid, abscisic acid, indole-3-acetic acid, alpha-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin-6-furfurylaminopurine and N6-benzyladenine. The influences of some crucial parameters including buffer concentration, pH value, micelle concentration and applied voltage on electrophoretic separation were investigated. Under optimum conditions (50 mM borate as the running buffer containing 50 mM sodium dodecylsulfate, pH 8.0; separation voltage: -15 kV; injection: hydrodynamic injection, 5 s at 50 mbar; temperature: 25 degrees C), a complete separation of seven plant hormones was accomplished within 30 min. Emphasis was placed on improving detection sensitivity in order to detect small amounts of hormones in plant tissue. Multiple wavelength detection and expanded bubble cell capillary were used with enrichment factors of 2 and 3, respectively. In addition, an on-line concentration method of large volume sample stacking was designed. Enrichment factors of up to approximately 10-600 were achieved for these hormones with detection limits down to 0.306 ng/ml. The method was successfully applied to analyzing abscisic acid in flowers of transgenic tobacco.     

Determination of three bioactive constituents in moutan cortex by capillary electrophoresis with electrochemical detection.

Capillary electrophoresis with electrochemical detection has been employed for the separation and determination of the three active constituents (paeonol, benzoyloxypaeoniflorin, and oxypaeoniflorin) in traditional Chinese medicine, Moutan Cortex (root cortex of Paeonia suffruticosa Andrews). The effects of several important factors, such as the concentration of running buffer, the separation voltage, the injection time, and the detection potential, were investigated to determine the optimum conditions. The detection electrode was a 300 microm diameter carbon-disc electrode at a working potential of +0.90 V (versus SCE). The three analytes could be well separated within 7 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2). The relation between the peak current and the analyte concentration was linear over 3 orders of magnitude with detection limits (S/N = 3) ranging from 0.4 to 0.7 microM for all analytes. The proposed method has been successfully applied to the determination of paeonol, benzoyloxypaeoniflorin, and oxypaeoniflorin in real plant samples with satisfactory assay results.     

高效毛细管电泳紫外检测法分离检测射干苷和鸢尾黄素

建立了毛细管电泳法测定射干药材中的射干苷和鸢尾黄素.探讨了检测波长、缓冲体系、缓冲液浓度和pH、分离电压等对分离的影响.在最优条件为20 mmol/L硼砂(pH 9.0),分离电压20 kV,被测物在10 min内实现基线分离.射干苷和鸢尾黄素的回收率分别为94.4%、93.5%,RSD分别为3.3%、2.8%.该法已...     

Method development and validation for the simultaneous determination of four coumarins in Saussurea superba by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the determination of four coumarins--skimmin, scopolin, scopoletin, and umbelliferone-in Saussurea superba with UV detection at 254 nm. The capillary temperature was kept constant at 25 degrees C. Effects of buffer pH, electrolyte concentration, organic modifier, and applied voltage on migration behavior were studied systematically. The optimum conditions for separation were achieved by using 30 mM borate buffer at pH 9.02 containing 15% (v/v) methanol as the electrolyte and 25 kV as the applied voltage. For all analytes a good linear regression relationship (r > 0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, and accuracy. The validated method was successfully applied to the simultaneous determination of the four analytes in S. superba.     

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).     

Far infrared-assisted extraction followed by MEKC for the simultaneous determination of flavones and phenolic acids in the leaves of Rhododendron mucronulatum Turcz

A method based on micellar electrokinetic chromatography with amperometric detection and far infrared‐assisted extraction has been developed for the simultaneous determination of two flavones (rutin and farrerol) and three phenolic acids (syringic acid, vanillic acid, and 4‐hydroxybenzoic acid) in the dried leaves of Rhododendron mucronulatum Turcz., a commonly used traditional Chinese medicine. The effects of some important factors such as the voltage applied on the infrared generator, irradiation time, the concentration of borate and sodium dodecylsulfate (SDS), separation voltage, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300‐μm diameter carbon disc electrode. The five analytes could be well separated within 8 min in a 40 cm‐long capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2) containing 50 mM SDS. The relationship between peak current and analyte concentration was linear over about three orders of magnitude with the detection limits (S/N=3) ranging from 0.20 to 0.46 μM. The results indicated that far infrared irradiations significantly enhanced the extraction efficiency. The extraction time was substantially reduced to 6 min compared with 3 h for conventional hot solvent extraction.     

Simultaneous Determination of Four Major Iridoid Glycosides in Scrophularia ningpoensis by CE

A simple, accurate and reproducible capillary electrophoresis method with UV detection has been developed for the simultaneous determination of four iridoid glycosides, 6-O-methyl-catalpol, aucubin, harpagide, and harpagoside, in Scrophularia ningpoensis (Xuan-shen). The running buffer was 100 mM borate (pH 9.3) containing 20% methanol. Applied voltage was 20 kV and temperature was 25 °C. Diphylloside A was used as an internal standard (IS) and detection was at 200 nm. The effects on separation of buffer pH, buffer concentration, and organic modifiers were investigated. The extracts of S. ningpoensis were well separated within 45 min.

     

Simultaneous Determination of Four Major Iridoid Glycosides in Scrophularia ningpoensis by CE

A simple, accurate and reproducible capillary electrophoresis method with UV detection has been developed for the simultaneous determination of four iridoid glycosides, 6-O-methyl-catalpol, aucubin, harpagide, and harpagoside, in Scrophularia ningpoensis (Xuan-shen). The running buffer was 100 mM borate (pH 9.3) containing 20% methanol. Applied voltage was 20 kV and temperature was 25 °C. Diphylloside A was used as an internal standard (IS) and detection was at 200 nm. The effects on separation of buffer pH, buffer concentration, and organic modifiers were investigated. The extracts of S. ningpoensis were well separated within 45 min.     

糖类衍生物在毛细管区带电泳下的分离研究

以新合成的1-萘基-3-甲基-5-吡唑啉酮(NMP)为柱前衍生试剂,采用毛细管区带电泳模式考察并优化了糖类衍生物的分离条件。实验采用58.5cm×50μmi.d.毛细管(有效柱长50cm),55mmol/L硼酸盐缓冲溶液(pH9.46),柱温20℃,分离电压22kV,进样10s,在不加任何添加剂的情况下,高效、快速地实现了9种糖的基线分离,并在最优化条件下进行了唐古特白刺实际样品的分离分析,结果令人满意。     

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.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Original paper application of cyclodextrins as modifiers in electrophoretic separation of metalloporphyrins

Several metallocomplexes of tetrakis-carboxyphenylporphyrin (TCPP) were separated on fused-silica capillary using CZE with UV-VIS detection. Metalloporphyrins of Co(II), Cu(II), Mn(II), Ni(II), and Zn(II) were formed directly in TCPP solution with addition of Cd(II) to increase the formation reaction rate. The composition of BGE, its concentration, and pH were optimized to ensure the stability of complexes and proper resolution. In particular, the problem of signals' shape was investigated and discussed. The presence of beta-CD in borate buffer significantly improved separation efficiency and signal shapes due to formation of inclusion complexes. Under the best separation conditions (50 mM borate running buffer at pH 9 with addition of 2 mM beta-CD, 30 kV applied voltage) a separation of metal complexes with TCPP was accomplished in 16 min.     

Capillary electrochromatographic analysis of barbiturates in serum

A capillary electrochromatographic method was developed for the separation of barbiturates. The separation was optimized in a 75 microm ID capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effect of buffer pH, buffer concentration, and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 40% methanol provided the baseline separation of barbital, phenobarbital, secobarbital, and thiopental (internal standard) in less than 4.5 min. The method was successfully applied to the analysis of barbiturates in human serum. Under the optimal conditions, good repeatability and linearity were obtained in the range of 2.90-43.29 microg/mL for barbital, phenobarbital, and secobarbital.     

Analysis of cerebrospinal fluid γ‐aminobutyric acid by capillary electrophoresis with laser‐induced fluorescence detection

The measurement of γ‐aminobutyric acid (GABA) is suitable for investigating various neurological disorders. In this study, a sensitive and selective method for free GABA quantification in cerebrospinal fluid (CSF) has been standardised. This method is based on CE with LIF detection using 4‐fluoro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐F) as a derivatisating agent. The reaction conditions (NBD‐F concentration, pH, temperature and reaction time) and the electrophoretic parameters (run buffer composition and pH and separation voltage) were optimised to obtain the maximum derivatisation efficiency and electrophoretic resolution. The best resolution was obtained using 200 mM sodium borate, 10 mM SDS, 8.5 mM β‐CD, pH 10 and 20 kV voltage. The method was linear in the concentration range of 2.5–1000 nM with good inter‐ and intra‐assay precision values. The effects of CSF handling on free GABA concentrations were also evaluated. Our results show that the time delay between CSF collection and freezing strongly increases the CSF GABA values. Age‐related reference values were established in 55 paediatric controls. The influence of antiepileptic therapy on free CSF GABA was studied in 38 neuropaediatric patients. Significantly, higher GABA values were obtained in patients taking valproic acid or vigabatrin therapy, which are antiepileptic drugs that modulate GABA metabolism.