Characterization of paromomycin sulfate by capillary electrophoresis with UV detection after pre-capillary derivatization

Summary A capillary electrophoretic method has been developed for determination of the components of the paromomycin mixture salt. Paromomycin was detected at 330 nm after pre-capillary derivatization witho-phthaldialdehyde and thioglycolic acid. The electrophoretic separation was performed in a fused-silica capillary at 25°C and 18kV with a background electrolyte comprising 40mm sodium tetraborate, 3mm β-cyclodextrin, and 12.5% (v/v) methanol. Although the analysis time was reduced to 10min, five peaks could be separated to baseline. The relative standard deviation of the ratios (peak area/internal standard peak area) was <5% for all peaks.     

Development and validation of a simple capillary zone electrophoresis method for the analysis of kanamycin sulfate with UV detection after pre-capillary derivatization

Capillary zone electrophoresis was successfully applied to separate eight related substances of kanamycin and several minor unknowns from the main component. Strategies to enhance derivatization and selectivity and to optimize separation parameters involved the application of experimental designs. This chemometrical approach considers main effects as well as interactions of the influential parameters, thus conducting a more thorough investigation of the method than the common step-by-step approach. Central composite face centered designs established optimal separation conditions: 30 mM borax buffer, pH 10.0 containing 16.0% (v/v) methanol and optimal composition of derivatization reagent: 27 mg/ml 1,2-phthalic dicarboxaldehyde and 25 microl/ml mercaptoacetic acid in borate buffer, pH 10.4. The standard curves were linear over the concentration range of 0.007-1.01 mg/ml for the main component and 0.003-0.1 mg/ml for the related substances. The limit of quantitation was 0.14% (m/m) for the related substances and impurities (S/N= 10). The assay method was used to determine the composition of several commercial samples. Quantitative analysis indicates potential usefulness of capillary electrophoresis as an alternative to the assay method prescribed in the European Pharmacopoeia and the United States Pharmacopeia.     

Electrophoretically mediated microanalysis of gentamicin with in-capillary derivatization and UV detection.

This paper describes a system for integration of a one-step-microscale chemical derivatization and analysis by a methodology known as electrophoretically mediated microanalysis (EMMA). Differential electrophoretic mobility between an analyte, reagent, and their product offers EMMA a unique capability to selectively carry out electrophoretic mixing, control product formation, and separation. This system was successfully applied to perform derivatization and separation of the multicomponent aminoglycoside antibiotic gentamicin using 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid as labeling reagents. A multivariate approach based on central composite experimental design was used to optimize the derivative yield. Full automation of the derivatization and analytical procedure, high derivatization efficiency, high sample throughput, and precision are the excellent features of the present method. In addition, this methodology offers short analysis time, as well as selectivity and sensitivity suitable for impurities determination. Separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin, and several minor components was achieved. For the first time separation and identification of three impurities, namely garamine, 2-deoxystreptamine, and paromamine are described.     

Capillary gel electrophoresis of single-stranded DNA fragments with UV detection

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal “fading” effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.     

Capillary electrophoresis of organic and inorganic cations with indirect UV detection

Summary The determination of alkali and alkaline earth metal ions by capillary electrophoresis using indirect UV detection is described. With the identical system it is also possible to determine short chain aliphatic amines and alkanol amines within 4 minutes. Indirect UV detection is achieved at 214 nm with a background electrolyte containing 5 mmol L^–1 imidazole. Linear calibration curves could be obtained for peak areas between 0.5 and 10 ppm. The detection limits are around 0.1 ppm (corresponding to 10 fmol in about 10 nL sample volume) for all cations and amines and 0.05 ppm for lithium. Practical applications demonstrate the applicability of this system in routine analysis.     

Capillary zone electrophoresis with pre-column NDA derivatization and amperometric detection for the analysis of four aliphatic diamines

A method was developed for the analysis of four aliphatic diamines by capillary zone electrophoresis using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA)/CN⁻ and amperometric detection. The pre-column derivatization reaction conditions including the molar ratio of NDA to amines, the cyanide concentration, the pH value of derivatization buffer, and the reaction time, were investigated. The separation of four derivatives of aliphatic diamines has been optimized by capillary zone electrophoresis (CZE) using end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.7 V versus SCE. The optimum conditions for the separation were 10 mM Tris-H₃PO₄ (pH 4.0) for the running buffer solution, 15 kV for the separation voltage. The detection limits for diaminopropane, putrescine, cadaverine, diaminohexane were 6.7×10⁻⁸, 5.1×10⁻⁸, 1.9×10⁻⁷ and 3.8×10⁻⁷ M, respectively (S/N=3). The proposed method was applied to the determination of aliphatic diamines in a lake water sample by the standard addition method. The recovery of these amines in water was 89.9-107%.     

Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection

The separation of 14 lanthanides by capillary zone electrophoresis was studied in the background electrolyte containing hydroxyisobutyric acid as complexing counter-ion and creatinine as a UV absorbing coion for indirect detection of lanthanide zones. A complete separation was achieved in less than 5 min and the applicability of the method for the analysis of real samples was demonstrated.     

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。     

Development and validation of capillary electrophoresis method for tobramycin with precapillary derivatization and UV detection

One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.     

Capillary electrophoresis and capillary flow injection analysis with electrochemical detection

Measurements by capillary flow injection analysis (CFIA) and capillary electrophoresis (CE) in conjunction with electrochemical detection are described. The detection is based on an end-column electrode arrangement. Several novel electrodes, such as a spherical gold electrode and a dual-microdisk electrode, are presented and characterized regarding their analytical utility. In order to improve the selectivity of CFIA, dual-electrode and multiple-pulse detection are studied using couples of cyanometallates or metallocenes. Capillary electrophoretic experiments with amperometric detection are performed using 50 m i.d. capillaries without any electrical-field decoupler. The practicality and analytical characteristics of this detection strategy are illustrated for the separation of serotonin and some biological precursors and metabolites of neurotransmitter substances.     

Capillary electrophoresis of nonprotein and protein amino acids without derivatization

An efficient separation of eleven nonprotein amino acids (NPAAs) and three protein amino acids containing aromatic moieties was achieved by capillary electrophoresis without derivatization. The fourteen amino acids were well separated with a 100 mM sodium phosphate run buffer (pH 2.0) using a 57 cm fused-silica capillary (50 microm ID, 50 cm effective length) at 20 degrees C. With an electric field of 351 V/cm, the time needed for the separation was less than 20 min. Under optimum conditions, excellent linear responses were obtained in the concentration range of 5-100 microM, with the linear correlation coefficient ranging from 0.9785 or greater. The relative standard deviations of the migration times and the corrected peak areas were found to be 1.5-3.9% and 8.0-11.5%, respectively. In order to improve the limit of detection (LOD), simple stacking and large volume stacking using an EOF pump (LVSEP) methods were used. Improved LODs were about 300 nM in stacking and below 15 nM for five small NPAAs in LVSEP.     

Determination of amoxicillin in trout by liquid chromatography with UV detection after derivatization

A liquid chromatographic (LC) method based on solid-phase extraction was developed for determination of amoxicillin in muscle tissue of rainbow trout. The compound was extracted in an aqueous solution by precipitation of organic material with a mixture of sulfuric acid and sodium tungstate. The extract was processed by solid-phase extraction on an end-capped phenyl sorbent, and concentrated on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. The extract was derivatized and analyzed by reversed-phase gradient LC on a C18 column with UV detection at 323 nm. The method detection limit was 2.9 micrograms/kg. Mean recovery in muscle was 80.5% (range 10-200 micrograms/kg). The method was applied to fillets from trout offered feed containing amoxicillin in an aquaculture pilot plant. Amoxicillin was detected in muscle tissue shortly after administration but not 3 weeks later. The relative repeatability standard deviation for incurred residues in muscle tissue was 6.4% (range 11-143 micrograms/kg).     

Capillary electrophoresis and microchip capillary electrophoresis with electrochemical and electrochemiluminescence detection

Recent advances and key strategies in capillary electrophoresis and microchip CE with electrochemical detection (ECD) and electrochemiluminescence (ECL) detection are reviewed. This article consists of four main parts: CE-ECD; microchip CE-ECD; CE-ECL; and microchip CE-ECL. It is expected that ECD and ECL will become powerful tools for CE microchip systems and will lead to the creation of truly disposable devices. The focus is on papers published in the last two years (from 2005 to 2006).     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

非水体系毛细管电泳-紫外检测法测定苯甲酸和苯甲醛

建立了用非水相体系高效毛细管电泳-紫外检测法同时测定苯甲酸和苯甲醛的新方法，考察了运行电压、非水相介质和电解质等因素的影响，在25℃下，以V（乙腈）：V（碳酸丙烯酯）=1：1的混合液为溶剂，缓冲体系中含15mmol／L十六烷基三甲基溴化铵体积分数1％乙酸，重力进样30S，运行电压20kV，毛细管总长45cm有效长度30cm，φ75μm，检测波长285nm。苯甲酸线性范围为5～40μg／mL，线性方程为：Y=13．473ρ＋13．336，相关系数r=0．9985，检出限为0．92μg／mL，RSD为3．8％。苯甲醛的线性范围75～1125μg／mL线性方程为：Y=5．2449ρ＋564．01，相关系数r=0．9997，检出限为15．60μg／mL，RSD为3．5％。已用于经空气氧化后的苯甲醛中苯甲酸和苯甲醛的测定。     

Capillary zone electrophoresis with indirect UV detection: Determination of sodium dodecyl sulfate in simulated stream water

SDS (sodium dodecyl sulfate) has been quantitatively determined by capillary zone electrophoresis using a fused silica capillary and a 5 mM dihydroxybenzoic acid / sodium hydroxide buffer in 5% methanol solution at a pH of 8.1. The ion was detected by indirect UV absorption at 250 nm. Detection range was from 0.8 to 50 mg SDS/L. This rapid method requiring only small sample volumes was developed in support of an aquatic toxicology study in a simulated stream water and is applicable to waters containing common inorganic ions.     

Capillary electrophoresis with electrochemiluminescence detection for the analysis of quinolone drugs and pharmacokinetics study

A novel method for the determination of two quinolone drugs norfloxacin （NOR） and levofloxacin （LVX） was described by capillary electrophoresis with electrochemiluminescence detection. The good relationship （r ≥ 0.9991） between peak area and concentration of analytes was established over two orders of magnitude. The limits of detection （LOD, S/N = 3） in standard solution are 4.8 × 10^-7 mol/L for NOR and 6.4 × 10^-7 mol/L for LVX, respectively. The limits of quantitation （LOQ, S/N = 10） in real human urine samples are 1.2 × 10^-6 mol/L for NOR and 1.4 × 10^-6 mol/L for LVX, respectively. The present method was successfully applied to the determination of NOR and LVX in human urine and the studv of oharmacokinetics of NOR.     

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.     

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%.