非水介质毛细管电泳电导检测罗红霉素及其制剂

采用甲醇为分离介质，三(羟甲基)氨基甲烷-硼酸(Tris—H3BO3)为支持电解质，采用负高压，使用电导检测，对罗红霉素及其制剂进行了毛细管电泳分离检测，对电泳介质的种类、浓度、表观pH、以及操作电压和进样时问对分离的影响进行了研讨，在选定的条件下，罗红霉素的线性范围为19．0—142．0mg/L，检出限为0．8mg/L(S／N≥3)，峰面积的相对标准偏差RSD(n=6)为4．3％。3种供试品中罗红霉素的平均加标回收率分别为97．7％、94．8％、93．6％。     

Determination of therapeutic oligonucleotides using capillary gel electrophoresis

Oligonucleotides have developed into highly versatile and selective therapeutics over the past 20 years. More than five discrete mechanisms of action have been reported and more than 10 different chemical modifications have been used to extend their in vivo half-life and reduce their toxicity. Capillary gel electrophoresis (CGE) has been used extensively for the quantitative analysis of oligonucleotide therapeutics in both preclinical and clinical studies since the 1990s. The success of CGE is based on its extraordinary resolving power, which allows for the simultaneous determination of the parent drug and its metabolites. More recently, capillary gel electrophoresis has seen renewed interest with the emergence of replaceable gels with single-base resolving power and new capillary electrophoresis-mass spectrometry interfaces. This review discusses the bioanalysis of therapeutic oligonucleotides showing the evolution of the field over the past two decades leading to the current new approaches. Included in this review are topics such as different gel types, sample introduction modes, sample extraction procedures, separation conditions and detection methods used in CGE, along with discussions of the successes and limitations associated with each.     

Nonaqueous capillary electrophoresis using a titania-coated capillary

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.     

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes’ mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20 mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.     

高速毛细管电泳安培法检测甲巯咪唑及其制剂

采用高速毛细管电泳安培法对甲巯咪唑(TMZ)及其制剂的测定进行了研究; 通过优化检测电位、毛细管长度和内径、分离电压、缓冲溶液等实验参数, TMZ在60 s内可以得到较好的分离, 线性范围在2.00×10-3～2.80×10-5 mol/L, 检出限为3.50×10-6 mol/L; 峰面积和迁移时间的相对标准偏差分别为2.7%、 1.2% (n＝8); 该法可用于制剂中TMZ的检测.     

Temperature effects in capillary electrophoresis. 1: Internal capillary temperature and effect upon performance

Summary In isothermal CE the migration velocity of analytes and the number of theoretical plates delivered are expected to be proportional to the field strength. In reality ohmic heating of the capillary causes distortions: the migration velocity increases more rapidly while the plate count increases less rapidly, and may even fall at high values of the field. These distortions are worse the larger the bore of the capillary and the higher the concentration of buffer. A detailed investigation of these effects using capillaries cooled by natural convection has confirmed that self heating of the capillary is indeed largely responsible. The extent of self heating has been determined by three independent methods and to a first approximation is proportional to the power dissipation in the capillary. Decreasing viscosity with temperature is responsible for the nonlinearity of the dependence of velocity upon field strength while increase in the diffusion coefficient of analytes is responsible for the poorer than expected performance at high field strengths.     

Protein-aptamer binding studies using microchip affinity capillary electrophoresis

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.     

Pre-equilibrium capillary zone electrophoresis or frontal analysis: advantages of plateau peak conditions in affinity capillary electrophoresis

The feasibility of using the affinity CE methodologies pre-equilibrium CZE and CE frontal analysis was tested on interaction systems exhibiting rapid on-and-off kinetics. Experimentally, the methodologies differ only with respect to the volume of sample introduced into the capillary. Pre-equilibrium CZE has been considered amendable to interactions with slow on-and-off kinetics only; however, it has recently been applied in studies of interactions with fast on-and-off kinetics. The effect of varying the sample volume introduced hydrodynamically into the capillary on the apparent degree of complexation was studied. For two different binding systems, the fraction of free analyte was found to be overestimated using pre-equilibrium CZE as compared to volumes providing plateau peak conditions as used with frontal analysis. Results indicate that frontal analysis conditions lead to more robust binding assays and thus more reliable data. The validity of data obtained by pre-equilibrium CZE may be low, thus the use of an experimental setup providing plateau peaks is highly recommended. It is suggested that the effect of altering the sample volume on the degree of binding should be investigated as part of method development and validation.     

Determination of enoxacin and ofloxacin by capillary electrophoresis with electrochemiluminescence detection in biofluids and drugs and its application to pharmacokinetics

A novel and sensitive method for the simultaneous determination of enoxacin and ofloxacin has been established using capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection based on the ECL enhancement of tri(2,2‐bipyridyl)ruthenium(II). The conditions for sample solvent type, CE separation and ECL detection were investigated systematically. The analytes were well separated and detected within 7 min. The limits of detection (S/N = 3) of enoxacin and ofloxacin are 9.0 × 10^?9 and 1.6 × 10^?8 mol/L, respectively. The precisions (RSD%) of intraday and interday are less than 2.1 and 4.0%, respectively. The limits of quantitation (S/N = 10) of enoxacin and ofloxacin are 3.2 × 10^?7 and 5.4 × 10^?7 mol/L in human urine samples and 4.1 × 10^?7 and 6.9 × 10^?7 mol/L in human serum samples, respectively. The recoveries of enoxacin and ofloxacin at different concentration levels in human urine, serum and eye drop samples are between 94.0 and 106.7%. The proposed method was successfully applied to the determination of the enoxacin and ofloxacin in human urine, serum and eye drop samples and the monitoring of pharmacokinetics of ofloxacin in human body. Copyright © 2009 John Wiley & Sons, Ltd.     

The study of polyoxometalates formation using capillary zone electrophoresis

The formation process of polyoxometalates [PMo₁₂O₄₀]^3? and [PMo_{12 ? x}V_xO₄₀]^?3?x has been studied in aqueous solutions of 0.1 M malonate buffer at pH 2.8–3.0 using CZE. Two different approaches, pre‐capillary and in‐capillary, were examined and compared. In precapillary mode, the reaction mixture of the reactants and reaction products was injected into the capillary followed by the separation procedure. In in‐capillary mode, the sequential input of the reagents and running electrolyte into the capillary and the species separation occurs simultaneously. The optimal parameters of in‐capillary separation were established as functions of applied voltage and the length of the intermediate buffer zone between the reagents in the capillary. As a result the best‐compromise conditions for the separation of the mixtures containing the reactants, intermediates, and reaction products, in order to achieve the best efficiency, symmetry, and peak areas, were achieved at ?18 kV and the input parameter of 900 mbar·s. It was also shown that in‐capillary mode is more informative than pre‐capillary mode for studying the complex compound formation process.     

毛细管电泳分析中手性化合物的定性检测

毛细管电泳(CE)作为一种新型分离分析技术,具有分离效率高、分析速度快、样品用量少、分离模式灵活多样等众多优势,在手性物质分离等领域应用广泛。在以往的工作中,手性化合物的CE分离模式、手性拆分剂选择及提高分离度等研究已作了详尽报道,而成功分离后的手性物质定性、对映体出峰顺序确认等问题也至关重要。该文以CE手性化合物分离分析中是否依赖标准品分类,及其定性检测方法进行了总结。利用CE分离分析手性样品,若待测物有手性对映体标准品时,其定性通常通过比较标准品迁移时间或标准加入法完成。基于检测器的不同,依赖标准品的CE分析主要分为光学、质谱和电化学3类检测模式。其中光学检测又包含紫外-可见(UV)、激光诱导荧光(LIF)、化学发光(CL)等多种检测方式。不同的检测方式决定了样品前处理方式的差异,随之形成的谱图及手性化合物定性方式也大相径庭。当缺少手性对映体标准品时,可用的CE手性分离分析方法主要有酶消解和抗体添加法、计算法等。前两种方法主要依据对映体与特定消解酶或抗体间的相互作用,完成手性物质的选择定性,适用范围均较局限。相比较,借助理论计算,使CE结合圆二色光谱(CD)的计算法操作简单,定性准...     

Temperature effects in capillary electrophoresis 2: Some theoretical calculations and predictions

Summary The dependence of the temperature excess, , in the capillary bore on the applied power, EI, is considered for both natural and forced convective cooling, using classical heat equations. The dependence of on EI is found to be linear for forced convection but not for natural convection. Use of forced convective cooling and capillaries of large outer diameter reduces . Direct comparison of the performance of different systems can be achieved by consideration of . Column performance is ultimately limited by thermal gradients across the capillary bore.     

Rectangular capillary electrophoresis: Some theoretical considerations

Summary A theoretical study of the use of rectangular columns in capillary electrophoresis (CE) is presented. It was done employing some of the most important parameters that normally define the performance of CE separations, i.e. thermal effect, analysis speed, efficiency and sample capacity. Theoretical results from rectangular and cylindrical capillaries are compared in terms of the aforementionated parameters. Also, an estimate of the additional zone broadening that arises from the noninfinite dimension in the y-direction, in which the channel has its largest dimension, is presented.     

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R_f), which provided information about the binding strength and the overall charge of the protein‐ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.     

毛细管电泳-安培检测法测定3种拟肾上腺素药物

建立了毛细管电泳-安培检测法测定盐酸去氧肾上腺素(phenylephrine hydrochloride, PHE)、重酒石酸间羟胺(metaraminol bitartrate, MR)和盐酸异丙肾上腺素(isoprenaline hydrochloride, IP)3种拟肾上腺素药物的方法。检测电位为0.950 V(Ag/AgCl为参比电极),硼酸盐浓度为50 mmol/L(pH 10.00),分离电压为18 kV,进样时间为10 s。在最佳实验条件下,3种物质在18 min内达到基线分离,在2～100 μmol/L浓度范围内峰面积与浓度呈良好的线性关系,线性相关系数不小于0.9991。盐酸去氧肾上腺素、重酒石酸间羟胺和盐酸异丙肾上腺素的检出限分别为0.8、0.8和1.0 μmol/L。将所建立的方法应用于针剂样品的分析,结果令人满意。     

免疫亲和毛细管电泳的研究进展

免疫亲和毛细管电泳方法结合了免疫分析的高特异性和毛细管电泳分离的高效、快速、样品用量少等优点,是复杂样品中特定组分分析的重要方法之一。激光诱导荧光检测器的使用以及毛细管电泳分离前免疫预富集过程的引入,可以进一步提高分析测定的灵敏度,使其能够用于痕量物质的高灵敏测定。本文结合作者所在课题组的工作,对免疫亲和毛细管电泳的两种主要模式,即均相的毛细管电泳免疫分析(CEIA)和非均相的免疫亲和毛细管电泳(IACE)的研究进展进行了综述。     

水相体系毛细管电泳与非水体系毛细管电泳分离手性药物的比较

将非水毛细管电泳(NACE)与传统的水相毛细管电泳从有机溶剂的添加、离子强度的改变以及pH的改变等方面作了一系列的对比性研究。在有机组分从10%增加到100%的过程中,被分离样品的迁移速率逐渐降低,其分离效率先呈升高的趋势,达到一个最大值后,在呈下降的趋势。这主要是由于缓冲介质粘度和介电常数的变化以及管壁Zeta电位的变化所造成的。在非水体系中,最佳分离pH要比在水相体系中高,其分离效率也是随着离子强度的增大而升高。     

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.