On-line post-capillary affinity detection of immunoglobulin G subclasses and monoclonal antibody variants for capillary electrophoresis

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-₁-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.     

On-line solution stability determination of pharmaceuticals by capillary electrophoresis

Summary Good agreement between the impurity levels in a batch of a related impurity of ranitidine were obtained by CE and HPLC. A solution of the impurity was positioned on the CE autosampler and analysed sequentially. The extent of degradation was monitored by loss of main peak and the formation of two principal degradation products. It was found that after 9.25 hours only 2% area/area of the original impurity remained. Buffering of the sample solution to pH 7 was shown to minimise this degradation.Unattended in-situ stability testing of an solution of the impurity in water was performed by CE.     

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.     

A technique for capillary joining in capillary electrophoresis

Summary Aspects of cracking and joining capillaries have been investigated. Capillary coupling was achieved using various methods. The most successful used hydrofluoric acid-etched capillaries to form male and female ends which were then joined together. This type of joint was used to connect sections of capillary of similar and different internal diameters with minimal loss in resolution, peak width and number of theoretical plates. (Uridine and hypoxanthine was used as a test mixture). For hypoxanthine on a 50 m/50 m etched joined capillary 10 cm from the detector window the number of theoretical plates was 96.6% of that for hypoxanthine on an unbroken capillary. Following the relative success of capillary joining, a coupled capillary flowcell (50 m/200 m) was produced and evaluated.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

On-line preconcentration capillary electrophoresis for purity profiling of synthetic peptides

In this work, an on-line preconcentration capillary electrophoresis method was optimized and evaluated for the purity control of the biologically active synthetic peptide fas-F (a 28-residue long fragment of fasciculin-1) and applied for the purity profiling of angiotensin I, oxytocin, bradykinin and luteinizing hormone releasing hormone. The laboratory-made device of the analyte concentrator cartridge consisted of a fused-silica capillary piece (150 μm i.d.×8 mm in length) packed with silica-based C18 reversed-phase chromatographic material and coupled on-line near the inlet of the separation capillary (bare fused-silica capillary, 75 μm from the concentrator to the detector). Separation of impurities present in assayed samples was achieved by using 25 mM potassium dihydrogen phosphate, pH 3.5, as running buffer and a mixture of acetonitrile: running buffer, 75:25 (v/v), as elution buffer. The intra-day relative standard deviation (R.S.D.) values for migration times ranged from 3.4 to 4.2 and 2.2-2.6% for peak areas. The inter-day R.S.D. values were 5.6-7.1 and 4.6-5.1% for migration times and peak areas, respectively. The impurity profiles obtained for each peptide were compared by CZE and on-line preconcentration CE. The proposed method allowed the preconcentration and separation of impurities with greater selectivity and higher sensitivity (100-200-fold) with respect to capillary zone electrophoresis without on-line preconcentration.     

Purity control of oxytetracycline by capillary electrophoresis

The applicability of capillary electrophoresis for the purity control of oxytetracycline (OTC) was investigated. OTC is a broad-spectrum antibiotic belonging to the group of the tetracyclines. Several related substances can be present due to fermentation or degradation, such as 4-epioxytetracycline, -apooxytetracycline, β-apooxytetracycline, anhydrooxytet racycline, 2-acetyl-2-decarboxamidooxytetracycline, tetracycline and 4-epitetracycline. Using fused-silica capillaries, the influence of buffer type, buffer pH and buffer concentration were investigated. In all cases 1 mM EDTA was added to prevent metal-ion complexation. The influence of the buffer counter-ion type was examined. Consequently, some instrumental parameters were changed such as capillary length and diameter as well as capillary temperature and applied voltage. The following method is finally proposed: fused-silica capillary, l (effective length) = 38 cm, L (total length) = 44 cm, 50 μm I.D.; buffer, sodium carbonate 20 mM-EDTA 1 mM, pH 11.25; voltage, 10 kV; temperature, 10°C. Linearity, limit of detection and limit of quantitation were determined as well as the relative standard deviations for all the analytes involved. This method is less selective then existing liquid chromatographic methods but it may be used as a complementary tool in purity control and stability studies.     

Investigation of multivalent interactions between conjugate of quantum dots with c‐Myc peptide tag and the anti‐c‐Myc antibody by capillary electrophoresis with fluorescence detection

Herein, we report an assay for detecting the binding of a multivalent peptide and antibody within a capillary with the use of fluorescence coupled capillary electrophoresis. Quantum dots and a c‐Myc tag containing peptide EQKLISEEDLG₄H₆ were injected sequentially and formed a multivalent quantum dot‐EQKLISEEDLG₄H₆ assembly within the capillary. The efficiency of the quantum dot‐peptide self‐assembly was affected by the peptide/quantum dot molar ratio, sampling time, and interval time. Finally, the binding of the monoclonal anti‐c‐Myc antibody and the multivalent quantum dot‐EQKLISEEDLG₄H₆ ligand was studied using an in‐capillary assay. The microscopic dissociation constant for the self‐assembly of monoclonal anti‐c‐Myc antibody and quantum dot‐EQKLISEEDLG₄H₆ was determined to be 14.1 μM with a stoichiometry of the peptide‐antibody complex of 1.7 determined after fitting this to the Hill equation. This method can be further extended to detect a wide range of biomolecule–biomolecule binding interactions.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

On-line microheterogeneity analysis and rapid phenotyping of haptoglobin by capillary electrophoresis using sodium dodecyl sulfate as additive

To improve the detection sensitivity and determine phenotypes of haptoglobin (Hp), a prefilling technique was developed and tested in capillary electrophoresis (CE) with UV–vis absorbance detection. Adding 0.01% sodium dodecyl sulfate (SDS) to the protein sample and 0.1% SDS to the prefilling buffer solution, on-line stacking and microheterogeneity separation of Hp were achieved. In addition, the influences of pH, buffer concentration, sample and prefilling buffer SDS concentration upon resolution were examined. Under optimized conditions, Hp-microheterogeneity was well resolved and two phenotypes of Hp (Hp 1-1 and Hp 2-2) were differentiated. This method was applied to the analysis of sera from normal individuals and β-Thalassemia patients. After the depletion of albumin (HSA) and immunoglobulin G (IgG), this method allowed to determine two phenotypes in different individuals and to detect the decrease of Hp in β-Thalassemia patients. Featuring high efficiency, speed and simplicity, the proposed method shows great potential for use in clinical diagnosis and proteome research.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

High-performance capillary electrophoresis using open tubes and gels

Summary This paper overviews several aspects of high performance capillary electrophoresis (HPCE), a promising new method of analytical and micropreparative separation of biochemically important samples. The basic migration equations of electrophoresis are first presented and the benefit of high fields for rapid analysis and high performance emphasized. Since power is generated with high voltages, Joule heating results and this heat must be dissipated. The use of capillary columns is shown to be important in efficient heat removal and in minimizing the temperature differences within the column. The various factors influencing band broadening are next described, and it is shown how plate counts close to 10⁶ can be achieved. Various results from our laboratory on open tube and gel columns are then presented to illustrate the potential of this method. Chiral resolution of dansylated amino acids using a chiral metal chelate micelle in open tube HPCE is shown. With the gel columns, the baseline separation of a 2-chain variant from methionine growth hormone (met-hGH) under non-denaturing conditions at fields close to 1000 V/cm is presented. Finally, the micropreparative purification of a 20-mer oligonucleotide using the gel column is described.     

Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis

Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogeneity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.     

Analysis of doxycycline by capillary electrophoresis

Summary Doxycycline is a semi-synthetic broad spectrum antibiotic with improved serum half-lie. Potential impurities are 4-epidoxycycline, 6-epidoxycycline, 4,6-epidoxycycline, metacycline and 2-acetyl-2-decarboxamidodoxycycline. Method development has been undertaken to investigate the potential of capillary electrophoresis for the analysis of doxycycline. The influence of buffer type, buffer pH and concentration was systematically examined, then that of capillary temperature and applied voltage. All the potential impurities could be separated at 15 °C on a 44 cm × 50 μm I.D. fused silica capillary (effective length to detector, 38 cm) with sodium carbonate (70 mM) - EDTA (1 mM), pH 10.50, as background electrolyte and with a voltage of 12 kV. The relative standard deviation was 2.2 % for doxycycline. The limit of detection and quantification for doxycycline were 0.2 and 0.4 %.     

On-line pre-concentration and UV determination of DNA fragments by dynamic coating capillary electrophoresis and its application to detection of genetically modified oilseed rape based on PCR

In this work, it was demonstrated that on-line pre-concentration and separation of DNA fragments within bared silica column by dynamic coating capillary electrophoresis and UV detection. The DNA fragments were pre-concentrated with long electrokinetic injecting time (99 s), peak height increased dramatically as a function of injection time, especially for shorter length DNA. The concentration sensitivity of DNA fragments can be improved from 20- to 100-fold relative to a normal injection (5 s). The electro-osmotic flow (EOF) and DNA-wall interactions within the capillary were eliminated effectively by dynamic coating method. Employing 0.5% poly(ethylene oxide) (PEO) in Tris-phosphate-EDTA (TBE) buffer as sieving matrix, DNA fragments, ranging from 11 to 657 bp, were separated within 20 min. The linear coefficient of linear relation between the migration and DNA length is 0.999. The DNA fragments amplified from transgenic oilseed rape by polymerase chain reaction (PCR) were separated and detected by this method, demonstrating the potential use of this method for effective DNA analysis and detection of genetically modified organisms (GMO).     

Speciation of oxidation states of elements by capillary electrophoresis

Progress made in the last five years in the application of capillary electrophoresis methods to chemical speciation of elements is reported on the basis of over 100 literature references. The main trends observed include development of new on‐ and off‐capillary derivatization methods, application of new detection methods, and especially coupling of CE separation systems to powerful atomic spectroscopy and mass spectrometry instruments with various ionization techniques, providing either a sensitive element‐specific detection method or a third dimension for high performance separation. Besides numerous CZE and MEKC capillary electrophoresis methods only very few examples of CE speciation with capillary electrochromatography can be found. Concerning the chemical forms of elements determined, the new procedures developed are mostly focused on redox speciation of various oxidation states of elements, metal‐bound high molecular compounds, and organometallic species.