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.     

Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.     

小分子大豆多肽的分离检测研究

最近的研究表明,多肽特别是二肽和三肽更易被人体吸收,此外小分子大豆肽还具有很好溶解性、稳定性、低粘度和许多生理活性,如降血压,降胆固醇,抗疲劳等,因此大豆肽广泛应用在食品、化妆品、药品等领域 [1,2].     

High-performance capillary electrophoresis of unsaturated oligosaccharides derived from glycosaminoglycans by digestion with chondroitinase ABC as 1-phenyl-3-methyl-5-pyrazolone derivatives.

This paper proposes a new method for simultaneous analysis of unsaturated disaccharides derived from glycosaminoglycans by enzymatic digestion with chondroitinase ABC, based on high-performance capillary electrophoresis (HPCE) of their 1-phenyl-3-methyl-5-pyrazolone derivatives. The O-sulphate group is stable in this derivatization, and this method allows reproducible microdetermination of glycosaminoglycans. This paper also demonstrates the applicability of this method to estimation of urinary chondroitin sulphates. Urinary creatinine as an inherent internal standard could also be estimated by HPCE, though in another mode of separation, i.e. ion-exchange electrokinetic chromatography.     

Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.     

高效毛细管电泳电化学检测器的研制

本文提出了一种用于高效毛细管电泳的新型安培电化学检测器设计，使用Ｎａｆｉｏｎ溶液制作的ＨＰＣＥ／ＥＤ接口，可有效地隔开两化学系统的干扰，且不引入附加体积，经对有机酚类化合物的胶束电动毛细管色谱分离与电化学检测知，该系统性能优良，对对苯二酚的检出限为３０ａｍｏｌ。     

寡糖的毛细管电泳分析

多种寡糖经α－萘胺衍生化后，用硼砂作为电泳介质，实现了高效毛细管电泳分离。比较了毛细管区带电泳和胶束毛细管电动色谱分离寡糖α－萘胺衍生物的电泳行为，对影响分离度的诸因素进行了考察，选择了最佳分离条件。     

Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with L- and D-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.     

Separation of phosphorylated histone H1 variants by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone H1 variants. With an untreated capillary (50 cm x 75 microns I.D.) the electrophoresis was performed in about 15 min. Inconvenient interactions of these highly basic proteins with the capillary wall were eliminated by using 0.1 M sodium phosphate buffer (pH 2.0) containing 0.03% hydroxypropylmethylcellulose. Under these experimental conditions the histone H1 variants H1b and H1c obtained from mitotic enriched NIH 3T3 fibroblasts and isolated by reversed-phase high-performance liquid chromatography were clearly separated in their non-phosphorylated and different phosphorylated forms. This result was confirmed by acid-urea gel electrophoresis, comparison with non-phosphorylated histones H1b and H1c, isolated from quiescent NIH 3T3 cells, and incubation of multi-phosphorylated histone H1b with alkaline phosphatase and subsequent acid-urea and capillary electrophoresis. The results illustrate that the application of HPCE to the analysis of histone modifications provides a new alternative to traditional gel electrophoresis.     

对映异构体的高效毛细管电泳分离与测定

高效毛细管电泳是８０年代发展起来的一种高效、快速的新型分离技术，在对映异构体分离方面有着广泛的应用前景，本文介绍了这一新型分离技术用于对映体分离的基本原理，并列举了一些对映异构体分离的实例。     

Comprehensive two-dimensional chromatography and capillary electrophoresis coupled with tandem time-of-flight mass spectrometry for high-speed proteome analysis

A comprehensive two-dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high-performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve-free hydrodynamic sampling interface. The CZE effluents were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix sheath flow via CE-MALDI interface, and then directly deposited on the MALDI target at a 3 s time-interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high-throughput analysis and application in proteomics.     

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE).O-andN-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection.N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.     

Determination of thiopental in human serum and plasma by high-performance capillary electrophoresis-micellar electrokinetic chromatography.

The quantitation of thiopental in human serum and plasma was investigated using high-performance capillary electrophoresis (HPCE) in a micellar configuration and the results were compared with reversed-phase high-performance liquid chromatography (HPLC). Thiopental and an internal standard (carbamazepine for HPCE and thiamylal for HPLC) were extracted from serum or plasma using pentane and a phosphate buffer (pH 6.4). HPCE analysis took place in a phosphate-borate buffer with 50 mM sodium dodecyl sulphate using an automated instrument and HPLC was performed with a C8 column and a mobile phase of phosphate buffer-acetonitrile (65:35, v/v). HPCE and HPLC data from 66 patient samples compared well based on linear regression analysis. However, estimates obtained with the inclusion of the internal standard were lower than those based on the sample peak only. This example allows the elucidation of the advantages of using HPCE as an assay methodology for the therapeutic monitoring of thiopental and other drugs.     

Purification of the Escherichia coli K5 capsular polysaccharide and use of high-performance capillary electrophoresis to qualitative and quantitative monitor the process

A rapid, highly sensitive, and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K5 bacteria Bi8337/41 010:K5:H4. This natural polysaccharide having the structure of a desulfo-heparin composed of -4)-alphaGlcUA-(1,4)-alpha-GlcNAc-(1- is separated (GlcUA = D-glucuronic acid; GlcNAc = D-glucosamine) and qualitatively and quantitatively determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient > approximately 0.99) was found for the polymer over a wide range of concentrations, from approx. 60 to 1500 ng, with a detection sensitivity of < approximately 60 ng. Furthermore, this qualitative and quantitative HPCE approach was applied to the K5 extraction and purification process from cultured bacteria in several stages. HPCE was also able to separate several molecular species mainly due to the presence of polysaccharides of distinct and increasing mean chain lengths. A linear relationship was found for migration time and log molecular mass of different K5 polysaccharide species, and this model was used to calculate the molecular mass of the main K5 species producing a result of approx. 17,000, also confirmed by high-performance size-exclusion chromatography analysis, yielding approx. 17 200.     

High-performance capillary electrophoresis of proteins from the fluid lining of the lungs of rats exposed to perfluoroisobutylene.

Measurements of the biochemical constituents in the fluid lining of the lung can be used for diagnosing and assessing lung disorders. To facilitate such measurements, a high-performance capillary electrophoresis (HPCE) method has been developed by which the proteins in lung fluid can be analyzed. The lung fluid was obtained by a bronchoalveolar lavage procedure using 48 ml of physiological saline to wash out the lung fluid of rats. The proteins were precipitated from the fluid with 10 volumes of acetone and concentrated by dissolution in 2 ml of water containing 0.2% of trifluoroacetic acid. Aliquots of these samples (5 microliters) were then injected into a Bio-Rad HPE-100 capillary electrophoresis instrument fitted with a 50 cm x 50 microns I.D. coated capillary filled with 0.1 M phosphate buffer (pH 2.5). With phosphate buffer in the outlet electrode chamber (cathode) and water in the inlet electrode chamber (anode), the proteins were loaded into the capillary electrophoretically for 10 s at 10 kV constant voltage. The inlet electrode chamber was then filled with phosphate buffer and HPCE was performed at 8 kV constant voltage. Six major protein fractions were resolved in 35 min, and were detected by UV absorption at 200 nm. The procedure was used to compare the lung fluid proteins of normal untreated rats with those of rats exposed by inhalation to perfluoroisobutylene (PFIB) at a concentration of 100 mg/m3. It was found that PFIB induced pulmonary edema involving a translocation of blood compartment proteins into the lung's alveolar compartment. Comparison of the HPCE fractions with similar fractions obtained by high-performance liquid chromatography confirmed albumin, transferrin and IgG as three major proteins translocated into the alveolar space after PFIB exposure.     

Capillary electrophoresis for diagnosis and studies of human disease, particularly metabolic disorders.

High-performance capillary electrophoresis (HPCE) has been used in a multicomponent analytical system designed to diagnose and study human diseases, particularly metabolic disorders. Comparative analyses, using HPCE, high-performance liquid chromatography (HPLC) and an automated amino acid analyser, were carried out on urine and blood samples from patients with homocystinuria, cystinuria, glutathione synthetase deficiency and adenylosuccinase deficiency. HPCE of the sulphur-containing amino compounds, derivatized with monobromobimane and detected by fluorescence spectroscopy, was a quick and simple alternative to classical amino acid analysis. The detection of the characteristic succinylpurines associated with adenylosuccinase defect was equally well achieved with HPLC and HPCE (absorbance detector). Owing to the possible connection between deficiency of taurine (2-amino-1-ethanesulphonic acid) in the heart and the development of cardiomyopathy and heart failure, a simple HPCE method was developed for the determination of taurine in sub-milligram samples of biopsies of the myocardium. The homologue 3-amino-1-propanesulphonic acid was the internal standard, and derivatives of 9-fluorenylmethyl chloroformate and fluorescence detection were used. It is suggested that the potential of HPCE to analyse small volumes should be exploited in biomedicine and clinical diagnosis to analyse sub-milligram samples of tissue biopsies and cells.     

Comparison of ion-pair chromatography and capillary zone electrophoresis for the assay of organic acids as markers of abnormal metabolism

The abnormal organic acids in urine are closely related with physiological metabolism. To determinate the low-molecular-mass metabolites in human biological fluids, although there were some previous reports by both of capillary electrophoresis and ion-exchange high-performance liquid chromatography, but it was rarely found by reverse phase of liquid chromatography using ion pair reagent. The objective of this study was aimed to suggest and compare two methods, an additional chromatographic method-ion-pair chromatography (IPC) and a sharp capillary zone electrophoresis (CZE), to determinate organic acids, acting as the abnormal metabolic markers, namely uric acid, orotic acid, pyruvic acid, alpha-ketoglutaric acid, fumaric acid, and hippuric acid. The proposed method of IPC possessed both the extreme stability for column and the good results of reproducibility, linearity and detection limit. The optimum mobile phase was 22% methanol and 10 mM tetra-n-butyl ammonium hydrogen sulfate (pH 4) by gradient elution. As well as the optimum condition of CZE was 5% acetonitrile and 0.5 mM CTAB in phosphate buffer. From the results, CZE showed better recovery and sharp lucid electropherogram. Finally, the two proposed analytical methods were applied to assay human urine with direct and spiked analysis. CZE showed good potency to overcome the sample-to sample variation with standard deviation less than 10%. By comparison results of urinary spiked analysis between IPC and CZE by statistical paired t-test, the results were evaluated no significant difference under P < 0.05. The quantitative linearity of both methods was fitted in application of clinical biological analysis even with 50-fold dilution.     

Analysis of linear alkylbenzenesulfonates by capillary zone electrophoresis with large-volume sample stacking

A systematic investigation of optimal conditions for determining the homologues of linear alkylbenzenesulfonates (LAS) by capillary zone electrophoresis (CZE) using the large-volume sample stacking technique was presented. The most effective sample stacking and separation conditions was 20 mM borate buffer with 30% acetonitrile at pH 9.0, and the sample hydrodynamic injection of up to 90 s at 4 p.s.i. (1 p.s.i. = 6,892.86 Pa) (around 711 nl). Under such conditions, approximately a 100-fold enrichment factor was achieved based on peak heights. The reproducibility of migration time and quantitative results of stacking CZE can be improved by using internal standards. Quantitation limits of the homologues of LAS were 0.002-0.01 mg/l under these enrichment conditions. The analysis of real samples of laundry and dishwashing detergents was performed. The established high-performance liquid chromatography method was applied to evaluate the stacking CZE method, and compatible results were obtained.     

Separation of capsular polysaccharide K4 and defructosylated K4 by high-performance capillary electrophoresis

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. The two polyanions, K4 and defructosylated K4, are separated and readily determined within 30 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship was found for the two polysaccharides over a wide range of concentrations, from approximately 30 ng (0.5 microg/microL) to 210 ng (3.5 microg/microL). The described method was used to evaluate the defructosylation process of K4 under drastic acid conditions.     

Determination of nonsteroidal anti-inflammatory drugs in human specimens by capillary zone electrophoresis and micellar electrokinetic chromatography

Therapeutic drug monitoring of anti-inflammatory drugs is necessary for the identification of the agents that cause toxic events and for the decision on the treatment for intoxication. Recently, capillary electrophoresis (CE) has been developed for the simple and rapid analyses of a variety of chemical agents. Micellar electrokinetic chromatography (MEKC) can separate acidic, neutral and basic anti-inflammatory drugs in serum. Furthermore, serum samples are directly applied to the CE system without any pretreatments, and some anti-inflammatory drugs can be separated from serum albumin in the MEKC analysis. On the other hand, capillary zone electrophoresis (CZE) enables us to determine a few microg/mL levels of acidic anti-inflammatory drugs with simple running buffer and stacking technique. A rapid and simultaneous determination of several analgesic anti-inflammatory agents, including ibuprofen, acetaminophen, indomethacin, and salicylic acid in human serum has been developed by using CZE. Therefore, the CZE and MEKC analysis may become a potentially useful alternative to high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring, particularly in serum of patients suffering from intoxication by overdosage of nonsteroidal anti-inflammatory agents.