Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

Protein adsorption in fused-silica and polyacrylamide-coated capillaries

The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.     

A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients.We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA),satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.     

Enantiomeric analysis of rivastigmine in pharmaceuticals by cyclodextrin-modified capillary zone electrophoresis

Chiral separation method development is usually very time-consuming due to the diversity in chemical structures of pharmaceutical drug substances as well as the suitable separation conditions and the problem to choose the appropriate chiral selector. This paper shows capillary zone electrophoresis (CZE) which was developed for chiral separation of a basic compound - rivastigmine (RIV) using 30 cm × 50 μm i.d. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm), amine-modified phosphate buffer of pH 2.5 and sulfated-β-CD (S-β-CD) as chiral selector. Other selected native or derivatized cyclodextrins (CDs) were also tested: β-CD (5, 30 mM), carboxymethyl-β-CD (5, 30 mM), dimethyl-β-CD (15 mM), hydroxypropyl-β-CD (5, 30 mM), hydroxypropyl-α-CD (5, 30 mM) and hydroxypropyl-γ-CD (5, 30 mM). Complete enantiomeric separation of RIV was achieved at 20 kV, 18 °C and detection at 200 nm within 8 min with R.S.D. for the absolute migration time reproducibility of less than 2.1%. Rectilinear calibration range was 5.0-500.0 μM of each enantiomer (r = 0.9994-0.9995). The CZE method proposed was used for the control of chiral purity of pharmaceutically active S-RIV and for the analysis of Exelon caps preparation.     

Capillary Zone Electrophoresis of Eleven Priority Phenols with Amperometric Detection

A scheme for separation and detection of eleven priority phenols using capillary zone electrophoresis (CZE) coupled with amperometric detection is described. With a capillary of I.D. 50 μm and length 62.5 cm at 9 kV and an electrophoretic buffer of 20 mM CHES (pH 10.1), complete separation of the eleven compounds was achieved in less than 17 min. Amperometric detection was carried out using a carbon fiber microelectrode of diameter 9 μm inserted into the end of the detection capillary. Linearity over two orders of magnitude was generally obtained for the eleven priority phenols. With an electrode potential+1.10 V (vs. Ag/AgCl reference), the concentration limits of detection were in the sub-ppm (10^?6 M) level. This method was successfully applied to analysis of priority phenols in industrial waste water.     

合并带流动注射分光光度法研究硫羟乳酸掩蔽性能

本文运用合并带流动注射分析技术，建立了以Ｙｂ＾３＋ＸＯ显色体系为参考体系研究硫羟乳酸掩蔽性能的方法，在ｐＨ５．６硫羟乳酸能掩蔽Ｓｎ＾４＋，Ｂｉ＾３＋，Ｔｌ＾３＋，Ｈｇ＾２＋，Ｃｕ＾２＋，Ｃｒ＾３＋和Ｃｄ＾２＋测定Ｙｂ＾３＋的线性范围为１．３６×１０＾－６～２．７２×１０＾－５ｍｏｌ／Ｌ，采样频率可达１２０次／ｈ。     

Chiral Recognition of Phenylacetic Acid Derivatives by Aminated Cyclodextrins

Chiral recognition of mandelic acid ( 1), acetylmandelic acid ( 2), 1-methoxyphenylacetic acid ( 3), phenylsuccinic acid ( 4), 2-phenylpropanoic acid ( 5) and ibuprofen ( 6) in their anionic forms by protonated 6A-amino-6A-deoxy--cyclodextrin (mono-NH3+--CD) and 6A,6D-diamino-6A,6D-dideo xy--cyclodextrin (di-NH3+--CD) has been studied by means of capillary zone electrophoresis (CZE) and 1H NMR spectroscopy. Both methods show the preferable guests for mono-NH3+--CD to be the (R)-enantiomers of 1, 3 and 5 and the (S)-enantiomers of 2, 4 and 6. Cooperative work of Coulomb interactions and inclusion is essential for chiral recognition of these anionic guests.     

Determination of rimantadine in pharmaceutical preparations by capillary zone electrophoresis with indirect detection or after derivatization

Summary Rimantadine is synthetic analog of amantadine; both are antiviral agents used for prophylaxis and treatment of influenza A. A capillary zone electrophoretic (CZE) procedure for the determination of rimantadine has been developed. As the direct determination of rimantadine is poorly sensitive because the compound is almost transparent in the UV/Vis range, several indirect methods were studied. Two were found to be the particularly useful: (a) indirect detection using 5 mM 4-methylbenzylamine in 1:4 methanol-water as absorbing background electrolyte, with detection at 210 nm, and (b) derivatization of rimantadine with 1,2-naphthoquinone-4-sulfonic acid in alkaline medium and subsequent determination of the derivative by CZE (40 mM tetraborate, pH 9.2, detection at 280 nm). Uncoated capillary tubing, 44 cm length ×75 μM i.d., was used for both determinations. The detection limits were 0.1 and 2 ppm for methods a and b, respectively. The methods were used to determine rimantadine in pharmaceutical products and for dissolution testing of Flumadin^? tablets.     

A quick method for the simultaneous determination of ascorbic acid and sorbic acid in fruit juices by capillary zone electrophoresis

A method of quickly determining ascorbic acid and sorbic acid by capillary zone electrophoresis with ultraviolet detection was developed. The choice of background electrolyte, wavelength, injection time and applied voltage were discussed. Ascorbic acid and sorbic acid were well separated in 80 mmol L⁻¹ boric acid-5 mmol L⁻¹borax (pH = 8.0) in 5 min at the detecting wavelength of 270 nm. Under the optimum condition, the method has linear ranges of 2.54-352.00 mg L⁻¹ for ascorbic acid and 1.08-336.39 mg L⁻¹ for sorbic acid with the detection limit of 1.70 mg L⁻¹ for ascorbic acid and 0.54 mg L⁻¹ for sorbic acid, respectively. Other organic acids in fruit juices have no effect on the detection. This method is very feasible and simple and can be used to detect ascorbic acid and sorbic acid in fruit juices.