Analysis of sialo-N-glycans in glycoproteins as 1-phenyl-3-methyl-5-pyrazolone derivatives by capillary electrophoresis

A method for the analysis of the sialo-N-glycans in glycoproteins was established by the electrokinetic chromatography mode of capillary electrophoresis (CE) in sodium dodecyl sulfate (SDS) micelles as 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives, using sialo-N-glycans in fetuin as a model. Six major and some minor peaks were observed for the N-glycans in fetuin, which were well separated from each other using 50 mM phosphate buffer, pH 6.0, containing SDS to a concentration of 30 mM in an uncoated fused-silica capillary, and these peaks were assigned to sialo-N-glycans having either of the biantennary or β1-3/β1-4 linked galactose-containing complex type triantennary N-glycans as the basic structures, by an indirect method based on the assignment of the peaks in high-performance liquid chromatography separated in parallel with CE and peak collation between these two separation methods. The attaching position of the sialic acid residue was determined using the linkage preference of neuraminidase isozymes. The established system is considered to be useful for routine analysis of microheterogeneity of the carbohydrate moiety of this model glycoprotein from the following reasons: (1) the derivatization with PMP proceeds quantitatively under mild conditions without causing release of the sialic acid residue, (2) the derivatives can be sensitively detected by UV absorption, (3) the procedure is simple, rapid and reproducible. Preliminary results of N-glycan analysis for several other glycoproteins under these conditions are also presented.     

Determination of synthetic food dyes by capillary electrophoresis

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm × 75 μm I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of β-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.     

Rapid determination of drugs in biofluids by capillary electrophoresis Measurement of antipyrine in saliva for pharmacokinetic studies

A micellar electrokinetic capillary chromatography method was developed that permitted the resolution of antipyrine from endogenous compounds and its quantitation in neat saliva in as little as 1 min. Final conditions were: SpectraPhoresis 1000, 30(23) cm × 50 μm silica capillary, 50 mM sodium phosphate pH 9.6, 50 mM SDS, 10 s hydrodynamic load, detection scanning 200–300 nm or 260 nm, run 25 kV. To overcome the effects of Joule heating the capillary was cooled to 15°C. Sensitivity was <10 μM and linearity extended to 350 μM. Comparison with an HPLC assay demonstrated that hydrodynamic injection gave a loading bias unless samples and standards were of equal viscosity. For 75 samples from five subjects the correlation of CE vs. HPLC was then r = 0.99.     

Determination of melatonin in biological samples by capillary electrophoresis

The determination of melatonin (MLT) in physiological samples was investigated using capillary electrophoresis (CE). Mouse blood was collected in tubes containing EDTA, centrifuged at 1500 g for 20 min at 4°C, and stored at −20°C. Plasma samples were extracted with dichloroethane, centrifuged and the aqueous phase was discarded. Then the organic phase was evaporated to dryness. The residue was dissolved in deionized water and filtered with a microfilter (0.22 μm). Separations were carried out using a CE system equipped with a fused silica capillary [80 cm (effective length 52 cm)×75 μm I.D.] and an ultraviolet–visible detector (200 nm), and programmed to provide 25 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.7). Injection was performed hydrostatically by elevating the sample by 10 cm at the cathodic side of the capillary. The calibration curve, reproducibility, recovery and limit of detection were examined, and validation of the method was performed. The result showed that MLT in blood could be easily determined with the new method.     

银杏种子糖蛋白的SDS-PAGE分离及N-糖链的质谱分析

利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离了银杏种子中的糖蛋白组分, 进一步用氨水催化释放N-糖链, 并采用电喷雾离子质谱(ESI-MS)及在线液相色谱-质谱联用(LC-UV-MS/MS²)等技术对胶条上释放的N-糖链进行了定性定量分析. 结果表明, 从银杏种子中分离得到11种糖蛋白, 共检测到11条N-糖链, 包括高甘露糖型(4.88%)和复杂型(95.12%) 2种类型, 其中分子量为21000, 36000和50000的糖蛋白所释放的核心α-1,3-岩藻糖和β-1,2-木糖修饰的N-糖链所占比率较高, 分别为68.23%, 64.37%和75.09%. 本研究对进一步研究银杏糖蛋白功能具有重要意义.     

Separation of charged and neutral isotopic molecules by micellar electrokinetic chromatography in coated capillaries

Some unique separations are reported of pairs of deuterated and non-deuterated compounds by capillary zone electrophoresis (CZE) in coated capillaries in the absence and presence of surfactant micelles. Pyridine (pyridine-h₅) and [² H₅]pyridine (pyridine-d₅) could be separated in plain buffer (R = 1.1) and in 2% Nonidet P-40 (R = 1.5). Owing to the good separation obtained, it was possible to assess the degree of cross-contamination when “pure” isotopes were analysed. A 1:1 mixture of benzoic-h₅ and -d₅ acid was poorly separated in the absence of detergent (R = 0.39) but well separated in 50 mM sodium dodecyl sulphate (SDS) (R = 1.14). Benzyl-h₅ and -d₅ alcohol were reasonably well separated in a micellar system containing 50 mM SDS (R = 1.02) (a separation previously unreported in the literature. Benzene-h₆ and -d₆ were well separated again in presence of 50 mM SDS (R = 1.59). It appears that CZE offers a unique environment for the separation of positively and negatively charged and neutral isotope mixtures, previously reported only using RP-HPLC and GC.     

Synthesis of a LEC14 nonasaccharide, a core-fucosylated, biantennary N-glycan with a novel GlcNAc residue in the core region

The recently found core substitution of N-glycans termed LEC14 is characterized by a GlcNAc residue linked β(1,2) to the central β-mannoside. Starting from a pentasaccharide building block functionalized for core-fucosylated N-glycans the total synthesis of a protected LEC14 nonasaccharide was accomplished. The key step of the synthesis was the introduction of the additional β(1,2)-linked GlcNAc residue that was highly dependent on the solvent and appears to proceed via an amide acetal intermediate.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

Capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples from hydrothermal springs and fumaroles

A new application of capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples is presented. Ten fluid samples were collected from hydrothermal springs and fumaroles located in a volcanic zone of Deception Island, Antarctica. Anion separation was achieved in less than 6 min using indirect UV detection at 254 nm with a negative power supply (−15 kV). The electrolyte consisted of 4.7 mM sodium chromate, 4.0 mM electroosmotic flow modifier (OFM) hydroxide, 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid and 0.1 mM calcium gluconate (pH 9.1). Major anions (Cl⁻, SO₄², PO₄H²⁻, and CO₃H⁻) were measured using hydrostatic injection (10 cm for 30 s) at 25°C. Trace amounts of anions (F⁻, Br⁻, and NO₃⁻) were better determined by electromigration injection (4 kV, 10 s) at 15°C. Good reproducibility of the migration times (<0.72% RSD), a satisfactory linear response and accuracy as well as acceptable detection limits were successfully obtained.     

Analysis of water extracts from airborne dust samples by capillary isotachophoresis

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM β-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl⁻, K⁺, Na⁺, Ca²⁺) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.     

Ion chromatographic separation of alkali metal and ammonium cations on a C18 reversed-phase column

The separation of alkali metal (Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺) and ammonium cations on a C₁₈ reversed-phase column using three anionic surfactants [sodium 1-eicosyl sulphate, sodium dodecyl benzenesulphonate and sodium dodecyl sulphate (SDS)] is described. Two methods were examined: (a) “permanent” coating, with the use of a C₁₈ reversed-phase column previously coated with the surfactants; and (b) dynamic coating, with addition of the surfactants to the mobile phase. With method (a) the separation of the six cations was achieved with SDS. However, the retention times gradually decreased owing to dissolution of the SDS coating. Good separation was obtained with method (b), where 10 mM HNO₃ containing 0.1 mM SDS was used as the mobile phase with conductivity detection, and it was applied satisfactorily to real samples. The effect of system peaks on determination is also discussed.     

Sample stacking in laboratory-on-a-chip devices

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in cyclodextrin (CD)-modified capillary zone electrophoresis were investigated using a phosphate buffer (40 mM) at pH 3.0. We focussed on the separation of phenothiazines with the use of CDs at low concentrations. Three different CDs, including β-CD, hydroxypropyl-β-CD (HP-β-CD) and γ-CD, were chosen as chiral selectors. The results indicate that effective enantioseparation of phenothiazines, except for methotrimeprazine, is simultaneously achievable with addition of γ-CD at a concentration of 2.5–6.0 mM. The enantiomers of ethopropazine and trimeprazine are effectively separated with addition of HP-β-CD at low concentrations, in the range 0.4–6.0 mM, whereas those of promethazine and trimeprazine are baseline resolved with β-CD at much lower concentrations (0.02–3.0 mM) than with HP-β-CD. The results also confirm that the separation window is greatly enlarged at low CD concentrations. Moreover, the drastic variations of the electrophoretic mobility of phenothiazines as a function of CD concentration reveal that phenothiazines interact very strongly with CDs in the order γ-CD相似文献   

Comparison of standard capillary and chip separations of sodium dodecylsulfate-protein complexes

Conditions for converting a set of five standard proteins to electrochemically active sodium dodecylsulfate (SDS) complexes were worked out with the aim of using such complexes for conductivity detection with a a chip electrophoresis system. The results obtained were compared with standard capillary electrophoresis (37 cm (effective length 30 cm)×75 μm I.D. capillary, 10 kV, negative polarity at the inlet). The chip separations were run at 500 V per chip (100 V/cm) as compared to the standard capillary arrangement, which was run at 266.6 V/cm. For the capillary set-up the protein complexes were prepared in aqueous solution (Milli-Q water) made 10 mM with respect to SDS. If the SDS concentration was increased to 50 mM, the separation in the capillary was incomplete. On the other hand with the chip system both approaches yielded acceptable results. The chip separations were slightly (but not distinctly) shorter and offered better separations than the standard set-up. The concentration of the surfactant used for the preparation the complexes results in alternations of the elution sequence, which is preserved if the chip separation is used instead of the capillary set-up. Apparently the full capacity of protein–SDS binding is not exploited for the preparation of the adducts.     

Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-performance liquid chromatography with cyclodextrins and heparin as chiral selectors

The methods of separation of the enantiomers of the chiral drug oxamniquine are compared, between HPLC with either cyclodextrins and their related derivatives as chiral selectors in the mobile phase or immobilisedin a chiral stationary phase (as Cyclobond I and II) and between capillary zone electrophoresis (CZE) where the cyclodextrins are added to the buffer solution. The HPLC experiments, which included structured method optimisation were largely unsuccessful in resolving the enantiomers, with the exception of when a Chiral-AGP protein stationary phase was introduced into the programme. However although this chiral stationary phase provided baseline resolution of the enantiomers the stability of the method was suspect to small changes in the pH (0.2 units). In contrast the CZE method developed for both cyclodextrins and their derivatives gave good resolution of the enantiomers and method stability (R.S.D. <1%, N = 10 on precision). The basis of the interaction mechanism between selector and selectand was shown as a 1:2 relationship of cyclodextrin to analyte by NMR. In addition the polysaccharide, heparin was investigated as a chiral additive and excellent resolution of the oxaminiquine was achieved with 3 mM heparin in 50 mM sodium dihydrogenphosphate (pH 3.0) as buffer in CZE, which also gave a stable procedure. This method allowed the detection of each of the enantiomers in the presence of the other down to 0.23% (m/m). The overall composition of the heparin material from different sources can however be slightly variable and this can result in small differences in resolution capability.     

Highly sensitive capillary electrophoresis analysis of N-linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser-induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).     

Capillary electrophoresis of methylmercury with injection by sample stacking

A procedure for separation and quantitation of methylmercury by capillary electrophoresis using sample stacking as the injection technique is presented. The CE conditions have been optimized in order to separate the methylmercury from the excess cysteine peak and to concentrate large volumes of sample obtaining a low detection limit. Under the proposed operational conditions, the detection limit (S/N = 3) was 12 ng g⁻ and the limit of quantitation (S/N = 10) was 20 ng g⁻¹ with a linear range of 20–100 ng g⁻¹ (as methylmercury in samples). The method was tested using different reference materials with a certified methylmercury content.     

Ion-pair chromatography of polythionates and thiosulfate with detection based on their catalytic effects on the postcolumn azide–iodine reaction

The reduction of iodine with azide, catalyzed by polythionates (tri-, tetra-, penta- and hexathionate) and thiosulfate, has been utilized as a postcolumn reaction for chromatographic determination of these sulfur oxyanions. The method is based on the separation of polythionates and thiosulfate on an octadecylsilica column with an acetonitrile–water (20:80, v/v) mobile phase (pH 5.0) containing 3 mM tetrapropylammonium hydroxide and 6 mM acetic acid, followed by photometric measurement of the residual iodine (as triiodide) from the catalytic postcolumn azide–iodine reaction after mixing a reaction solution containing azide and iodine with the column effluent. Chromatograms obtained for the sulfur oxyanions showed negative peaks as a result of the decrease in absorbance of background. The conditions for the catalytic postcolumn reaction of the sulfur oxyanions in the column effluents were established by varying the concentrations of azide, iodine, iodide and acetic acid in the reaction solution, and varying the flow-rate, reaction temperature and length of the reaction tube. The detection limits (defined as S/N=3) were 4.3 μM for trithionate, 0.10 μM for tetrathionate, 2.7 nM for pentathionate, 5.0 nM for hexathionate and 1.1 nM for thiosulfate. When compared with earlier methods, the proposed method gave a much higher sensitivity for the determination of two polythionates (penta- and hexathionate) and thiosulfate. This method was applied successfully to the analysis of polythionates and thiosulfate added to hot-spring water samples.     

Separation of coumarins by micellar electrokinetic capillary chromatography

Nine coumarins were successfully separated simultaneously using micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard. A carrier composed of buffer solution (20 mM sodium dodecyl sulfate-15 mM sodium borate-15 mM sodium dihydrogenphosphate)-acetonitrile (24:1) was found to be the most suitable electrolyte for this separation. The analysis time (22 min) was shorter than that using high-performance liquid chromatography (47 min). Contents of coumarins in the crude drug of Angelicae Tuhou Radix could be easily determined by the proposed method.     

Simultaneous separation of the enantiomers of cizolirtine and its degradation products by capillary electrophoresis

The simultaneous enantioselective separation of (±)-cizolirtine and its impurities: (±)-N-desmethylcizolirtine, (±)-cizolirtine-N-oxide and (±)-5-(-hydroxybenzyl)-1-methylpyrazole was investigated by capillary electrophoresis. Electrokinetic chromatography with carboxymethyl-β-CD (CM-β-CD) and sulfobutyl-ether-β-CD was tried, showing good enantioseparation but poor chemical selectivity. The four racemic pairs were baseline separated, in a single run, by cyclodextrin-modified micellar electrokinetic chromatography. The migration buffer composition was: (60 mM hydroxypropyl-β-cyclodextrin–150 mM sodium dodecyl sulfate–50 mM disodium tetraborate, pH 9.2, in water)–butanol (95:5, v/v). Work was done to determine the effect of buffer components and their optimal concentration on selectivity. The method was validated with respect to enantioselectivity of cizolirtine as well as its degradation products and separation selectivity between the different components. Linearity, limit of detection, limit of quantitation and precision were also determined. This method is suitable for the enantiomeric purity determination and stability control of cizolirtine (racemic mixture or enantiomers) and its degradation products. Examples of electropherograms of (R)-cizolirtine degraded under stressed conditions are shown.     

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.