Determination of meningococcal polysaccharides by capillary zone electrophoresis

Meningococcal polysaccharides are medically important molecules and are the active components of vaccines against Neisseria meningiditis serogroups A, C, W135, and Y. This study demonstrates that free solution capillary zone electrophoresis (CZE) using simple phosphate/borate separation buffers is capable of separating intact, native polysaccharides from these four serogroups. Separation appeared to be robust with respect to variations in test conditions and behaved in expected ways with respect to changes in temperature, ionic strength, and addition of an organic modifier. Serogroups W135 and Y are composed of sialic acid residues alternating with either galactose or glucose, respectively. Separation of these serogroups could be achieved using phosphate buffer and was therefore not dependent on differential complexation with borate. Addition of sodium dodecyl sulfate to the separation buffer (i.e., MEKC) resulted in peak splitting for all four serogroups. Changes in polysaccharide size did not affect migration time for the size range examined, but serogroup C polysaccharide (a sialic acid homopolymer) was separable from sialic acid monosaccharide. CZE quantification of multiple lots of each of the four serogroups was compared to wet chemical determination by phosphorus or sialic acid measurement. Results from CZE determination showed good agreement with the wet chemical methods.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate-35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length x 75 microm I.D.) and the operating conditions were: 15 kV applied voltage, 30 degrees C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0-120.0 microg ml(-1) for both compounds, with a detection limit of 0.2 microg ml(-1) for grepafloxacin and 0.3 microg ml(-1) for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.     

Determination of malondialdehyde in rat brain by capillary zone electrophoresis

A method for determination of malondialdehyde with capillary electrophoresis using UV detection at 267 nm has been developed. The buffer system consisted of 10 mM borax and 0.5 mM CTAB at pH 9.3. Malondialdehyde migrated as the first peak in the electropherogram at 2.6 min. Limit of detection was 1.2 μM corresponding to 7.8 pg. Malondialdehyde was determined before and after stimulating lipid peroxidation with the addition of ferrous ammonium sulphate to homogenates of rat brain tissue. Proteins were precipitated by boiling and removed from the brain homogenates with centrifugation. No further pretreatment was made before injecting the homogenates on the CE system. Non-precipitated homogenates could also be analyzed, but this required washing of the capillary with 0.1 M NaOH before introduction of the next sample.     

Determination of midecamycin by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of midecamycin using an end-column amperometric detection with a carbon fiber micro-disk bundle electrode at a constant potential of +1.15 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 1.00x10(-3) mol l(-1) Na(2)HPO(4)-3.49x10(-4) mol l(-1) NaOH (pH 11.4) for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The limit of detection is 5.0x10(-7) mol l(-1) or 0.41 fmol (S/N=3). The linear range of the calibration curve is 1.00x10(-6)-1.00x10(-3) mol l(-1). The relative standard deviation is 1.4% for the migration time and 4.9% for the electrophoretic peak current. The method could be applied to the determination of midecamycin in human urine. In this case, a separation voltage of 14 kV was used.     

Determination of cimetidine in human plasma by free capillary zone electrophoresis

The separation of cimetidine from the metabolites cimetidine amide and cimetidine sulfoxide, endogenous creatinine and the internal standard ranitidine was achieved by capillary electrophoresis in less than 5 min. All compounds were well separated from cimetidine, including possible plasma ingredients, as the UV spectra of cimetidine standard and cimetidine from the plasma extract match. Plasma levels of cimetidine were determined in the range 250–3000 ng/ml in plasma and higher concentrations were determined by dilution of the sample with blank plasma.     

Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon fruits, CZE was successfully applied in all the analyzed samples. The proposed CZE protocols give lower detection limits (<0.4 microg ml(-1)); are cheaper, quicker, and highly reproducible; and can be applied to analyze large series of samples (ca. 50 samples per day) which is utmost importance, not only in screening trials for internal quality and tomato breeding programs, but also in systematic and routine characterization of Lycopersicon fruits.     

Determination of carbohydrates as their p-sulfophenylhydrazones by capillary zone electrophoresis.

p-Hydrazinobenzenesulfonic acid was explored as an ultraviolet labeling reagent for capillary electrophoresis of mono-, di- and trisaccharides. The labeling reaction that produces p-sulfophenylhydrazines took less than 8 min, and introduced both chromphore and charged groups into the carbohydrate molecules. The derivatives of nine mono- and disaccharides were completely separated in 9 min using a 100 mM borate buffer at pH 10.24. On-column UV detection at 200 nm allowed the detection of glucose with a mass detection limit of 17.6 fmol or a concentration limit of 3.6 microM. Reproducible quantification of carbohydrates was achieved in the concentration range of 0.1-9.1 mM in reaction solution. The method was applied successfully to determine the monosaccharide composition of laminaran.     

Determination of urinary orotic acid and uracil by capillary zone electrophoresis

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperornithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.     

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.     

Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis

Ferulic andp-coumaric acid can be separated from their corresponding aliphatic methyl esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl andp-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester from wheat bran was also efficiently separated and used as substrate for the enzyme assays.Penicillium expansum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture.The authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK; A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

Determination of purity degree and counter-ion content in lecirelin by capillary zone electrophoresis and capillary isotachophoresis

Capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) were applied for the determination of peptide purity degree and counter-ion content in lecirelin, the synthetic analogue of luteinizing hormone-releasing hormone (LHRH). CZE analyses were carried out in acidic background electrolyte (100 mM H3PO4, 50 mM Tris, pH 2.25) in bare fused silica capillary using UV-absorption detection at 206 nm. CITP analyses were performed in the electrophoretic analyzer with column coupling, equipped with contactless conductivity detectors both in preseparation capillary and in analytical capillary, and with UV-absorption detector (220 and 254 nm) in analytical capillary. Determinations of peptide purity were carried out in cationic mode with leading electrolyte (LE), 10 mM KOH/AcOH, pH 4.5, and terminating electrolyte (TE), 10 mM beta-alanine (BALA)/AcOH, pH 4.4. Degree of peptide purity determined by both CZE and CITP was in the range 60.1-80.9% for crude preparations of lecirelin and in the range 96.4-99.9% for HPLC purified batches. Concentrations of contaminating counter-ions, the anions of trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA) and acetic acid (AcOH), were determined by CITP analyses in anionic mode with LE 10 mM HCl/His, pH 6.0, and TE 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.0, by the calibration curve method. Mass percentages of the counterion contents in the analyzed lecirelin batches varied from zero to ca. 9% (TFMSA), 3% (TFA) and 11% (AcOH), respectively.     

Antibody fragment separations by capillary zone electrophoresis

This study investigated methods of improving the separation and identification of an IgA antibody, McPC603, and its pepsin fragments. The problem presented by purification of antibody fragments (Fabs) and the antibody light chain required accurate and informative analysis of highly hydrophobic proteins, which can polymerize and fold to form secondary structures. Capillary zone electrophoresis (CZE) permits the separation of peptides and small proteins by a method which is orthogonal to the traditional method of reversed-phase HPLC. To facilitate planned studies of the antibody's biological activity, our buffer composition was kept as simple as possible. During CZE analysis, if the buffer pH is below the isoelectric point of the protein, or the protein is large (with a heterogeneous distribution of surface charges), it can irreversibly bind to the capillary wall unless the capillary is coated. We found that C₁-coatings in RP-capillaries at pH 9.5 adequately prevented the antibody fragments from binding to the wall. However, the coating did not remain stable at such high pH, so different conditions were sought. We achieved adequate separations in several buffers at nearly physiological pH, in a bare silica capillary which had been coated once with a soluble cationic polymer coating (Micro-Coat applied during column conditioning). Antibody electropherograms changed depending on the type of inorganic buffer salt used in a separation. Phosphate binds to the antigen-binding site of the IgA with low affinity, and interesting effects were observed in separations using phosphate buffer. These effects will be discussed.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used for determination of dissociation constant of phosphinate group in phosphinic pseudopeptides, i.e. peptides where one peptide bond is substituted by phosphinic acid moiety -PO2--CH2-. The dissociation constants were determined for a set of newly synthesized pseudopeptides derived from a structure N-Ac-Val-Ala(psi)(PO2--CH2)Leu-His-NH2 by nonlinear regression of experimentally measured pH dependence of their effective electrophoretic mobilities. CZE experiments were carried out in Tris-phosphate background electrolytes in the pH range 1.4-3.2. The pseudopeptides were synthesized as a mixture of four diastereomers, the separation of which was achieved in most cases. Moreover, differences of the effective mobilities of the pseudopeptide diastereomers enabled simultaneous determination of the dissociation constant of their phosphinate group without necessity of previous isolation of individual isomers.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

The combination of capillary isotachophoresis and capillary zone electrophoresis may enhance greatly the performance of analytical capillary electrophoresis with respect to both separation power and the concentration sensitivity. The concentrating effects and the separation power of isotachophoresis allow the analysis of diluted samples and the elimination of interferences due to bulk components. The separation process of zone electrophoresis enables one to resolve the stack of trace analytes and detect the resulting individual zones with high sensitivity. The transition of isotachophoresis into zone electrophoresis plays the key role in the overall performance of this hyphenated technique. This article describes the dynamics of the conversion of isotachophoresis into zone electrophoretic mode and shows that the key role is played by the segments of the leading and terminating zones from the isotachophoretic stage. The magnitude of these segments directly effects the detection time as well as the separation width of the peaks of analytes. It is shown that these effects are also important in the analyses by capillary zone electrophoresis where isotachophoresis is induced by the sample itself. Finally, the paper presents a list of recommended, user-friendly, electrolyte systems which enable one to simply predict the performance of the combination isotachophoresis-zone electrophoresis.     

Pharmaceutical-induced cell apoptosis characterized by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) with a non-gel-sieving system was employed to characterize actinomycin D-induced cell apoptosis by measuring cellular DNA damage, termed DNA ladder, which proved to be thoroughly different from the DNA damage pattern of cell necrosis. The results by CZE analyses were identical to those obtained by conventional slab gel electrophoresis, demonstrating that CZE would be a reliable and more convenient technique for the identification of cell death with advantages of higher performance, high-speed, minute sample requirement, and advanced automation.     

Determination of three major catecholamines in human urine by capillary zone electrophoresis with chemiluminescence detection

A sensitive simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detection. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of 20.0mM sodium borate and 1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than 8 min. Detection limits of 7.9 × 10(-8)M, 1.0 × 10(-7)M, and 6.9 × 10(-8)M were observed for EP, NE, and DA, respectively. Relative standard deviation (RSD) values for the peak height were 4.7% to 5.4% (n = 5). Our proposed method was applied to the determinations of the catecholamines in urine samples from 12 healthy individuals and 26 pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.