Separation of hyaluronic acid by ultrahigh-voltage capillary gel electrophoresis

Hyaluronic acid was separated using 95 kV applied potential in a polyacrylamide gel-filled capillary. The results of this separation were compared to those obtained using a capillary electrophoresis instrument operated at a more conventional potential of 15 kV. For lower-molecular-weight oligomers, the separation efficiency was found to improve by about tenfold, and the resolution by about threefold. However, the improvement in resolution declined as the polymer molecular weight increased.     

Lactone formation of N-acetylneuraminic acid oligomers and polymers as examined by capillary electrophoresis.

We examined lactone-formation reaction of oligomers and polymers of N-acetylneuraminic acid in diluted hydrochloric acid solution and found that the time course of the lactonization reaction was easily traced by capillary electrophoresis. The reaction proceeded more rapidly with increasing the molecular mass of oligomers because the conformation of inner units became rigid and more favorable for the formation of lactone linkage. Present results obtained using capillary electrophoresis will be useful in understanding of physical and chemical properties of oligo/polysialic acids.     

Separation of quaternary ammonium diastereomeric oligomers by capillary electrophoresis

The separation of novel diastereomeric trimers (3M) and pentamers (5M), derived from quaternary ammonium salts, was studied in conventional, uncoated and coated capillaries using capillary zone electrophoresis (CZE) with a variety of buffers and additives. Resolution of 5M diastereomers was best achieved using gamma-cyclodextrin (gamma-CD) as a chiral selector, while no diastereomeric resolution was realized for the 3M material.     

Enantiomer migration order in chiral capillary electrophoresis

Enantiomer migration order (EMO) in chiral capillary electrophoresis (CE) represents a challenging issue, referred to in less than 20% of the articles on CE enantioseparations. This review article will (i) illustrate the actuality of the topic, (ii) discuss some technical problems related to EMO in CE enantioseparations, (iii) examine the principal differences between CE and other separation techniques from the viewpoint of enantiomer elution order, (iv) demonstrate the potential for a designed reversal of EMO in CE, and (v) emphasize the importance of studying EMO for better understanding of chiral CE as well as its more effective application. Along with CE, the results obtained by other instrumental techniques such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography, as well as molecular modeling calculations will be shortly discussed. Rather than referring to all published examples of the opposite migration order of enantiomers in CE, the emphasis will be on general aspects. Recently, the reversal of the EMO was described in detail in a book chapter (Chankvetadze, B., Capillary Electrophoresis in Chiral Analysis, Wiley & Sons, Chichester, UK 1997, Chapter 12) as well as in three review articles.     

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.     

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis.

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.     

Comparison of the sensitivity of 11 crosslinked hyaluronic acid gels to bovine testis hyaluronidase

Crosslinked hyaluronan gels are used in various applications where their stability is a prerequisite. The sensitivity of such gels to hyaluronidase can be determined as an index of stability by several approaches: chromatography, electrophoresis, and viscometry. We describe here a test based on the colorimetric determination of the N-acetyl-d-glucosamine released by hyaluronidase in standardized conditions. The sensitivities to bovine testicular hyaluronidase of 11 different gels used to fill skin wrinkles (Restylane; Perlane; Juvéderm 18, 24, 24HV, 30, and 30HV; Surgiderm 18, 24XP, 30, and 30XP) were compared.The method was reproducible, easy to perform, not time-consuming and allowed us to demonstrate that the sensitivity to testicular hyaluronidase was dependent on the degree of crosslinking of the gels and also on their monophasic/biphasic nature. Under our conditions, Surgiderm 30, 24XP and 30XP were the most resistant gels.We propose to retain the hyaluronidase test to predict the in situ stability of a crosslinked gel used to fill skin wrinkles.     

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.     

Determination of thiodiglycolic acid in urine by capillary electrophoresis.

A sensitive capillary electrophoretic method was developed for the determination of thiodiglycolic acid (TDA) in urine which avoids the pretreatment of the urine sample. Several carrier electrolytes were examined. The most suitable carrier electrolyte system consisted of potassium hydrogen phthalate (5 mM), 2-(N-morpholino)ethanesulfonic acid (50 mM) and tetradecyltrimethylammonium bromide (0.5 mM), pH 5.2. Ten times diluted fresh midstream void urine was used for the determination. In this way, the concentrations of TDA between 5 and 50 mg/l in undiluted urine samples can be determined.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 microg/mL (0.2-1.0 microg/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.     

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis: reversal of migration order

Enantioseparations of phenothiazines with gamma-cyclodextrin (gamma-CD) as a chiral selector were investigated using citrate and phosphate buffer electrolytes at pH 3.0. Reversal of the enantiomer migration order of promethazine, ethopropazine, and trimeprazine was observed by varying gamma-CD concentration in the range of 5-9 mM, 2.5-4.5 mM and 1.5-2.8 mM, respectively, using 100 mM citrate buffer at pH 3.0. As in the case of beta-CD, the (+)-enantiomers of phenothiazines possess greater binding strength to gamma-CD than the (-)-enantiomers. The evaluation of the binding constants and limiting mobility of the complexes formed between the enantiomers of phenothiazines and gamma-CD reveals that the binding strength of phenothiazines to gamma-CD and the differences in the binding constants and limiting mobility of the complexes are responsible for the enantiomer migration reversal. Both the binding constants and limiting mobility of the complexes between the (+)-enantiomers of phenothiazine and gamma-CD are greater than those of the corresponding (-)-enantiomers in a citrate buffer, while the binding constants of the complexes primarily determined the migration order of the enantiomers in a phosphate buffer. Compared with the results obtained using a phosphate buffer, we may conclude that citrate buffer which involves competitive complexation with chiral selector plays a significant role in the enantiomer migration reversal.     

脱氧核糖核酸的毛细管无胶筛分电泳迁移规律研究

本文研究了不同因素对脱氧核糖核酸(DNA)毛细管电泳迁移行为的影响.采用毛细管无胶筛分电泳法,对不同片段长度DNA进行分离检测,考察DNA片度长度、电场强度、聚合物浓度及分子量等因素对DNA迁移行为的影响.实验结果显示,DNA迁移时间随其长度的增加而延长;电场强度越高,DNA迁移时间越短,分离效果变差;DNA在高浓度聚...     

Influence of pH on the migration properties of oligonucleotides in capillary gel electrophoresis.

The effect of pH on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary gel electrophoresis was investigated. Different homooligodeoxyribonucleotides of equal chain length showed significant differences in relative migration when the pH of the gel buffer was varied from pH 6 to 8, parallel with the running buffer. A similar variation in migration order was observed during the electrophoretic equilibration of a pH 8 gel-filled capillary column with a pH 6 running buffer. In the latter instance, the current reached the new level after 20 min of electrophoretic equilibration with the pH 6 running buffer. However, it was observed that the migration order characteristic of the pH 6 gel was achieved only after 4 h of electrophoretic equilibration. To avoid this time-consuming equilibration process, these results suggest that gel-filled capillary columns should be prepared with the same buffer (composition and pH) that will be used as the running buffer during the separations.     

Assay of synovial fluid hyaluronic acid using high-performance liquid chromatography of hyaluronidase digests

A high-performance liquid chromatographic method for the determination of hyaluronic acid levels in synovial fluids has been developed. The hyaluronidase sample digests, containing an internal standard (benzoic acid), were separated on a reversed-phase octadecylsilyl column eluted with 0.01 M tetrabutylammonium phosphate-acetonitrile (83:17, v/v) at pH 7.35. The determination was made on 1:10 diluted samples, by using a calibration curve from 50 to 500 micrograms/ml of human umbilical cord hyaluronic acid. For validation, the synovial fluids were simultaneously analysed by this method and a radiometric method: a high correlation was found between the two (correlation coefficient 0.94). The proposed method can be used to determine specifically the high hyaluronic acid levels of synovial fluids without interferences from other glycosaminoglycans or non-steroidal anti-inflammatory drug treatment.     

Quantitative analysis of hyaluronic acid by high-performance liquid chromatography of streptomyces hyaluronidase digests

The separation and quantitative analysis of streptomyces hyaluronidase ( HAase ) degradation products of hyaluronic acid (HA) by high-performance liquid chromatography are described. The substituted 4,5-unsaturated tetrasaccharide and hexasaccharide which result from the digestion of HA are quickly separated on a silica gel (Zorbax SIL) column. The assay of HA is linear between 5 and 250 micrograms of HA. This procedure is suitable for some weakly acidic glycosaminoglycan contaminants having similar properties to those of HA.     

A new method for determination of hyaluronidase activity in biological samples using capillary zone electrophoresis

The aim of the study was to develop a new capillary zone electrophoresis (CZE) method for determination of enzymatic activity of hyaluronidase. The method permits monitoring of the process of hyaluronic acid digestion by hyaluronidase. Studies were performed using CZE instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter 75 µm. Separation was performed in the phosphate buffer (pH 8.10) in the electric field of 20 kV, λ = 220 nm. The procedure was based on mixing a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate the activity of hyaluronidase originating from different sources with high precision and accuracy. t‐Tests showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The developed method is characterized by a short duration of analysis, low volume of analyzed sample, small amount of buffers used and low cost of analysis. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of unexpected migration behavior of adenosine in capillary zone electrophoresis

Summary The capillary zone electrophoresis of two common nucleosides, adenosine and inosine, was investigated. Both compounds were resolved in a 0.1 M sodium phosphate buffer, pH 7.5. Contrary to expectations, adenosine behaved at this pH— 5 pH units lower than the literature pK_a— as a negative ion, migrating behind mesityloxide (neutral marker) when working in normal polarity mode. To confirm the migration order, peaks were identified from absorption maxima, by high-speed scanning detector. The change in electrophoretic mobility with pH was investigated for the nucleosides, and 10 other background electrolytes were tried to match the separation capabilities of the sodium phosphate buffer. Most inorganic buffers showed comparable separation, while organic, Good-type buffers lacked selectivity.     

Stereoselective analysis of acid herbicides in natural waters by capillary electrophoresis.

A capillary electrophoretic method for the stereoselective analysis of aryloxypropionic and aryloxyphenoxypropionic acidic herbicides in ground water and river water was performed. Vancomycin and gamma-cyclodextrin were added to the background electrolyte (BGE) as chiral selectors. Water sample preconcentration was accomplished by solid-phase extraction on styrene-divinylbenzene packed cartridges (2 L of ground water and 1 L of river water). The analytical method allowed for the resolution of mecoprop, fenoprop, fluazifop and haloxyfop racemic mixtures in natural water samples spiked with enantiomer concentration levels in the range 0.1-0.13 ppb for ground water and 0.4-0.54 ppb for river water.     

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.