Quantitation of hyaluronic acid and chondroitin sulphates in rabbit synovial fluid by high-performance liquid chromatography of oligosaccharides enzymatically derived thereof

A high-performance liquid chromatographic (HPLC) method for quantifying unsaturated hexasaccharide and tetrasaccharide from Streptomyces hyaluronidase enzyme digestion products of hyaluronic acid was developed using a gel-permeation column packed with a sulphated polystyrene-divinylbenzene gel. For the oligosaccharides, the separation was accomplished in less than 7 min with a detection limit of 65 ng. An unsaturated non-sulphated disaccharide prepared from hyaluronic acid (delta Di-HA) and an unsaturated sulphated disaccharide (delta Di-4S) were analyzed by a HPLC method using a combination of two different gel-permeation columns. The separation of the disaccharides required less than 17 min at a flow rate of 0.7 ml/min with detection limits of as little as 4 ng for delta Di-HA and 5 ng for delta Di-4S. Both chromatographic methods were used for assay of a major component of hyaluronic acid and trace amounts of chondroitin sulphates in rabbit synovial fluid. The resulting contents of hyaluronic acid were compared to the values of polymeric hyaluronic acid directly measured by a HPLC method using two gel-permeation columns packed with a poly(hydroxyalkyl methacrylate) gel and the amounts of hyaluronic acid converted from uronic acid content determined by a colorimetric method.     

Analytical method of chondroitin/dermatan sulfates using high performance liquid chromatography/turbo ionspray ionization mass spectrometry: application to analyses of the tumor tissue sections on glass slides

We established a highly sensitive quantitative analytical method for chondroitin/dermatan sulfates by LC/MS method. By this method, the unsaturated disaccharides produced after the enzymatic digestion of chondroitin/dermatan sulfates can be determined in the amounts as low as 0.5 pmol levels. The use of tetrabutylammonium hydroxide as an ion-pair reagent for LC/MS allowed us to separate unsaturated 4-sulfated disaccharide and unsaturated 6-sulfated disaccharide. Furthermore, the peak areas of unsaturated disaccharides were increased almost 10 times by the postcolumn addition of acetonitrile. We applied this LC/MS method to the analyses of unsaturated disaccharides from chondroitin/dermatan sulfates in the tissues sections on glass slides, which were prepared from MethA tumor-bearing mice. This method brought about considerable reduction in the time distance from sample collection to preparation of analytical results.     

Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate.

A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.     

Chondroitin sulphate proteoglycans in the vitreous gel of sheep and goat

In the present study, the amounts and the fine structural characteristics of chondroitin sulphate proteoglycans (CSPGs) present in sheep and goat vitreous gels were determined. The results showed that in both examined species hyaluronan was the predominant glycosaminoglycan (GAG), whereas CSPGs were present in minor amounts. CSPGs were identified as versican and collagen IX with versican being the predominant PG type. Fine structural characterization indicated that the CS chains of versican in both mammalian species were of smaller size than those found in collagen IX. The difference in the sulphation pattern of CS chains between versican and collagen IX was also of particular interest. The results indicated that the predominant disaccharide type in CS side chains of versican and collagen IX from both sheep and goat vitreous gels was the 4-sulphated disaccharide. CS chains of versican were found to be richer in 4-sulphated disaccharide units than those in collagen IX, which also contained a significant proportion of non-sulphated disaccharides. These findings showed that, firstly, the CS content and the hydrodynamic size of the CS chain and, secondly, the sulphation pattern of CS chains from versican and collagen IX in both sheep and goat vitreous gels are PG type-dependent.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Dynamic modification of poly(methyl methacrylate) chips using poly(vinyl alcohol) for glycosaminoglycan disaccharide isomer separation

We describe a microchip electrophoresis (MCE) method for the assay of unsaturated disaccharides of chondroitin sulfates, dermatan sulfates, and hyaluronic acid (HA). Poly(vinyl alcohol) (PVA) could be irreversibly adsorbed onto poly(methyl methacrylate) (PMMA) substrates and this approach was applicable for dynamic coating. The characteristics of the PMMA surface with PVA coating were evaluated in terms of the wettability, EOF, and adsorption of 2-aminoacridone (AMAC)-labeled disaccharide. The water contact angle decreased from 73 degrees on a pristine PMMA surface to 37.5 degrees on a PVA-coated surface, indicating that the PVA coating increased hydrophilicity. EOF was reduced approximately twofold and was relatively stable. Scanning electron microscopy and fluorescence microscopy images showed that adsorption of AMAC-labeled disaccharides was dramatically suppressed. Using the PVA coating, baseline separation of two pairs of glycosaminoglycan (GAG) disaccharide isomers, DeltaDi-diS(B)/DeltaDi-diS(D) and DeltaDi-0S/DeltaDi-HA, was achieved in Tris-borate buffer within 130 s by MCE.     

High-performance liquid chromatographic identification of disaccharides generated from heparan sulphate isomers using heparitinases

Specific heparan sulphate-lyases, heparitinases I and II, were used to identify unsaturated disaccharide constituents generated from heterogeneous heparan sulphate isomers. All determinations were made using high-performance liquid chromatography with a column containing a sulphonized styrene-divinylbenzene copolymer. Unsaturated disaccharides generated from variously sulphated heparan sulphate isomers after simultaneous digestion with heparitinases I and II facilitated separation of the individual disaccharides, based on sulphate groups at the specific position of the uronic acid and glucosamine residues. The simultaneous digestion with heparitinases I and II produces unsaturated disaccharides from heparan sulphate isomers with the structure of 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-deoxy-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-aminodeoxy-6-O-sulpho-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose, 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-2-deoxy-6-O-sulpho-D-glucose and 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose.     

Negative Ion Fast-Atom Bombardment Tandem Mass Spectrometry To Determine Sulfate and Linkage Position in Glycosaminoglycan-Derived Disaccharides

Negative ion fast-atom bombardment tandem mass spectrometry has been used in the analysis of monosulfated disaccharides. These commercially obtained disaccharides have been enzymatically prepared from glycosaminoglycans using polysaccharide lyases. Three disaccharides from chondroitin sulfate and dermatan sulfate and two disaccharides from heparan sulfate and chemically derivatized heparin were analyzed. All five disaccharides were isomeric, with differences in sulfate position and linkage position. The full-scan mass spectra are useful in differentiating isomers when the sulfate group resides on different saccharide units. This structural information was obtained from fragment ions produced through cleavage at the glycosidic linkage. the full-scan mass spectra of each monosulfated disaccharide also produced intense molecular anions having long lifetimes. Collisional activation of these resulted in tandem mass spectra rich in significant product ions. Some of these fragment ions were formed through ring cleavage and were useful in the determination of both sulfate and linkage position.     

A novel LIF-CE method for the separation of hyaluronan- and chondroitin sulfate-derived disaccharides: Application to structural and quantitative analyses of human plasma low- and high-charged chondroitin sulfate isomers

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.     

Profile analysis of chondroitin sulfates in human urine and serum

A highly sensitive high-performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for the profile analysis of chondroitin sulfates (ChS) in normal human urine and serum. Over-sulfated disaccharide units such as di- or trisulfated unsaturated disaccharides in urine were estimated and unsaturated 6-sulfated disaccharide (delta Di-6S) was found as a major component from ChS in urine, although only small amounts of delta Di-6S from ChS were present in serum.     

High-performance liquid chromatographic analysis of g;ycosaminoglycan-derived oligosaccharides

High-performance liquid chromatography of glycosaminoglycan (GAG)-derived oligosaccharides has been employed for structural analysis and measurement of hyaluronan, chondroitin sulphate, dermatan sulphate, keratan sulphate, herapan sulphate and heparin. Recent developments in the separation and detection of unsaturated dissacharides and oligosaccharides derived from GAGs by enzyme or chemical degradation are reviewed.     

A novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencer

Analysis of glycosaminoglycans (GAGs) is of increasing importance concerning alterations in extracellular matrix composition and selectivity of glomerular basement membrane. In this report we describe the analysis of chondroitin sulfate disaccharides as an example of GAG delta disaccharide analysis using standard DNA sequencing equipment (DNA sequencer-assisted GAG disaccharide separation, DSA-GAGS). The presented methodology allows nanomolar quantification of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-derived GAG disaccharides. In comparison to RP-HPLC the established method is much more sensitive, showing detection limits of 38 fmol/microL. Variation coefficients were approximately 10%, enabling exact quantifications after run times of 17 min at 30 degrees C and an electrophoresis voltage of 15 kV; using a capillary DNA sequencer, available in many molecular laboratories, presented advantages like automated sample injection, opportunity of high-throughput analyses, separation of even sulfated disaccharide epimers, and the possibility of using APTS-derived fucose as an internal standard. Furthermore, highly reproducible retention times rendered easy identification of specific signals (SD 0.02). With regard to these results, the described method is a useful tool for the quantification of GAG disaccharides in low amounts, indicating advantages of obverse RP-HPLC and slab gel polyacrylamide electrophoresis in sensitivity, error-proneness, automation, and handling.     

Microdetermination of hyaluronic acid in human urine by high performance liquid chromatography.

A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (delta Di-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of delta Di-HA and delta Di-0S, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows--column: a stainless steel tube (4 mm i.d. x 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCl buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2-cyanoacetamide in alkaline medium was used for post-column detection. The calibration curve for delta Di-HA was linear in the range 5 pmol-5nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for delta Di-HA and delta Di-0S were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.     

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2‐aminoacridone and subjected to reversed polarity CE‐LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE‐LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE‐LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.     

Determination of chondroitin sulfate content in raw materials and dietary supplements by high-performance liquid chromatography with ultraviolet detection after enzymatic hydrolysis: single-laboratory validation

A method to quantify chondroitin sulfate in raw materials and dietary supplements at a range of about 5 to 100% (w/w) chondroitin sulfate has been developed and validated. The chondroitin sulfate is first selectively hydrolyzed by chondroitinase ACII enzyme to form un-, mono-, di-, and trisulfated unsaturated disaccharides; the resulting disaccharides are then quantified by ion-pairing liquid chromatography with ultraviolet detection. The amounts of the individual disaccharides are summed to yield the total amount of chondroitin sulfate in the material. Single-laboratory validation has been performed to determine the repeatability, accuracy, selectivity, limit of detection, limit of quantification, ruggedness, and linearity of the method. Repeatability precision for total chondroitin sulfate content was between 1.60 and 4.72% relative standard deviation, with HorRat values between 0.79 and 2.25. Chondroitin sulfate recovery from raw material negative control was between 101 and 102%, and recovery from finished product negative control was between 105 and 106%.     

Proton nuclear magnetic resonance characterisation of glycosaminoglycans using chemometric techniques

Various types of glycosaminoglycans (GAGs) including heparins, chondroitin sulfates, dermatan sulfate and hyaluronic acid were studied from their proton nuclear magnetic resonance (1H NMR) spectra using chemometric techniques. Despite the complexity of the 1H NMR signals, data analysis using principal component analysis enabled the different GAG classes to be distinguished and permitted their classification according to their chemical structure. The analysis of the composition of the major disaccharide unit and other relevant chemical structures in the heparin samples was performed using partial least squares regression.     

Online preconcentration and determination of chondroitin sulfate,dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis

Capillary electrophoresis with large‐volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and ‐16 kV. Exploiting large‐volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.     

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.     

糖胺多糖酶解产物—不饱和二糖的高效液相色谱分析

介绍了一种糖胺多糖的二糖组成成分分析方法－高效液相色谱法。该方法柱前处理简单。应用该方法对人体尿液中糖胺多糖的二糖组成进行了分析     

Fluorometric analysis of urinary chondroitin sulfate isomers by HPLC using dansylhydrazine as a prelabeling reagent

Chondroitin sulfate isomers (ChS isomers) were digested with chondroitinase ABC, and the unsaturated disaccharides produced were converted into fluorescent dansylhydrazine derivatives and analyzed by HPLC. The proposed method was applied to the determination of relative amounts of ChS isomers in normal human urine and analytical results showed that chondroitin-6-sulfate and chondroitin-4-sulfate were excreted in large amounts. Moreover, this method was used for the identification of urinary ChS isomers which were extracted from a cellulose acetate strip after carrying out electrophoresis.