A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.     

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H₂O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of ³H by reduction with NaB³H₄ permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.     

Separation of anionic oligosaccharides by high-performance liquid chromatography

We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the amnionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (alpha 2.3 vs alpha 2.6) and/or location of alpha 2,3- and alpha 2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.     

Analysis of polysulfated chondroitin disaccharides by high-performance liquid chromatography

A high-performance liquid chromatography method for analyzing disaccharides derived from chondroitin sulfate glycosaminoglycans has been developed which employs a Whatman Partisil-10 PAC amino-cyano column and an acetonitrile/methanol/ammonium acetate solvent to resolve disulfated, monosulfated, and unsulfated disaccharides in a chromatographic run of less than 20 min. The single known trisulfated chrondroitin disaccharide can be eluted in an alternate solvent system containing the same mobile phase components in different proportions. Disaccharides were prepared for chromatography from glycosaminoglycans and proteoglycans of known compositions by digestion with chondroitinase ABC, with the exception of king crab cartilage glycosaminoglycan which was incubated sequentially with hyaluronidase and chondroitinase ABC. Disaccharides were extracted from the digestion mixtures in 80% ethanol, dried over nitrogen, resuspended in the HPLC solvent, and chromatographed at a flow rate of 1 ml/min. Unsaturated disaccharides in the column eluate were detected by continuous ultraviolet absorbance monitoring at 232 nm; alternatively, fractions were collected and assayed for uronic acid content or radioactivity. By utilizing the HPLC technique in conjunction with chondroitinase ABC and AC digestion and sulfatase hydrolysis, the epimeric structures of chondroitin sulfates E and H were confirmed. With this technique, rapid and reproducible analyses of chondroitin sulfate disaccharides generated from mouse mast cell proteoglycan and from glycosaminoglycans of squid cranial cartilage, shark skin, hagfish skin, and hagfish notocord were in close agreement with compositions obtained by other techniques.     

A high-performance liquid chromatography method for the analysis of picomole amounts of oligosaccharides

A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintillation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Fractionation of human milk oligosaccharides by high-performance liquid chromatography

An ion-exchange chromatographic system was used to isolate several human milk oligosaccharides, the elution being carried out with a linear gradient of a sodium borate buffer. Lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, II and III, lacto-N-difucohexaose I and 2'-alpha-fucosyllactose can be separated by this method.     

Characterization of oligosaccharides by lectin affinity high-performance liquid chromatography

Alterations of the oligosaccharide structures of glycoproteins are associated with differentiation, malignant transformation, and expression of the same protein in different cell types. The potential biological importance of oligosaccharides has resulted in a growing need for detailed structural information. When glycoproteins are available in limited quantities and/or bear highly heterogeneous oligosaccharides, characterization of their oligosaccharides is difficult. We have developed an efficient approach for obtaining detailed information about oligosaccharides by determining structural 'fingerprints' using lectin affinity high-performance liquid chromatography.     

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation.The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.     

Analysis by high-performance liquid chromatography of hyaluronic acid and chondroitin sulfates

The use of high-performance liquid chromatography for the quantification of glycosaminoglycan disaccharides has been hampered by the inability to isocratically resolve the chondroitinase digestion products 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (delta Di-HA) and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS). To overcome this limitation, we have developed a solvent system capable of resolving delta Di-HA, delta Di-OS, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S). Integrator responses were linear from 1 microgram down to 25 ng for delta Di-HA, delta Di-OS, and delta Di-4S and down to 100 ng for delta Di-6S. This method was used to examine changes in the content of urinary hyaluronic acid and chondroitin sulfates isolated from normal individuals and from patients with Lowe Syndrome, Werner Syndrome, and Hutchinson-Gilford Progeria Syndrome. We confirmed that the HPLC method gave results comparable to colorimetric methods.     

The application of chondro-2-sulfatase for identification of the products generated from chondroitin sulfate isomers by high-performance liquid chromatography

A specific chondroitin sulfate-lyase, chondro-2-sulfatase, was first used for identification of the unsaturated disaccharide constituents (delta Di-S) generated from variously sulfated chondroitin sulfate and dermatan sulfate isomers by a high-performance liquid chromatographic (HPLC) method. delta Di-S generated from oversulfated chondroitin sulfate and dermatan sulfate isomers following digestion with chondroitinases were further digested by the chondro-2-sulfatase, which led to the release of one sulfate from a specific 2-position of the uronic acid residue, as judged with the new HPLC system using a resin made from a sulfonized styrene-divinylbenzene copolymer. It was also found that the chondro-2-sulfatase digests not only delta Di-S with the structure of D-uronic acid 2 sulfate 1-3-N-acetyl-D-galactosamine but also other sulfated delta Di-S with partially the same constituents, i.e., unsaturated di-sulfated disaccharide B, unsaturated di-sulfated disaccharide D or G, and unsaturated tri-sulfated disaccharide.     

Size fractionation of anionic oligosaccharides and glycopeptides by high-performance liquid chromatography

A rapid method for the fractionation of anionic oligosaccharide and glycopeptide species on the basis of net carbohydrate content utilizing high-performance liquid chromatography has been developed. Amine-bearing bonded-phase columns are eluted with a mobile phase consisting of a water:acetonitrile gradient containing 3% acetic acid titrated to pH 5.5 with triethylamine. Phosphorylated and sialylated oligosaccharides within various charge classes differing in their hexose or hexosamine contents but bearing the same number of anionic species can be resolved without prior removal of the anionic moieties. Glycopeptides containing at least as many as six amino acids are also well fractionated on the basis of carbohydrate content. A variety of detection methods may be used and sensitivity in the subnanomole range is possible with fluorescent or radiolabeling techniques. This method offers a significant improvement in the rapidity and resolution attainable for the size fractionation of anionic complex carbohydrates.     

Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography

An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 mM ammonium phosphate and 1 mM tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously (M.J. Bienkowski and H.E. Conrad (1985) J. Biol. Chem. 260, 356-365). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.     

Rapid isolation of triosephosphate isomerase utilizing high-performance liquid chromatography

A new method for the isolation of homogeneous triosephosphate isomerase (TPI, EC 5.3.1.1) has been developed. The method utilizes high-performance liquid chromatography on DEAE 5PW and Hydrophase-polyethyleneimine columns, which results in the rapid isolation and essentially quantitative recovery of the enzyme. The procedure is superior to previous methods with respect to specificity, recovery, and time. In addition, this rapid process minimizes the potential for postsynthetic modifications of the protein. Milligram quantities of TPI can be isolated from 100 g of tissue.     

Liquid chromatography/mass spectrometry sequencing approach for highly sulfated heparin-derived oligosaccharides

Liquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of -32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10000 Da and a net charge of -60.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%).This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.