Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

OBJECTIVE: Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations. MATERIALS AND METHODS: In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR. CONCLUSION AND RESULTS: The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

Glycomics by ion mobility tandem mass spectrometry of chondroitin sulfate disaccharide domain in biglycan.

Biglycan (BGN), a small leucine-rich repeat proteoglycan, is involved in a variety of pathological processes including malignant transformation, for which the upregulation of BGN was found related to cancer cell invasiveness. Because the functions of BGN are mediated by its chondroitin/dermatan sulfate (CS/DS) chains through the sulfates, the determination of CS/DS structure and sulfation pattern is of major importance. In this study, we have implemented an advanced glycomics method based on ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS) to characterize the CS disaccharide domains in BGN. The high separation efficiency and sensitivity of this technique allowed the discrimination of five distinct CS disaccharide motifs, of which four irregulated in their sulfation pattern. For the first time, trisulfated unsaturated and bisulfated saturated disaccharides were found in BGN, the latter species documenting the non-reducing end of the chains. The structural investigation by IMS MS/MS disclosed that in one or both of the CS/DS chains, the non-reducing end is 3-O-sulfated GlcA in a rather rare bisulfated motif having the structure 3-O-sulfated GlcA-4-O-sulfated GalNAc. Considering the role played by BGN in cancer cell spreading, the influence on this process of the newly identified sequences will be investigated in the future.

Molecular Dynamics-Based Comparative Analysis of Chondroitin and Dermatan Sulfates.

Glycosaminoglycans (GAGs) are a class of linear anionic periodic polysaccharides containing disaccharide repetitive units. These molecules interact with a variety of proteins in the extracellular matrix and so participate in biochemically crucial processes such as cell signalling affecting tissue regeneration as well as the onset of cancer, Alzheimer's or Parkinson's diseases. Due to their flexibility, periodicity and chemical heterogeneity, often termed "sulfation code", GAGs are challenging molecules both for experiments and computation. One of the key questions in the GAG research is the specificity of their intermolecular interactions. In this study, we make a step forward to deciphering the "sulfation code" of chondroitin sulfates-4,6 (CS4, CS6, where the numbers correspond to the position of sulfation in NAcGal residue) and dermatan sulfate (DS), which is different from CSs by the presence of IdoA acid instead of GlcA. We rigorously investigate two sets of these GAGs in dimeric, tetrameric and hexameric forms with molecular dynamics-based descriptors. Our data clearly suggest that CS4, CS6 and DS are substantially different in terms of their structural, conformational and dynamic properties, which contributes to the understanding of how these molecules can be different when they bind proteins, which could have practical implications for the GAG-based drug design strategies in the regenerative medicine.

Simultaneous determination of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan glycosaminoglycan disaccharides by high-performance liquid chromatography using a reverse-phase column with adamantyl groups.

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.

Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Swim Bladder: Isolation, Structural Analysis, and Anticoagulant Activity.

Glycosaminoglycans (GAGs) with unique structures from marine animals show intriguing pharmacological activities and negligible biological risks, providing more options for us to explore safer agents. The swim bladder is a tonic food and folk medicine, and its GAGs show good anticoagulant activity. In this study, two GAGs, CMG-1.0 and GMG-1.0, were extracted and isolated from the swim bladder of Cynoscion microlepidotus and Gadus morhua. The physicochemical properties, precise structural characteristics, and anticoagulant activities of these GAGs were determined for the first time. The analysis results of the CMG-1.0 and GMG-1.0 showed that they were chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains with molecular weights of 109.3 kDa and 123.1 kDa, respectively. They were mainly composed of the repeating disaccharide unit of -{IdoA-α1,3-GalNAc4S-ß1,4-}- (DS-A). The DS-B disaccharide unit of -{IdoA2S-α1,3-GalNAc4S-ß1,4-}- also existed in both CMG-1.0 and GMG-1.0. CMG-1.0 had a higher proportion of CS-O disaccharide unit -{-GlcA-ß1,3-GalNAc-ß1,4-}- but a lower proportion of CS-E disaccharide unit -{-GlcA-ß1,3-GalNAc4S6S-ß1,4-}- than GMG-1.0. The disaccharide compositions of the GAGs varied in a species-specific manner. Anticoagulant activity assay revealed that both CMG-1.0 and GMG-1.0 had potent anticoagulant activity, which can significantly prolong activated partial thromboplastin time. GMG-1.0 also can prolong the thrombin time. CMG-1.0 showed no intrinsic tenase inhibition activity, while GMG-1.0 can obviously inhibit intrinsic tenase with EC50 of 58 nM. Their significantly different anticoagulant activities may be due to their different disaccharide structural units and proportions. These findings suggested that swim bladder by-products of fish processing of these two marine organisms may be used as a source of anticoagulants.

Capillary Zone Electrophoresis-Electrospray Ionization Tandem Mass Spectrometry for Total Analysis of Chondroitin/Dermatan Sulfate Oligosaccharides.

Proteoglycans are heavily glycosylated proteins, covalently linked to one or more glycosaminoglycan (GAG) chains, abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play an essential role at the ECM level; however, the composition of the hybrid CS/DS as well as the distribution of the sulfate groups along the chain were also shown to influence biological activities in brain. The elevated structural diversity of CS/DS motifs, in which sulfation may occur at GalNAc and/or IdoA/GlcA in various combinations, requires the development of specific high performance analytical methods for reliable elucidation. Due to its sensitivity, reproducibility, and efficiency, capillary zone electrophoresis (CZE) for separation of CS/DS oligosaccharides coupled to electrospray ionization mass spectrometry (ESI-MS) for their structure determination contributed an essential progress to this field.In the present chapter, two powerful methods based on CZE for separation and ESI-MS for identification and structural analysis of CS/DS are presented. The first part is devoted to offline CZE-ESI-MS based on fraction collection, screening by negative ion mode nanoESI, and fragmentation analysis in tandem MS using collision-induced dissociation (CID) at low ion acceleration energies. In the second part of the chapter, a strategy for online CZE-ESI-MS in normal polarity and negative mode ESI followed by tandem MS in real-time data-dependent acquisition mode for CS/DS separation, screening, and fragmentation is described in detail. The latter method entails the in-laboratory manufacturing of a simple yet sturdy interface for the online CZE coupling to ESI-MS and the optimization of the coupled system for total analysis of regularly sulfated and irregularly, i.e., under- and oversulfated CS/DS domains.

Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore.

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/ß anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.

Effect of Sulfation on the Conformational Dynamics of Dermatan Sulfate Glycosaminoglycan: A Gaussian Accelerated Molecular Dynamics Study.

Glycosaminoglycans (GAGs) are anionic biopolymers present on cell surfaces as a part of proteoglycans. The biological activities of GAGs depend on the sulfation pattern. In our study, we have considered three octadecasaccharide dermatan sulfate (DS) chains with increasing order of sulfation (dp6s, dp7s, and dp12s) to illuminate the role of sulfation on the GAG units and its chain conformation through 10 µs-long Gaussian accelerated molecular dynamics simulations. DS is composed of repeating disaccharide units of iduronic acid (IdoA) and N-acetylgalactosamine (N-GalNAc). Here, N-GalNAc is linked to IdoA via ß(1-4), while IdoA is linked to N-GalNAc through α(1-3). With the increase in sulfation, the DS structure becomes more rigid and linear, as is evident from the distribution of root-mean-square deviations (RMSDs) and end-to-end distances. The tetrasaccharide linker region of the main chain shows a rigid conformation in terms of the glycosidic linkage. We have observed that upon sulfation (i.e., dp12s), the ring flip between two chair forms vanished for IdoA. The dynamic cross-correlation analysis reveals that the anticorrelation motions in dp12s are reduced significantly compared to dp6s or dp7s. An increase in sulfation generates relatively more stable hydrogen-bond networks, including water bridging with the neighboring monosaccharides. Despite the favorable linear structures of the GAG chains, our study also predicts few significant bendings related to the different puckering states, which may play a notable role in the function of the DS. The relation between the global conformation with the micro-level parameters such as puckering and water-mediated hydrogen bonds shapes the overall conformational space of GAGs. Overall, atomistic details of the DS chain provided in this study will help understand their functional and mechanical roles, besides developing new biomaterials.

Structural analysis of glycosaminoglycans from Oviductus ranae.

Oviductus ranae (O.ran.) has been widely used as a tonic and a traditional animal-based Chinese medicine. O.ran. extracts have been reported to have numerous biological activities, including activities that are often associated with mammalian glycosaminoglycans such as anti-inflammatory, antiosteoperotic, and anti-asthmatic. Glycosaminoglycans are complex linear polysaccharides ubiquitous in mammals that possess a wide range of biological activities. However, their presence and possible structural characteristics within O.ran. were previously unknown. In this study, glycosaminoglycans were isolated from O.ran. and their disaccharide compositions were analyzed by liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). Heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate (DS) and hyaluronic acid (HA) were detected in O.ran. with varied disaccharide compositions. HS species contain highly acetylated disaccharides, and have various structures in their constituent chains. CS/DS chains also possess a heterogeneous structure with different sulfation patterns and densities. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of O.ran..

Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates.

Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology.

Structural analysis of glycosaminoglycans from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a traditional animal-based Chinese medicine. Chondroitin sulfate (CS), dermatan sulfate (DS) and hyaluronic acid (HA) are structurally complex classes of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their possible structural characteristics in CCA are not clear. In this study, GAG fractions containing CS/DS and HA were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). The result showed that CS/DS/HA disaccharides were detected in the three lower salt fractions from anion-exchange chromatography. The sulfation patterns and densities of CS/DS chains in these fractions differed greatly, while HA chains varied in their chain lengths. The quantitative analysis first revealed that the amount of GAGs in CCA varied significantly in total and in each fraction. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of CCA.

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.

Fine structural characterization of sulodexide.

Sulodexide is a heparinoid which combines the properties of its components heparin and dermatan sulfate and is used not only for the prophylaxis and treatment of thromboembolic diseases but also for the treatment of diabetic nephropathy. Despite many clinical studies have been conducted to investigate its activity and safety, no data are available on the fine chemical characterization of its components. In this work, the in-depth investigation on the structural features of both the whole mixture and the isolated components was accomplished, involving the analysis of molecular weight distribution and of their mono, di and oligosaccharide composition by HP-SEC/TDA, 2D-NMR and HPLC-MS techniques. Moreover, also the separation of fractions endowed of graded affinity to antithrombin was achieved followed again by detailed structural analysis. The combination of different techniques permits to profile in depth the structural features of such a drug and offers a useful tool for possible analysis of batch production.

Analysis of Sulfate Patterns in Glycosaminoglycan Oligosaccharides by MSn Coupled to Infrared Ion Spectroscopy: the Case of GalNAc4S and GalNAc6S.

We report distinctive spectroscopic fingerprints of the monosaccharide standards GalNAc4S and GalNAc6S by coupling mass spectrometry and ion spectroscopy in the 3-µm range. The disaccharide standards CSA and CSC are used to demonstrate the applicability of a novel approach for the analysis of sulfate position in GalNAc-containing glycosaminoglycans. This approach was then used for the analysis of a sample containing CSA and CSC disaccharides. Finally, we discuss the generalization of the coupling of mass spectrometry with ion spectroscopy for the structural analysis of glycosaminoglycans on a tetrasaccharide from dermatan sulfate source. Graphical abstract ᅟ.

Concentration of sulfated glycosaminoglycans in the mammary tissue of female rats with the aging and about hormonal influence.

It was to evaluate the concentration of sulfate glycosaminoglycans (GAG) in mammary tissue of the young and adult female rats and ovariectomized females rats after hormonal stimulation. For this purpose, 60 female rats were divided into six groups with 10 animals/each: nonovariectomized groups: G1 (5 months), and G2 (15 months) and ovariectomized groups: OG (vehicle); EG: (estradiol, 7 days of treatment), PG (progesterone acetate, 23 days of treatment) and EPG: (estradiol (7 days of treatment) and next progesterone acetate (23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary tissue removed, processed for biochemical evaluation and quantification of the GAG. The comparison between groups showed that the concentration dermatan sulfate (DS) G1 was lower compared to G2, OG, EG (p < .05) and G2 was lower compared to OG (p < .05), and OG was higher compared to EG, GP, EPG (p < .05); and heparan sulfate (HS) G1 was higher compared to G2 (p < .05), and G2 was higher compared to OG, EP, PG and EPG (p < .05). These changes in the extracellular matrix might explain, at least in part, hormonal influence about sulfated glycosaminoglycans in response to physiological state/age, and in response to hormonal treatment in the mammary tissues.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients. METHODS: In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS). RESULTS: Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine. CONCLUSIONS: Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

Characterization of Danaparoid Complex Extractive Drug by an Orthogonal Analytical Approach.

Danaparoid sodium salt, is the active component of ORGARAN, an anticoagulant and antithrombotic drug constituted of three glycosaminoglycans (GAGs) obtained from porcine intestinal mucosa extracts. Heparan sulfate is the major component, dermatan sulfate and chondroitin sulfate being the minor ones. Currently dermatan sulfate and chondroitin sulfate are quantified by UV detection of their unsaturated disaccharides obtained by enzymatic depolymerization. Due to the complexity of danaparoid biopolymers and the presence of shared components, an orthogonal approach has been applied using more advanced tools and methods. To integrate the analytical profile, 2D heteronuclear single quantum coherence (HSQC) NMR spectroscopy was applied and found effective to identify and quantify GAG component signals as well as those of some process signatures of danaparoid active pharmaceutical ingredient (API) batches. Analyses of components of both API samples and size separated fractions proceeded through the determination and distribution of the molecular weight (Mw) by high performance size exclusion chromatographic triple detector array (HP-SEC-TDA), chain mapping by LC/MS, and mono- (¹H and 13C) and bi-dimensional (HSQC) NMR spectroscopy. Finally, large scale chromatographic isolation and depolymerization of each GAG followed by LC/MS and 2D-NMR analysis, allowed the sequences to be defined and components to be evaluated of each GAG including oxidized residues of hexosamines and uronic acids at the reducing ends.

Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin.

Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL(-1) at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL(-1), exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL(-1).

Development of a rapid method for simultaneous separation of hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparin by capillary electrophoresis.

This study reports the use of diethylenetriamine as background electrolyte for the simultaneous separation of hyaluronan acid, chondroitin sulfate, dermatan sulfate and heparin. The analytes were baseline separated by using an uncoated fused silica capillary at 37°C with a run time of 23min. The migration order, with hyaluronan acid at first and heparin at last, was related to the sulfation degree. The effect of salt concentration on resolution and migration order was also investigated. The developed method was applied to the simultaneous determination of hyaluronan acid and chondroitin sulfate in mouse plasma. Interferences in plasma were removed by protein precipitation and glycosaminoglycans were further purified by ethanol precipitation. The method was validated over the concentration range from 50 to 600µg/mL for hyaluronan acid and 500 to 6000µg/mL for chondroitin sulfate in mouse plasma. Results from assay validations showed that the method was selective and robust.