Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus).

Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5- 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2- 3.1 and 0.9- 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised ß-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).

Multicomponent analysis of dietary supplements containing glucosamine and chondroitin: comparative low- and high-field NMR spectroscopic study.

With the prevalence of glucosamine- and chondroitin-containing dietary supplements for people with osteoarthritis in the marketplace, it is important to have an accurate and reproducible analytical method for the quantitation of these compounds in finished products. NMR spectroscopic method based both on low- (80 MHz) and high- (500-600 MHz) field NMR instrumentation was established, compared and validated for the determination of chondroitin sulfate and glucosamine in dietary supplements. The proposed method was applied for analysis of 20 different dietary supplements. In the majority of cases, quantification results obtained on the low-field NMR spectrometer are similar to those obtained with high-field 500-600 MHz NMR devices. Validation results in terms of accuracy, precision, reproducibility, limit of detection and recovery demonstrated that the developed method is fit for purpose for the marketed products. The NMR method was extended to the analysis of methylsulfonylmethane, adulterant maltodextrin, acetate and inorganic ions. Low-field NMR can be a quicker and cheaper alternative to more expensive high-field NMR measurements for quality control of the investigated dietary supplements. High-field NMR instrumentation can be more favorable for samples with complex composition due to better resolution, simultaneously giving the possibility of analysis of inorganic species such as potassium and chloride.

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.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Adherence of Candida albicans and Malassezia Species to Skin Cells Induces Changes in the Expression of Genes Responsible for Heparan and Chondroitin Sulfate Chain Synthesis. / La unión de Candida albicans y Malassezia spp. a células de piel promueve cambios de expresión en los genes responsables de la síntesis de las cadenas de heparán y condroitín sulfato.

Superficial fungal infections are common in dermatology and are often caused by opportunistic species in the Candida and Malassezia genera. The aim of this study was to analyze changes in the expression of genes coding for enzymes involved in the biosynthesis of glycosaminoglycans (GAGs) chains following the adherence of Candida and Malassezia yeasts to skin cell lines. Gene expression was analyzed using reverse transcriptase-quantitative polymerase chain reaction assays. Interactions between the yeasts and the skin cells induced the following changes in genes involved in the biosynthesis of heparan sulfate and chondroitin sulfate: downregulation of CHPF in keratinocytes and downregulation of EXT1, EXT2, CHSY3, and CHPF in fibroblasts. Adherence to fibroblasts had an even greater effect on GAG biosynthetic enzymes, inducing the downregulation of 13 genes and the upregulation of two (CHST15 and CHST7). Interactions between yeasts and skin cells might affect the binding affinity of GAG chains, possibly changing their ability to function as receptors for pathogens and interfering with a key stage at the start of infection.

A general strategy for the structural determination of carbohydrates by multi-dimensional NMR spectroscopies.

Two-dimensional NMR spectroscopies are one of the most frequently used techniques for the structural determination of carbohydrates. However, the data analysis is challenging because of the signal overlap in the 1H homonuclear correlation spectra. We attempted to explore a general strategy for the structural determination of carbohydrates by combined multi-dimensional spectroscopies. The strategy was applied to a human milk oligosaccharide lacto-N-difucohexaose I, that has been previously studied by conventional two-dimensional NMR spectroscopy. Assignment of the intra-residue resonances of the hexasaccharide using the three-dimensional spectrum was straightforward. Consequently, data analysis of the multi-dimensional spectra was significantly simplified, leading to a quicker determination of the intra- and inter-residue connections in the hexasaccharide. Application of the NMR strategy to chondroitin sulfate from bovine cartilage revealed two repeating disaccharide regions of the A and C units of chondroitin sulfate, indicating the high potential of this technique for the structural determination of complex polysaccharides.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

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..

Vulnerability to shear stress caused by altered peri-endothelial matrix is a key feature of Moyamoya disease.

Moyamoya disease (MMD) is characterized by progressive bilateral stenotic changes in the terminal portion of the internal carotid arteries. Although RNF213 was identified as a susceptibility gene for MMD, the exact pathogenesis remains unknown. Immunohistochemical analysis of autopsy specimens from a patient with MMD revealed marked accumulation of hyaluronan and chondroitin sulfate (CS) in the thickened intima of occlusive lesions of MMD. Hyaluronan synthase 2 was strongly expressed in endothelial progenitor cells in the thickened intima. Furthermore, MMD lesions showed minimal staining for CS and hyaluronan in the endothelium, in contrast to control endothelium showing positive staining for both. Glycosaminoglycans of endothelial cells derived from MMD and control induced pluripotent stem cells demonstrated a decreased amount of CS, especially sulfated CS, in MMD. A computational fluid dynamics model showed highest wall shear stress values in the terminal portion of the internal carotid artery, which is the predisposing region in MMD. Because the peri-endothelial extracellular matrix plays an important role in protection, cell adhesion and migration, an altered peri-endothelial matrix in MMD may contribute to endothelial vulnerability to wall shear stress. Invading endothelial progenitor cells repairing endothelial injury would produce excessive hyaluronan and CS in the intima, and cause vascular stenosis.

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

Reliable and sensitive detection of glycosaminoglycan chains with immunoblots.

Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.

Offline Selective Extraction Combined with Online Enrichment for Sensitive Analysis of Chondroitin Sulfate by Capillary Electrophoresis.

A capillary electrophoresis (CE) method combined with online and offline enrichment for improving the detection sensitivity of chondroitin sulfate (CS) is established. The online enrichment method is based on the field-amplified sample stacking and large volume electrokinetic injection, and offline enrichment is based on the association between cetyltrimethylammonium chloride and CS. Experimental parameters affecting CE method such as the type and pH of background electrolyte, the injection mode and time and the steps of offline enrichment were optimized. Under optimum conditions, the calibration plot between CS concentration and peak area was linear in the range of 1 ~ 100 µg/mL. The enrichment factor was 130 times and the limit of detection was 50 ng/mL. The average recovery was 103.5% and the relative standard deviation of peak area was <2.0%. The method was successfully applied to the quantitative analysis of CS in drugs.

Rapid Differentiation of Chondroitin Sulfate Isomers by Gas-phase Hydrogen-deuterium Exchange.

Chondroitin sulfate (CS)-glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides attached to CS proteoglycans that make up a major component of biological matrices throughout both central and peripheral tissues. The position of their attached sulfate groups to the CS disaccharide is predicted to influence protein-glycan interactions and biological function. Although traditional immunohistochemical analysis of CS-GAGs in biological tissues has provided information regarding changes in GAG abundance during developmental and disease states, quantitative analysis of their specific sulfation patterns is limited due to the inherent complexity of separating CS isomers. While methods have been developed to analyze and quantify sulfation isomers using liquid phase separation, new techniques are still needed to elucidate the full biology of CS-GAGs. Here, we examine ion mobility spectrometry and gas-phase hydrogen-deuterium exchange to resolve positional sulfation isomers in the most common sulfated 4S- and 6S-CS disaccharides. The mobilities for these two isomers are highly similar and could not be resolved effectively with any drift gas tested. In contrast, gas-phase hydrogen-deuterium exchange showed very different rates of deuterium uptake with several deuterium exchange reagents, thereby presenting a promising novel and rapid approach for resolving CS isomers.

Structure characterization of a heavily fucosylated chondroitin sulfate from sea cucumber (H. leucospilota) with bottom-up strategies.

Two bottom-up strategies, disaccharide and oligosaccharide analyses, were applied to elucidate the structure of a fucosylated chondroitin sulfate (FCS). The FCS was hydrolyzed with mild acid. The remained part was digested with CS lyase for disaccharide analysis. The products from each step were analyzed and the results revealed that mild sulfuric acid mainly released sulfated fucose branches, but also affected some residues and sulfo-groups on the backbone. Over 140 oligosaccharide fragments were generated by catalytic oxidation and identified by HPSEC-MS, including sulfated fucose oligosaccharides exclusively from branches, sulfated backbone fragments, and junctional fragments. Based on the results provided by these two methods, the proposed backbone of the FCS is mainly composed of GlcA→GalNAc4S6S and GlcA→GalNAc6S, and the branch is mainly located at GalNAc. The longest branch observed is nonasaccharide, and most of the fucose on the branches are mono and/or di-sulfated. NMR results supported the conclusion.

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.

Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate.

Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it were mostly inclusion bodies. In this study, a signal peptide (pelB) was used for the soluble form expression of ChSase ABC I in E. coli. Then the culture condition for ChSase ABC I expression was optimized through response surface methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L fermentor (29.03 mL-1) was approximately 1.65-fold higher than that of the shake flask level (17.55 mL-1). The enzymatic properties and kinetic constants of recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body, but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.

Tools for the Quality Control of Pharmaceutical Heparin.

Heparin is a vital pharmaceutical anticoagulant drug and remains one of the few naturally sourced pharmaceutical agents used clinically. Heparin possesses a structural order with up to four levels of complexity. These levels are subject to change based on the animal or even tissue sources that they are extracted from, while higher levels are believed to be entirely dynamic and a product of their surrounding environments, including bound proteins and associated cations. In 2008, heparin sources were subject to a major contamination with a deadly compound-an over-sulphated chondroitin sulphate polysaccharide-that resulted in excess of 100 deaths within North America alone. In consideration of this, an arsenal of methods to screen for heparin contamination have been applied, based primarily on the detection of over-sulphated chondroitin sulphate. The targeted nature of these screening methods, for this specific contaminant, may leave contamination by other entities poorly protected against, but novel approaches, including library-based chemometric analysis in concert with a variety of spectroscopic methods, could be of great importance in combating future, potential threats.

European chondroitin sulfate and glucosamine food supplements: A systematic quality and quantity assessment compared to pharmaceuticals.

Chondroitin sulfate and glucosamine, commercialized as anti-osteoarthritis food supplements, do not undergo the strict quality controls of pharmaceuticals. In this paper a systematic multi-analytical approach was designed to analyse 25 food supplements from 8 European countries compared to 2 pharmaceuticals by using high performance anion-exchange chromatography with pulsed amperometric detection, size exclusion chromatography with triple detector array, capillary electrophoresis, mono and bi-dimensional NMR. Furthermore the biological activity was assessed on in vitro human synoviocyte and chondrocyte primary cell models. Most of the samples (over 19 out of 25) showed lower condroitin sulfate and glucosamine contents than the declared ones (up to -60.3%) while all of them showed a KS contamination (up to 47.1%). Mixed animal origin chondroitin sulfate and multiple molecular weight species were determined in more than 32% of the samples. Only 1 on 5 biologically screened samples had an effective action in vitro almost comparable to the pharmaceuticals.

Bottom-up analysis using liquid chromatography-Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers.

Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure-activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography-Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.