Analysis of the mandibular molars root canals morphology. Study by computed tomography.

This study aimed to analyze the root and root canal morphology of mandibular first and second molars using CBCT images. A total of 2,400 mandibular molars exams were selected from 600 patients aged between 18 and 75 years. The number of roots, number of root canals, and root canal configuration according to the Vertucci classification were verified. Overall, 94.92% of mandibular first molars and 90.17% of mandibular second molars had two separate roots. Among the biradicular molars, the first molars showed a greater incidence of type IV canals in the mesial root and type I in the distal root. In the second molars, the most common canal form was type IV in the mesial root and type I in the distal root. In the triradicular molars, the type IV and type I configurations were the most common in the mesial root of the first molar and second molar, respectively. In both triradicular molars, there was a prevalence of type I canal in the distal and DL roots. Statistical analysis was performed at a significance level of 0.05. The number of roots was correlated with gender (Spearman test), and the canal's configuration with gender and bilaterality (Wilcoxon test). The subpopulation studied has a high incidence of bilateral symmetry and mandibular molars with two roots with two distinct mesial canals and one distal canal. The bilateral configuration is possible to estimate the number of canals, especially in images that are difficult to visualize, such as atresic canals.

Analysis of the mandibular molars root canals morphology. Study by computed tomography

Abstract This study aimed to analyze the root and root canal morphology of mandibular first and second molars using CBCT images. A total of 2,400 mandibular molars exams were selected from 600 patients aged between 18 and 75 years. The number of roots, number of root canals, and root canal configuration according to the Vertucci classification were verified. Overall, 94.92% of mandibular first molars and 90.17% of mandibular second molars had two separate roots. Among the biradicular molars, the first molars showed a greater incidence of type IV canals in the mesial root and type I in the distal root. In the second molars, the most common canal form was type IV in the mesial root and type I in the distal root. In the triradicular molars, the type IV and type I configurations were the most common in the mesial root of the first molar and second molar, respectively. In both triradicular molars, there was a prevalence of type I canal in the distal and DL roots. Statistical analysis was performed at a significance level of 0.05. The number of roots was correlated with gender (Spearman test), and the canal's configuration with gender and bilaterality (Wilcoxon test). The subpopulation studied has a high incidence of bilateral symmetry and mandibular molars with two roots with two distinct mesial canals and one distal canal. The bilateral configuration is possible to estimate the number of canals, especially in images that are difficult to visualize, such as atresic canals.

Glycosaminoglycan signatures in body fluids of mucopolysaccharidosis type II mouse model under long-term enzyme replacement therapy.

Mucopolysaccharidosis type II (MPS II) is a neurometabolic disorder, due to the deficit of the lysosomal hydrolase iduronate 2-sulfatase (IDS). This leads to a severe clinical condition caused by a multi-organ accumulation of the glycosaminoglycans (GAGs/GAG) heparan- and dermatan-sulfate, whose elevated levels can be detected in body fluids. Since 2006, enzyme replacement therapy (ERT) has been clinically applied, showing efficacy in some peripheral districts. In addition to clinical monitoring, GAG dosage has been commonly used to evaluate ERT efficacy. However, a strict long-term monitoring of GAG content and composition in body fluids has been rarely performed. Here, we report the characterization of plasma and urine GAGs in Ids knock-out (Ids-ko) compared to wild-type (WT) mice, and their changes along a 24-week follow-up, with and without ERT. The concentration of heparan-sulfate (HS), chondroitin-sulfate (CS), and dermatan-sulfate (DS), and of the non-sulfated hyaluronic acid (HA), together with their differentially sulfated species, was quantified by capillary electrophoresis with laser-induced fluorescence. In untreated Ids-ko mice, HS and CS + DS were noticeably increased at all time points, while during ERT follow-up, a substantial decrease was evidenced for HS and, to a minor extent, for CS + DS. Moreover, several structural parameters were altered in untreated ko mice and reduced after ERT, however without reaching physiological values. Among these, disaccharide B and HS 2s disaccharide showed to be the most interesting candidates as biomarkers for MPS II. GAG chemical signature here defined provides potential biomarkers useful for an early diagnosis of MPS II, a more accurate follow-up of ERT, and efficacy evaluations of newly proposed therapies. KEY MESSAGES : Plasmatic and urinary GAGs are useful markers for MPS II early diagnosis and prognosis. CE-LIF allows GAG structural analysis and the quantification of 17 different disaccharides. Most GAG species increase and many structural features are altered in MPS II mouse model. GAG alterations tend to restore to wild-type levels following ERT administration. CS+DS/HS ratio, % 2,4dis CS+DS, and % HS 2s are potential markers for MPS II pathology and ERT efficacy.

Human milk glycosaminoglycans inhibit cytomegalovirus and respiratory syncytial virus infectivity by impairing cell binding.

BACKGROUND: The antiviral role of glycosaminoglycans in human milk (HM-GAGs) has been poorly investigated. They are highly sulfated polysaccharides, which were proposed to act as decoy receptors according to their structure. The aim of this study is to evaluate the antiviral potential and the mechanism of action of total and individual HM-GAGs against three pediatric clinically relevant viruses: respiratory syncytial virus (RSV), cytomegalovirus (HCMV), and rotavirus. METHODS: HM-GAGs were isolated from HM and a library of individual GAGs, structurally related to HM-GAGs, was prepared. The antiviral activity of HM-GAGs and the impact of thermal treatment were investigated in vitro by specific antiviral assays. RESULTS: We demonstrated that HM-GAGs are endowed with anti-HCMV and anti-RSV activity and that they act by altering virus attachment to cell. We clarified the contribution of individual HM-GAGs, showing a specific structure-related activity. We did not observe any alteration of HM-GAG antiviral activity after thermal treatment. CONCLUSIONS: We showed that HM-GAGs contribute to the overall antiviral activity of HM, likely exerting a synergic action with other HM antiviral agents. HM-GAGs can now be added to the list of endogenous factors that may reduce breast-milk-acquired HCMV symptomatic infections and protecting infants from respiratory tract infections by RSV. IMPACT: HM-GAGs have been poorly investigated for their antiviral action so far. We demonstrated that HM-GAGs are endowed with significant anti-HCMV and anti-RSV activity and that they are able to alter virus binding to the cell. The contribution of individual HM-GAGs is mainly exerted by the FMHep and is not based on a simple charge interaction between the virus and sulfate groups but involves a specific GAG structural configuration. Our results contribute to identifying the multiple factors synergically acting in mediating HM antiviral properties and to clarifying their specific mechanism of action.

Capillary Electrophoresis Separation of Artepillin C: Determination in Brazilian Green Propolis.

Propolis is important in complementary and alternative medicine having well-known therapeutic applications. Artepillin C, a main component of Brazilian (green) propolis, has attracted great attention for its anticancer action. Consequently, the synthesis of artepillin C has been reported but, due to the limited yield and elevated costs, this biomolecule is largely produced from Brazilian propolis. We report the capillary electrophoresis (CE) separation of artepillin C in Brazilian propolis also comparing the results with those of HPLC-UV-MS. Optimal separation was obtained with a simple buffer constituted of sodium tetraborate 30 mM pH 9.2 and detection at 210 nm. Artepillin C and the polyphenols of propolis were fully separated with a voltage gradient of 30 to 8 kV and a current of 300 µA for a total run of 50 min. The sensitivity of CE-UV was 22 times greater than HPLC-UV and 100 times more than HPLC-MS with also a stronger reduction in the run time and a greater robustness and reproducibility. The development of CE as an effective and reliable method for the analysis of artepillin C is desired as the standardized quality controls are essential before propolis or its biomolecules can be adopted routinely in nutraceuticals, food ingredients and therapeutic applications.

Structural definition of terrestrial chondroitin sulfate of various origin and repeatability of the production process.

We report results on the structure, physicochemical characteristics and purity of chondroitin sulfate (CS) samples derived from three largely available and common biological sources such as bovine and porcine trachea and chicken keel bones with the aim to define their structural signatures. Many lots of CS produced by a manufacturer at industrial scale were characterized with a view to assess the reproducibility of the process as not controlled extractive procedures may produce final products with variable structure and biological contaminants as well as not constant clinical efficacy and safety. By using standardized source animal tissues and manufacturing procedure, highly pure CS (∼92 %) products with constant structure and characteristics were obtained. Bovine CS showed a lower molecular weight (MWw of ∼21,500 Da) than porcine (MWw of ∼26,000 Da) and chicken (MWw of ∼35,900 Da) products with a CV% of ∼2.0-7.5 and a polydispersity variability of 0.7-2.7 %. The ratio between the sulfate groups main located in position 4 and 6 of N-acetyl-galactosamine (4/6 ratio) was ∼1.70 for bovine CS versus a value of 3.60 for porcine and ∼2.70 for chicken samples with a overall charge density of 0.92-0.93 and a CV% of 2.1-2.5. The final products also showed the presence of a very low and constant content of other co-purified bio(macro)molecules (hyaluronic acid, keratan sulfate, dermatan sulfate, heparan sulfate, nucleic acids and proteins), calcium and sodium, and the absence of versican. Finally, a high reproducibility of molecular weight values, disaccharide composition, specific optical rotation and particle dimension was observed. The observed parameters are structural signatures useful to specifically identify the origin of CS and obtained by a standardized and highly reproducible manufacturing process. The compositional profile determined from this study provides a measure of the norm and range of variation in CS samples of terrestrial origin produced under standardized production protocol to which future pharmaceutical/nutraceutical final products can be compared. Moreover, the physicochemical properties including molecular weight, disaccharide composition, presence of natural contaminants and particle dimension were characterized to provide the basis of CS of high quality for application as pharmaceutical/nutraceutical active agents.

Short- and Long-Term Effectiveness of Supplementation with Non-Animal Chondroitin Sulphate on Inflammation, Oxidative Stress and Functional Status in Obese Subjects with Moderate Knee Osteoarthritis before and after Physical Stress: A Randomized, Double-Blind, Placebo-Controlled Trial.

It has recently been demonstrated that chronic supplementation with nonanimal chondroitin sulfate (nonanimal CS) in overweight subjects with knee osteoarthritis (OA) improves the function, pain and inflammation, but there are no studies of its effectiveness in an acute setting. In 48 obese subjects with moderate knee OA, we investigated the effectiveness of nonanimal CS supplementation for eight weeks on the inflammation, functional status, oxidative stress, cartilage catabolism markers, metabolic profile and body composition, by Dual-Energy X-ray Absorptiometry (DXA) at the baseline, after 15 days and at the end of the eight-week study. To evaluate the acute effectiveness on inflammation, 15-min cycle training sessions were done 15 days after the start of the study and at the end. C-reactive protein (CRP) was assayed in blood samples collected before and after the two cycling exercises. The 48 obese subjects (M and F, 20-50 years, body mass index (BMI) 30-35 kg/m2) were randomly assigned to an experimental group (N = 24, 600-mg tablet of nonanimal CS/day) or the control group (N = 24, placebo). The between-groups analysis of covariance showed a significant effect on the Western Ontario and McMaster Universities Arthritis index (WOMAC) scale (p = 0.000) and CRP (p = 0.022). For intra-group differences, the result was significant in the CS group for BMI, WOMAC, CRP, total cholesterol and Homeostasis Model Assessment (HOMA). In these obese adults with OA, nonanimal CS improved the inflammation, knee function, metabolic profile and body composition.

Purification, compositional analysis, and anticoagulant capacity of chondroitin sulfate/dermatan sulfate from bone of corb (Sciaena umbra).

Chondroitin sulfate/dermatan sulfate (CS/DS) were isolated and purified for the first time from the bone of corb (Sciaena umbra) (CBG) and their chemical composition and anticoagulant activity were assessed. Infrared spectrum and agarose-gel electrophoresis for extracted CS/DS were also investigated. The results showed that the purified CS/DS obtained at a yield of 10% contains about 31.28% sulfate and an average molecular mass of 23.35 kDa. Disaccharide analysis indicated that CBG was composed of monosulfated disaccharides in positions 6 and 4 of the N-acetylgalactosamine (8.6% and 40.0%, respectively) and disulfated disaccharides in different percentages. The charge density was 1.4 and the ratio of 4:6 sulfated residues was equal to 4.64. Chondroitinase AC showed that the purified CS/DS contained mainly 74% CS and 26% DS. Moreover, the new CS/DS extracted from bone of corb showed a strong anticoagulant effect through activated partial thrombosis time (aPTT), thrombin time (TT) and prothrombin time (PT). In fact, CBG prolonged significantly (p < 0.05), aPTT and PT about 2.62 and 1.26 fold, respectively, greater than that of the negative control at a concentration of 1000 µg/mL. However, TT assay of CBG was prolonged 3.53 fold compared with the control at 100 µg/mL. The purified CS/DS displayed a promising anticoagulant potential, which may be used as a novel and soothing drug.

Oral Bioavailability and Pharmacokinetics of Nonanimal Chondroitin Sulfate and Its Constituents in Healthy Male Volunteers.

The pharmacokinetic profile of a new 800-mg tablet of nonanimal chondroitin sulfate (CS) (Mythocondro®, 800-mg tablets, Gnosis S.p.A., Italy) was investigated vs an animal CS in healthy volunteers for a total period of 48 hours. After a single 2400-mg dose of the test and the reference formulation, total CS, the compositional disaccharides (ΔDi6S, ΔDi4S and ΔDi0S), and the overall charge density were quantified in plasma. The safety and tolerability profile after a single dose of this new nonanimal CS tablets was excellent. After baseline-corrected concentrations, an overall greater plasma concentration was observed after 24 hours of ∼44% and after 48 hours of ∼45% from administration of nonanimal when compared to animal-derived CS. Moreover, nonanimal CS increases the specific sulfation in the 6-position of N-acetyl-galactosamine in human plasma CS and, as a consequence, the overall charge density, reaching double values (0.91), after 48 hours compared to bovine CS and to endogenous CS. In conclusion, nonanimal CS, possessing a lower molecular weight than an animal-derived sample, produces a greater CS concentration for a more prolonged period of time in plasma and an increase in charge density and specific 6-sulfation of endogenous plasma CS.

Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis.

Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 µg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.

Isolation, Purification and Structural Characterestics of Chondroitin Sulfate from Smooth hound Cartilage: In vitro Anticoagulant and Antiproliferative Properties.

Chondroitin sulfate was extracted from the cartilage of smooth hound (CSSH) and then purified by anion exchange chromatography. The structual characteristic of CSSH was evaluated by acetate cellulose electrophoresis, FTIR, 13C NMR and SAX-HPLC. Molecular weight of CSSH was average 68.78 KDa. Disaccharide analysis indicated that CSSH was predominately composed of monosulfated disaccharides in position 6 and 4 of the N-acetylgalactosamine (45.34% and 32.49%, respectively). CSSH was tested for in vitro anticoagulant activity using the three classical coagulation assays (activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests). The finding showed that CSSH prolonged significatively (p < 0.05), aPTT, TT and PT about 1.4, 3.44 and 1.21 fold, respectively, greater than that of the negative control at a concentration of 100 µg/ml. The CSSH caused a significant antiproliferative activity against HCT116 cell, which was 79% of cell proliferation inhibition at the concentration of 1000 µg/ml. Further, CSSH presented no toxicity against the normal cells and no hemolysis towards bovine erythrocytes for all concentrations tested. CSSH demonstrated hopeful antiproliferative and anticoagulant potential, which may be used as a novel and effective drug.

Chondroitin sulfate/dermatan sulfate from corb (Sciaena umbra) skin: Purification, structural analysis and anticoagulant effect.

In this study, chondroitin sulfate/dermatan sulfate was isolated and purified from the skin of corb (Sciaena umbra) (CSG) with a yield of 6.2%. Chemical and structural analysis showed that CSG consisted of high sulfate content 28.74% and an average molecular weight of 15.46 KDa. The separation of CSG by agarose-gel electrophoresis revealed the presence of DS and CS. Structural analysis of the purified CS/DS by means of SAX-HPLC after treatment with specific chondroitinases showed that this polymer was composed of nonsulfated disaccharide, monosulfated disaccharides and disulfated disaccharides in various percentages. The results also suggest that the percentage of CS and DS recovred in CSG were 24% and 76%, respectively. Anticoagulant activity in vitro was measured in plasma using classical anticoagulation tests: activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. The findings thus indicated that the purified CS/DS exhibits a remarkably high anticoagulant effect.

Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides.

This article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE.

Purification and structural elucidation of chondroitin sulfate/dermatan sulfate from Atlantic bluefin tuna (Thunnus thynnus) skins and their anticoagulant and ACE inhibitory activities.

Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus) skin (SGAT) and was purified and characterized. SGAT was characterized by acetate cellulose electrophoresis, FTIR spectroscopy, 13C NMR spectroscopy and SAX-HPLC. According to the results obtained for specific chondroitinases (ABC and AC) and the SAX-HPLC separation of generated unsaturated repeating disaccharides, the polymer was found to contain a disaccharide monosulfated in positions 6 and 4 of GalNAc and disulfated disaccharides in different percentages. These results were confirmed by 13C NMR experiments. The average molecular mass was 24.07 kDa, as determined by PAGE analysis. SGAT was evaluated for its in vitro anticoagulant activity via activated partial thromboplastin time, thrombin time and prothrombin time tests. The polymer showed strong inhibitory activity against angiotensin I-converting enzyme (IC50 = 0.25 mg mL-1). Overall, the results suggest that this newly extracted CS/DS can be useful for pharmacological applications.

Purification, structural characterization and antiproliferative properties of chondroitin sulfate/dermatan sulfate from tunisian fish skins.

Chondroitin sulfate/dermatan sulfate GAGs were extracted and purified from the skins of grey triggerfish (GTSG) and smooth hound (SHSG). The disaccharide composition produced by chondroitinase ABC treatment showed the presence of nonsulfated disaccharide, monosulfated disaccharides ΔDi6S and ΔDi4S, and disulfated disaccharides in different percentages. In particular, the nonsulfated disaccharide ΔDi0S of GTSG and SHSG were 3.5% and 5.5%, respectively, while monosulfated disaccharides ΔDi6S and ΔDi4S were evaluated to be 18.2%, 59% and 14.6%, 47.0%, respectively. Capillary elecrophoresis analysis of GTSG and SHSG contained 99.2% and 95.4% of chondroitin sulfate/dermatan sulfate, respectively. PAGE analysis showed a GTSG and SHSG having molecular masses with average values of 41.72KDa and 23.8KDa, respectively. HCT116 cell proliferation was inhibited (p<0.05) by 70.6% and 72.65% at 200µg/mL of GTSG and SHSG respectively. Both GTSG and SHSG demonstrated promising antiproliferative potential, which may be used as a novel, effective agent.

Analytical Methods for Assessing Chondroitin Sulfate in Human Plasma.

Chondroitin sulfate (CS) is a linear heteropolysaccharide of repeating disaccharide units bearing sulfate groups in various positions, commonly at C4 and/or C6 of galactosamine. CS plays important roles in various (patho)physiological processes also performing intriguing biological and therapeutical activities. Plasmatic CS is mainly composed of nonsulfated and 4-sulfated disaccharides. To obtain samples for the determination of CS amount and composition in blood/plasma, dried blood spot (DBS) could be used. DBSs have many advantages over other laboratory methods, allowing for large-scale population screening. Many analytical techniques may be used for the determination of CS. In particular, CE has proved to be a very attractive alternative separation technique for complex polysaccharide characterization. In this work, we compared CS levels between plasma and DBS samples, using CE equipped with the highly sensitive laser-induced fluorescence detector. CS from DBS differs from plasma CS owing to the high content of disaccharides sulfated in C4 and C6. This is due to the presence of the more sulfated CS derived from blood cellular fraction, in particular leukocytes. The identification and quantification of CS in blood plasma could be a useful prognostic and diagnostic tool in pathological conditions and for pharmacological applications.

Chondroitin Sulfate and Glucosamine as Disease Modifying Anti- Osteoarthritis Dru gs (DMOADs).

Osteoarthritis is a disabling affliction expected to increase in the coming decades, and disease- modifying osteoarthritis drugs (DMOADs) would be highly desirable adjuncts to symptomatic relief and structure reconstruction as they may delay the disease process. Chondroitin sulfate and glucosamine have been observed to exert beneficial effects on the metabolism of various cells involved in osteoarthritis as well as in animal models and clinical trials. Clinical trials have reported beneficial effects of both these biological agents, alone or in combination, on pain and functions as well as their structure-modifying capacity reported and analyzed in recent meta-analyses. Nonetheless, the effectiveness of these bioactive (macro)molecules as DMOADs reported from randomized trials is mismatched. Current studies with varying levels of evidence suggest that chondroitin sulfate and glucosamine can modify the disease progression but at the same time there are not absolute certainties on their efficacy in modifying the course of the disease. This comprehensive review aims to clarify the role of these compounds in the therapeutic molecules/ drugs useful to patients affected by osteoarthritis.

Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns.

In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10-18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity.

Recent advances on separation and characterization of human milk oligosaccharides.

Free human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography, off-line and on-line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state-of-the-art techniques used in HMO analysis.