Analysis Of Oligosaccharides Research Articles

Structure analysis and immunomodulatory activity of novel oligosaccharide from Nicotiana tabacum roots

A novel oligosaccharide (NTRP60-W-2) with an average molecular weight of 1377 Da was isolated and purified from Nicotiana tabacum roots. Its structural characteristics and immunomodulatory properties were investigated. Structural analysis revealed that NTRP60-W-2 was composed exclusively of glucose, featuring →1)-α-D-Glcp-(6→ backbone. Immunological assays demonstrated that NTRP60-W-2 significantly enhanced cell viability, nitric oxide production and cytokine secretion (IL-6 and TNF-α) in RAW264.7 cells. These findings provide a foundation for further exploration of Nicotiana tabacum carbohydrates and their potential biological activities.

A Comparative Analysis of Milk Oligosaccharides via LC-MS: Globally Distributed Cattle Breeds and Native Northern Finncattle

Milk oligosaccharides are complex carbohydrates composed of various monosaccharide units linked together by glycosidic bonds. They play an essential role in promoting gut health by fostering beneficial bacteria, supporting the development of the immune system, and protecting against infections and diseases. This work compared the oligosaccharide profiles in widely utilized breeds such as Holstein and Ayrshire (Nordic Red), with the native Northern Finncattle, which is considered an endangered breed. Oligosaccharides were extracted from milk and analyzed by liquid chromatography–mass spectrometry. The composition and relative abundance of the identified oligosaccharides were characterized and compared. The statistical analyses showed that neutral, sialylated, and fucosylated oligosaccharides vary among the breeds. Ayrshire and Northern Finncattle oligosaccharides formed a cluster, while Holstein’s profile shared features with both Ayrshire and Northern Finncattle. Holstein had the lowest abundance of fucosylated OS among the three breeds, with Ayrshire having the highest content followed by Northern Finncattle. The relatively higher sialylated over neutral content of Northern Finncattle is an important feature that should be preserved. Ayrshire is a good candidate to recover more diverse oligosaccharides with potential gut health implications for consumers.

Analysis of human milk oligosaccharides from women with gestational diabetes mellitus

Human milk oligosaccharides (HMOs) are bioactive components which play an important role in infant health. HMO composition is vulnerable to changes of maternal conditions including lactation stages and maternal phenotypes. Pregnant diseases such as gestational diabetes mellitus (GDM) are commonly found in women during pragnancy, and may cause disorder in maternal physiological metabolism which is harmful to infants. Unfortunately, anlysis of oligosaccharides from women with GDM is limited. To address this issue, we analyzed HMO compositions and profiles in breast milk from women with GDM using an established 96-well plate permethylation platform and MALDI-TOF-MS. We enrolled 127 women with GDM, and investigated HMO abundances in colostrum, transition milk, and mature milk respectively. We found that GDM affected HMO compositions in breast milk, and the level of fucosylation became higher over the course of lactation for all the women with GDM. Interestingly, the relative abundances of fucosylated HMOs in different lactation stages were affected differentially by GDM, with the most pronounced effect in colostrum. In particular, the relative abundances of H3N1F1 and H3N1F2 sharply decreased over time, showing very low levels in late lactation. These differences in our findings need further investigation to develop optimal feeding for mothers with GDM.

Application of an α-galactosidase from Bacteroides fragilis on structural analysis of raffinose family oligosaccharides

Raffinose family oligosaccharides (RFOs) have diverse structures and exhibit various biological activities. When using RFOs as prebiotics, their structures need to be identified. If we first knew whether an RFO was classical or non-classical, structural identification would become much easier. Here, we cloned and expressed an α-galactosidase (BF0224) from Bacteroides fragilis which showed strict specificity for hydrolyzing α-Gal-(1 → 6)-Gal linkages in RFOs. BF0224 efficiently distinguished classical from non-classical RFOs by identifying the resulting hydrolyzed oligo- and mono-saccharides with HPAEC-PAD-MS. Using this strategy, we identified a non-classical RFO from Pseudostellaria heterophylla (Miquel) Pax with DP6 (termed PHO-6), as well as a classical RFO from Lycopus lucidus Turcz. with DP7 (termed LTO-7). To characterize these RFO structures, we employed four other commercial or reported α-galactosidases in combination with NMR and methylation analysis. Using this approach, we elucidated the accurate chemical structure of PHO-6 and LTO-7. Our study provides an efficient analytical approach to structurally analyze RFOs. This enzyme-based strategy also can be applied to structural analysis of other glycans.

Physicochemical analysis of chitosan oligosaccharide revealed its usefulness in effective delivery of drugs

Chitosan oligosaccharides are biopolymers with a wide range of potential applications in various fields. This biopolymer is diverse and promising, and current research is investigating its capabilities for improved drug delivery. As chitosan oligosaccharide has the potential to be used as a drug delivery option, the purpose of this study was to examine its physicochemical characteristics and its potential for drug delivery. In this study, the pharmacokinetic properties of chitosan oligosaccharide were studied through Insilco investigation, which revealed that it is an extremely soluble and effective drug delivery candidate because it does not inhibit CYP isoenzymes and has a log Kp of −12.10 cm/s. It belongs to toxicity class 6 for acute oral toxicity, with an average similarity of 87.5% and a prediction accuracy of 70.97%. Additionally, XRD peak analysis revealed that the material was amorphous, as the peak appeared at 2θ = 24.62°, indicating the absence of well-defined crystalline areas. This characteristic makes the material more suitable for customization in many applications such as drug delivery and tissue engineering. FTIR, SEM, and TGA analysis were performed to gain a better understanding. These findings also emphasize the distinctive qualities and benefits of the oligosaccharides in this domain. Application of chitosan oligosaccharides in the development of efficient drug delivery systems. In the future, it would be more effective, targeted, and safe, with potent therapeutic efficacy for drug delivery.

Isolation and characterization of feruloylated oligosaccharides from Phyllostachys acuta and in vitro antioxidant activity

Feruloylated oligosaccharides (FOs) generated by decomposing plant hemicellulose, offer a wide range of potential applications in both the food and biomedical areas. As a graminaceous plant, bamboo is rich in hemicellulose. However, the structural composition and activity studies of FOs from it were rarely reported. In this study, FOs from Phyllostachys acuta (pFOs) obtained by enzymatic hydrolysis were isolated by AmberliteXAD-2 and C18 SPE columns. Then, pFOs were qualitatively and quantitatively analyzed by UPLC-ESI-MS/MS after labeled by 3-Amino-9-ethyl-carbazole (AEC), and the chemical antioxidant activity of pFOs and effects of pFOs on H2O2-induced oxidative damage were investigated. Finally, 14 of pFOs isomers were distinguished and identified, of which 10 did not contain hexoses and 4 did, and the three most abundant pFO structures were 12 (Iso 7, F1A1X2H2-AEC, 29.04 %), 11 (Iso 6, F1A1X1H2-AEC, 17.96 %), and 4 (Iso 3-1, F1A1X3-AEC, 15.57 %). The results of antioxidant studies showed that pFOs possessed certain reducing power, scavenging DPPH radicals, scavenging superoxide anion radicals, and scavenging hydroxyl radicals. Among them, the ability to clear DPPH radicals was particularly significant. pFOs significantly reduced the viability of RAW264.7 cells after H2O2 induction, whereas pFOs had a significant protective effect (p < 0.001). pFOs increased the viability of T-AOC and SOD enzymes in oxidatively damaged cells, as well as had a significant inhibition effect on ROS elevation (p < 0.001). This study lays the foundation for the structural analysis and antioxidant activity evaluation of bamboo-derived feruloyl oligosaccharides for their application in food and pharmaceutical fields.

Application of on-line sample preconcentration by large-volume dual preconcentration by isotachophoresis and stacking (LDIS) on straight-channel microchips.

In this study, large-volume dual preconcentration by isotachophoresis and stacking (LDIS) which is an on-line sample preconcentration technique coupling large-volume sample stacking with an electroosmotic flow pump (LVSEP) with transient isotachophoresis (tITP) was applied to microchip electrophoresis (MCE) for improving both detection sensitivities and peak shapes. To realize LDIS in MCE, we investigated experimental procedures for injecting a short plug of a leading electrolyte (LE) solution into a straight microchannel without any sophisticated injector apparatus. We found that a short LE plug could be injected into a sample-filled straight-channel only by making the liquid level of the LE solution in an outlet reservoir higher than that in an inlet one. By applying a reversed-polarity voltage to the microchip, anionic analytes injected throughout the microchannel were first enriched by LVSEP, followed by tITP. Through the second preconcentration effect by tITP in LDIS, sensitivity enhancement factor (SEF) and asymmetry factor for a standard dye were improved from 878 and 0.62 to 1330 and 1.14, respectively, relative to those in conventional LVSEP. It should be noted that more viscous running buffer containing sieving polymers could be employed to the LDIS analysis, which was effective for improving the SEF and the separation efficiencies, especially for bio-polymeric compounds. Finally, LDIS was applied to the oligosaccharide and protein analyses in MCE, resulting in the SEFs of 1410 and ca. 50 for maltotriose and bovine milk casein, respectively.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Malting and Brewing Performance of β-Amylase-Deficient Barley

β-Amylase (α-1,4-glucan maltohydrolase; EC 3.2.1.2) is known to be an important enzyme in beer brewing. To identify the contribution of β-amylase in more detail, two β-amylase-deficient barley, ‘Backcrossed line (BCL)-Haruna’ and ‘Nittakei 81’, were used for the investigation of malt quality and brewing characteristics. ‘BCL-Haruna’ was bred by the backcross method for the introduction of the β-amylase-deficient trait from landrace barley (‘Tibet Violet 1’) to Japanese traditional malting barley ‘Haruna Nijo’. ‘Nittakei 81’ was bred by crossing between Japanese malting barley ‘Sukai Golden’ and ‘Tibet Violet 1’. With regards to the malting quality under 41.0% target cast moisture in micro-malting, the diastatic power (DP) and apparent attenuation limit (AAL) of ‘BCL-Haruna’ and ‘Nittakei 81’ were 408 oWK and 416 oWK and 12.4% and 14.8% lower than those of the control variety ‘Haruna Nijo’. Oligosaccharide analysis indicated that the maltose concentration of the ‘Nittakei 81’ wort was 19.7 g/L lower in comparison to a standard European malting barley ‘Propino’ wort, although the maltotetraose and maltopentaose concentrations of the ‘Nittakei 81’ wort were 5.6 g/L and 2.7 g/L higher than those of the ‘Propino’ wort. After fermentation, the alcohol concentration of the ‘Nittakei 81’ beer was 0.67% lower compared with the ‘Propino’ beer, and the extract and concentrations of maltotetraose and maltopentaose of the ‘Nittakei 81’ beer were 5.0 g/L and 2.5 g/L higher than those of the ‘Propino’ beer. The results indicate that the β-amylase-deficient barley has a unique malting quality, especially lower DP and AAL, which might induce inadequate starch degradation or fermentation. However, this trait caused no serious issues during the brewing process, suggesting the possibility of β-amylase-deficient barley as a unique brewing material including low-alcoholic and non-alcoholic beer production with a specific saccharide composition.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary Oligosaccharides and Glycoamino Acids for the Diagnosis of Mucopolysaccharidosis and Glycoproteinosis.

Mucopolysaccharidosis (MPS) and glycoproteinosis are 2 groups of heterogenous lysosomal storage disorders (LSDs) caused by defective degradation of glycosaminoglycans (GAGs) and glycoproteins, respectively. Oligosaccharides and glycoamino acids have been recognized as biomarkers for MPS and glycoproteinosis. Given that both groups of LSDs have overlapping clinical features, a multiplexed assay capable of unambiguous subtyping is desired for accurate diagnosis, and potentially for severity stratification and treatment monitoring. Urinary oligosaccharides were derivatized with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) together with the underivatized glycoamino acids. Novel biomarkers were identified with a semi-targeted approach with precursor mass scanning, the fragmentation pattern (if applicable), and the biochemical basis of the condition. A UPLC-MS/MS analysis with improved chromatographic separation was developed. Novel biomarkers for MPS-IIIA, IIIB, IIIC, and VII were identified and validated. A total of 28 oligosaccharides, 2 glycoamino acids, and 2 ratios were selected as key diagnostic biomarkers. Validation studies including linearity, lower limit of quantitation (LLOQ), and precision were carried out with the assay performance meeting the required criteria. Age-specific reference ranges were collected. In the 76 untreated patients, unambiguous diagnosis was achieved with 100% sensitivity and specificity. Additionally, the levels of disease-specific biomarkers were substantially reduced in the treated patients. A multiplexed UPLC-MS/MS assay for urinary oligosaccharides and glycoamino acids measurement was developed and validated. The assay is suitable for the accurate diagnosis and subtyping of MPS and glycoproteinosis, and potentially for severity stratification and monitoring response to treatment.

Difference analysis of oligosaccharides in different varieties of Rehmannia glutinosa

The oligosaccharide and monosaccharide contents of different varieties of Rehmannia glutinosa were compared to explore the difference in the oligosaccharide and monosaccharide contents in different varieties and to provide a scientific basis for formulation of product quality standards of Rehmannia glutinosa. The contents of fructose, glucose, sucrose, raffinose, and stachyose were determined via high-performance liquid chromatography. The total sugar content in the fresh Rehmannia glutinosa varieties in descending order was as follows: Beijing Tuodu 3 &gt; Huaifeng &gt; Jinjiu &gt; Huaizhong 1 &gt; Beijing 3. The total sugar content in the raw Rehmannia glutinosa varieties in descending order was as follows: Jinjiu &gt; Huaifeng &gt; Beijing Tuodu 3 &gt; Beijing 3 &gt; Huaizhong 1. The stachyose content was the highest among the five oligosaccharides. The total sugar content in the Beijing Tuodu 3 variety was the highest among the fresh Rehmannia glutinosa varieties, and the total sugar content in the Jinjiu variety was the highest among the raw Rehmannia glutinosa varieties. There were significant differences in the oligosaccharide and monosaccharide contents among the different varieties.

UPLC-MS/MS analysis of urinary oligosaccharides for the diagnosis of mucopolysaccharidosis and glycoproteinosis

UPLC-MS/MS analysis of urinary oligosaccharides for the diagnosis of mucopolysaccharidosis and glycoproteinosis

Sensitivity and specificity of serum oligosaccharide analysis for the diagnosis and treatment monitoring of patients with alpha-mannosidosis

Sensitivity and specificity of serum oligosaccharide analysis for the diagnosis and treatment monitoring of patients with alpha-mannosidosis

Combining Liquid Chromatography and Cryogenic IR Spectroscopy in Real Time for the Analysis of Oligosaccharides.

While the combination of liquid chromatography (LC) and mass spectrometry (MS) serves as a robust approach for oligosaccharide analysis, it has difficulty distinguishing the smallest differences between isomers. The integration of infrared (IR) spectroscopy within a mass spectrometer as an additional analytical dimension can effectively address this limitation by providing a molecular fingerprint that is unique to each isomer. However, the direct interfacing of LC-MS with IR spectroscopy presents a technical challenge arising from the mismatch in the operational time scale of each method. In previous studies, this temporal incompatibility was mitigated by employing strategies designed to slow down or broaden the LC elution peaks of interest, but this workaround is applicable only for a few species at a time, necessitating multiple LC runs for comprehensive analysis. In the current work, we directly couple LC with cryogenic IR spectroscopy by acquiring a spectrum in as little as 10 s. This allows us to generate an orthogonal data dimension for molecular identification in the same amount of time that it normally takes for LC analysis. We successfully demonstrate this approach on a commercially available human milk oligosaccharide product, acquiring spectral information on the eluting peaks in real time and using it to identify both the specified constituents and nonspecified product impurities.

Preparation, Structural Analysis, and Intestinal Probiotic Properties of a Novel Oligosaccharide from Enzymatic Degradation of Huangshui Polysaccharide.

Huangshui polysaccharide (HSP) has attracted more and more interest due to its potential health benefits. Despite being an excellent source for the preparation of oligosaccharides, there are currently no relevant research reports on HSP. In the present study, a novel oligosaccharide (HSO) with a molecular weight of 1791 Da and a degree of polymerization of 11 was prepared through enzymatic degradation of crude HSP (cHSP). Methylation and NMR analyses revealed that the main chain of HSO was (1 → 4)-α-d-glucose with two O-6-linked branched chains. Morphological observations indicated that HSO exhibited smooth surface with lamellar and filamentary structure, and the glycan size ranged from 0.03 to 0.20 μm. Notably, HSO significantly promoted the proliferation of Bifidobacterium, Bacteroides, and Phascolarctobacterium, thereby making positive alterations in intestinal microbiota composition. Moreover, HSO markedly increased the content of short-chain fatty acids during in vitro fermentation. Metabolomics analysis illustrated the important metabolic pathways primarily involving glucose metabolism, amino acid metabolism, and fatty acid metabolism.

Evaluation of solid-phase extraction sorbents for purification of oligosaccharides and glycans derivatized by positively charged labels followed by capillary electrophoretic analysis.

The sample preparation including labeling and clean-up represents a key analytical step in the analysis of oligosaccharides and glycans by either chromatographic or electrophoretic separation methods. Although the majority of labeling has been performed by neutral and/or negatively charged tags, the introduction of a positive charge into the saccharide molecule can significantly improve the analysis, especially with mass spectrometry detection. In this work, we present the evaluation of five solid-phase extraction sorbents differing in extraction chemistry for the clean-up and concentration of positively labeled maltooligosaccharides from the reaction mixtures. Maltooligosaccharides containing four to seven glucose units were labeled by cationic tags (2-aminoethyl)trimethylammonium chloride and (carboxymethyl)trimethylammonium chloride hydrazide and the extraction conditions were optimized followed by electrophoretic analysis with conductivity detection. The effects of the solid-phase extraction sorbent chemistry, extraction conditions, and sample composition are discussed. All tested sorbents were capable of cleaning up maltooligosaccharides from the reaction mixtures to some extent after optimization of the solid-phase extraction procedure (51.9%-98.9% recovery). The best-rated amide-based sorbent was used to process the sample of N-linked glycans enzymatically released from ribonuclease B.

Synthesis and application of BODIPY-based fluorescent labeling tag for oligosaccharide and N-linked glycan analysis by high-performance liquid chromatography with fluorescence detection

BackgroundGlycosylation analysis is still challenging, not only because of the extreme structure complexity and conjugation diversity of glycans but also because of instrumental aspects such as the sensitivity limits of analyses. Therefore, glycan analysis by chromatographic methods is very often combined with fluorescence detection in addition to MS. The majority of fluorescent labeling employed before LC separation is based on 2-aminobenzamide, which has several disadvantages such as low labeling yield, poor fluorescence properties, and MS ionization efficiency. Therefore, even after several decades of development of new labels, there is still a need for new labeling tags with improved characteristics. ResultsWe present the application of a newly synthesized fluorescent label designed for oligosaccharide and glycan analysis by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). The novel hydrazide derivative of dipyrrometheneboron difluoride (BODIPY) was synthesized from 2,4-dimethylpyrrole, methyl succinyl chloride, and boron trifluoride etherate followed by a reaction with hydrazine. The synthesized label was characterized by several analytical methods including NMR, UV/Vis and fluorescence spectroscopy, and mass spectrometry. The labeling reaction via hydrazone formation chemistry was optimized by labeling of maltooligosaccharide standards. The analysis of maltohexaose labeled by BODIPY-hydrazide followed by HPLC/FLD analysis provided the limit of detection in the low tens of femtomole. The presented method based on fluorescence detection is at least 30 times more sensitive than the standard approach employing labeling by 2-aminobenzamide. In addition, the labeling method by BODIPY-hydrazide was used for N-linked glycan profiling of several glycoproteins (ribonuclease B, immunoglobulin G) by RP-HPLC/FLD as well as HILIC/FLD analysis. SignificanceThis work represents the design, synthesis, and application of a new fluorescent label based on the BODIPY core and hydrazone formation chemistry for oligosaccharide and glycan analysis by HPLC/FLD. The proposed approach significantly improved the oligosaccharide and glycan analysis in comparison to the commonly used procedure employing 2-aminobenzamide.

On-line sample preconcentration by LVSEP-FASI with simple voltage control on Y-channel chips.

To eliminate complicated voltage controls for highly sensitive microchip electrophoresis (MCE) analyses on the basis of combining two online sample preconcentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), cross-channel microchips and a multichannel high-voltage power supply were replaced to Y-channel chips and a conventional power supply designed for capillary electrophoresis, respectively. By simple switching of the electric circuit after the LVSEP-FASI sample enrichments, the focused analytes could be separated during anodic migration in a separation channel. In the LVSEP-FASI analysis of fluorescein using the Y-channel microchip, the maximum sensitivity enhancement factor (SEF) of 7400 was achieved, resulting in a 30-fold detectability increase compared to the conventional LVSEP. The developed method was applied to the oligosaccharide analysis in MCE. As a result, the SEF for maltotriose was improved from 450 to 2300 and the baseline separation of the oligosaccharides was achieved without any complicated voltage control in LVSEP-FASI on the Y-channel chips.

Optimization of preparation conditions, molecular structure analysis and antitumor activity of sulfated sodium alginate oligosaccharides

Sodium alginate is a kind of marine polysaccharide extracted from brown algae, and its degradation products have been widely used in food, medicine and other fields. Sodium alginate oligosaccharide (AOS) was degraded by hydrogen peroxide from sodium alginate and modified by sulfonic acid-chlorosulfonic acid-pyridine to obtain sulfated alginate oligosaccharide (S-AOS). The main research contents included the optimization of the preparation process of S-AOS and the study of its structure characterization and biological activity. First, the optimal reaction conditions of S-AOS were optimized by single factor analysis and response surface analysis. The sulfated derivatives were characterized using Fourier transform infrared spectrum (FTIR), High performance liquid chromatography (HPLC), Nuclear magnetic resonance Hydrogen (1H NMR) spectrum and Electrospray ionization mass spectrometry, and molecular formula and substitutions were determined. Molecular docking, GO and KEGG enrichment analysis were used to predict S-AOS biological activity and possible action targets, and the mechanism of tumor suppression activity of S-AOS was preliminarily investigated. The results showed that, the optimal reaction conditions for S-AOS was finally determined as: Chlorosulfonic acid - pyridine volume ratio was 7:1. Sulfation reagent – AOS volume ratio was 3:1. Reaction time was 3 h. Reaction temperature Was 40 ℃. Under this condition. In contrast to AOS, S-AOS showed two new absorption peaks in the FTIR spectrum and two new chemical shift peaks in the 1H NMR spectrum, suggesting that the sulfate group selectively reacts with the hydroxyl groups on the third carbon and the fourth on the mannoconide. The relative molecular mass of S-AOS was 1566 g/mol and a degree of sulfate substitution was 0.931. Molecular docking screening, GO and KEGG enrichment analysis of S-AOS revealed that it may have a role in apoptosis, oxidative stress, thrombosis, and cellular autophagy. In vitro experiments showed that S-AOS could inhibit the expression of apoptosis and proliferation related proteins, and showed certain anti-tumor activity. S-AOS had more or stronger biological activity by introducing sulfuric acid groups, and its application in the field of biomedicine is expanded.

Purification free N-glycan analysis by capillary zone electrophoresis: Hunt for the lost glycans

Capillary gel electrophoresis is a widely used method for rapid separation of fluorophore labeled carbohydrates. Even though, many publications conferred about this popular technique, no report yet investigated the possible sample losses during the purification process of the fluorophore labeling reaction mixture. In the present work, normal polarity capillary zone electrophoresis separation mode was applied to take advantage of the opposite migration directions of the electroosmotic flow and the negatively charged sample components using Tris-hexanoic acid running buffer at basic pH. For purification free oligosaccharide analysis, the separation parameters were designed in such a way that the triple charged labeling reagent of aminopyrenetrisulfonate (APTS) could not enter the separation capillary in contrary to the labeled sample components of interest, therefore, the APTS did not have to be removed before analysis. The method was used to show electrophoretic profile differences possibly caused by the cleanup process that was immediately apparent by comparing the electropherograms of the purified and non-purified APTS labeled maltooligosaccharides. Furthermore, qualitative and quantitative N-glycosylation profile alterations were revealed during CZE separation of the fluorophore labeling reaction mixtures before and after purification along with the analysis of the consecutively used washing solutions for the well characterized standard glycoproteins of IgG, ribonuclease B and fetuin.