scholarly journals Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

2019 ◽  
Author(s):  
Hao Liu ◽  
Apoorva Joshi ◽  
Pradeep Chopra ◽  
Lin Liu ◽  
Geert-Jan Boons ◽  
...  
Keyword(s):  
Structure Function ◽  
Structural Complexity ◽  
Reversed Phase ◽  
Separation Method ◽  
Size Exclusion ◽  
Reaction Products ◽  
Exclusion Chromatography ◽  
Function Characterization ◽  
Function Relationship ◽  
Isomeric Mixtures

AbstractHeparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

scholarly journals Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hao Liu ◽  
Apoorva Joshi ◽  
Pradeep Chopra ◽  
Lin Liu ◽  
Geert-Jan Boons ◽  
...  
Keyword(s):  
Structure Function ◽  
Structural Complexity ◽  
Reversed Phase ◽  
Separation Method ◽  
Size Exclusion ◽  
Reaction Products ◽  
Exclusion Chromatography ◽  
Function Characterization ◽  
Function Relationship ◽  
Isomeric Mixtures

Abstract Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

scholarly journals Living with Breakthrough: Two-Dimensional Liquid-Chromatography Separations of a Water-Soluble Synthetically Grafted Bio-Polymer

Separations ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 41
Author(s):  
H.C. van de Ven ◽  
J. Purmova ◽  
G. Groeneveld ◽  
Tijmen S. Bos ◽  
A.F.G. Gargano ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Reversed Phase ◽  
Water Soluble ◽  
Size Exclusion ◽  
Two Dimensional ◽  
Reaction Products ◽  
Grafting Reaction ◽  
Exclusion Chromatography ◽  
The One ◽  
Strong Injection

In this study, we evaluate the use of various two-dimensional liquid chromatographic methods to characterize water-soluble, synthetically grafted bio-polymers, consisting of long poly(acrylic acid) chains and short maltodextrin grafts. The confirmation of the presence of grafting and the estimation of its extent is challenging. It is complicated by the limited solubility of polymers, their structural dispersity and chemical heterogeneity. Moreover, the starting materials (and other reagents, reaction products and additives) may be present in the product. Reversed-phase liquid chromatography (RPLC), hydrophilic-interaction liquid chromatography (HILIC) and size-exclusion chromatography (SEC) were used to characterize the product, as well as the starting materials. Additionally, fractions were collected for off-line characterization by infrared spectroscopy and mass spectrometry. The one-dimensional separation methods were found to be inconclusive regarding the grafting question. Breakthrough (the early elution of polymer fractions due to strong injection solvents) is shown to be a perpetual problem. This issue is not solved by comprehensive two-dimensional liquid chromatography (LC × LC), but information demonstrating the success of the grafting reaction could be obtained. SEC × RPLC and HILIC × RPLC separations are presented and discussed.

scholarly journals Characterization and Antioxidant Activity of Products Derived from Xylose–Bovine Casein Hydrolysate Maillard Reaction: Impact of Reaction Time

Foods ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 242 ◽  
Author(s):  
Kun Chen ◽  
Jiajia Zhao ◽  
Xiaohan Shi ◽  
Qayum Abdul ◽  
Zhanmei Jiang
Keyword(s):  
Antioxidant Activity ◽  
Reaction Time ◽  
Maillard Reaction ◽  
Radical Scavenging ◽  
Casein Hydrolysate ◽  
Size Exclusion ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Exclusion Chromatography ◽  
Initial Ph

The characterization and antioxidant activity on Maillard reaction products (MRPs) derived from xylose and bovine casein hydrolysate (BCH) was investigated at 100 °C and initial pH 8.0 as a function of reaction time. The pH values and free amino groups contents of xylose–BCH MRPs remarkably decreased with the reaction time up to 8 h, whereas their browning intensities significantly increased (p < 0.05). After 4 h of heat treatment, the fluorescence properties of xylose–BCH MRPs reached the maximum. There was a production of higher and smaller molecular substances in xylose–BCH MRPs with an increased reaction time, as analyzed by size exclusion chromatography. The 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging capacity and ferrous reducing activity of xylose-BCH MRPs gradually increased with the reaction time extended from 0 to 8 h.

scholarly journals Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays

Toxins ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 35 ◽  
Author(s):  
Bruna Xavier ◽  
Rafaela Perobelli ◽  
Maurício Walter ◽  
Francielle da Silva ◽  
Sérgio Dalmora
Keyword(s):  
Botulinum Neurotoxin ◽  
Type A ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Mouse Bioassay ◽  
Forced Degradation ◽  
Mean Values ◽  
Botulinum Neurotoxin Type A ◽  
Exclusion Chromatography ◽  
Cell Culture Assay

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T−47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.

Separation of Liquiritin by Two-Dimensional Liquid Chromatography

2012 ◽  
Vol 455-456 ◽  
pp. 1232-1238 ◽  
Author(s):  
Jing Xiang Cong ◽  
Shao Yan Wang ◽  
Hong Gao
Keyword(s):  
Liquid Chromatography ◽  
Reversed Phase ◽  
Normal Phase ◽  
Size Exclusion ◽  
Two Dimensional ◽  
Target Compound ◽  
Reversed Phase Chromatography ◽  
Complex Samples ◽  
Exclusion Chromatography ◽  
Licorice Extract

Two-dimensional liquid chromatography (2DLC) is an important technology for the separation and analysis of complex samples. Liquiritin, an important active component in licorice, was chosen as the target compound and it was separated by three kinds of off-line 2DLC, i.e. size exclusion chromatography × reversed phase chromatography, normal phase × reversed phase chromatography and reversed phase chromatography × reversed phase chromatography (SEC×RP, NP×RP and RP×RP). The chromatographic conditions were selected and the 2D systems were combined. The results show that it is feasible to separate Liquiritin from licorice extract using 2DLC. Among the 2D modes mentioned above, the highest purity of Liquiritin was obtained in the RP×RP mode, and the concentration of Liquiritin was increased most significantly in the NP×RP mode.

scholarly journals Coupling of Size-Exclusion Chromatography to Liquid Chromatography/Mass Spectrometry for Determination of Trace Levels of Thifensulfuron-Methyl and Tribenuron-Methyl in Cottonseed and Cotton Gin Trash

2000 ◽  
Vol 83 (3) ◽  
pp. 651-659 ◽  
Author(s):  
James J Stry ◽  
Jennifer S Amoo ◽  
Steve W George ◽  
Teresa Hamilton-Johnson ◽  
Ellen Stetser
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Size Exclusion Chromatography ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Liquid Chromatography Mass Spectrometry ◽  
Exclusion Chromatography ◽  
Tribenuron Methyl ◽  
Cotton Gin

Abstract Size-exclusion chromatography (SEC) was coupled to reversed-phase liquid chromatography/mass spectrometry for the determination of thifensulfuron-methyl and tribenuron-methyl in cottonseed and cotton gin trash. The limit of quantitation was 20 parts per billion (ppb), and the limit of detection was 6 ppb. The analytes were extracted by homogenization in a buffer solution. The extracts underwent a solvent exchange into methanol and were injected onto an SEC column. As the analytes eluted from the SEC column, the eluate was diverted onto a reversed-phase column for additional separation of the analytes and their detection via mass spectrometry. This method is unique because the samples are not cleaned up before analysis, the analytes are injected in methanol, and the entire analysis is completed in 30 min. Average recoveries and standard deviations for thifensulfuronmethyl and tribenuron-methyl in cotton gin trash were 91 ± 6% and 88 ± 5%, respectively. Average recoveries and standard deviations for thifensulfuron-methyl and tribenuron-methyl in cottonseed were 91 ± 11% and 99 ± 12%, respectively. This is an effective method for the detection and determination of thifensulfuron-methyl and tribenuronmethyl in cotton.

Effect of dextran and enzymatically decomposed dextran on the sucrose crystal shape

2019 ◽  
pp. 588-596
Author(s):  
Karin Abraham ◽  
Henriette Brykczynski ◽  
E.S.J. Rudolph-Flöter ◽  
Karl Schlumbach ◽  
A. Schäfer ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Molecular Mass Distribution ◽  
Size Exclusion ◽  
Crystal Shape ◽  
Reaction Products ◽  
Mass Distributions ◽  
Crystallization Experiments ◽  
Exclusion Chromatography ◽  
Molecular Masses ◽  
Sucrose Crystals

The effect of dextran’s molecular mass distribution on the sucrose crystal shape was key to this study. Therefore, sucrose crystals were produced by evaporating crystallization experiments using synthetic thick juices in the form of pure sugar syrups containing high (T2000) and low (T40) molecular mass dextran fractions as well as enzymatically decomposed dextran. The combined analysis of molecular mass distributions by size exclusion chromatography and sucrose crystal shapes by static image analysis were used to identify the least harmful reaction products resulting from the enzymatic decomposition of dextran. The combined evaluation of two shape parameters, circularity and width-to-length ratio, has shown that three different shape modifications can be related to the presence of dextran, namely cube-shaped crystals, elongated needle-shaped crystals and agglomerates. In the main, the data indicated that high T2000 contents and generally all T40 dextran contents led to an increased occurrence of agglomerated and occasionally elongated crystals. The latter was especially found for high T2000 dextran contents. In contrast, low T2000 dextran contents predominantly increased the amount of cube-like crystals. The enzymatic decomposition of dextran resulted in a gradual reduction of the molecular mass. It was shown that an insufficient decomposition to broadly distributed low molecular mass dextran fragments, which are realistic to assume for technical cane and beet juices, still dramatically affected the sucrose crystal shape. Once dextran was decomposed to molecules with molecular masses of less than 5 kDa, no dextran-related effects on the sucrose crystal shape were found.

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