Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

Melmer, Michael; Stangler, Thomas; Premstaller, Andreas; Lindner, Wolfgang

doi:10.1016/j.chroma.2010.06.069

Cited by 18 publications

(16 citation statements)

References 22 publications

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“…The strong adsorption of polar analytes on PGC compared to conventional reversed-phases even allows for the analysis of native and reduced oligosaccharides [13,14]. In previous experiments we identified the nature of the organic modifier, the column temperature and the redox state of the PGC material as important parameters impacting retention of oligosaccharides [15,16].…”

Section: Introductionmentioning

confidence: 97%

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Melmer

Stangler

Premstaller

et al. 2011

Journal of Chromatography A

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Section: Introductionmentioning

confidence: 97%

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Melmer

Stangler

Premstaller

et al. 2011

Journal of Chromatography A

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150

115

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“…6 Porous graphitized carbon has also been investigated as an alternative stationary phase for liquid phase glycan separation due to its unique and attractive selectivity 7 , but issues of recovery especially for highly sialylated glycans can arise. 8,9 Capillary electrophoretic analysis of intact glycoproteins and released glycans offers an alternative and complementary approach to chromatographic based techniques. 10,11,12 Mass spectrometry using either matrix assisted laser desorption ionization or electrospray ionization has also been routinely employed for the characterization of oligosaccharides.…”

Section: Introductionmentioning

confidence: 99%

2D-LC Analysis of BRP 3 Erythropoietin N-Glycosylation using Anion Exchange Fractionation and Hydrophilic Interaction UPLC Reveals Long Poly-N-Acetyl Lactosamine Extensions

et al. 2011

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Post translational modifications, in particular glycosylation, represent critical structural attributes that govern both the pharmacodynamic and pharmacokinetic properties of therapeutic glycoproteins. To guarantee safety and efficacy of recombinant therapeutics, characterization of glycosylation present is a regulatory requirement. In the current paper, we applied a multidimensional strategy comprising a shallow anion exchange gradient in the first dimension, followed by analysis using the recently introduced 1.7 μm HILIC phase in the second dimension for the comprehensive separation of complex N-glycans present on the European Biological Reference Preparation (BRP) 3 erythropoietin standard. Tetra-antennary glycans with multiple sialic acids and poly-N-acetyl lactosamine extensions were the most abundant oligosaccharides present on the molecule. Site-specific glycan analysis was performed to examine microheterogeneity. Tetra-antennary glycans with up to four sialic acids and up to five poly-N-acetyl lactosamine extensions were observed at asparagine 24 and 83, while bi-antennary glycans were the major structures at asparagine 38. The combined AEC × UPLC HILIC allows for the rapid and comprehensive analysis of complex N-glycosylation present on therapeutic glycoproteins such as BRP3 erythropoietin.

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“…Solvent C was acetonitrile, and solvent D consisted of 80 % ( v / v ) acetonitrile-water containing 0.10 % ( v / v ) trifluoroacetic acid. Before starting analytical measurements, a column regeneration procedure was performed to establish a defined redox state of the Hypercarb PGC stationary phase, which is required to achieve stable elution and separation of highly polar compounds [14]. The column regeneration method consisted of 180 min flushing with solvent D. Afterwards, re-equilibration was performed by rinsing for 90 min with 53 % A, 40 % B, and 7.0 % C. This method was used as a sequence “primer”, meaning that each sequence of measurements started with this column regeneration phase.…”

Section: Methodsmentioning

confidence: 99%

Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation

et al. 2014

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An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (HypercarbTM) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L−1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants.FigureᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7746-3) contains supplementary material, which is available to authorized users.

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Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

Cited by 18 publications

References 22 publications

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

2D-LC Analysis of BRP 3 Erythropoietin N-Glycosylation using Anion Exchange Fractionation and Hydrophilic Interaction UPLC Reveals Long Poly-N-Acetyl Lactosamine Extensions

Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation

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