Quantification of hyaluronan in pharmaceutical formulations using high performance capillary electrophoresis and the modified uronic acid carbazole reaction

Plätzer, Manuela; Ozegowski, Jörg‐Hermann; Neubert, Reinhard H.H.

doi:10.1016/s0731-7085(99)00120-x

Cited by 39 publications

(22 citation statements)

References 11 publications

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“…The sensitivity of our carbazole assay was 5-fold higher than that of the conventional assay [21,22] due to a 5-fold reduction in the reaction volume. It can detect as little as 1.25nmol (1.25nmol × 194 = 243ng) GlcA (Fig.…”

Section: Discussionmentioning

confidence: 89%

“…The carbazole assay is based on the quantitative concentration-dependent color reaction between carbazole and uronic and can be as reliable as the HPLC assay for quantifying uronic acid containing GAGs [21,22]. However, the carbazole assay cannot detect KS, which lacks uronic acid residues.…”

Section: Discussionmentioning

confidence: 99%

“…Three direct GAG quantification methods, including HPLC [20], carbazole [21,22], and Alcian Blue [23] assays have been established. However, a systematic comparison of these three assays in terms of sensitivity, reliability, and limitations has not been reported.…”

Section: Introductionmentioning

confidence: 99%

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The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Frazier

Roodhouse²,

Hourcade³

et al. 2008

TOGLYJ

111

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Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes.

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Frazier

Roodhouse²,

Hourcade³

et al. 2008

TOGLYJ

111

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“…The collected sample from DEAE-52 was then separated by preparative electrophoresis according to the method described by elsewhere (Platzer et al, 1999). Spacer gel (5%): Acrylamide 4.75 g/100 mL, bisacrylamide 0.25 g/100 mL, adjust pH to 6.3 using HCl.…”

Section: Purification Of Low-molecular-weight Chondroitin Sulfate By mentioning

confidence: 99%

Separation and purification of low-molecular-weight chondroitin sulfates and their anti-oxidant properties

Wang

Lian

Jin

2016

Bangladesh J Pharmacol

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Low-molecular-weight chondroitin sulfate was obtained by degradation of chondroitin sulfate using hyaluronidase. Then, separated with sephadex G25, DEAE-52, and finally purified with AKATA superpeptide separation system and fluorescence-assisted carbohydrate electrophoresis. The main compo-nents detected by high performance gel-filtration chromatography were disaccharide, tetrasaccharide, hexasaccharide with molecular weight of 521, 1024 and 1527 Da, respectively. The anti-oxidant activity of these three oligosaccharides in vitro showed that the reducing power (maximum value at 10 mg/mL) and superoxide anion radical scavenging abilities were increased (maximum value at 4 mg/mL) with an increased in their concentration. There were no significant differences of the anti-oxidant properties between those three oligosaccharides.

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“…Abnormal concentration of HA can also be demonstrated by HPLC and CE in the exfoliation syndrome of the eye (Lamari et al, 1998) and in pathological conditions of the vitreous body (Grimshaw et al, 1994). Determination of intact and/or large-sized HA gels by CE may also be of importance for quality control in pharmaceutical industry (Platzer and Neubert, 1999), where several applications, including surgical and therapeutical ones, have been found for this macromolecule.…”

Section: Diagnostic and Biopharmaceutical Importance Of Gags/pgs Analmentioning

confidence: 99%

Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance

Karamanos

Hjerpe

1999

Biomed. Chromatogr.

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Proteoglycans are key biological macromolecules that, via their glycan constituents, participate and regulate several cellular events and physiopathological processes. Refined structures of their highly anionic glycan chains, involving sulphation pattern and uronic acid distribution through the polymeric chain, determine the interactions of proteoglycans with matrix effector molecules and are responsible for numerous effects. Analysis and structural characterization of glycans are, therefore, essential in understanding the biological functions of proteoglycans. Capillary electrophoresis with its high resolving power and sensitivity may successfully be used for the fine chemical characterization of components present in low amounts and to overcome limitations due to low amount/volume of biologic samples available. This technique is also friendly to the user and to the environment, since a very small amount of solvents (a few µL) is required. In this review the strategies used to analyse and characterize the structure of glycan chains of proteoglycans are summarized. They involve capillary electrophoretic analysis of depolymerized acidic glycan chains using specific enzymes and analysis of intact chains. The importance of this type of analysis in biologic samples and tissues and its possible diagnostic and biopharmaceutical use are also discussed. Copyright © 1999 John Wiley & Sons, Ltd.

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Quantification of hyaluronan in pharmaceutical formulations using high performance capillary electrophoresis and the modified uronic acid carbazole reaction

Cited by 39 publications

References 11 publications

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Separation and purification of low-molecular-weight chondroitin sulfates and their anti-oxidant properties

Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance

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