A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

Abstract

Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core.

A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

“…Nonetheless, the inferior quality and heterogeneity of raw materials and the laborious downstream purification process heavily limit the availability of commercial CS. Moreover, the potential risk of interspecies disease and the keratin sulfate contamination also further restrict its applications (Krasnova & Wong, ; Restaino, Finamore, et al, ). For example, keratin sulfate as a contaminant will result in negative assessments of CS efficacy in clinical trials on osteoarthritis (Rainsford, ).…”

Systematic Analysis of Pharmaceutical Preparations of Chondroitin Sulfate Combined with Glucosamine

“…In addition to the structural heterogeneity of CS obtained from different animal sources, some pharmaceutical preparations also present contaminations with other GAGs due to flaws during the purification process [5,6,7]. A common contaminant is keratan sulfate (KS), a conspicuous component of proteoglycans from cartilages [5,6,7].…”

“…A common contaminant is keratan sulfate (KS), a conspicuous component of proteoglycans from cartilages [5,6,7]. Even the international standards of CS differ in their structures and purities.…”

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

“…Analyses were performed at concentrations ranging from 0.1 to 0.4 g/L for HA, and from 0.5 to 4 g/L for chondroitin, to have a column load for each sample (injection volume × sample concentration × intrinsic viscosity) of approximately 0.2 dL, and runs were performed at 40 °C with a running time of 50 min. The fragment molecular weight distribution, molecular size distribution, polydispersity, hydrodynamic radius, and intrinsic viscosity were determined as described by La Gatta et al (2010) and Restaino et al (2017). …”

“…About 7 mg of powder (obtained after lyophilisation) were hydrolyzed in 1 M HCl for 18 h at 100 °C and analysed as described by Restaino et al (2017). …”