Monitoring the Purity of Pharmaceutical Heparin Preparations by High-Field 1H-Nuclear Magnetic Resonance Spectroscopy

Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains‐All

“…In the course of its extraction, varying amounts of other GAGs are also isolated. Recent reports [22,23] of high levels of the related GAG DS in Hep preparations of diverse origin suggest that less attention is being paid by the industry than in former years to its quality control of Hep products. By using the ability of heparin lyase to specifically degrade Hep, a very low amount of GAG contaminant, i.e., HA, CS and DS, about 0.1%, can be revealed and detected with great precision in Hep preparation.…”

Pharmacology of Heparin and Related Drugs

“…The use of NMR for analytical purposes grew as equipment of the necessary sensitivity and resolving power became generally available (Neville et al, 1989;Tachibana et al, 1990), and 13 C NMR was incorporated into the European Pharmacopoeial monographs for LMWH products. NMR has been recognized as a particularly powerful analytical tool, allowing the simultaneous determination of the major and minor structural motifs that characterize LMWHs (Casu and Torri, 1999;Guerrini and Bisio, 2012).…”

Capillary Electrophoresis

“…CE was applied for the determination of GalN (and GlcN) in several samples-CSA, CSC, DS, OSCS, noncontaminated Hep, contaminated raw Hep, and a formulated contaminated Hep sample-in the absence and presence of previous treatment with chondroitin ABC lyase (also considering that there are no known contaminants in Hep samples able to interfere with enzymatic digestion [11]). As expected, CS samples and DS were found to produce 100% GalN with no presence of GlcN, whereas almost no GalN was detected by CE after treatment with chondroitinase ABC due to the complete degradation of these GAGs by this lyase.…”

“…This approach was also applied for the sensitive quantitative determination of OSCS in Hep raw material and formulations as well as in the presence of natural Hep contaminants, in particular DS [11], by performing a simple pretreatment step with chondroitinase ABC that is able to degrade CS/DS (and hyaluronic acid) but unable to act on OSCS. Furthermore, GlcN and GalN showed same calibration curves with quite similar absorbance capacities after derivatization with AA that is useful for direct and simple quantification of OSCS in Hep samples by just considering their relative percentages.…”

“…In this article, a validated CE method has been developed for the quantitative determination of OSCS in Hep preparations after degradation of the polysaccharides to produce hexosamine units (i.e., GlcN from Hep and GalN from OSCS), their derivatization with anthranilic acid (AA, 2-aminobenzoic acid), and separation by means of CE at 214 nm. Furthermore, a preliminary step of specific enzymatic treatment may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate (CS)/DS [11]. Finally, this analytical approach has been performed on raw material such as Hep final formulations and is also potentially applicable to LMW Hep preparations.…”