Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches

Determination of the degree of acetylation and the distribution of acetyl groups in chitosan by HPLC analysis of nitrous acid degraded and PMP labeled products

“…However, recently published studies demonstrate that 1 H NMR fails to detect up to 50% polysaccharides in chitosan and PEGylated chitosan copolymers [33] It has also been reported that even though the same M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 amount of oversulfated chondroitin sulfate (OSCS) and oversulfated dermatan sulfate (OSDS), two types of highly resembling chemically sulfated animal polysaccharides, possess the same numbers of acetyl groups, the 1 H NMR proton signal intensities reflecting the acetyl groups in OSDS are only half of that observed in OSCS. [34] Furthermore, 1 H NMR could detect 0.1% OSCS in heparin based on the proton signal intensity of the acetyl groups in OSCS but could not detect 30% oversulfated heparan sulfate (OSHS) in heparin because the proton signal of the acetyl groups in OSHS is totally disappeared when mixed with heparin, [35,36] which suggest that 1 H NMR invisible or non-quantitative phenomena might be associated with polysaccharide structural analysis in general. Although 1 H NMR-based chitin/chitosan analysis produces consistent spectra demonstrating great reproducibility towards polysaccharide analysis and has been chosen as a standard method by the American Standard Test Method organization to determining the DA for chitosan, [37] the invisible and un-quantitative phenomena observed in chitosan and other polysaccharides by 1 H NMR analysis raise questions about the reliability of using 1 H NMR analysis alone for chitin/chitosan DA measurement and for comprehensive structure analysis.…”

Ion chromatography for the separation of heparin and structurally related glycoaminoglycans: A review

“…SAX-HPLC has proven to be a more sensitive method of detecting OSCS than NMR spectroscopy and is able to successfully separate heparin from OSCS as well as other [17] contaminants. Detection limits for SAX-HPLC were reported to be in the range of 0.02-0.03% w/w with recoveries between 82.9 and 111.0% for OSCS, while the LOD of DS was 0.1% with a 96.0-103.0% recovery [10,11]. Table. 1 summarizes SAX-HPLC methods that have been reported in literature for the separation of heparin and heparin-like compounds.…”

“…SAX is the most common LC technique for the separation intact GAGs, oligosaccharides, and disaccharides [7][8][9] and has been selected as the FDA method for QC of pharmaceutical heparin [10][11][12][13]. The mechanism of ion exchange is based on the reversible ionic interaction between a charged analyte and oppositely charged stationary phase [14,15].…”

Quantitative NMR spectroscopy of biologically active substances and excipients

“…Until then, contaminants like OSCS could not be identified using conventional screening tests described in various pharmacopoeias. Therefore, new methods such as NMR spectroscopy [28,[51][52][53][54], capillary electrophoresis [55][56][57], strong-anion-exchange high-performance liquid chromatography [58], infrared (IR), near-infrared reflectance (NIR), and Raman spectroscopy [59][60][61], one-dimensional cellulose acetate plate electrophoresis and polyacrylamide gel electrophoresis [62,63], potentiometric polyanion sensors [64], and anticoagulant time assays [65][66][67] were developed and partly included in the international pharmacopoeias for identification and quantification purposes of potential contaminants in heparin.…”