Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations.
A simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately 10 min with high sensitivity detection at 214 nm (limit of detection [LOD] of approximately 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity, allowing direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination given that chondroitin ABC lyase is unable to act on this semisynthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD, and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS, whereas a formulated contaminated Hep was calculated to have 39.7% OSCS.
Volpi
Nicola
N
Department of Biologia Animale, Section of Biochemistry, University of Modena and Reggio Emilia, 41100 Modena, Italy. volpi@unimo.it
Maccari
Francesca
F
Linhardt
Robert J
RJ
D000925:Anticoagulants / Q000032:analysis / Q000737:chemistry*; D002809:Chondroitin Sulfates / Q000032:analysis*; D003871:Dermatan Sulfate / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006493:Heparin / Q000032:analysis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013056:Spectrophotometry, Ultraviolet; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000925:Anticoagulants; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D003871:Dermatan Sulfate; D006493:Heparin; D002809:Chondroitin Sulfates