chondroitin-sulfates and anthranilic-acid

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.

Topics: Anticoagulants; Chondroitin Sulfates; Dermatan Sulfate; Electrophoresis, Capillary; Heparin; Magnetic Resonance Spectroscopy; ortho-Aminobenzoates; Spectrophotometry, Ultraviolet

Urinary trypsin inhibitor (ulinastatin) is a characteristic protein pharmaceutical which contains both glycosaminoglycans and N-linked glycans in its molecule and has been used for treatment of acute pancreatitis. The comparability of ulinastatin preparations of different lots or from different companies was studied by using conventional analytical approaches such as SDS-PAGE, cellulose acetate membrane electrophoresis, and HP size-exclusion chromatography (SEC) and also by using newly developed techniques such as CE and MALDI-TOF MS. The methods using SEC and SDS-PAGE according to The Japanese Pharmacopoeia showed similar molecular masses for two different preparations, and the estimated molecular masses were significantly different from those observed with MALDI-TOF MS. We also showed that the electrophoretic methods using cellulose acetate membrane electrophoresis and CE can be used for comparability assessments of ulinastatin preparations. In addition, we analyzed the unsaturated disaccharides derived from glycosaminoglycan (chondroitin 4-sulfate chain) and N-linked oligosaccharides attached to ulinastatin by CE after releasing them by enzymatic digestion followed by fluorescent labeling with 2-aminoacridone and 2-aminobenzoic acid, respectively. The results indicated that carbohydrate chains are important as markers for comparability assessments of ulinastatin pharmaceutical preparations.

Topics: Amino Acid Sequence; Aminoacridines; Chondroitin Sulfates; Electrophoresis; Fluorescent Dyes; Glycoproteins; Molecular Sequence Data; Oligosaccharides; ortho-Aminobenzoates; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization