Determination of N-acetylneuraminic acid and N-glycolylneuraminic acid in glycoproteins by high-performance liquid chromatography without derivatization.
A simple and sensitive method for the determination of N-acetylneuraminic acid and N-glycolylneuraminic acid in glycoproteins by high-performance liquid chromatography has been investigated. The best condition to release sialic acid from glycoproteins involved incubation at 80 degrees C for 1 h in vacuo in 0.01 M hydrochloric acid with removal of the hydrochloric acid by evaporation without decomposing the sialic acids. Of the substances examined, N-acetylglycine was the best internal standard, because it is absent from glycoproteins, has an appropriate retention time on high-performance liquid chromatography (HPLC) and is stable during the procedures. The sample solution containing the internal standard was analysed directly by isocratic HPLC without pre-column or post-column labelling, and elution was monitored by ultraviolet absorbance at 205 nm. This method can determine 0.08-10 nmol of sialic acid accurately within 12 min and was successfully applied to bovine vitronectin.
Ogawa
H K
HK
Department of Chemistry, Faculty of Science, Ochanomizu University, Tokyo, Japan.
Takeuchi
Y
Y
Uchibori
H
H
Matsumoto
I
I
Seno
N
N
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000031:analogs & derivatives / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006868:Hydrolysis; D019158:N-Acetylneuraminic Acid; D009438:Neuraminic Acids / Q000032:analysis*; D012015:Reference Standards; D012794:Sialic Acids / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D019096:Vitronectin
D006023:Glycoproteins; D009438:Neuraminic Acids; D012794:Sialic Acids; D019096:Vitronectin; C032592:N-glycolylneuraminic acid; D019158:N-Acetylneuraminic Acid; D005998:Glycine; C006368:aceturic acid