A Method to Quantitate Total Sialic Acid, Glucosamine, and Galactosamine in Blood Serum and Glycoconjugates by HPLC

• Published in: Journal of liquid chromatography & related technologies Vol. 21; no. 19; pp. 3031 - 3045
• Main Authors: Makatsori, E., Karamanos, N. K., Anastassiou, E. D., Hjerpe, A., Tsegenidis, T.
• Format: Journal Article
• Language: English
• Published: Colchester Taylor & Francis Group 01-01-1998; Taylor & Francis
• Subjects: Biological and medical sciences; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Medicin och hälsovetenskap; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Galactosamine; Human; Biological fluid; Glucosamine; Sialic acid; Chemical analysis; HPLC chromatography; Glycoproteins; Glycoconjugate; Derivatization; Blood; Glycolipid; Reversed phase chromatography; Ketoaldonic acid; Ultraviolet detector; Analysis method; Aminoglycoside; Animal; Clinical biology; Neuraminic acid; Glycosaminoglycan; Blood serum; Tosylation; Quantitative analysis
• ISSN: 1082-6076; 1520-572X
• DOI: 10.1080/10826079808006884

A sensitive method for determining the total amount of sialic acid, glucosamine, and galactosamine in blood serum and glycoconjugates using reversed phase high performance liquid chromatography is described. Sialic acids present in glycoconjugates are released by acidic hydrolysis with 2 M TFA at 80°C for 2 h, whereas hexosamines are released following hydrolysis with 6 M HCl at 100°C for 8 h. The sialic acids released under these conditions are identified as a decarboxylated and deacylated product bearing a primary amino group (amino-product). Following removal of neutral monosaccharides and amino acids by ion-exchange chromatography on Dowex 50x8, the obtained amino-product and hexosamines are converted to Tosyl-derivatives. The obtained derivatives of amino-product, glucosamine, and galactosamine, are completely separated and determined on a Supelcosil LC-18 column by means of isocratic elution using a mobile phase of water-acetonitrile (85:15, v/v) and detection at 231 nm. The method shows a linearity range up to 25 nmol of sialic acids and hexosamines, has a detection limit ranging from 6 to 12 pmol, and is highly accurate when compared with literature values. The present method has the advantage of permitting determination of total sialic acid, glucosamine, and galactosamine in glycoconjugates and blood serum using the same HPLC conditions.