Analysis of carbohydrates in biological fluids by high performance liquid chromatography

A method for analyzing for reducing saccharide compounds in a biological fluid comprising charged reducing saccharide compounds and neutral reducing saccharide compounds, which comprises:

• (i) filtering the biological fluid through a membrane or a filtration device, thereby obtaining a filtered biological fluid;
• (ii) separating said charged reducing saccharide compounds from said neutral reducing saccharide compounds by contacting said filtered biological fluid with an anion exchange medium, thereby causing said charged reducing saccharide compounds to be retained on said anion exchange medium;
• (iii) eluting said neutral reducing saccharide compounds from said anion exchange medium to provide a first eluate;
• (iv) eluting said charged reducing saccharide compounds from said anion exchange medium to provide a second eluate;
• (v) derivatizing said second eluate with a 3-alkyl-5-pyrazoline derivative of the formula ##STR3## wherein R.sub.1 is a C.sub.1-12 alkyl group;
• and R.sub.2 is a C.sub.1-12 alkyl group;
• a substituted C.sub.1-12 alkyl group, wherein said substitutions are C.sub.1-8 alkyl, C.sub.1-8 alkoxy, or halogen;
• or a chromophore selected from the group consisting of phenyl, naphthyl, benzyl and pyridyl, wherein said chromophore is unsubstituted or substituted with C.sub.1-8 alkyl, C.sub.1-8 alkoxy or halogen;
• (vi) extracting the derivatized eluate with an organic solvent to provide an aqueous phase and an organic phase;
• and
• (vii) analyzing said aqueous phase by HPLC analysis.