High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides.

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods. However, available methods for quantifying nitrogen and phosphorus are not sensitive enough and laborious. We report a highly sensitive high-performance anion-exchange chromatography-conductivity detector (HPAEC-CD) method for quantifying nitrogen and phosphorus present in pneumococcal capsular polysaccharides. The method is reliable, robust and reproducible with no interference. The LOQ for nitrogen and phosphorus of 3.125 and 62.5 ng/mL, respectively, is highly critical for estimating low levels of total nitrogen and total phosphorus. We have implemented this method to quantify total nitrogen in Typhoid Vi polysaccharide and phosphorus in Haemophilus influenzae type-b polysaccharide. This method has greater application for quantification of nitrogen and phosphorus present in low concentrations in polysaccharide vaccines/biologicals.

2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification.

Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulphuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm. Though anthrone reacts well with polysaccharides containing hexoses (such as glucose and galactose) and rhamnose, it is less reactive with uronic acids (such as glucuronic acid and galacturonic acid) and hexosamines (such as fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). Here, we report a novel reagent, 2-Phenoxyethanol, which reacts with furfural or hydroxymethyl furfural resulting in higher absorptivity. This method is rapid, sensitive, simple and direct, and can be used for quantitative determination of any type of carbohydrate that contains neutral sugars and uronic acids. For these saccharides, the sensitivity of the assay using 2-Phenoxyethanol (2-PE) is twice over anthrone method. Uronic acids show improved sensitivity using 2-PE over Phenol and it is more than twice with glucuronic acid. 2-PE reagent method has greater application for quantification of carbohydrates when present in low concentration in vaccines/biologicals.