This article from Issue 14 of the Analytix Reporter reveals a sensitive quantitative LC-MS/MS method under reversed phase (RP) conditions using a Supel™ Carbon LC column. This was developed for the simultaneous analysis of eleven highly polar nucleotide sugars, having similar structures and physicochemical properties.
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Introduction
Nucleotide sugars, or nucleotide activated sugars, are highly energetic forms of monosaccharides that act as key metabolites in glycosylation reactions, during which the glycosyl group from the activated sugars is transferred to an acceptor molecule, e.g. a protein.
In mammals, the most common nucleotide for sugar activation is uridine diphosphate (UDP), which is found in combination with Glucose (Glc), Galactose (Gal), N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), Glucuronic acid (GlcA) and Xylose (Xyl). Furthermore, Guanosine diphosphate (GDP) linked to Mannose (Man) and Fucose (Fuc) as well as Cytidine monophosphate (CMP) linked to sialic acid (Neu5Ac) are used for glycan assembly. Plants and bacteria utilize an even larger variety of nucleotides and sugars.
Understanding the nucleotide sugar metabolism is of interest in different scientific areas, e.g. for the production of glycosylated therapeutic proteins in cell culture, where a sufficient glycosylation needs to be ensured in order to obtain a product of high quality and efficacy.1 For this purpose, a sensitive, quantification method is needed that is capable of simultaneously analyzing a set of polar analytes with similar structures and physicochemical properties. In this application note, an LC method using a Supel™ Carbon LC column in combination with selective MS/MS detection is described.
Experimental Conditions
The used conditions for the analysis of 11 nucleotide activated sugars in a standard solution with a concentration of 80 µM each are described in Table 1 & 2 (see full article).
Results
The 5 cm x 3 mm I.D. Supel™ Carbon LC column provided, under reversed phase conditions, good retention of the 11 activated nucleotide sugar compounds in the applied standard solution (Figure 1 & Table 3) (see full article).
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