Sialic acids (SAs), a family of 9-carbon containing acidic monosaccharides also known as neuraminic acids are very important carbohydrates terminating cell surface glycoconjugates that are involved in many physiological and pathological processes. SAs also exist in both free and conjugated forms such as glycoproteins and glycolipids in tissues and fluids such as serum, urine and saliva related to certain disease status. In addition, therapeutic glycoprotein sialylation and the identity of the SAs are very critical to the glycoproteins' efficacy, pharmacokinetics, and potential immunogenicity. Therefore, identification and quantitation of SAs are very important for biomedical research and therapeutic glycoprotein production and applications. Quinoxaline derivatization of SAs has been the most useful approach for sialic acid quantitation of biological samples and glycoproteins by either HPLC or LC-MS/MS. However, stability of SA quinoxaline derivatives and phenyldiame reagents and their cost prevent its practical applications. In this report, we examined SA quinoxaline derivatives formation features with commercially available phenyldiamines and their SAs quantitation competency. We found a stable and inexpensive 4,5-dimethylbenzene-1,2-diamine (DMBA), which selectively reacts with SA to form a stable quinoxaline derivative with high UV and fluorescence properties, proving a convenient and cost-effective HPLC analysis of SAs. DMBA derivatization of both N-acetyl-D-neuraminic acid (Neu5Ac) and N-glycolyl-D-neuraminic acid (Neu5Gc) and their HPLC quantitation in FBS and glycoprotein fetuin were successfully demonstrated. In addition, DMBA derivatization of SA provides a stable derivative of SA with high MS response, proving a convenient and cost-effective LC-MS/MS analysis of free SAs. The reported DMBA derivatization method provides a robust and affordable approach for quantitation of SAs in both biomatrices and therapeutic glycoproteins.

Quantification of sialic acid based on a robust and affordable quinoxaline derivatization

248th National Meeting (2014)

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