The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.
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Gennaro et al. (2003) studied this question.
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