A Dual-Detection Strategy in the Chromatographic Analysis of 2-Aminoacridone-Derivatized Oligosaccharides

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Abstract
A protocol has been developed involving the derivatization of glycan mixtures with 2-aminoacridone and co-injection with a dextran ladder derivatized with methyl 4-aminobenzoate (M-4AB). These two derivatizing agents have very different ultraviolet absorbance and fluorescence characteristics. A chromatographic separation using a normal-phase column support followed by in-series UV and fluorescence detection allowed simultaneous analysis of the two mixtures of the separately derivatized carbohydrates without any interference. This new approach uses the M-4AB dextran ladder derivatives as internal standards spanning the whole chromatogram, allowing an accurate and detailed comparison of glycosylation profiles. It also saves much time by avoiding the necessity of “sandwiching” an unknown glycan mixture between two chromatographic runs of a dextran ladder. The use of this technique has been demonstrated in the case of glycans released from ribonuclease B and human IgG.