Quantification of N-acetyl- and N-glycolylneuraminic acids by a stable isotope dilution assay using high-performance liquid chromatography–tandem mass spectrometry

The present report describes a method for the quantification of N-acetyl- and N-glycolylneuraminic acids without any derivatization, using their 13C3-isotopologues as internal standards and a C18 reversed-phase column modified by decylboronic acid which allows for the first time a complete chromatographic separation between the two analytes. The method is based on high-performance liquid chromatographic coupled with electrospray ion-trap mass spectrometry. The limit of quantification of the method is 0.1 mg/L (2.0 ng on column) for both analytes. The calibration curves are linear for both sialic acids over the range of 0.1–80 mg/L (2.0–1600 ng on column) with a correlation coefficient greater than 0.997. The proposed method was applied to the quantitative determination of sialic acids released from fetuin as a model of glycoproteins.