Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC–MS/MS using isotope-labeled standard calibration

•A robust SA derivatization was developed for its LC–MS/MS quantification.•An isotope-labeled standard calibration was used for quantification of free SA.•The method provided a LLOQ of 20 ng/mL of SA and a total run time of 4.2 min.•The method was applied to the quantification of free SA in human plasma samples.

We report an accurate quantification of free sialic acid (SA) in human plasma using LC–MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC–MS/MS analysis of free SA. In addition, the use of 13C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20 ng/mL and a total running time of 4.2 min, which is the most sensitive and quick measurement for free SA in biomatrices.