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.