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

Abstract

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 (13)C3-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 20ng/mL and a total running time of 4.2min, which is the most sensitive and quick measurement for free SA in biomatrices.

Keywords: 12-diamino-4,5-methylenedioxybenzene; 3,4-diaminotoluene; CV; DAT; DMB; ESI; Free sialic acid; Human plasma; ILSC; IS; Isotope-labeled standard calibration; KDN; LLOQ; Liquid chromatography–tandem mass spectrometry; MRM; PAD; PPT; QC; Quinoxaline derivatization; RE; RT; SA; UHPLC; coefficient of variation; electrospray ionization; internal standard; isotope-labeled standard calibration; ketodeoxynonulosonic acid; lower limit of quantification; multiple reaction monitoring; protein precipitation; pulsed amperometric detection; quality control; relative error; room temperature; sialic acid; ultra high performance liquid chromatography.