A novel, sensitive and convenient method for determination of sialic acids in human serum utilizing ultrasonic-assisted closed in-syringe hydrolysis and derivatization prior to high performance liquid chromatography

Abstract

A novel, sensitive and convenient method, utilizing ultrasonic-assisted closed in-syringe hydrolysis and derivatization (UCSHD) prior to high performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD) and online mass spectrometry (MS) identification, has been developed for determination of sialic acids. The pivotal parameters affecting the release of sialic acids from serum and the derivatization were investigated using a response surface methodology (RSM). Under optimized conditions, the maximum yield of sialic acids release were achieved and they were labeled successfully in a relatively short time of 72 min (traditional time > 3 h) for the reason of the combination of hydrolysis steps with derivatization in a closed system with the assistance of ultrasonication. Excellent linearity (R² > 0.9991) in the calibration range of 0.5–16 μmol mL⁻¹ and quite low detection limits (LODs) (0.30 pmol for Neu5Ac and 0.21 pmol for Neu5Gc) were achieved. When the established UCSHD-HPLC-FLD-MS method was applied to the analysis of sialic acids in various human sera, low relative error (RE: −3.4% to 2.5%), high recoveries (90–96%) and intra- and inter-day precisions (RSD, 0.9–2.2% for Neu5Ac and 1.4–2.8% for Neu5Gc) were also obtained, demonstrating obvious advantages for the accurate, sensitive and convenient determination of sialic acids in bio-samples.