Determination of sialic acids in infant formula by chromatographic methods: a comparison of high-performance anion-exchange chromatography with pulsed amperometric detection and ultra-high-performance liquid chromatography methods

Abstract

Sialic acid determination in an infant formula presents many challenges, including efficient sialic acid release from glycoconjugates, effective sample preparation, and rugged chromatography. This work compares 2 chromatographic assays developed for determination of sialic acids in infant formula. Prior to chromatography, both assays release sialic acids by acid hydrolysis and treat the hydrolysate with a subsequent anion-exchange sample preparation. Both high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and fluorescence ultra-high-performance liquid chromatography (UHPLC) sample analysis methods were evaluated to compare assay performance and convenience. Calibration ranges were chosen to encompass the expected amounts of 2 sialic acids in infant formula: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Response was linear by either method with coefficients of determination of 1.00 by HPAEC-PAD between 5.0 and 100pmol of Neu5Ac and between 0.34 and 6.8 pmol of Neu5Gc and >0.99 by UHPLC between 5.0 and 260 pmol of Neu5Ac and between 0.20 and 9.8 pmol of Neu5Gc. Both methods had sufficient sensitivity to determine these sialic acids in infant formula. Three infant formulas were analyzed to evaluate accuracy and precision of the assays. The HPAEC-PAD assay was found to be faster overall and the UHPLC assay was more sensitive. Reaction efficiency, and therefore sensitivity, was dependent on the sample matrix. This work illustrates sample-specific complexity that must be considered in choosing an assay.