Rapid and sensitive chromatographic determination of free sialic acid in complex bio-pharma fermentation media samples

Abstract

This paper describes a rapid, sensitive and reproducible liquid chromatographic method specifically for the quantitative determination of total free sialic acid, employing thiobarbituric acid as pre-column tagging agent, following the oxidation of sialic acid with periodic acid. The derivatised sialic acid was separated from 2-deoxy-D-ribose and the other components utilising a short C₁₈ monolithic column, with total run-times of under 90 s per sample. The method was successfully applied to quantify sialic acid in a range of complex samples, e.g. yeastolate powders, basal media and in-process samples supplied from the biopharmaceutical industry. The selective method was highly reproducible (%RSD for method (n = 6) = <2, for peak area (n = 6) = 1.3, for retention time (n = 6) = 0.6), linear (R² = 0.9994, range 0.25–25 μmol L⁻¹) and sensitive (LOD = 842 fmol based upon a 100 μL inj. vol.).