Comparison of spectrophotometric and HPLC methods for determining sialic acid in infant formulas

Abstract

Two methods for determining sialic acid in infant formulas – spectrophotometry and HPLC with fluorescence detection – have been optimised and validated, the first one allows to determine total sialic acid while the second allows to differentiate the two main forms of sialic acid ( N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)). A common sample preparation procedure (hydrolysis and purification) for both methods has been proposed. The linearity (from 6 to 150 μg of total sialic acid in the assay for spectrophotometry, and from 12.5 to 250 ng and 1 to 5 ng of Neu5Ac and Neu5Gc, respectively, for HPLC) is adequate. The detection and quantification limits (0.29 and 0.97 mg of total sialic acid/L of reconstituted sample, respectively, for spectrophotometry, and 0.03 and 0.08 mg Neu5Ac/L; 0.003 and 0.009 mg Neu5Gc/L of reconstituted sample, respectively, for HPLC) are low enough for the determination of sialic acid in infant formulas. The precision of both methods, expressed as relative standard deviation, is less than 6%, and the accuracy evaluated by recovery assays show 104% recovery for spectrophotometry; 95% for Neu5Ac and 109% for Neu5Gc for HPLC. Samples analysed show no significant differences ( α < 0.05) attributable to the method used; consequently, both of them could be applied after common sample preparation, the choice of technique depending on the facilities available in the laboratory.