Method validation for the measurement of sialic acid and sialyllactose in human milk using high performance anion exchange chromatography with pulsed amperometric detection

Abstract

Sialic acid (SA) is an important component of human milk and occurs both as free SA and bound to oligosaccharides (OSs), proteins and complex lipids. To provide accurate and sensitive analytical methods for determination of SA and sialyllactose, one of the most prevalent sialyllated OSs in human milk, two independent method validations were conducted. The analytes in the first validation were free SA, 3′‐sialyllactose (3′SL) and 6′‐sialyllactose (6′SL). The second validation consisted of measurement of SA after an acid hydrolysis sample treatment to provide the level of human milk SA that occurs as part of OSs, glycoproteins, and complex lipids (total SA). All analytes were quantified using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Method precision for all analytes is represented by relative standard deviation of less than 11.7% for six replicate analyses on same day, and less than 7.3% for six replicates on three days. Accuracy was demonstrated in pooled quality control samples fortified with analytes at three levels, low, middle and high, where recoveries ranged from 93.9 to 116%. The limits of quantitation are 8.00, 8.00, 3.20 and 40 mg/L; and the limits of detection are 2.89, 2.33, 1.12 and 0.34 mg/L for 3′SL, 6′SL, free SA and total SA, respectively. The validated methods are accurate and sensitive.Funded by Pfizer Nutrition.