Determination of residual protein in commercial human milk oligosaccharides

A method for determining residual protein in commercial human milk oligosaccharides is described. Size exclusion chromatography is used to separate any protein from the oligosaccharides, and UV absorbance is used to estimate its concentration. The analysis is calibrated with casein-spiked oligosaccharide solutions, with protein mass fractions at 5 mg/kg–100 mg/kg of oligosaccharide (or when expressed as mass/volume of calibration solution, 0.75 mg/L–15 mg/L). Method performance was defined by assessments of linearity (correlation coefficient ≥ 0.998; residuals from -2.9 % to -5.6 %); reproducibility (analyses of multiple lots of oligosaccharides, with and without a quantifiable protein presence); accuracy (verification by three alternate methods); selectivity (reagent blanks and peak area ratio); and limits of quantitation and detection (~ 5 mg/kg and ~ 2 mg/kg, respectively, as mass fraction protein/oligosaccharide). The method provides a simple means for determining residual protein in oligosaccharides, capable of quantifying low protein levels (e.g., 5 mg/kg oligosaccharide) in the presence of a high oligosaccharide concentration (150 g/L, the HMO mass/volume in the prepared sample) by conventional LC/UV.