Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with...

Defining the biological roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon high-performance liquid chromatography (HPLC) with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights, allowing 11 peaks to be fully resolved and quantified by monitoring mass-to-charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/ml (R(2)>0.998). Precision (coefficient of variation) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, and accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation.