Qualitative and quantitative analysis of N-acetyllactosamine and lacto-N-biose, the two major building blocks of human milk oligosaccharides in human milk samples by high-performance liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column

Abstract

This study presents a validated, porous graphitic carbon stationary phase-based LC-MS/MS method for the identification and quantification of lacto-N-biose (LNB) and N-acetyllactosamine (LacNAc). These compounds are the major building blocks of human milk oligosaccharides, however the presence of their unbound form in human milk has not been examined so far. The separation of these highly related structures in their alditol form was accomplished by a gradient LC method and multiple reaction monitoring (MRM) analysis after appropriate sample preparation including size-exclusion chromatography and solid-phase extraction. Baseline separation of the components provides the selectivity for the method. Validation was performed according to the European Medicines Agency (EMA) Guidelines and the method was found to be precise and accurate. Using our developed and validated method we were able to identify and quantify both saccharides in human milk for the first time. Based on our results the LacNAc concentration is in the range of 6.7-31μg/mL while LNB concentration decreased from 26μg/mL below the detection limit during the first week of lactation. The presence of LNB and LacNAc in human milk also implies new biological functions which can lead us closer to the understanding of the various functions of this complex biofluid.

Keywords: HPLC–MS/MS; Human milk oligosaccharide; Lacto-N-biose; N-Acetyllactosamine; Porous graphitic carbon.