Nano‐HPLC–mass spectrometry and MEKC for the analysis of oligosaccharides from human milk

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Abstract
The separation of oligosaccharides derivatized with various esters of aminobenzoic acid by means of reversed‐phase nano‐HPLC (nHPLC) with on‐line ESI mass spectrometry and off‐line MALDI‐TOF mass spectrometry as well as MEKC is described. For this purpose methyl, ethyl and butyl aminobenzoates and heptyloxyaniline were used as derivatization agents for homologous maltodextrins and oligosaccharides from human milk. Four different C18 stationary phases were tested for this purpose because the type of stationary phase was shown to have a dramatic effect on the performance of the separation. Optimal results were obtained using n‐butyl aminobenzoate as label and an encapsulated ODS stationary phase. The on‐line coupling of nHPLC to ESI MS allowed to separate and identify various oligosaccharides from human milk. This technique enabled the exact attribution of the molecular structure to a signal in the chromatogram. In a second approach oligosaccharides were separated by nHPLC and subsequently fractionated. The fractions were analyzed by MALDI‐TOF mass spectrometry. The results obtained by this approach confirmed the ESI MS data. An analogous separation profile was obtained by using sodium dodecyl sulfate in MEKC, which proves that the retention mechanisms of both techniques are identical. Copyright © 2002 John Wiley & Sons, Ltd.