Label-free absolute quantitation of oligosaccharides using multiple reaction monitoring

Qiuting Hong, L. Renee Ruhaak, Sarah M. Totten, Jennifer T. Smilowitz, J. Bruce German, Carlito B Lebrilla

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2′-fucosylation in their milk.

Original languageEnglish (US)
Pages (from-to)2640-2647
Number of pages8
JournalAnalytical Chemistry
Volume86
Issue number5
DOIs
StatePublished - Mar 4 2014

ASJC Scopus subject areas

  • Analytical Chemistry

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