Rapid-Throughput Analysis of Human Milk Oligosaccharides from Filipino Breastmilk

Abstract

Human milk oligosaccharides (HMOs), although considered indigestible to the infant, are the third most abundant solid component of breastmilk. They serve many health-related functions benefitting the infant, such as nourishment of gut bacteria, inhibition of pathogenic infection, and aiding in brain development. In this study, a rapid-throughput analytical method is validated for Filipino breastmilk HMOs. The method entails lipid extraction by centrifugation, protein precipitation using ethanol, alditol sugar reduction using sodium borohydride, and solid phase extraction purification using porous graphitized carbon, all in a 96-well plate format. Nano-HPLC Chip/TOF-MS, coupled with the use of Agilent MassHunter programs for filtering datasets using an in-house library, is then used to analyze purified HMOs. The results evidently show that the rapid-throughput method minimizes sample loss, ensures reproducible MS signals, and considerably shortens sample handling time. Furthermore, the method significantly reduced analysis time 5-fold and eliminated the individual sample preparation, which enabled simultaneous analysis of 386 samples (four 96-well plates) in a single run.  Thus, the rapid-throughput analysis is highly efficient and effective for the profiling and structural determination of Filipino breastmilk HMOs even in large sample population sets.