A new dilution-enrichment sample preparation strategy for expanded metabolome monitoring of human breast milk that overcomes the simultaneous presence of low- and high-abundance lipid species.

The complex nature of human breast milk (HBM) makes samples difficult to analyze, requiring several extraction techniques and analytical platforms to obtain high metabolome coverage. In this work, we combined liquid-liquid extraction (LLE) and solid-phase extraction (SPE) techniques to prepare HBM samples to overcome the challenge of low- and high-abundance lipid species, enabling the semiquantitative analysis of total HBM lipids in one liquid chromatography-mass spectrometry (LC-MS) run. A nonorganic fraction obtained during the LLE step was used to analyze small polar metabolites. This analytical approach allows extensive metabolome coverage, especially for low-abundance glycerophospholipids and sphingolipids. The method was applied to monitor short-term metabolome changes in HBM composition within individual mothers and the results showed variable metabolite composition patterns. Simultaneous detection of high-abundance glycerolipids and low-abundance but not less significant phospholipids in one LC-MS run saves time, decreases cost, and enables comprehensive insight into the dynamics of HBM composition.