Abstract: In this study, an untargeted metabolomics method for the analysis of breast milk samples from different lactation stages was established using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The method combined protein precipitation and concentration for sample pretreatment. The reconstitution solvent composition was optimized to enhance the extraction efficiency and detection sensitivity of metabolites. A combined strategy utilizing reversed-phase (T3) and hydrophilic interaction liquid chromatography (HILIC) was employed to balance the separation efficiencies of polar and non-polar metabolites, thereby broadening metabolite coverage. To evaluate their performance, we systematically compared protein precipitation, single-phase extraction, and biphasic extraction methods in terms of the number and chemical classification of metabolites identified, as well as the capacity for identifying lipid and non-lipid compounds. The results demonstrated that protein precipitation combined with concentration treatment was simple, efficient, and suitable for high-throughput analysis, and outperformed the other two methods in terms of metabolite coverage. However, the appropriate selection of extraction methods should be guided by the specific research objectives. Chromatographic comparison revealed the complementary advantages of the T3 and HILIC columns: 78.16% of metabolites were uniquely detected on the T3 column, 21.84% on the HILIC column, and only 7.32% were detected on both columns, indicating substantial complementarity between them. This dual-column approach significantly increased metabolite coverage.

Key words: breast milk; high coverage; metabolomics; ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry