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The multicomponent analysis of human breast milk (BM) by metabolic profiling is a new area of study applied to determining milk composition, and is capable of associating BM composition with maternal characteristics, and subsequent infant health outcomes. A multiplatform approach combining HPLC‐MS and ultra‐performance LC‐MS, GC‐MS, CE‐MS, and ¹H NMR spectroscopy was used to comprehensively characterize metabolic profiles from seventy BM samples. A total of 710 metabolites spanning multiple molecular classes were defined. The utility of the individual and combined analytical platforms was explored in relation to numbers of metabolites identified, as well as the reproducibility of the methods. The greatest number of metabolites was identified by the single phase HPLC‐MS method, while CE‐MS uniquely profiled amino acids in detail and NMR was the most reproducible, whereas GC‐MS …