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To identify the impact of maternal vitamin D status on neuroactive compounds, we conducted a post hoc analysis of breast milk and matched infant stool samples from mothers categorized as sufficient or deficient for vitamin D. Neuroactive metabolites were quantified using both targeted and nontargeted liquid chromatography with tandem mass spectrometry (LC\u2010MS\/MS). Our findings revealed that breast milk from mothers with sufficient vitamin D levels contained significantly higher concentrations of tryptophan, phenylalanine, and tyrosine compared to milk from mothers with lower vitamin D levels. No significant differences were observed in tryptamine, kynurenine, kynurenic acid, anthranilic acid, quinolinic acid, tyramine, dopamine, epinephrine, or norepinephrine between the two groups. Among SCFAs and other acids, only hexanoic acid was significantly elevated in the breast milk of mothers with sufficient vitamin D. 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