Analytical Strategies for Trace-Level Determination of Hormones in Infant Milk Formula: Advances, Challenges, and Future Perspectives.

Infant milk formula (IMF) is designed to support infants during a highly sensitive stage of development, yet its trace-level bioactive composition remains less explored than its nutritional profile. Among these minor constituents, hormones are of particular interest because they link infant nutrition with endocrine regulation, food safety, and analytical chemistry. Hormones in IMF may originate naturally from milk-derived ingredients, result from veterinary practices, or occur as hormone-like endocrine-active residues. However, their determination is challenging due to their low concentrations and the complexity of the formula matrix, which can interfere with extraction, clean-up, separation, and detection. This review critically summarizes recent advances in analytical strategies for trace-level hormone determination in IMF, focusing on sample preparation, chromatographic and mass spectrometric techniques, method validation, analytical challenges, and future perspectives. It also critically evaluates the reported analytical strategies from a White Analytical Chemistry perspective, emphasizing the balance between analytical performance, environmental sustainability, robustness, cost-effectiveness, and routine applicability. Despite progress in extraction, purification, and sensitive detection approaches, gaps remain in occurrence data, method harmonization, validation consistency, and exposure assessment. To the best of our knowledge, this is the first review dedicated specifically to hormones analysis in IMF.