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The fatty acid (FA) composition of human milk (HM) from N = 9 Italian healthy donors following a free diet exhibited FA-dependent ranges of variability, as assessed by GC-FID. The possible short-term changes in the FA profile were monitored in the milk of lactating mothers (three) collected at five time points over a 6 h period, following an oral load (200 mL) of bovine milk. An array of techniques was exploited, including UHPLC-ESI-MS/MS of intact lipids and MALDI-TOF MS before and after chemical hydrogenation or bromination, in addition