Human milk is the only source of vitamin K for exclusively breastfed neonates. This vitamin is crucial both
for blood coagulation (vitamin K1) and for the normal neurological and skeletal development of the
foetus and newborn (vitamin K2). Since vitamin K is ubiquitous in foods, deficiency is not common in
adults, but plasma levels and hepatic storage are very low at birth. In light of the importance of this
valuable micronutrient, a non-invasive method for verifying that exclusively breastfed infants are
receiving an adequate supply of the vitamin is clearly a topic of great significance. In spite of this, the
determination of the several vitamin K homologues in human milk has still not been completely
elucidated. This paper presents an HPLC–MS/MS method for the simultaneous determination of
phylloquinone, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human milk after a simple and
effective isolation procedure. Overnight cold saponification and extraction of the analytes with hexane
provided yields above 75%. This procedure, combined with high performance liquid chromatography–
tandem mass spectrometry (HPLC–MS/MS), made it possible to achieve limits of detection (LODs) below
0.8 ng/mL. After a complete validation study, the method was applied to measure vitamin K congeners in
several human milk samples; results showed vitamin K1 concentrations comparable with those reported
in the literature. In addition, this is the first study performed for the determination of MK-4 and MK-7 in
the maternal milk of Italian women.
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