Liquid chromatography-tandem mass spectrometry method for the determination of vitamin K homologues in human milk after overnight cold saponification

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.