Vitamin K in milk and infant formulas: determination and distribution of phylloquinone and menaquinone-4

A method is described for the determination of phylloquinone and menaquinones following enzymatic digestion, extraction and a single-stage HPLC technique utilizing post-column reduction with zinc and fluorescence detection. The technique is applicable to both routine compliance control of phylloquinone supplemented infant formula powders (30-150 micrograms per 100 g) and fundamental studies of the K vitamins at endogenous levels in fluid milks (0-5.0 micrograms per 100 g). Analytical figures of merit include a detection limit of 30 micrograms on-column, recoveries greater than 98% for both K1 and MK4, an RSDR of 2.35% (K1) and 2.32% (MK4) and a regression correlation of 0.9932 for a wide range of infant formulas when compared against an alternative HPLC-UV technique. MK4 and 2',3'-dihydrophylloquinone, both with undefined bioactivity, were detected at measurable levels in a range of infant formulas. Although the higher menaquinones were found to be essentially absent in the milk of several species, the significant presence of MK4 relative to K1 has been confirmed in all milks examined, with both dominant forms correlated during early lactation in the cow. These observations suggest an as yet unrecognized physiological function for MK4 in infant nutrition.