Enzymatic sample hydrolysis and HPLC in a study of phylloquinone concentration in human milk

Phylloquinone (vitamin K) is essential to prevent hemorrhagic diseases in newborns. We were interested in determining the concentration of phylloquinone in human milk and in elucidating the factors that influence that concentration. Human milk contains lipid constituents encapsulated with phylloquinone in the fat globules. To eliminate the lipids, we combined sonication and an enzymatic treatment with lipase to prevent degradation by drastic hydrolysis. Despite the low phylloquinone concentrations and the procedure's complexity (lipase treatment, two HPLC steps, and an on-line thermoinduced postcolumn reduction followed by fluorescence detection), within-day and day-to-day CVs of 5.2% and 5.8% were obtained (n = 8, mean = 1.86 micrograms/L, and n = 7, mean = 1.74 micrograms/L, respectively). The mean concentration of phylloquinone in 126 samples was 1.15 +/- 0.82 micrograms/L (mean +/- SD); no correlation was observed between the vitamin concentration and the postpartum date of collection. There was a positive correlation between phylloquinone concentration and phospholipid and cholesterol content (r = 0.5578 and 0.6020, respectively).