Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co‐migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K 1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post‐partrum. Vitamin K 1 was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.