Abstract: Key technology research was developed for the determination of vitamin K₁ in infant formula by high performance liquid chromatography(HPLC). The sample was enzymatic hydrolysis by lipase,saponified with alkali,and then extracted with hexane,washed with NaCl saturated solution(containing phenolphthalein solution),and finally,Vitamin K₁ was redissolved in methanol.The vitamin K₁ were separated on a symmetry C₁₈ column(contecting to the zinc riduction column),and detected with fluorescence detection by the mobile phases of methanol dissolving Zinc chloride,sodium acetate and glacial acetic acid. The result show that the linearity of vitamin K₁ was in the concentration ranges of 0.2 to 5 μg/mL,with the good correlation coefficients(r ≥ 0.999). When the concentration of VK,was 5~100 μg/100 g,The average recoveries for the target compounds were between 90%~110%,6 independent determinations of samples with different contents were carried out with RSD less than 5%.The limit of detection was 1 μg/100 g. The method was easy,accurate and highly reproducible,which could provide strong technical support for enterprise quality control and government regulation.