Abstract: A method for simultaneous determination of two kinds of vitamin K₂ in 8 kinds of food,including pork,pork liver,pure milk,yogurt,butter,eggs,lobster sauce and natto,by high performance liquid chromatography-fluorescence detector(HPLC-FLD)was developed. After enzymatic hydrolysis,saponification,extraction,nitrogen blowing and resolving treatment,the sample was separated by an Agilent ZORBAX SB-C₁₈column(150 mm×4.6 mm,5 μm)and derivatized with a post-column zinc powder reduction column(35 mm×4.6 mm). Methanol(containing 10% dichloromethane,0.03%glacial acetic acid,1.5 g/L zinc chloride and 0.5 g/L anhydrous sodium acetate)was used as mobile phase for detection by fluorescence detector. Results showed that the chromatographic peaks of menatetrenone 4(MK-4)and menatetrenone7(MK-7)were found within 15 min. A good respective linearity of two kinds of vitamin K₂was obtained with the correlation coefficient(r=1.0000).When the sample size was 2 g and the volume was 2 mL,the detection limit of MK-4 was 0.01 μg/100 g and the quantitative limit was 0.04 μg/100 g.The detection limit of MK-7 was 0.06 μg/100 g and the quantitative limit was 0.2 μg/100 g. When the sample size was 10 g and the volume was 2 mL,the detection limit of MK-4 was 0.002 μg/100 g and the quantitative limit was 0.008 μg/100 g.The detection limit of MK-7 was 0.012 μg/100 g and the quantitative limit was 0.04 μg/100 g.The relative standard deviation(RSD)was no more than 3.56%,and the recoveries ranged from 80.6% to 95.5%.Among the 8 kinds of food,pork,pure milk,yogurt,butter and eggs only contained MK-4,among which the content of pure milk was the lowest(0.34±0.02) μg/100 g and the content of egg was the highest(25.38±0.85) μg/100 g.There was only MK-7 in pork liver,lobster sauce and natto,among which the content of pork liver was low(25.45±0.83) μg/100 g and the content of natto was the most abundant,up to(923.20±11.45) μg/100 g.The method is accurate,reliable,efficient,and can be used for high-throughput determination of vitamin K₂ in food.