Determination of Vitamin K₁ and Vitamin K₂ in Foods by HPLC-FLD

An analytical method was developed for determining vitamin K₁ and vitamin K₂ in foods by high performance liquid chromatography with Fluorescence Detector(HPLC-FLD). Milk powder,yoghurt,natto freeze-dried powder and chicken gizzard freeze-dried powder were hydrolyzed by enzyme,dispersed by isopropanol,extracted by n-hexane,evaporated by rotation and redissolved. The separation of vitamin K₁(VK₁),menaquinone-4(MK-4),menaquinone-7(MK-7)and menaquinone-9(MK-9)were achieved on Thermo Acclaim^TM120 C₁₈ column(150 mm×4.6 mm,5 μm)and derivatived by zinc powder reduction column(50 mm×4.6 mm)using 900 mL methanol:100 mL tetrahydrofuran(containing 0.3 mL glacial acetic acid,1.5 g zinc chloride,0.5 g anhydrous sodium acetate)as the mobile phase at excitation wavelength was 243 nm,emission wavelength was 430 nm. The results showed that the linear range of VK₁、MK-4、MK-7、MK-9 were 20~1000 μg/L. The relative standard deviation(RSD)of VK₁,MK-4,MK-7,MK-9 in dried milk,yoghurt,natto freeze-dried and chicken gizzard were less than 5%,indicating high precision. The recovery rate of VK₁,MK-4,MK-7 and MK-9 in dried milk,yoghurt,natto freeze-dried were 85.0%~107.8%,85.8%~109.6%,85.1%~102.7% and RSD were less than 5%,indicating high recovery rate and small relative standard deviation. Therefore,this developed method is high reproducibility and accuracy. It can be applied to accurate determine of vitamin K₁ and vitamin K₂ in foods.