Determination of vitamin K1 (phylloquinone) and K2 (menaquinones 4-10) in a broad range of matrices by HPLC with electrochemical reduction and fluorescence detection.

An analytical method for simultaneous determination of cis-/trans-forms of vitamin K1 (phylloquinone), β, γ-dihydro K1 and K2 (menaquinones 4-10) was developed and thoroughly validated for applicability to a broad range of food matrices, tissue fluids, and supplements. The extraction procedures and post-liquid-liquid extraction solvents were optimized to maximize recovery that ranged from 80 to 118%. A longer HPLC column and a gradient were applied for improved separation of vitamers as well as cis/trans-forms. External standard curves for the vitamin K forms were efficiently injected by an autosampler (R2 ≥ 0.999). Vitamin K was efficiently reduced to hydroquinone by an online post-column electrochemical reduction cell before fluorescence detection. By analyzing reference materials and spiked samples the average LOQs were approximately 0.1 μg/kg for all vitamers except for 0.2 μg/kg for MK-6 and MK-10. The intra- and inter-day precision, expressed as 2RSD, were typically around 8% for K1 and 10% for the K2 forms.