Measurement of menadione in urine by HPLC

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C30 column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H2O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R2) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.