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 C(30) 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} {H(2)O.} Menaquinone-2 {(MK-2)} was used as an internal standard. The standard calibration curve was linear with a correlation coefficient {(R(2))} of 0.999 for both menadione and {MK-2.} The lower limit of quantification {(LLOQ)} was 0.3pmole {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.