ARS | Publication request: Measurement of Menadione in Urine by HPLC

Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Abstract
Publication Acceptance Date: November 5, 2008
Publication Date: April 28, 2009
Citation: Al Rajabi, A., Choi, S., Peterson, J., Booth, S.L. 2009. Measurement of Menadione in Urine by HPLC. Journal of Federation of American Societies for Experimental Biology. Abstract No. 566.4.

Technical Abstract: Mammals convert phylloquinone to MK-4, with menadione as a possible intermediate. We developed and validated a method measuring urinary menadione. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed. The mobile phase was composed of 95 % methanol and 5 % DI H2O. Aqueous solution (2 M zinc chloride, 1 M acetic acid, and 1 M sodium acetate) was added to both methanol and H2O. MK-2 was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R^2) of 0.99 for both menadione and MK-2. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Menadione was then extracted using iso-octane. Drying extraction solvent prior to HPLC injection resulted in high menadione losses. This was resolved by avoiding complete evaporation. We were able to detect < 0.05 pmole menadione/ injection. Menadione was detected in archived urine samples from subjects receiving 500 ug/d phylloquinone. The assay was validated by "spiking recovery" and by removing the zinc column and observing the disappearance of the menadione peak. This HPLC method presents a sensitive and reproducible way to detect menadione in urine, and can be used to elucidate the role played by menadione in MK-4 formation. Research support: USDA ARS Cooperative Agreement 58-1950-7-707.