Premium
Measurement of Menadione in Urine by HPLC
Author(s) -
Al Rajabi Ala,
Choi SangWoon,
Peterson James,
Booth Sarah L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.566.4
Subject(s) - menadione , chromatography , high performance liquid chromatography , chemistry , urine , zinc , methanol , aqueous solution , chloride , biochemistry , organic chemistry , enzyme
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 H 2 O. Aqueous solution (2 M zinc chloride, 1 M acetic acid, and 1 M sodium acetate) was added to both methanol and H 2 O. 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 µg/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. Grant Funding Source USDA ARS Cooperative Agreement 58‐1950‐7‐707