Measurement of menadione in urine by HPLC

Abstract

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.

Trial registration: ClinicalTrials.gov NCT00183001.

Copyright (c) 2010 Elsevier B.V. All rights reserved.

Figures

Figure 1

Chromatograms of menadione (MD) in a urine sample collected from a healthy volunteer: 1.a. Sample subjected to acid treatment prior to measurement. 1.b. Sample not subjected to acid treatment.

Figure 1

Chromatograms of menadione (MD) in a urine sample collected from a healthy volunteer: 1.a. Sample subjected to acid treatment prior to measurement. 1.b. Sample not subjected to acid treatment.

Figure 2

Bland-Altman Plot: Comparison of average % recoveries of each simultaneous injection (n=19) of menadione (MD) and menaquinone-2 (MK-2). Average % recovery= [(MK-2 + MD)/2] × 100. Difference in % Recovery= (MK-2 − MD) × 100. Paired student’s t test P value=0.382. ○: MD concentration = 13.3 pmol/mL, MK-2 concentration = 13.0 pmol/mL X: MD concentration = 8.7 pmol/mL, MK-2 concentration = 11.1 pmol/mL □: MD concentration = 7.0 pmol/mL, MK -2 concentration = 8.9 pmol/mL

Figure 3

Effect of H2O content in mobile phase on sensitivity. Exponential trend line was fitted to the data.