Measurement by reversed-phase high-performance liquid chromatography of malondialdehyde in normal human urine following derivatisation with 2,4-dinitrophenylhydrazine.
A selective and sensitive method based on derivatisation with 2,4-dinitrophenylhydrazine (DNPH) and consecutive HPLC gradient separation is described for the determination of malondialdehyde (MDA) in urine. Preparation of urine samples involved a one-step derivatisation/extraction procedure. Separation was achieved using a Waters SymmetryC(18) column (3.9 x 150 mm) and linear gradient of acetonitrile in water (from 30% to 70% in 30 min). The overall detection limit of the method was 56 nM of MDA in urine. The recovery of MDA was 94.3+/-8.6%. MDA in urine of healthy volunteers, measured using the method of standard additions, was 0.019+/-0.012 microM/mmol creatinine. MDA in the same samples measured using the 2-thiobarbituric acid (TBA) assay was 0.181+/-0.063 microM/mmol creatinine. We demonstrate that the commonly used TBA assay in conjunction with HPLC may overestimate the MDA concentration in human urine by almost 10-fold.
Korchazhkina
Olga
O
Centre for Science and Technology in Medicine, University of Keele, Hartshill, ST4 7QB, Stoke-on-Trent, UK. o.v.exley@keele.ac.uk
Exley
Christopher
C
Andrew Spencer
Stephen
S
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008315:Malondialdehyde / Q000737:chemistry / Q000652:urine*; D010659:Phenylhydrazines / Q000737:chemistry*; D015203:Reproducibility of Results; D013858:Thiobarbiturates / Q000032:analysis
D010659:Phenylhydrazines; D013858:Thiobarbiturates; C004787:2,4-dinitrophenylhydrazine; D008315:Malondialdehyde; C029684:thiobarbituric acid