HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PEAK IDENTIFICATION OF 2,4-DINITROPHENYLHYDRAZINE DERIVATIVES OF LIPID-PEROXIDATION ALDEHYDES BY PHOTODIODE-ARRAY DETECTION

Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas . In this study we report the photodiode array detection of the 2,4-di nitrophenylhydrazine (DNPH) derivatives of MDA using HPLC. Oxidative s tress was produced by injecting (i.p.) bacterial lipopolysaccharide (L PS) into rats at a dose of 100 mu g/kg, or i.v. into rabbits (1 mu g/k g), or added to freshly drawn human blood (200 ng/ml). Blood was colle cted at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma. The supe rnatants were derivatized with DNPH, and the aldehyde-DNPHs were extra cted with pentane. After evaporation, aliquots of 10 mu l in acetonitr ile were injected onto a Beckman Ultrasphere C-18 (3 mu m) column, chr omatographed with an acetonitrile-water-acetic acid gradient mobile ph ase and scanned using Waters 996 photodiode array detector. Peak ident ification and homogeneity was determined by comparing the experimental peaks and Uv scans with those of authentic standards. A significant i ncrease in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehy de (ADA), acetone and propionaldehyde (PDA) was seen over the first ho ur after LPS administration in anesthetized rats, while in conscious r abbits this trend lasted up to 3 h. The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards . Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress. This technique coul d accurately measure pmol amounts of MDA-DNPH indicating the usefulnes s of photodiode array detection method for estimating small changes in the oxidative stress. (C) 1998 Elsevier Science B.V.