Quantification of Malondialdehyde in Human Urine by HPLC‐DAD and Derivatization with 2,4‐Dinitrophenylhydrazine

Abstract

This study demonstrated that human urine can be derivatized in a simple manner and the proposed method can be suitable for the routine and noninvasive evaluation of oxidative stress. A rapid and sensitive method for the determination of malondialdehyde (MDA) concentrations in human urine has been developed using high‐performance liquid chromatography. The MDA concentration in pooled human urine samples was determined after its conversion with 2,4‐dinitrophenylhydrazine (DNPH) to a hydrazone (MDA‐DNPH). The MDA‐DNPH was separated on C18 column and quantified by diode array detection at 310 nm. The method was validated by the use of spiked human urine. The calibration curves of analyte showed good linearity (r 2 > 0.999) in the ranges. The limit of detection was estimated as 0.16 µg/mL. In addition, accuracy was from 104.0 to 112.7% and the precision was less than 3.2%. In this study, the developed method was applied to the analysis of real human urine samples. The level of MDA in urine of adults was 1.3 ± 0.6 µg/mg creatinine.

Simultaneous Analysis of Seven Biomarkers of Oxidative Damage to Lipids, Proteins, and DNA in Urine

“…Labeled ISs of 8-OHdG, diY, and 8-PGF 2α were spiked into samples for the quantification of concentrations. Isotopically labeled MDA was not commercially available, and some authors have recommended glutaraldehyde (pentanedial, PDA) as an IS due to its structural similarity to MDA. , Stoichiometry of reaction with DNPH is different between PDA and MDA, however, as MDA reacts with one DNPH molecule and PDA reacts with two molecules, yielding products with different properties. Further, PDA-DNPH is strongly retained in the chromatographic column (elutes in Period 2), and negative ionization is preferred in contrast to MDA, which is analyzed in the positive ionization mode.…”

“…Traditionally, MDA was quantified by a 2-thiobarbituric acid (TBA) assay, and the MDA–TBA product was detected by fluorescence or spectrophotometry . However, the TBA assay lacks specificity, as the reaction can take place with other aldehydes in the sample, yielding unknown TBA derivatives in addition to MDA–TBA, which leads to overestimation of MDA. ,, Analyses of MDA in urine after O -(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (O-PFB) derivatization followed by GC with electron capture detection (GC-ECD), pentafluorobenzyl bromide (PFB-Br) derivatization followed by GC-MS analysis, TBA derivatization followed by LC-fluorescence detection, , 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by liquid chromatography with ultraviolet detection (LC-UV), , and solid phase extraction (SPE)-LC-MS have been reported.…”

Modeling and optimization of malondialdehyde (MDA) absorbance behavior through response surface methodology (RSM) and artificial intelligence network (AIN): An endeavor to estimate lipid peroxidation by determination of MDA

“…Previous studies presented MDA in free form and covalently bound to proteins in human blood specimens and tissue samples 4,7,9 . To determine the free or total level of MDA, scientists have devised several analytical methods such as mass spectrometric‐based proteomics, liquid chromatography with tandem mass spectrometry (LC‐MS/MS), electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS), and liquid chromatography (LC)‐ and gas chromatography (GC)‐based analysis 3,7,9,11,12,17,20,22,26,30–32,34–40 . Most research has used thiobarbituric acid (TBA) as a color reagent through colorimetric or fluorometric approaches to detect MDA, usually under acidic media and harsh heating at 95–100°C.…”

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis

“…formaldehyde, can be determined as a trace in various matrices, the resulting hydrazone having an absorbing maximum (λ max ) at 360 nm (Soman, Qiu, & Li, 2008). Other examples of determining simple carbonylic compounds in biomatrices by means of this derivatization reaction are malondialdehyde in human urine (Kim, Jung, & Kho, 2017), acetaldehyde in hepatoma cell culture medium, blood and plasma (Guan, Rubin, & Anni, 2012) and acetone in blood samples (Kalkan, Sahiner, Cakir, Alpaslan, & Yilmaz, 2016;Fujii et al, 2014).…”