Micro Assay for Malondialdehyde in Human Serum by Extraction-Spectrophotometry Using an Internal Standard

Abstract.

Malondialdehyde (MDA) is a widely accepted biomarker of lipid peroxidation. Thus, the measurement of MDA in clinical samples is useful in the evaluation of oxidative stress. In this study, a micro-extraction-spectrophotometric assay was developed, based on the formation of MDA–thiobarbituric acid (TBA) adduct. To enhance the analytical performance, Erioglaucine A was used as an internal standard (IS), and the first derivative spectra were obtained. The volume of serum sample was 20 µL and the total volume of aqueous phase 420 µL (200 µL of 0.6% TBA in acetic acid, pH 2.5 and 200 µL of 6.25·10⁻⁴% IS). The extraction of adduct and IS was carried out with 300 µL of aliquat 336 (0.06%) in ethyl acetate. The analytical signal S was defined as the ratio between the first derivative absorbances measured at 543.1 nm (adduct) and 644.4 nm (IS). In the calibration range up to 10 µmol L⁻¹ MDA, the linear regression coefficient was 0.9998. The quantification limit was 0.19 µmol L⁻¹ and the CV values for 2 µmol L⁻¹ and 5 µmol L⁻¹ MDA, respectively, were 0.8% and 0.7%. The procedure was applied to the analysis of diabetic sera and the results compared with those obtained by HPLC (derivatization with 2,4-dinitrophenylhydrazine). Lower HPLC results (about 15%) indicated that interferences from other TBA reactive substances had not been completely eliminated by the extraction procedure and derivatization of spectral data. On the other hand, the micro-procedure presents important advantages: it is simple, precise and environmentally friendly (small amounts of reagents), which makes it readily adaptable to the analysis of large sample series. The feasibility of micro-assay in the evaluation of lipid peroxidation status was demonstrated in the analysis of 156 serum samples from diabetic patients divided into three groups according to the stage of development of typical complications.