HPLC analysis of malonic dialdehyde in plasma and saliva with the purification of derivatives on super-cross-linked polystyrene (Puroxep-270)
Malondialdehyde (MDA) is a marker of oxidative stress, but its determination in biological fluids is quite a challenge. In its native form, malonic dialdehyde practically does not determine. The main problem is the choice of the method of hydrolysis of plasma and the method of deproteinization, which significantly affects the yield of malonic dialdehyde. The aim of this work was to develop a simple and reproducible way to determine free MDA in blood plasma and saliva. The investigations were carried out by HPLC: a high pressure pump (Shimadzu LC-20AT Prominence), a spectrophotometric detector (Shimadzu SPD-20A Prominence), a manual injector (Rheodyne 7725i), a chromatographic column Luna 150x4.6 mm, C18 (2), 5 μm (Phenomenex, USA) with an efficiency of about 8500 TT. For protection, a 0.2 micron pre-column filter (Supelco, USA) was used. Data processing was carried out using software («Multichrome» version 3.0, Ampersand). As a derivatization reagent, a solution of 2-thiobarbituric acid was proposed. The optimal way of deproteinization was chosen. A technology of solid-phase extraction / purification of derivatives has also been developed. This procedure allows not only to clean, but concentrate the extract, which increases the sensitivity of the determination. Cartridges with super-cross-linked polystyrene Purosep-270 were used for solid-phase extraction. The obtained chromatographic results showed that the yield of MDA after the step of solid-phase purification was 80-84% (n=33). The use of the solid-phase purification procedure made it possible to compensate for the dilution effect of the extracts of the bioassay resulting from the sample preparation, thereby increasing the sensitivity of the assay. As a result, there is no need for fluorometric detection and, as it follows from chromatograms, detection in the visible range at 530 nm is possible. The limit of quantitative detection (LOQ) was 0.05 ng per injection at a signal-to-noise ratio of 10. The results of the analysis can be used for diagnosis since the concentration of MDA in saliva can be affected not only by systemic but local diseases of the oral cavity. For this aim it is necessary to accumulate a database and take into account the current conditions (storage of biomaterial, conditions for preparation of the standard and derivatization reagent, conditions for derivatization and solid-phase cleaning) Thus, the developed sorption-chromatographic method for determining total malonic dialdehyde in blood plasma and saliva after derivatization with 2-thiobarbituric acid and solid phase purification / concentration (if it is necessary) is simple, well reproducible and can be recommended for routine clinical analyzes.