3.5. Determination of Malondialdehyde by Reverse-Phase High-Performance Liquid Chromatography

MDA is not commercially available. MDA is an unstable compound and the only possibility to obtain it is by hydrolysis of its stable derivative, bisdiethyl acetal (TMP).

MDA standard was prepared as a hydrolyzed product of TMP (Figure 3A). A volume of 10 µL of TMP was diluted in 10 mL of 0.1 M HCl and incubated in a 100 °C water bath for 5 min, then quickly cooled with tap water (solution X). After hydrolysis, a working solution of MDA was prepared by diluting 1.0 mL of obtained solution X with 0.1 M HCl up to 100 mL in a volumetric flask. The resulting MDA standard of 4.37 µg/mL was then diluted with 0.1 M HCl to yield the final concentration of 21.5, 215, 430, 860, 1290 and 1720 ng/mL to obtain the calibration curve. The MDA standard working solution was stored at 5 °C in a dark place and was freshly prepared on a weekly basis. Calibration standards were prepared at the beginning of each analytical run.

(A) The preparation of MDA by acidic hydrolysis of TMP; (B) the proposed structure of TBA pigment as a colored adduct between TBA and malondialdehyde, MDA-TBA; and (C) the formation of the DNPH derivate of MDA, MDA-DNPH.

The DNPH reagent was prepared by dissolving 31 mg of DNPH in 10 mL of 2.0 M HCl and incubated for 30 min at room temperature in the dark. This solution of the derivatising reagent of DNPH was freshly prepared on the day of use. The derivatising procedure for the standards of MDA was the same as for the derivatization of meat sample. A volume of 20.0 µL of derivatized standard solution of MDA-DNPH was injected into the column for HPLC analysis. The formation of the DNPH derivate of MDA, 1-(2,4-dinitrophenyl) pyrazole and its chemical structure are shown in Figure 3.

Sample (5.0 g ground meat or meat products) was weighed in a 50 mL centrifuge tube, and 0.5 mL 0.3% EDTA was added immediately. After gently stirring, 2.5 mL 0.8% BHT in hexane was added, and the tube was gently blended again. Before homogenization, 4.0 mL ice-cold 20% TCA was added to the tube and homogenization was performed at 10,000 rpm for 1 min. After the centrifugation (5 min at 3500 rpm, 4 °C), the top hexane layer was removed, and the bottom layer was filtered through Whatman filter paper no. 4. For derivatization, aliquots of the filtrate (500 µL) were transferred into a vial and 50 µL DNPH reagent was added and mixed. Samples were stirred and incubated at room temperature for 30 min in the dark. Finally, sample aliquots (20 µL) of a resulting solution was injected into to column for chromatographic analysis.

Separation and HPLC analysis of MDA as well MDA-DNPH adduct were performed using high-performance liquid chromatography (HPLC). The HPLC system Dionex UltiMate 3000 RS (Thermo Fisher Scientific, Braunschweig, Germany) consisted of a quaternary pump, degasser, automated injector, column oven, and diode array detector (DAD). The DAD detector was set to collect signals within the spectral range of 190–800 nm. Chromatographic separation was achieved on the chromatographic column Polaris C18-A (particle size, 5 μm; column size 250 mm × 4.6 mm; Varian, Santa Clara, CA, USA). Samples were isocratically eluted with a mixture of 0.2% (v/v) glacial acetic acid in deionized water, and acetonitrile (61:39, v/v) at a flow rate of 1 mL/min at 25 °C. The injection volume was 20 µL, and the DAD detector was set at 307 nm. Analyses were performed with Chromeleon Chromatography Data System, Version 7.2 (Thermo Fisher Scientific, Braunschweig, Germany) for collecting and processing data. Each analysis was performed in three replicates. All solvents were filtered through a Whatman filter paper no. 4 before use. A calibration curve was prepared mixing a 500.0 µL volume of each of the above mentioned concentrations of standard of MDA and 50 µL of DNPH was added into a vial, and the resulting solution was incubated at room temperature for 30 min in the dark. The clear solution was transferred into a vial and then 20 µL of the resulting solution was injected onto to a column for chromatographic analysis. Triplicate 20 µL injections were made for each standard solution to see the reproducibility of the detector response at each concentration level. The peak area of MDA-DNPH was plotted against the concentration to obtain the calibration graph. The six concentrations of MDA were subjected to regression analysis to calculate the calibration equation and correlation coefficients.

The HPLC-DAD system proved to be a good option for the determination of MDA in real meat samples, allowing analysis with good sensitivity and in a total time of 20 min. We used an even higher DNPH solution of 15.6 mM in this study for derivatization to complete the derivatization reaction. The MDA-DNPH peak was identified by the elution profile of the authentic standard. Typical chromatograms for derivatized MDA (MDA-DNPH) in meat products and standard are demonstrated in Figure 3, where it is possible to see that no interferences are present in the region of the retention time of MDA. Peak identification in meat samples was performed by comparison of the retention time. Under the chosen chromatographic conditions, MDA-DNPH showed a retention time of 13.6 ± 0.1 min. For the purpose of peak identification, a 430 ng/mL MDA standard was analyzed and its chromatogram was overlaid with that of the sample chromatograms matched perfectly, indicating that the major peak from MDA was at 13.58 min.

Six calibration standards were analyzed, and their peak areas were plotted against concentration. The equation of linear regression obtained for the six concentration levels, each one injected three times, was: y = 0.0027x − 0.0004, where y is the peak area, and x is the MDA concentration (ng/mL). The regression coefficient R² was 0.9979, indicating good linearity. From the analytical curve, the linearity of the method was evaluated, demonstrating a linear interval in the range of 21.5 to 1720 ng/mL.