Analysis of MDA

We used a fluorescence-generating agent, thiobarbituric acid (TBA), to derivatize MDA; and then analyzed the MDA-TBA adduct using an HPLC-fluorescence technique described previously (11). Compared to the conventional colorimetric analysis method for MDA-TBA adduct detection, the use of HPLC separation of MDA-TBA adduct in the present study minimized interference from TBA-reactive substances (TBARS) and achieves improved specificity and accuracy of MDA analysis (33).

For free MDA analysis, we started with TBA derivatization by mixing a 150 µL sample aliquot with 750 µL phosphoric acid (440 mM) and 250 µL TBA solution (42 mM). The mixture was then incubated at 80 °C for one hour to allow for the formation of MDA-TBA₂. This TBA derivatization process was identical for free MDA and total MDA, while total MDA measurement used an extra step of alkaline hydrolysis before TBA derivatization. The alkaline hydrolysis procedure for the serum and urine samples involved incubating a 20 µL sample aliquot with 65 µL 1N NaOH at 60 °C for 30 min, followed by mixing it with 65 µL 1N HCl. The hydrolysis procedure for the EBC samples, however, was slightly different as follows. A 120 µL EBC aliquot was incubated with 15 µL 5N NaOH at 60 °C for 30 min, followed by adding 15 µL 5N HCl. After TBA derivatization, 20 µL of the resultant mixture was injected into the HPLC system where MDA-TBA₂ adduct was isolated using a Nova-Pak C18 column (Waters, USA). The mobile phase of the HPLC system consisted of 40% (v/v) methanol and 60% (v/v) 50 mM KH₂PO₄ solution, its pH was adjusted to 6.8, and then the solution was filtered using a 0.22 µm nylon membrane (Catalog # 58522-N47, Microsolv Tech, USA). The flow rate of mobile phase was set at 0.8 mL/min. The MDA-TBA₂ adduct was quantified using a fluorescence detector with excitation wavelength at 532 nm and emission wavelength at 553 nm. The method detection limit, extraction recovery, and analytical precision (measured as relative standard deviation from eight replicate injections) were 1.8 nM, 75.9%, and 2.2%, respectively.