HPLC Quantification of Protein-Bound MDA

MDA was determined according to a previously described HPLC method after derivatization with 2,4-dinitrophenylhydrazine (DNPH) [30 (link)]. Protein-bound MDA was hydrolyzed and deproteinized as described previously [25 (link)]. The supernatant was mixed with 12.5 µL DNPH solution and injected into the HPLC system (injection volume: 40 µL). The MDA standard was prepared as previously described [31 (link),32 (link)]. The DNPH derivatives (hydrazones) were isocratically separated, and the utilized HPLC consisted of an L-2200 autosampler, L-2130 HTA pump, and L-2450 diode array detector (all: VWR Hitachi; Vienna; Austria). Detector signals (absorbance at 310 nm) were recorded, and the EZchrom Elite software (VWR) was used for data acquisition and analysis.