Free and total malondialdehyde assessment in biological matrices by gas chromatography mass spectrometry: What is needed for an accurate detection

A method to determine free and total (free and bound) malondialdehyde (MDA) in fresh human plasma, or in rat liver microsomes, using selected ion moni toring (SIM) gas chromatography-mass spectrometry in the electron impact mo de was set up,The dideuterated internal standard, 3-hydroxy[1,3-H-2(2)]-2-p ropenal (dMDA), was added to the biological samples before their analytical manipulation, To detect free MDA the samples were reacted under mild condi tions (25 degrees C, pH 4.0, 30 min) with phenylhydrazine (PH), affording 1 -phenyl-1H-pyrazole and its 3,5-dideuterated isotopomer, For the evaluation of total MDA level the plasma or microsomes were subjected, before the der ivatization step, to hydrolysis in the presence of 1 M NaOH under preestabl ished conditions. This method offers several advantages such specificity, p recision (within-day CV 2.0%, between-day CV 2.1%), linearity (0.01-15 mu M ) and high sensitivity (5 pmol injected). The recovery of known added MDA a mounts from plasma and microsomes, hydrolyzed or not, accounted for 98 +/- 0.6%. The free MDA levels found in the plasma and microsomes were 0.14 +/- 0.03 mu M and 0.048 +/- 0.006 nmol/mg protein, respectively. The total MDA levels were 1.3 +/- 0.07 mu M in plasma and 0.36 +/- 0.04 nmol/mg protein i n the microsomes. (C) 1999 Academic Press.