Abstract: The oxidative chemiluminescence of phenylhydrazines can be applied to the determination of carbonyl compounds by utilizing the attenuation of the signal which occurs on conversion to the corresponding phenylhydrazone. Using an assay for 2,4-dinitrophenylhydrazine, off-line derivatization for a period of 2 h is optimized for a temperature of 30°C and for a sulfuric acid concentration. of 0.05 M. The optimum conditions for 2,4-dinitrophenylhydrazone formation can be understood in terms of a stepwise mechanism of carbinolamine formation via a zwitterion intermediate. The log-log calibration for the combined derivatization/assay of hexanal in optimized conditions is linear (r = 0.9931, n = 5) from 1 x 10^-6 to 2 x 10^-5 M. The limit of detection calculated as above was 1.7 x 10^-7 M (1.7 pmol of hexanal). Applications to the oxidative deterioration of linoleic acid and to the effect of lipoxygenase on linoleic acid are described.

Abstract: Solution (10 mM) of linoleic (I) and α-linolenic (II) acids in Tween 20 were prepared as described by Axelrod et al. (Methods in Enzymology, 1981, 71, 441). Portions (20 µL) were injected into the FIA system and were carried in a stream (450 µL/min) of 0.2 M potassium borate buffer of pH 9 containing 0.02% Tween 20, to a column (5 cm x 3 mm i.d.) of lipoxygenase immobilized on CNBr-activated Sepharose 4B. The decrease in O2 concentration resulting from enzyme-catalyzed lipid peroxidation was measured using a Clark-type O2 electrode held at -600 mV vs. Ag/AgCl. The calibration graphs for I and II were linear from 0.1-1.2 mM. The RSD for determination of 1 mM I (n = 55) was 1.4%.