Factors affecting the 2,4-dinitrophenyl hydrazine reaction with lipid carbonyls

Morgan Kandrac
The basic reactions of lipid oxidation were first reported more than 50 years ago, yet measurement of lipid oxidation remains a challenge for both industry and academia. Particularly missing are sensitive and accurate methods for quantitating and identifying secondary non-volatile oxidation products such as monomer carbonyls, both saturated and unsaturated, and core aldehydes remaining on triacylglycerols. Methods for quantitating carbonyls by reaction with 2,4-dinitrophenylhydrazine (DNPH)
more » ... been plagued with inconsistencies and problems that have limited applications to analyses of lipid carbonyls. This thesis has re-evaluated the chemistry underlying reaction of 2,4-dinitrophenylhydrazine with lipid carbonyls to develop a robust method that is chemically accurate and quantitative as well as simple enough for both research and industrial quality control analyses.DNPH reaction conditions and characteristics were tested using pure aldehydes that are typical lipid oxidation products, including saturated, monounsaturated, and diunsaturated compounds. DNPH was dissolved in N,N-dimethylformamide (DMF), acidified with varying concentrations and types of acids to form the base reagent. This was then reacted with aldehydes diluted in acetonitrile, for a range of times, and reagent mixtures were applied to HPLC columns for separation of product hydrazones, unreacted DNPH, and unreacted aldehyde (if any); this procedure also detected any side products generated. Peaks were detected by diode array detection and quantitated by comparison peak areas to standard curves generated from each aldehyde. Conditions under which saturated and unsaturated aldehydes reacted to completion, within 20 minutes, with minimal generation of hydrazone isomers and no carbonyl condensation products were identified as pH 2.52 with a molar ratio of 2.5:1 2,4-DNPH:Carbonyl. Reactions were incomplete at higher pH and were less efficient at lower pH. Reaction slopes for the various aldehydes were within 10% variation of each other in contrast to p [...]
doi:10.7282/t3-kffv-bv91 fatcat:cimg4amyfrcsnpbbysso4ckhxi