Diaminonaphtalene, a new highly specific regent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum

Abstract

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37°C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 ± 13 nM) for plasma and 6.6% (658 ± 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 ± 7.5 nM) for total MDA, 3.2% (16 ± 0.5 nM) for free MDA in plasma, and 1.6% (630 ± 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93–108% (mean = 100%). Plasma levels in healthy women ( n = 79, 45–51 years) are 162 ± 51 and 24 ± 15 nM for total and free MDA, respectively. In younger men ( n = 19, 21–37 years) total and free MDA are, respectively, 138 ± 28 and 19 ± 9 nM.