Measurement of 4-hydroxynonenal in small volume blood plasma samples: modification of a gas chromatographic–mass spectrometric method for clinical settings

Abstract

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC–MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 μl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC–MS 9,9,9- D 3-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring- d 5, 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at −80 °C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at −80 °C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.