Simultaneous Identification of 4-Hydroxy-2-Hexenal and 4-Hydroxy-2-Nonenal in Foods by Pre-Column Fluorigenic Labeling With 1,3-Cyclohexanedione and Reversed-Phase High-Performance Liquid Chromatography With Fluorescence Detection

In this study, we developed a reversed-phase high-performance liquid chromatographic method with fluorometric detection for the simultaneous quantitation of 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) in foods by using 1,3-cyclohexanedione reagent. This method provides good resolution and reproducibility. Chromatography is conducted with a gradient system using a mobile phase of methanol–water–tetrahydrofuran (75:25:1, v/v) (A) and acetonitrile (B). The gradient employed is 5% B to 25% B in 25 min; 25% B to 80% in 30 min; isocratic at 80% B in 40 min. The flow rate is 1.0 mL/min. Fluorescence detection is performed at an exciting wavelength of 385 nm and an emission wavelength of 450 nm. The calibration curves for HHE and HNE concentrations are linear from 0.08 to 14 and 0.08 to 5.2 µmol/mL, respectively. The limits of detection and quantitation for HHE are 27 and 160 nmol/mL and those for HNE are 30 and 149 nmol/mL, respectively. Validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of HHE and HNE. This rapid method is, therefore, appropriate for quantitating HHE and HNE in fatty foods such as fish and animal meats.