Significant improvements in targeted UHPLC-ESI-MS/MS analysis of the reactive aldehydes 4-hydroxy-2(E)-nonenal and 4-hydroxy-2(E)-hexenal and application to rat serum.

The elevated risk of colorectal cancer (CRC) induced by red or processed meat rich diets is now established. Those haem‑iron rich diets induce luminal lipid peroxidation, one of the most recognised hypotheses explaining CRC promotion. Due to their known toxic properties, quantification of reactive aldehydes such as 4-hydroxy-2(E)-nonenal (HNE) and 4-hydroxy-2(E)-hexenal (HHE) as lipid peroxidation end-products in biological fluids is of upmost importance. Following previous works on faecal waters, an UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in rat serum, using deuterated internal standards (ISs). After protein precipitation (PP) and solid phase extraction (SPE), LC-ESI-MS/MS analysis was achieved by MRM. The use of a brominated derivatisation reagent allowed using the bromine isotopes for selective detection of both HNE and HHE based on diagnostic transitions. This new method was validated according to the European Medicines Agency (EMA) guidelines. Our method proved to efficiently determine HNE and HHE serum concentrations with the required sensitivity (nM range) in serum of rats fed diets rich or not in red meat and different fatty acid compositions.