Shotgun lipidomics analysis of 4-hydroxyalkenal species directly from lipid extracts after one-step in situ derivatization.

4-Hydroxyalkenal species are a class of peroxidative products of polyunsaturated fatty acids, which serve as "toxic second messengers" in cellular systems. Investigation of their cellular role is hindered due to the lack of sensitive, reliable, robust method for identification and quantification of these metastable metabolites. Herein, we explored the facile Michael adduct of carnosine with 4-hydroxyalkenal species and developed a sensitive, facile, shotgun lipidomics-based method for quantification of these compounds directly from organic solvent lipid extracts of biological samples. In the study, we extensively examined the factors that may affect the accurate quantification of 4-hydroxyalkenal species and found that this method possessed high reproducibility (<8%) and nearly 3 orders of linear dynamic range with a limit of quantification at lower than 0.56 fmol/μL. Mass levels of 4-hydroxyalkenal species in various biological samples, including mouse heart, kidney, liver, and skeletal muscle, were determined by this developing method. In addition, the effects of sample collection methods and sample storage time on 4-hydroxyalkenal mass levels were also determined. We believe that development of this novel methodology should provide a powerful tool for us to better understand the role of 4-hydroxyalkenal species in biological processes.