Direct and indirect high-performance liquid chromatography enantioseparation of trans-4-hydroxy-2-nonenoic acid.

trans-4-Hydroxy-2-nonenoic acid (HNEA) is a marker of lipid peroxidation resulting from the metabolism of trans-4-hydroxy-2-nonenal (HNE). Direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were developed and compared. The indirect method involved pre-column derivatization with a chiral amino agent, (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol, and subsequent separation of diastereomers on a Spherisorb ODS2 column. The direct separation of HNEA enantiomers was performed using the chiral stationary phase, Chiralpak AD-RH. Validation parameters including limit of quantification, linear range, accuracy and precision were determined. The indirect separation method was successfully applied for the determination of enantiomeric ratio of HNEA in rat brain mitochondrial lysate, and showed that HNEA was formed (R)-enantioselectively from HNE.