Indirect enantioseparation of selenomethionine by reversed‐phase high‐performance liquid chromatography using a newly synthesized chiral derivatizing reagent based on (S)‐naproxen moiety

Abstract

(S)-Naproxen was reacted with N-hydroxyphthalimide in the presence of coupling reagent dicyclohexylcarbodiimide, and a new chiral derivatizing reagent, phthalimidyl-(S)-naproxen ester, was synthesized. It was characterized and was used for synthesis of diastereomers of selenomethionine via microwave irradiation or vortexing. The reaction conditions were optimized. Diastereomeric pairs synthesized by two approaches were successfully separated by reversed-phase high-performance liquid chromatography using binary mixtures of aqueous triethylammonium phosphate and acetonitrile. Detection was carried out at 231 nm. The limit of detection was found to be 0.11 and 0.10 pmol/mL for diastereomers of d- and l-SeMet, respectively. The method was validated for accuracy, precision and limit of detection. The new chiral derivatizing reagent was capable of enantioseparation of dl-SeMet in the form of diastereomers having higher stability, enhanced resolution and lower limits of detection in comparison to the diastereomers prepared with other chiral derivatizing reagents reported in the literature. Optimized structures of the two diastereomers were drawn using the Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G basis set to explain the separation mechanism.