Development of a liquid chromatographic method for enantioseparation of Eflornithine using (S)-α-ethyl benzylamine as a chiral derivatizing agent

ABSTRACT Eflornithine (2-fluoromethyl-DL-ornithine) is a potent and irreversible inhibitor that selectively targets ornithine decarboxylase, a key enzyme in the polyamine biosynthesis pathway. Due to the lack of chromophoric moiety in the eflornithine structure, its detection via UV detector is difficult. Thus, direct high-performance liquid chromatographic (HPLC) separation of enantiomers of Eflornithine is not feasible, and pre-column derivatization is required for its determination through chiral HPLC columns. We developed an indirect HPLC method using (S)-α-ethyl benzylamine as a chiral derivatizing agent for the enantioseparation of Eflornithine. The diastereomers thus produced were then separated via LiChrospher C18 column (5 μm particle size, L × I. D. 25 cm × 4.6 mm). The mobile phase used was a mixture of acetonitrile and 0.1% aq. TFA and varied in linear gradients of 30–70% of acetonitrile for 30 min run at a flow rate of 1.0 mL min−1 and UV detection at 320 nm. The separation parameters were optimized by altering the mobile phase composition and flow rate. The findings revealed that the chromatographic separation was accomplished within 15 min, with resolution values greater than 4.5 for Eflornithine enantiomers. The detection and quantitation limits were 9.26 ng mL− 1 and 18.52 ng mL− 1 for Eflornithine enantiomers.