Exploring lactoferrin as an innovative covalently immobilized chiral selector for the selective separation of pharmaceutical enantiomers using HPLC

Abstract

Background: Molecular chirality has a profound impact on pharmaceutical drugs' efficacy and safety. Thus, chiral recognition plays a vital role in drug development throughout all process stages. LF is a globular glycoprotein composed of approximately 700 amino acids, that's capable of providing multiple interactions through its amino acid residues. Results: In the proposed research, lactoferrin (LF) was covalently immobilized for the first time using a monolithic epoxy stationary phase via a Schiff's base formation as chiral selector (CS). LF-CS was evaluated for the enantioselective HPLC separation of 18 racemic pharmaceutical drugs. The chromatographic conditions were optimized for optimum enantio-separation. LF-CS proved its potential as a CS enabling the enantioseparation of 10 drug racemates: cetirizine, omeprazole, lansoprazole, dapoxetine, doxazosin, nebivolol, atenolol, bisoprolol, chlorthalidone and ofloxacin. Moreover, in-silico molecular docking studies were conducted to help understand the separation modes. LF as a CS reported hydrogen bonding, hydrophobic interactions, π bonding, van der Waals forces, and electrostatic interactions. Finally, the novel LF-CS was applied to determine the enantiomeric purity of marketed single-enantiomer pharmaceutical products, demonstrating its ability to verify their composition and identify impurities. Significance: HPLC remains the primary choice for all pharmaceutical research, as it offers higher sensitivity, reliability, and reproducibility. This work introduces LF as a novel, multifunctional CS for HPLC, covalently immobilized for the first time, expanding the toolbox of protein-based chiral stationary phases. Moreover, the study also offers a critical insight into the limitations of relying solely on computational predictions, empirically demonstrating that solvent effects can override binding affinities, a phenomenon not captured by standard docking simulations.