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.