Splitting and separation mechanism of tenofovir alafenamide fumarate chiral isomers based on indirect chiral ligand exchange chromatography.

The isolation and analysis of chiral isomers are critical parts of the drug development process to ensure effective and safe drug administration to patients. Indirect chiral ligand exchange chromatography (ICLEC) was developed to separate and determine tenofovir alafenamide fumarate (TAF) and its diastereoisomer GS-7339, with a hypothesized separation mechanism. The effect of using a chiral column versus a standard C18 column on the separation of the TAF chiral isomer mixture was investigated. Various factors in ICLEC, including ligand type, ligand ratio, mobile phase composition, and column temperature, were optimized. The separation of TAF and GS-7339 was successfully achieved by selecting L-phenylalanine as the chiral selective agent and Cu(II) as the central metal ion, using a C18 column as the analytic column and a mobile phase of 20 mM ammonium dihydrogen phosphate buffer (pH = 4.0)-acetonitrile (79 : 21, v/v). The corresponding linearity range for TAF and GS-7339 indicated a good correlation with R² > 0.9960. The average recoveries of TAF and GS-7339 ranged from 98.2% to 106.9%. None of the eight manufacturers detected GS-7339, and the percentage of TAF-labeled amounts in the drugs ranged from 95.0% to 98.5%. TAF tablets from eight manufacturers were of satisfactory quality. The separation mechanism of TAF and GS-7339 by ICLEC is due to the different spatial configurations of the two ternary complexes formed by the two chiral isomers, leading to differences in their thermodynamic stability and retention behavior. The established ICLEC method is economical, simple, and flexible, providing an effective strategy for studying chiral drug separation and analysis.