Separation of enantiomers and conformers of Tofisopamon

Importance of Topic

Chiral separation of drug candidates is essential to distinguish physiological activities of enantiomers and conformers. Tofisopam, a benzodiazepine derivative used in anxiety and alcohol withdrawal therapy, exists as two enantiomers each adopting two boat conformations. Accurate analysis of its stereoisomeric purity supports drug development, quality control, and pharmacological studies.

Objectives and Overview

The study aimed to develop a single high speed method to resolve all four stereoisomers of Tofisopam. Automated screening of chiral stationary phases and mobile phase modifiers was conducted on an Agilent 1260 Infinity Analytical SFC system with Daicel immobilized polysaccharide derived columns.

Methods and Instrumentation

Chromatographic separation employed supercritical CO2 with modifiers including methanol, 2-propanol, and acetonitrile each containing a diethylamine additive. Four Daicel columns (CHIRALPAK IA, IB, IC, ID with immobilized amylose or cellulose derivatives) sized 4.6 × 150 mm, 5 µm, were evaluated.

• Agilent 1260 Infinity Analytical SFC System with Fusion A5 CO2 pre- and post-conditioning module
• 1260 Infinity Binary Pump for precise modifier delivery
• Standard autosampler and diode array detector configured for high pressure SFC flow cell

Main Results and Discussion

Methanol with 0.1–0.5 % diethylamine consistently yielded the highest resolution. CHIRALPAK IA and ID provided baseline separation of all four stereoisomers in under six minutes. Modifier type influenced elution order and peak resolution. Reproducibility was demonstrated over 120 injections on CHIRALPAK IC with retention time and resolution deviations within acceptable limits. System pressure remained stable throughout runs.

Benefits and Practical Applications

The robust SFC method enables rapid and reliable quantification of Tofisopam stereoisomers. Short analysis times support high throughput screening in pharmaceutical development and quality control laboratories. Use of immobilized polysaccharide columns ensures broad solvent compatibility and column durability.

Future Trends and Possibilities

Integration of SFC with mass spectrometry detection may enhance sensitivity for low-level impurities. Continued expansion of immobilized chiral stationary phase chemistries will improve selectivity for challenging stereoisomeric separations. Automation of method development workflows can further accelerate screening of novel chiral compounds.

Conclusion

A single SFC protocol on Daicel immobilized polysaccharide columns effectively resolves enantiomers and conformers of Tofisopam in a fast, reproducible, and high throughput manner. The approach supports critical analytical needs in drug discovery and regulatory environments.

Reference

• Separation of enantiomers and conformers of Tofisopam Using Daicel immobilized polysaccharide derived chiral columns and Agilent 1260 Infinity Analytical SFC System, Application Note, Agilent Technologies and Chiral Technologies Europe, 2011