Modernizing Chiral Separations with Glycopeptide-Based Chiral Columns

Importance of the Topic

Chiral separations play a vital role in pharmaceutical quality control and regulatory compliance. With an increasing number of optically active drugs requiring enantiomeric purity assessment, rapid, reliable, and sensitive analytical methods are essential. Glycopeptide-based columns offer unique selectivity and solvent compatibility, enabling faster enantiomeric resolution in both reversed-phase and polar ionic modes.

Study Objectives and Overview

The study aims to develop and optimize a chiral HPLC method for timolol maleate using an Agilent InfinityLab Poroshell 120 Chiral-T column. The proposed method is benchmarked against the current USP/EP normal-phase procedure, focusing on analysis time, resolution, and MS compatibility.

Methodology and Instrumentation

The separation was performed in polar ionic mode using methanol with varying concentrations of ammonium formate. Optimal conditions were determined by screening chiral columns and salt concentrations. UV detection at 260 nm, a flow rate of 1 mL/min, and column temperature of 30 °C were employed.

Used Instrumentation

• Agilent 1260 Infinity II LC system: binary pump, multisampler, multicolumn thermostat, and diode-array detector
• Agilent InfinityLab Poroshell 120 Chiral-T column (4.6 × 100 mm, 2.7 µm)
• OpenLab CDS software (version C.01.07)

Key Results and Discussion

• Effective baseline separation of timolol enantiomers was achieved using methanol with 0.2% w/v ammonium formate, yielding retention times of 2.257 and 2.467 min and a resolution (Rs) of 2.46.
• Resolution improved to Rs 4.12 at 0.025% ammonium formate, with longer retention but enhanced enantiomeric discrimination.
• The method outperforms the USP normal-phase approach in speed (under 3 min vs. 9–14 min) and offers MS compatibility without phase bleed or dedicated normal-phase hardware.

Benefits and Practical Applications of the Method

• Significantly reduced analysis time, increasing laboratory throughput.
• High enantiomeric resolution across a range of salt concentrations.
• Broad solvent compatibility and stability of the bonded glycopeptide phase, extending column lifetime.
• Fully compatible with MS detection, facilitating method transfer and forensic or troubleshooting applications.

Future Trends and Opportunities

Advances in superficially porous glycopeptide columns and incorporation of supercritical fluid chromatography may further decrease analysis times and solvent usage. The approach could be extended to other chiral APIs and integrated with real-time MS detection for comprehensive quality control workflows.

Conclusion

The optimized polar ionic HPLC method using an Agilent InfinityLab Poroshell 120 Chiral-T column provides a rapid, high-resolution, and MS-compatible solution for timolol maleate enantiomeric analysis, demonstrating clear advantages over traditional normal-phase techniques.

References

1. Bonner W A. Parity Violation and the Evolution of Biomolecular Homochirality. Chirality 2000, 12, 114–126.
2. FDA's Policy Statement for the Development of New Stereoisomeric Drugs. 1992, 57 Fed. Reg. 22249.
3. Long W J; Mack A E. Screening Chiral Compounds Using Superficially Porous Based Chiral Columns. Pittcon 2018, CO-1479.
4. Agilent InfinityLab Poroshell 120 Chiral Application Compendium, publication 5991-8450EN, 2017.
5. United States Pharmacopeia (USP 40) Timolol Maleate Monograph.
6. European Pharmacopeia, 4th ed. Addendum 2002.
7. European Pharmacopeia (Version 7.5): Timolol Maleate Monograph.
8. Instruction Manual for Chiralcel OD-H and OJ-H Chiral Technologies, 07/2013.
9. Marley A; Connolly D. Determination of (R)-Timolol in (S)-Timolol Maleate: Validation of a New SFC Method with Established Normal Phase LC. J Chromatogr A 2014, 1325, 213–220.