Achiral-Chiral Heart-Cutting 2D-LC Analysis of Chiral Pharmaceutical Substances

Significance of the Topic

Accurate profiling of achiral and chiral impurities and determination of enantiomeric composition are critical steps in pharmaceutical development and quality control. Regulatory guidelines such as ICH Q3A and FDA stereoisomeric drug guidance demand sensitive, reliable methods to detect impurities above 0.05% and to ensure stereochemical purity. A combined achiral–chiral heart-cutting two-dimensional liquid chromatography (2D-LC) approach streamlines impurity profiling and enantiomeric analysis in a single workflow, enhancing throughput and compliance in drug analysis.

Objectives and Study Overview

This study demonstrates a heart-cutting 2D-LC method using the Agilent 1290 Infinity system to:

• Separate achiral impurities from the active pharmaceutical ingredient (API) in the first dimension by reversed-phase chromatography.
• Transfer (“heart-cut”) the API fraction to a chiral second dimension column for enantiomeric separation.
• Validate performance using racemic ibuprofen and a mixture of R-(+)- and S-(–)-thalidomide, including enantiomeric excess (ee) determination and impurity discrimination under forced-degradation conditions.

Used Instrumentation

Agilent 1290 Infinity 2D-LC Solution comprising:

• Two binary pumps
• Autosampler with temperature control
• Thermostatted column compartment
• 2-Position/4-Port Duo valve with 60 µL loop
• Two diode array detectors with Max-Light cartridge cells (10 mm and 60 mm)

Methodology

First dimension:

• Reversed-phase C18 column (2.1×150 mm, 1.8 µm)
• Gradient from 5% to 95% organic phase over 20 min, flow rate 0.25 mL/min
• Detection at 264 nm (ibuprofen) or 254/295 nm (thalidomide)

Heart-cutting and transfer:

• Timed loop fill (0.20 min) at the peak apex
• Valve switches from waste to second dimension loop position

Second dimension:

• Chiral stationary phase column (4.6×250 mm, 5 µm)
• Water/methanol or methanol mobile phases with 0.1% formic or acetic acid, flow rate 1 mL/min
• Detection at same wavelengths, 20 Hz data rate

Main Results and Discussion

Ibuprofen system:

• First dimension separated nine achiral impurities from API peak.
• Heart-cutting delivered ibuprofen enantiomers with resolution Rs=1.25.
• Retention time RSD <0.1% and area RSD <0.6% over ten injections.
• Linearity over 0.1–1 mg/mL, R2=0.9999 for each enantiomer and their sum.

Thalidomide system:

• Chiral separation achieved Rs=8.3 for R-(+)- and S-(–)-thalidomide.
• Enantiomeric excess of 98.1% determined in spiked mixture.
• Forced‐degradation experiment under alkaline conditions showed baseline separation of degradation products in the first dimension.

Benefits and Practical Applications

• Streamlined workflow combining impurity profiling and chiral analysis in a single run.
• High reproducibility and sensitivity support regulatory compliance (ICH Q3A, FDA chiral guidelines).
• Flexible method adaptation for various APIs, degradation studies, and trace enantiomer detection.
• Suitable for pharmaceutical R&D, quality assurance, and generics testing.

Future Trends and Applications

• Integration with high-resolution mass spectrometry for structure elucidation of impurities.
• Automation enhancements and software-driven heart-cut optimization.
• Extension to three-dimensional LC or multidimensional formats for complex mixtures.
• AI-based data analysis and peak recognition to accelerate method development.

Conclusion

The heart-cutting 2D-LC approach using the Agilent 1290 Infinity platform effectively separates achiral impurities and enantiomers of APIs such as ibuprofen and thalidomide. The method delivers excellent reproducibility, linearity, and resolution, meeting stringent regulatory requirements. Its adaptability makes it a powerful tool for comprehensive impurity and stereochemical analysis in pharmaceutical development and quality control.

Reference

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