Simultaneous Impurity Analysis and Enantioseparation of Atenolol Using Achiral-Chiral 2D-LC

Importance of the Topic

Quality control of pharmaceuticals requires reliable separation of synthetic byproducts and chiral variants of active ingredients. Achieving both impurity profiling and enantioseparation in a single analytical run enhances efficiency, reduces sample handling, and ensures patient safety by verifying the purity and stereochemical composition of drugs.

Objectives and Study Overview

This work demonstrates a two-dimensional liquid chromatography (2D-LC) method for simultaneous analysis of atenolol impurities and baseline separation of its R and S enantiomers. The aim was to develop a streamlined workflow combining reversed-phase impurity screening in the first dimension with chiral separation in the second, using high-resolution heart-cutting and advanced data software.

Methodology

The sample containing racemic atenolol and known impurities was first resolved on a superficially porous C18 column under a gradient of aqueous ammonium formate and methanol. Time-based heart cuts of the atenolol peak were transferred to a chiral column operating in polar organic mode with acetonitrile/methanol mobile phase modified by triethylamine and acetic acid. Five consecutive 5.5-second cuts captured the entire racemate fraction for enantioseparation. Automated stop-time calculation and high-resolution sampling guided by chromatogram previews streamlined method setup.

Used Instrumentation

• Agilent 1290 Infinity II 2D-LC System with dual high-speed pumps, multisampler, multicolumn thermostat, diode array detectors, and valve drives with active solvent modulation and multiple heart-cutting valves
• Agilent OpenLab CDS 2 Acquisition and Data Analysis Software with 2D-LC Add-On
• Agilent InfinityLab Poroshell 120 EC-C18 column (2.1 × 150 mm, 2.7 µm) for achiral separation
• Agilent InfinityLab Poroshell 120 Chiral-T column (4.6 × 100 mm, 2.7 µm) for enantioseparation

Main Results and Discussion

The C18 column achieved clear separation of atenolol impurity standards within a 20 min run at moderate backpressure. Heart-cut sampling and chiral second-dimension analysis yielded baseline resolution (Rs > 2.1) of S- and R-atenolol across cuts 2–4. Ten replicate injections demonstrated high precision: 2D retention time RSDs of 0.15–0.16 % and peak area RSDs of 0.19–0.25 %, while 1D retention time and area precision were 0.03 % and 0.2 %, respectively. System suitability parameters, including selectivity and plate counts, were readily reviewed within the software’s peak table.

Benefits and Practical Applications

This achiral-chiral 2D-LC approach consolidates impurity profiling and enantiomeric purity testing into a single automated workflow, reducing analysis time and solvent consumption. High reproducibility and integrated data review tools support robust quality control in pharmaceutical development, regulatory compliance, and stability studies.

Future Trends and Potential Applications

Further advancements may include coupling 2D-LC with high-resolution mass spectrometry for structural elucidation, implementation of faster heart-cutting strategies, and application to other chiral drug classes. Enhanced software automation and machine-learning‐driven method optimization will broaden accessibility for routine laboratory use.

Conclusion

A streamlined achiral-chiral 2D-LC method was established for simultaneous impurity analysis and enantioseparation of atenolol. The combined reversed-phase and chiral workflow, supported by high-resolution sampling and dedicated software, delivers precise, reproducible results suitable for pharmaceutical quality assurance.

References

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2. Put InfinityLab Poroshell 120 Chiral Innovation to Work for Your Challenging Separations. Agilent Technologies application compendium, 5991-8450EN, 2017.
3. Pearson A.; et al. A Stereoselective Central Hypotensive Action of Atenolol. J. Pharmacol. Exp. Ther. 1989, 250(3), 759–763.
4. Stoschitzky K.; et al. Stereoselective Features of R- and S-Atenolol: Clinical Pharmacological, Pharmacokinetic, and Radioligand Binding Studies. Chirality 1993, 5(1), 15–19.