Effective Determination of Pharmaceutical Impurities by Two Dimensional Liquid Chromatography

Importance of Topic

Effective determination of pharmaceutical impurities is critical for drug safety and regulatory compliance. Co-elution of low-level impurities with the active pharmaceutical ingredient (API) complicates analysis, requiring multiple methods and increasing time and resource demands. A two-dimensional liquid chromatography (2DLC) strategy combining heartcutting, at-column dilution and trap-elute addresses these challenges by delivering comprehensive impurity profiling in a single automated workflow.

Objectives and Study Overview

This study evaluates a targeted 2DLC platform—“Heartcut-ACD-Trap-Elute”—for quantifying glimepiride and its related impurities, including a cis-isomer that co-elutes with the API in a one-dimensional method. The goals were to:

• Develop a first-dimension reversed-phase UPLC method to resolve most impurities from the API peak.
• Implement a heartcut to transfer the API peak to a second dimension.
• Use at-column dilution and a trap column to focus the sample and improve chromatographic performance.
• Establish method repeatability and sensitivity sufficient to meet USP impurity limits.

Methodology and Instrumentation

A Waters ACQUITY UPLC H-Class Bio System with 2D Technology was configured with two 2-position/6-port valves, a quaternary pump (QSM) for the first dimension, a binary pump (BSM) for the second, and an isocratic pump (ISM) delivering at-column dilution.

• First dimension: CORTECS UPLC C+18 column (1.6 µm, 2.1 × 150 mm), isocratic 50 % acetonitrile/phosphate buffer (pH 2.1–2.7), 0.2 mL/min, PDA detection at 228 nm.
• Trap column: XBridge C8 (2.1 × 30 mm, 10 µm) captures the heartcut peak.
• At-column dilution: ISM pump at 0.8 mL/min of aqueous phosphate buffer (pH 7.0) with triethylamine, yielding a 5× dilution of organic content prior to trapping.
• Second dimension: ACQUITY UPLC BEH Phenyl column (1.7 µm, 2.1 × 150 mm), isocratic high-organic elution, flow 0.25 mL/min, TUV detection at 228 nm.

Main Results and Discussion

In the first dimension, six replicate injections of system suitability standards showed retention time RSD < 0.2 % and area RSD < 1 % for all related impurities. Analysis of glimepiride drug substance revealed GB and GD levels below USP limits. The heartcut-ACD-trap-elute approach successfully transferred the main peak containing the cis-isomer impurity to the second dimension. There, the impurity and API were fully resolved with retention time RSD < 0.1 % and area RSD < 0.5 %. A calibration curve for the cis-isomer was linear (R² > 0.995) over 0.1–2.5 % relative to API, with LOQ of 0.1 % (S/N > 5) and LOD of 0.07 % (S/N > 3). The cis-isomer content in drug substance was detected between LOD and LOQ.

At-column dilution significantly improved peak shape and retention in RPLC-RPLC mode by reducing the effective organic concentration from 50 % to 10 % before the second column.

Benefits and Practical Applications

The targeted 2DLC workflow integrates two impurity analyses into one system, reducing method development time, manual interventions and total analysis time. Heartcut trapping allows quantitative transfer of broad or asymmetric peaks without pressure spikes. At-column dilution enhances chromatographic performance for strong-solvent cuts. The platform meets USP impurity specifications, supports routine QA/QC, and can be adapted to other APIs and isomeric impurities.

Future Trends and Potential Applications

Advancements may include:

• Integration of mass spectrometry detectors for structural confirmation.
• Automated multi-heartcut scheduling to monitor several co-eluting zones.
• Gradient-capable at-column dilution schemes for broader applicability.
• Expansion to non-reversed-phase first dimensions and comprehensive LC×LC.

Conclusion

The Heartcut-ACD-Trap-Elute 2DLC strategy on an ACQUITY UPLC H-Class Bio System offers a robust, reproducible and efficient solution for comprehensive pharmaceutical impurity analysis. By consolidating two methods into a single automated run, it enhances throughput, data quality and regulatory compliance.

Reference

1. United States Pharmacopeial Convention. Glimepiride, USP38-NF33, United States Pharmacopeia, official from December 1 2015, p. 3671.
2. Khan MA; Sinha S; Vartak S; Bhartiya A; Kumar S. LC determination of glimepiride and its related impurities. Journal of Pharmaceutical and Biomedical Analysis 2005, 39: 928–943.
3. Wheat TE; Phoebe CH; Baynham MK; Neue UD; Fisk RP; Turner RC. Mobile phase dilution scheme for enhanced chromatography. US Patent US6790361 B2, Sep 14 2004.
4. Root DS; Wheat TE; McConville P. Routine MS Detection for USP Chromatographic Methods. Waters Application Note 720005306en, Feb 2015.