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Two-Dimensional Liquid Chromatography (2D-LC) in Pharmaceutical Analysis: Applications Beyond Increasing Peak Capacity


One-dimensional liquid chromatography (1D-LC) is not always capable of efficiently separating complex samples. This drawback is not solely due to the lack of column efficiency, but is mainly due to insufficient selectivity and the need to separate the analytes of interest with orthogonal retention mechanisms. In this regard, two-dimensional liquid chromatography (2D-LC) is currently attracting much interest for its markedly higher resolving power compared to one-dimensional separation. In this work, three applications of 2D-LC from the pharmaceutical industry are presented with the goal not only to increase peak capacity, but also to support investigations. In the first application, the retention times of peaks of interest are matched under different mobile phase conditions for the purpose of transferring the method from a mass spectrometry (MS) incompatible buffer to an MS compatible buffer. The second application includes developing a method for simultaneous detection and quantitation of degradants and aggregates in a biologics and small molecule combination product. The third application supports method development by confirming the purity of separated peaks using orthogonal separation conditions in the first and second dimensions and to investigate mass balance issues where some peaks are expected to elute in the solvent front.

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The authors would like to thank the Agilent for the provision of loan instrument. We would like also to acknowledge Bob Giuffre, Kritsin Swanson, and Lisa Zhang from Agilent Technologies for their helpful suggestions and discussions. We would like to thank members of the Analytical Network at Novartis Pharmaceuticals for their helpful discussions.

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Correspondence to Imad A. Haidar Ahmad.

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The research presented here did not involve human participants and/or animals.

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Ahmad, I.A.H., Blasko, A., Clarke, A. et al. Two-Dimensional Liquid Chromatography (2D-LC) in Pharmaceutical Analysis: Applications Beyond Increasing Peak Capacity. Chromatographia 81, 401–418 (2018).

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  • Two-dimensional liquid chromatography
  • 2D-LC
  • Selective 2D-LC
  • Peak capacity
  • Pharmaceuticals
  • Peak purity
  • Hydrophobic subtraction model
  • Mass-balance issues
  • Large biomolecule drug
  • Small molecule drug