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Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants

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Abstract

In this paper, we describe the findings of a study aimed at assessing the detection sensitivity of comprehensive two-dimensional high-performance liquid chromatography (LCxLC) separation of a degraded active pharmaceutical ingredient (API) with UV absorption as the detection technique. Specifically, we have examined the impact of the volume and solvent composition (referred to as “interface conditions”) of fractions of first-dimension column effluent transferred to the second dimension for further separation on the ability to resolve and detect low-abundance compounds. Historically, LCxLC has been perceived as being inferior to 1D-LC from the point of view of detection sensitivity. In this work, we demonstrate that LCxLC is sufficiently sensitive to be useful in the pharmaceutical context where in general impurities present at 0.05 % (relative to the API concentration) should be quantified. Moreover, we find that this level of sensitivity is only attained under certain conditions: dilution of the first column effluent with weak solvent (water in this case) prior to injection into the second-dimension column is very beneficial because it promotes focusing of the analyte band in the second column, thereby improving the detection sensitivity of the LCxLC system; and, quantitation limits are also a strong function of peak location in the second-dimension separation window, where baseline disturbances near the dead time of the second column can limit reliable detection of low-abundance compounds.

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Acknowledgments

E.T. was supported by a gift from Agilent Technologies University Relations. D.S. was supported by a grant from the National Science Foundation (CHE-1213364) and a gift from Agilent Technologies University Relations. The 1290 Infinity 2D-LC system and HPLC columns used in this work were provided by Agilent Technologies. The LC Image software used to produce the LCxLC chromatograms shown here was provided by GC Image.

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Authors and Affiliations

  1. Gustavus Adolphus College, 800 West College Avenue, St. Peter, MN, 56082, USA

    Dwight R. Stoll, Eric S. Talus & David C. Harmes

  2. Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA

    Kelly Zhang

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  1. Dwight R. Stoll
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Correspondence to Dwight R. Stoll.

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Published in the topical collection Multidimensional Chromatography with guest editors Torsten C. Schmidt, Oliver J. Schmitz, and Thorsten Teutenberg.

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Stoll, D.R., Talus, E.S., Harmes, D.C. et al. Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants. Anal Bioanal Chem 407, 265–277 (2015). https://doi.org/10.1007/s00216-014-8036-9

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