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The article presents the benefits of screening column and mobile phase combinations that generate differing chromatographic selectivities in reversed-phase gradient ultrahigh-pressure liquid chromatographic (UHPLC) method development strategies. Topics include the success of screening column and mobile phase combinations in a typical method development strategy, excellent accuracy retention predictions in reversed-phase liquid chromatography (RPLC), and anomalies retention predictions.

This review article summarizes the results obtained from the combined efforts of a joint academic and industrial initiative to solve the real-life challenge of determining low levels of peptide-related impurities (typically 0.05-1% of the drug substance) in the presence of the related biologically active peptide at a high concentration. A rational screening strategy for pharmaceutically important peptides has been developed that uses combinations of reversed-phase ultrahigh-pressure liquid chromatography (UHPLC) columns and mobile phases that exhibit complementary reversed-phase chromatographic selectivity using either UV- or mass spectrometry (MS)-compatible conditions. Numerous stationary and mobile phases were categorized using the chemometric tool of principal component analysis (PCA), employing a novel characterization protocol utilizing specifically designed peptide probes. This was successfully applied to the development of a strategy for the detection of impurities (especially isomers) in peptide drug substances using two-dimensional liquid chromatography coupled with MS detection (2D-LC-MS). [ABSTRACT FROM AUTHOR]

The retention of large molecules, such as peptides and proteins, is strongly dependent on mobile-phase composition and temperature. Consequently, the retention times (tR) of these molecules often fluctuate from one separation to the next. In two-dimensional liquid chromatography (2D-LC), this fluctuation can be challenging to deal with, especially if only one or a few specific peaks in the first dimension (1D) separation are targeted for further separation. In this instalment, we describe instrumental concepts that are useful for coping with this challenge. [ABSTRACT FROM AUTHOR]

The article offers information on to development and minimizing Fluctuating Peptide Retention in two-dimensional liquid chromatography (2D-LC) methods. It focuses on the need to take special precautions in mobile-phase preparation and operation of the LC system to reduce retention-time variation, particularly in the first dimension of the 2D-LC system.

Relatively small disparities in dwell volume, that is, the typical differences observed between ultrahigh-pressure liquid chromatography (UHPLC) and standard LC instrumentation, have been shown to have a profound effect on reversed-phase gradient chromatographic selectivity. These anomalous effects can be circumvented by either the addition of an isocratic hold (translating from a high to low dwell volume) or by performing a delayed injection (translating from a low to high dwell volume) after the gradient has been started. These selectivity discrepancies can be observed if the analytes are not highly retained at the column inlet during the initial isocratic phase of the gradient corresponding to the dwell volume. The effect of dwell volume on chromatographic selectivity can be successfully modelled using retention prediction software. Hence, the robustness of reversed-phase LC gradient methodologies, with respect to dwell volume, can be conveniently assessed. [ABSTRACT FROM AUTHOR]

Chromatographic principles and best practices for obtaining highly precise retention time, peak width, and resolution predictions for the optimization of reversed-phase liquid chromatography (LC) separations using retention modelling software will be discussed. The importance of fully characterizing the LC instrumentation, how to generate accurate input data, the selection of appropriate models, and peak tracking will be addressed along with a suggested workflow. Adhesion to a few basic rules and simple precautions and the use of modern retention modelling software programmes can assist the rapid development of highly accurate retention models to enable the development of robust and optimized reversed-phase LC separations using either ultrahigh-pressure liquid chromatography (UHPLC) or high performance liquid chromatography (HPLC) conditions. Examples of retention modelling for small and large molecules will be highlighted. [ABSTRACT FROM AUTHOR]

The selectivity of different combinations of organic modifiers, pH, and types of reversed-phase liquid chromatography (LC) materials has been characterized using Tanaka column characterization, linear solvent energy relationships (LSER), and selectivity correlations. The three characterization techniques highlighted the potential complementary selectivity of these phases and conditions as well as the type and dominancy of some of the retention mechanisms involved. Subsequently, selectivity differences were proven to be valid in the practical separation of acids, bases, and neutral analytes. This paper aims to help chromatographers to produce highly efficient method development strategies for reversed-phase LC separations in a relatively short time frame. [ABSTRACT FROM AUTHOR]

The numerous advantages of translating gradient chromatographic methods between the differing formats of liquid chromatography (LC) have been explored and discussed. Although translations in principle obey well-defined chromatographic theories, the authors investigate a number of potential pitfalls that may result in poor translations as exhibited by selectivity differences, changes in efficiency, and hence failure to meet resolution system suitability criteria. The consequences of these pitfalls are examined and the regulatory implications of method translation are explored. [ABSTRACT FROM AUTHOR]