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A new approach to numerical optimization of gradient separation conditions in liquid chromatography coupled with tandem mass spectrometric detection

Autoři: Česla Petr | Kašpar Michal | Kubát Miroslav | Tošovská Barbora

Rok: 2025

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	A new approach to numerical optimization of gradient separation conditions in liquid chromatography coupled with tandem mass spectrometric detection	High-performance liquid chromatography (HPLC) remains a cornerstone technique for the separation and analysis of a wide range of compounds, including bioactive substances, due to its versatility and broad applicability across various separation modes, such as reversed-phase liquid chromatography (RPLC) and normal-phase liquid chromatography (NPLC). Among these, RP-LC is currently the most widely used, with C18 stationary phases recognized as the gold standard. For highly polar compounds, which are common among bioactive substances, retention in RPLC is usually low, and thus aqueous NPLC, i.e., hydrophilic interaction liquid chromatography (HILIC), gained popularity in recent years. Method development and optimization typically involve selecting the appropriate separation mode, stationary phase, mobile phase composition, and gradient profile. Especially optimizing the gradient profile is a highly effective tool for increasing peak capacity, improving analyte resolution, and enhancing the quantitative characteristics of developed methods. In our previous work, we have shown that for both RPLC separations and HILIC separations of biologically relevant compounds, the numerical optimization approach using retention modelling can be successfully applied for achieving the best separation conditions. In coupling RPLC or HILIC with tandem mass spectrometry, the optimization procedure should, however, be extended with the selection of proper detection conditions. Therefore, in our recent work, we have developed a procedure for optimizing gradient profile and tandem mass spectrometric detection conditions in multiple-reaction monitoring mode, combining several statistical criteria, i.e., interquartile range of gradient retention times, probability of mass spectrometric time-window overlapping, and gradient time range. In the current contribution, both approaches are compared, and the extension of the latter approach to the multidimensional separations is considered as well.

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