Computer-driven optimization of complex gradients in comprehensive two-dimensional liquid chromatography.

Method development in comprehensive two-dimensional liquid chromatography (LC × LC) is a complicated endeavor. The dependency between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, renders method development by "trial-and-error" time-consuming and highly dependent on user experience. In this work, an open-source algorithm for the automated and interpretive method development of complex gradients in LC × LC-mass spectrometry (MS) was developed. A workflow was designed to operate within a closed-loop that allowed direct interaction between the LC × LC-MS system and a data-processing computer which ran in an unsupervised and automated fashion. Obtaining accurate retention models in LC × LC is difficult due to the challenges associated with the exact determination of retention times, curve fitting because of the use of gradient elution, and gradient deformation. Thus, retention models were compared in terms of repeatability of determination. Additionally, the design of shifting gradients in the second dimension and the prediction of peak widths were investigated. The algorithm was tested on separations of a tryptic digest of a monoclonal antibody using an objective function that included the sum of resolutions and analysis time as quality descriptors. The algorithm was able to improve the separation relative to a generic starting method using these complex gradient profiles after only four method-development iterations (i.e., sets of chromatographic conditions). Further iterations improved retention time and peak width predictions and thus the accuracy in the separations predicted by the algorithm.