A strategy for rapid optimization of liquid chromatography column temperature and gradient shape is presented. The optimization as such is based on the well established retention and peak width models implemented in software like e.g. DryLab and LC simulator. The novel part of the strategy is a highly automated processing algorithm for detection and tracking of chromatographic peaks in noisy liquid chromatography-mass spectrometry (LC-MS) data. The strategy is presented and visualized by the optimization of the separation of two degradants present in ultraviolet (UV) exposed fluocinolone acetonide. It should be stressed, however, that it can be utilized for LC-MS analysis of any sample and application where several runs are conducted on the same sample. In the application presented, 30 components that were difficult or impossible to detect in the UV data could be automatically detected and tracked in the MS data by using the proposed strategy. The number of correctly tracked components was above 95%. Using the parameters from the reconstructed data sets to the model gave good agreement between predicted and observed retention times at optimal conditions. The area of the smallest tracked component was estimated to 0.08% compared to the main component, a level relevant for the characterization of impurities in the pharmaceutical industry.

The polyphenols, for example stilbenes and flavonoids, are an important family of compounds present in grapes and wine. For the first time, eight natural stilbenes (trans-resveratrol, trans-piceid, cis-piceid, trans-astringin, trans-piceatannol, (+)-trans-Ɛ-viniferin, pallidol, and hopeaphenol), isolated and purified from Vitis vinifera, were simultaneously analysed by UHPLC coupled with photodiode-array detection. Separation of the stilbenes was optimized with the assistance of Quality-by-Design commercial software DryLab. Four different reversed-phase columns packed with 1.5-1.7 µm particles were tested and compared for their retention behaviour and separation efficiency. 

The optimization of chromatographic separation was achieved using DryLab Software (Molnár-Institute, Berlin, Germany). Specifically, the experiments were carried out in a systematic way. The contemporary societies of the developed countries are prone to use traditional far-east medicines as remedies for all diseases. Some of them, such as obesity, might be classified as civilization diseases. Combating the problem, people try not only several miraculous diets but also herbal infusions (teas) and variety of “herbal” preparations. All these believing that such treatment is healthy and harmless as far as it is “natural”. Leaving out of the way the question if herbal medicines can be taken safely without doctors’ control the query arises if the common preparations are strictly natural and herbal. 

A stepwise method development strategy has been employed to develop a robust HPLC method to resolve several closely eluting structurally related impurities in an active pharmaceutical ingredient (API). This strategy consisted of automated column screening, optimization of the most critical chromatographic parameters, DryLab modeling, and experimental verification of optimized separation conditions. DryLab was used to predict an optimized gradient profile and separation temperature and these predictions were verified experimentally. A discussion of the accuracy of these predictions is presented. The robustness of the method was verified and the ability of DryLab to predict, with reasonable accuracy, the outcome of such robustness studies was also examined. Once the robustness was established by the DryLab predictions the remainder of the subsequent verification by experiment becomes a simple reiterative exercise. It is critical to adopt a rational strategy, as demonstrated here, to evaluate the interplay of these factors, thereby greatly enhancing method development efficiency.

The current paper presents a novel approach to applying Quality by Design (QbD) principles to the development of high pressure reversed phase liquid chromatography (HPLC) methods using DryLab. Four common critical parameters in HPLC - gradient time, temperature, pH of the aqueous eluent, and stationary phase - are evaluated within the Quality by Design framework by the means of computer modeling software and a column database, to a satisfactory degree. This work proposes the establishment of two mutually complimentary Design Spaces to fully depict a chromatographic method, one Column Design Space (CDS) and one Eluent Design Space (EDS) to describe the influence of the stationary phase and of the mobile phase on the separation selectivity, respectively. The merge of both Design Spaces into one is founded on the continuous nature of the mobile phase influence on retention and the great variety of the stationary phases available.

This paper describes systematic method development strategies used with different instrumental set-ups according to Quality by Design principles for a sample of toxicological interest. Experiments for DryLab modeling were run in an automated fashion on three Shimadzu instruments, and the accuracy of all models was confirmed to be excellent. Generated models were employed to reduce the original method time of 45 minutes to just over 3 minutes. Additionally, DryLab models were shown to successfully emulate experimental changes typically encountered during a method transfer process (dwell volume, extra column volume, flow rate, column dimensions).