Highlights

• New stationary phases with low hydrophobicity were tested for protein separations
• The new ES-LH stationary phase showed excellent protein recovery
• The ES-LH and C1 materials were found to be less retentive than commercial phases
• The ES-LH phase showed novel selectivity due to its unique surface chemistry

In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called “Quality by Design” (QbD) principle is often being applied rather than “Trial and Error” approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG
, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.

Highlights:

• Phorbas amaranthus extract as natural products library
• DryLab® software for Design of Experiments in association with scouting systems
• Sixty-four metabolites chemically characterized by LC-HRMS
• New metabolites inferred based on Global Natural Product Social Molecular Networking (GNPS)

Abstract

The optimization of RP-HPLC separations of plant secondary metabolites can be quickly and easily carried out using DryLab^®, which predicts the chromatographic behavior of the analytes on the basis of a limited number of experiments. Such approach allows the development of robust and reliable RP-HPLC methods and reduces the consumption and waste of harmful and expensive organic solvents and, therefore, is both economic and ecologic.

Highlights:

• Quality-by-design based liquid chromatographic method for new chemical entity
• Exploration of design space using 2D and 3D chromatographic retention modeling
• Establishment of a methodology for achieving analytical method control space
• Demonstration of dependence of method control space on control strategy
• Use of DryLab^® to evaluate method robustness with acceptable accuracy