Characterization and Application of Mixed Mode Stationary Phases in Pharmaceutical and Biochemical Analysis using One- and Two-Dimensional Liquid Chromatography

Nowadays, the use of High-Performance Liquid Chromatography (HPLC) is indispensable in many analytical application fields. Thereby, Reversed Phase Liquid Chromatography (RP LC) is the most frequently used chromatographic mode as it is suitable for the majority of applications. However, the rising requirements in pharmaceutical quality control concerning comprehensive sample characterization as well as the increasing sample complexity rises the need of alternative, complementary chromatographic methods. Besides, the commercialization of two-dimensional liquid chromatography (2D-LC) systems further encouraged the popularity of separation methods complementary to RP LC. In this context, Mixed Mode Chromatography (MMC) gained growing importance alongside Hydrophilic Interaction Chromatography (HILIC). MMC stationary phases show alternative selectivities due to the surface functionalization of the separation material with multiple interaction sites of different species which are involved in the chromatographic process. In house synthesized silica gel based MMC stationary phases, namely N-Propyl-N’-2-pyridylurea and N-(10-undecenoyl)-3-aminoquinuclidine modified silica particles, were characterized concerning their flexible use under different elution conditions such as RP and HILIC conditions. The resulting retention data were used in order to comparatively classify the stationary phases by Principal Component Analysis (PCA) to elucidate similarities and dissimilarities. Such projections can support to make a fast-preliminary selection of potentially suitable columns before a specific sample is addressed. Additionally, the same procedure was used for the characterization of the zwitterionic chiral stationary phases ZWIX(+) and ZWIX(-) which can also be categorized as MMC stationary phases. In order to enhance the understanding of the contribution of the distinct molecule moieties to the chromatographic process, silica particles with immobilized selector fragments of the chiral ligands were characterized in the same manner. As a complementary measure, the conformations of the immobilized ligands were examined by molecular modelling. Furthermore, the surface charge state of the separation materials was characterized as it plays a crucial role in the separation and retention of charged analytes. In this context, zeta-potential determination as well as a chromatographic characterization proved to offer valuable information. The characterization of the zwitterionic separation materials ZWIX(+) and ZWIX(-) and the respective fragmented selectors was further supported by the characterization of the free ligands in capillary electrophoresis. In some cases, the use of high buffer concentrations is necessary in order to analyze multiply charged solutes. This limits the choice of compatible detection methods. Hence, the structure elucidation of impurities in pharmaceutical drug substances and drug products involve the use of high-resolution mass spectrometry (HR MS) which constrains the tolerated buffer concentrations. In order to address this problem, on the one hand the principle of immobilized counterions was realized and on the other hand, the basicity of the ion exchange site was adjusted to influence the charge state and polarity of the stationary phase. Milder elution conditions up to a umpolung of the surface charge in case of the latter one was the result of both strategies. Furthermore, column bleeding is an important factor in the routine analysis as well as in the field of structure elucidation as it detrimentally affects the detection sensitivity in universal detection methods and in mass spectrometry in case of ionizable ligands. Therefore, the attachment chemistry is of utmost importance also influencing the column lifetime. Previously, a new attachment chemistry was developed by Zimmermann et al. (J Chromatogr A, 1436 (2016) 73-83) where the chromatographic ligand is immobilized on a polysiloxane layer which is multiply linked to the silica particle. The stability of brush-type stationary phases compared to the polymer coated stationary phase was studied in a chromatographic stress test in this work. Challenging pharmaceutical example mixtures were separated using one dimensional (1D-LC) and two dimensional HPLC (2D-LC) in order to demonstrate the advantage which resulted from the improvement strategies. In 1D-LC nucleotides and synthetic oligonucleotides were analyzed demonstrating the beneficial use of both strategies aiming at the reduction of the utilized counterion concentration. The simultaneous analysis of fat- and water-soluble vitamins was addressed by selective comprehensive HILIC × RP with precedent optimization of the two chromatographic dimensions using design of experiments (DoE) strategy amongst others. However, the orthogonality of the two dimensions challenged their hyphenation in terms of solvent mismatch problems. Therefore, the active solvent modulation methodology was beneficially used in the 2D-LC method leading to sharp, well resolved peaks in 2D. Moreover, an impurity profiling set-up for polar analytes was developed. A set of polar stationary phases was screened under HILIC conditions analyzing proteinogenic amino acids. On the basis of these chromatograms, the most promising stationary phases for the HILIC × HILIC system were elucidated. The hyphenation of HILIC systems is challenging because of decreased refocusing possibilities in the beginning of the chromatogram as well as the comparable longer re-equilibration times. In order to prevent solvent mismatch issues, the 1D elution mode eschewed water which has a high elution strength under HILIC conditions. The instrumental set-up included besides the two chromatographic dimensions, the hyphenation of multiple detectors, namely diode array detector (DAD), charged aerosol detector (CAD) and HR MS. The UV detectors can be used to elucidate relations of compounds due to similar spectra and serve later on as an identification criterion when simpler instrumentations are used. The charged aerosol detector can be used for a quasi-universal quantification of the compound and high-resolution mass spectrometry (HR-MS and MS/MS) serves for proper identification of the peak of interest