Comprehensive profiling of conjugated fatty acid isomers and their lipid oxidation products by two-dimensional chiral RP×RP liquid chromatography hyphenated to UV- and SWATH-MS-detection.

This research reports on the development of a comprehensive two-dimensional liquid chromatography (2D-LC) method hyphenated to inline DAD-UV and ESI-QTOF-MS/MS-detection for the separation of conjugated polyunsaturated fatty acid isomers and structurally related (saturated, unconjugated, oxidized) compounds. In pharmaceutical lipid formulations conjugated fatty acids can be found as impurities, generated by oxidation of polyunsaturated fatty acids. Due to the structural complexity of resultant multi-component samples one dimensional liquid chromatography may be suboptimal for quality control and impurity profiling. The screened reversed-phase columns showed a lack of selectivity for the conjugated fatty acid isomers but the resolutions improved with the shape selectivity of the stationary phases (C18- < C30- < cholesteryl-ether-bonded). Further enhanced selectivity for the non-chiral conjugated FAs could be achieved with amylose/cellulose-based chiral stationary phases (CSPs) which harbor cavities for selective inclusion depending on E/Z configurations of the double bonds of the analytes. Amylose-based CSPs showed higher selectivity for conjugated fatty acids than the cellulose-based polysaccharide CSPs. Hyphenating the chiral and reversed-phase columns in a comprehensive 2D-LC-setup was favorable since they showed orthogonality and good compatibility, because both were operated under RP-conditions. The chiral dimension (1D) mainly separated the different isomers, while the reversed-phase dimension (2D) separated according to number of double bonds and degree of oxidation. Using this setup, advanced structural annotation of unknowns was possible based on UV-, MS1- and MS2-spectra. Data-independent acquisition (by SWATH) enabled differentiation of positional isomers of oxidized lipids by characteristic MS2-fragments and elucidation of co-eluted compounds by selective extracted ion chromatograms of fragment ions (MS2 EICs).

Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and mass spectrometric characterization of synthetic oligonucleotides.

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.

Fragment-based Design of Zwitterionic, Strong Cation- and Weak Anion-Exchange Type Mixed-mode Liquid Chromatography Ligands and their Chromatographic Exploration.

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.

Mixed-mode chromatography characteristics of chiralpak ZWIX(+) and ZWIX(-) and elucidation of their chromatographic orthogonality for LC × LC application.

Two-dimensional liquid chromatography requires orthogonal columns and/or separation principles in the first and second separation dimension. It is sometimes not straightforward to achieve. Chiral columns could expand the toolbox for 2D-LC, but are rarely exploited for this purpose, not least due to missing understanding of retention principles under non-chiral application conditions. To gain more insight, in this study Chiralpak ZWIX(+) and ZWIX(-), based on zwitterionic quinine and quinidine carbamate selectors, were carefully characterized by molecular dynamics simulations, lipophilicity/hydrophilicity measurements of selectors, pH-dependent ζ-potential determinations, and chromatographic characterization in RPLC and HILIC modes combined with unsupervised principal component analysis to extract classification of these columns in comparison to a number of commercial benchmarks (RP, HILIC and mixed-mode columns). The results showed that these chiral columns can be classified as mixed-mode chromatography phases with balanced lipophilic-hydrophilic surface character, excess of negative net charge due to sulfonic acid groups (in spite of weakly basic quinuclidine and quinoline rings), and multimodal applicability (RP, HILIC and polar organic elution modes). Orthogonality mapping in comparison to a number of modern HILIC and mixed-mode columns revealed that Poroshell HILIC-Z (with a zwitterionic ligand on 2.7 µm core-shell particles) can be beneficially combined as second dimension with the ZWIX column for comprehensive LC × LC. The online hyphenation of this 2D-LC system with complementary detection modalities including UV (DAD for chromophoric substances), charged aerosol detection (for universal detection and calibration of non-volatile analytes) and high-resolution mass spectrometry (ESI-QTOF-MS/MS for identification) provided an advanced method for comprehensive impurity profiling, applicable for instance for amino acid pharmaceutical products.

Cinchona-based zwitterionic stationary phases: Exploring retention and enantioseparation mechanisms in supercritical fluid chromatography with a fragmentation approach.

Chiralpak ZWIX(+) and ZWIX(-), are brush-type bonded-silica chiral stationary phases (CSPs), based on complex diastereomeric Cinchona alkaloids derivatives bearing both a positive and a negative charge. In the present study, we aimed to improve the understanding of retention and enantioseparation mechanisms of these CSPs employed in supercritical fluid chromatography (SFC). For this purpose, 9 other stationary phases were used as comparison systems: two of them are commercially available and bear only a positive charge (Chiralpak QN-AX and QD-AX) and the 7 others were designed purposely to be structurally similar to the parent ZWIX phases, but miss some portion of the complex ligand. First, cluster analysis was employed to identify similar and dissimilar behavior among the 11 stationary phases, where ionic interactions appeared to dominate the observed differences. Secondly, the stationary phases were characterized with linear solvation energy relationships (LSER) based on the SFC analysis of 161 achiral analytes and a modified version of the solvation parameter model to include ionic interactions. This served to compare the interaction capabilities for the 11 stationary phases and showed in particular the contribution of attractive and repulsive ionic interactions. Then the ZWIX phases were characterized for their enantioseparation capabilities with a set of 58 racemic probes. Discriminant analysis was applied to explore the molecular structural features that are useful to successful enantioseparation on the ZWIX phases. In particular, it appeared that the presence of positive charges in the analyte is causing increased retention but is not necessarily a favorable feature to enantiorecognition. On the opposite, the presence of negative charges in the analyte favors early elution and enantiorecognition. Finally, a smaller set of 30 pairs of enantiomers, selected by their structural diversity and different enantioseparation values on the ZWIX phases, were analyzed on all chiral phases to observe the contribution of each structural fragment of the chiral ligand on enantioselectivity. Molecular modelling of the ligands also helped in understanding the three-dimensional arrangement of each ligand, notably the intra-molecular hydrogen bonding or the possible contribution of ionic interactions. In the end, each structural element in the ZWIX phases appeared to be a significant contributor to successful enantioresolution, whether they contribute as direct interaction groups (ion-exchange functions) or as steric constraints to orientate the interacting groups towards the analytes.

Stable-bond polymeric reversed-phase/weak anion-exchange mixed-mode stationary phases obtained by simultaneous functionalization and crosslinking of a poly(3-mercaptopropyl)methylsiloxane-film on vinyl silica via thiol-ene double click reaction.

A polymeric reversed-phase/weak anion exchange (Poly-RP/WAX) mixed-mode stationary phase has been prepared by coating of a poly(3-mercaptopropyl)methylsiloxane film on vinyl-modified silica (100 Å, 5 µm) and simultaneous in situ functionalization with N-(10-undecenoyl)-3-aminoquinuclidine as well as crosslinking to the vinyl silica surface by solventless thiol-ene double click reaction. Such bonding chemistry showed greatly enhanced stability compared to brush-type analogs with bifunctional siloxane bonding to silica. Solid-state 29Si-CP/MAS NMR confirmed the immobilization of the siloxane layer. pH-Dependent ζ-potential determinations revealed a high anion-exchange capacity over the entire pH range with a maximum around pH 5. Oxidation of residual thiols yielded a zwitterionic Poly-RP/WAX/SCX mixed-mode phase with sulfonic acid endcapping and shifted the still net positive surface charge to lower ζ-potentials. It allowed a faster elution of strongly retained anionic species in particular of multiply negatively charged analytes such as oligonucleotides. Chromatographic tests under RPLC and HILIC elution mode with various test substances documented the multimodal utility and complementarity in retention profiles compared to RP, HILIC and commercial mixed-mode phases.

N-Propyl-N'-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal.

Herein, we present a novel silica-based stationary phase modified with N-propyl-N'-2-pyridylurea selector. Due to the weakly basic properties of the pyridine selector and the presence of residual silanols after selector immobilization, a zwitterionic surface with a pI observed at approximately pH 5.5 was measured by electrophoretic light scattering in pH-dependent ζ-potential determinations. The capability of the new N-propyl-N'-2-pyridylurea-modified silica to serve as mixed-mode stationary phase was investigated. For this purpose, it was characterized under RP and HILIC conditions using test mixtures. Subsequent classification of this stationary phase in comparison to in-house and commercial benchmarks was carried by principal component analysis of resultant retention factors from chromatographic tests. The results show a relatively unique mixed-mode character amongst the tested stationary phases. The chromatographic retention characteristics of acidic compounds matched well the ζ-potential determinations. The application of anion-exchange at low pH values (e.g. pH 5) and ion exclusion chromatography at pH 7 for the separation of uridine 5'-mono-, di- and triphosphate demonstrated a pH-dependent umpolung of the stationary phase surface. The combination of these separation principles in a pH gradient from 5 to 7 gave rise to weak anion-exchange selectivity with a charge-inducted elution due to repulsive interactions at higher pH and resulted in a significant faster separation with improved peak shape under mild elution conditions.