Search

A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico‐chemical evaluations in early‐phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa < 12). An RP‐HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so‐called "φ0 index" as the best correlating with the log D. The wspH${}_w^spH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n‐octanol, n‐butylamine, and n‐octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0. The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data. [ABSTRACT FROM AUTHOR]

Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D-amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion-exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure-selectivity relationships. It turned out that small di- and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol-tetrahydrofuran-water (49:49:2; v/v/v) with 25mM formic acid and 12.5mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. [ABSTRACT FROM AUTHOR]

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. Image 1 • New insights in physicochemical character of commercial Chiralpak ZWIX columns obtained. • Negative surface charge of zwitterionic stationary phases revealed by ζ-potential determination. • Benchmarking study with commercial RP, HILIC and mixed-mode phases classifies Chiralpak ZWIX(+/-) as mixed-mode chromatography columns with multiple elution modes. • LC × LC with Chiralpak ZWIX in polar organic elution mode as 1D and Poroshell HILIC-Z in HILIC as 2D established. • Full comprehensive LC × LC with three complementary detection modes (UV, CAD, QTOF-MS/MS) for amino acid impurity profiling. [ABSTRACT FROM AUTHOR]

Abstract Owing to their pronounced polarity, hydrophilic interaction liquid chromatography (HILIC) can be considered as the elective choice for the LC analysis of aminoglycoside (AG) antibiotics. In the present work, a gradient program was optimized for the first time with a diol-type stationary phase and an evaporative light scattering detector (ELSD), thus allowing the almost complete separation of the nine analysed AGs: spectinomycin, dihydrostreptomycin, streptomycin A, gentamicin C1, amikacin, kanamycin A, paromomycin, apramycin and neomycin. In the optimized analysis conditions, analyte retention was found to be governed by a multimodal mechanism encompassing electrostatic, partitioning and hydrophilic interactions. However, the gradient mode of elution complicated a deep understanding of the influence of each contribution on the retention behaviour. The developed HILIC-ELSD method was applied for the analysis of commercial tablets containing neomycin co-formulated with the polypeptide antibiotic bacitracin. The method was fully validated according to the guidelines enshrined in the International Conference on Harmonization (ICH). The use of the diol-type stationary phase was well suited for implementing a successful 2D-HPLC system. Indeed, in order to cope with the absence of chemoselectivity for the couples amikacin/kanamycin and paromomycin/apramycin, a successful 2D-HPLC method was implemented with the "heart-cut" approach and the use of either heptafluorobutyric (for the former) or perfluorooctanoic acid (for the latter) as the ion-pair reagent in the second RP-LC dimension. Graphical abstract Image 1 Highlights • A diol stationary phase allows the HILIC separation of aminoglycoside antibiotics. • Analyte retention is governed by a multimodal mechanism. • The optimized HILIC-ELSD method can be applied for quality control analyses. • The developed HILIC method is suited for implementing heart-cut 2D-HPLC systems. [ABSTRACT FROM AUTHOR]

• New mixed-mode RP/WAX, RP/SCX and RP/zwitterionic (ZWIX) ion-exchange stationary phases • Design of chromatographic ligands by fragmentation of Chiralpak ZWIX selectors • ζ-potentials revealed finely tuned surface charge by individual increments • Molecular dynamics ruled out intramolecular ion-pair formation in Chiralpak ZWIX selectors • Benchmarking study showed peculiarities compared to RP, HILIC and mixed-mode phases 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. [ABSTRACT FROM AUTHOR]

A key issue in enantioselective analysis is the determination of the enantiomeric elution order. To this aim, many computational modeling studies have been published in the last four decades. Docking and molecular dynamics simulations often supported by quantum mechanical and/or experimental spectroscopic and crystallographic analyses have been applied to gain a deeper insight into the main forces driving the enantiorecognition mechanism and, in turn, the enantiomer elution order. Very interestingly, almost all the relevant materials for enantioselective chromatography applications have been modeled over the years, spanning from high- to low-molecular weight chiral selectors together with the most relevant elution modes. In this review, examples from literature are presented to highlight the main improvements made in terms of accuracy, methodologies and computational performance. As concluding remarks, a critical evaluation of strength points and limitations of the treated in silico methods is discussed. • Docking and molecular dynamics simulations aid the explanation of the enantiomer elution order. • Low- and high-molecular weight chiral selectors can be computationally simulated. • All types of mobile phases can be implicitly and explicitly considered in silico. [ABSTRACT FROM AUTHOR]