Chiralpak ZWIX Research Articles

Chiral separation of dipeptides on Cinchona-based zwitterionic chiral stationary phases under buffer-free reversed-phase conditions.

Chiral zwitterion ion exchangers represent efficient chiral stationary phases for stereoselective resolution of various analytes including chiral acids, bases, and zwitterions. In this contribution, we have focused on utilization of chiral zwitterionic sorbents, denoted as ZWIX (+A) and ZWIX (-A). These are analogical chiral systems to commercially available columns, Chiralpak ZWIX (+) and Chiralpak ZWIX (-), which are usually operated with buffered mobile phases. In this contribution, we have studied the enantiorecognition power of the ZWIX (+A) and ZWIX (-A) columns on a series of dipeptides operated under buffer-free reversed-phase conditions. Retention characteristics of zwitterionic dipeptides are discussed using an electrostatically driven adsorption model, which provides a good fit with both monotonous and U-shaped curves.

Enantioselective high-performance liquid chromatographic separation of fluorinated ß- phenylalanine derivatives utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases: Enantioselective separation of fluorinated ß-phenylalanine derivatives

The enantioselective separation of newly synthesized fluorine-substituted β-phenylalanines has been performed utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases. Experiments were designed to study the effect of eluent composition, counterion content, and temperature on the chromatographic properties in a systematic manner. Mobile phase systems containing methanol or mixtures of methanol and acetonitrile together with acid and base additives ensured highly efficient enantioseparations. Zwitterionic phases [Chiralpak ZWIX (+) and ZWIX(–)] were found to provide superior performance compared to that by the anion-exchangers (Chiralpak QN-AX and QD-AX). A detailed thermodynamic characterization was also performed by employing van't Hoff analysis. Using typical liquid chromatographic experimental conditions, no marked effect of the flow rate could be observed on the calculated thermodynamic parameters. In contrast, a clear tendency has been revealed about the effect of the eluent composition on the thermodynamics for the zwitterionic phases.

Direct Stereoselective Method Development and Validation of Elagolix on Zwitterionic Chiral Stationary Phase by High-Pressure Liquid Chromatography

A specific, sensitive and accurate chiral technique has been developed for the quantification of elagolix enantiomer in a drug product. The present research work represents the development and validation of chromatographic technique for the separation of elagolix enantiomers on a chiral stationary phase (CSP) comprising a zwitterionic selector derived from cinchona alkaloid and sulfonic acid CHIRALPAK ZWIX (+) under isocratic condition containing 50mM Acetic acid and 25mM diethyl amine in water and methanol in the ratio of 02:98 v/v with 0.5 ml min-1 flow rate. Temperature of the column was monitored at 40°C with a resolving factor more than 3.2. The eluted compounds were monitored at 275 nm. The optimized method has high degree of selectivity with lower limiting values of detection and quantitation and were found to be 2.0 ng/ml and 7.5 ng/ml, respectively for the enantiomeric chiral impurity. The developed method was subjected for validation as per the ICH guidelines, produced good findings regarding specificity, accuracy, precision, robustness and linearity.

Gradient supercritical fluid chromatography coupled to mass spectrometry with a gradient flow of make-up solvent for enantioseparation of cathinones

In the past two decades, supercritical fluid chromatography has evolved from a niche application to a comprehensive technology and a fully-fledged alternative to conventional high-performance liquid chromatography.In this study, we have focused on chiral separation of synthetic cathinones in gradient supercritical fluid chromatography coupled to mass spectrometry using an inverse gradient of a make-up solvent. Synthetic cathinones possess an amphetamine-like effect and, therefore, are frequently being offered on the Internet as a replacement for illicit drugs. Cathinones are chiral compounds, however, they are usually marketed and used as racemic mixtures. Since the effect of individual enantiomers can significantly vary, there is a need for the development of enantioseparation methods enabling to study the biological effects of individual enantiomers. Since cathinones are basic molecules, they are easily protonated (positively charged) under weakly acidic mobile phase conditions, which is a typical feature of supercritical mobile phases with an alcohol as an organic modifier. The positively charged species represent ideal analytes for ion exchangers, such as chiral zwitterion ion exchangers Chiralpak ZWIX (+) and Chiralpak ZWIX (−), which possess a positively and negatively charged unit in the molecular structure of the selectors. The presence of the positive charge in the selector's structure, functioning as a counter-ion for the positively charged analytes, significantly reduces the required amount of a buffer, which is plausible for hyphenation of such a separation system with mass spectrometry. For mass spectrometry hyphenated to supercritical fluid chromatography, the use of a make-up solvent is required to avoid analyte precipitation when using a low concentration of an organic co-solvent (modifier) in the super-/subcritical mobile phase. Hereby, we introduce a unique approach, which is based on the gradient introduction of the make-up to the post-column effluent. Using this approach, it is possible to keep constant the overall amount of the organic solvent (modifier and make-up) introduced into the mass spectrometer when using a gradient of the organic modifier. We show that the developed gradient elution method facilitates the chiral separation of all employed analytes, while the mobile-phase gradient compensation by the inverse make-up gradient enables their detection with high signal intensities.

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.

Analysis of free amino acids with unified chromatography-mass spectrometry—application to food supplements

Amino acids are most often analyzed in reversed-phase liquid chromatography after a derivatization procedure to render them sufficiently hydrophobic and detectable with UV or fluorimetric detection. Simpler methods should be possible to avoid additional chemical reactions. We present an improved method to analyze free amino acids with unified chromatography, that is to say with a wide elution gradient starting with supercritical fluid chromatography (SFC) conditions (high percentage of carbon dioxide) and ending with high-performance liquid chromatography (HPLC) conditions (100% co-solvent). The mobile phase composition was carefully adjusted to permit the elution of 21 natural amino acids (among which 19 proteinogenic) with very good peak shapes from a zwitterionic cinchona-based stationary phase (Chiralpak ZWIX(+)). Chiral separation was not desired. The mobile phase finally selected comprised carbon dioxide and a co-solvent (methanol containing 2% water and 20 mM methanesulfonic acid), ranging from 10 to 100% in 7 min followed by 3 min re-equilibration at 25 °C. A reversed pressure gradient (15 to 11 MPa) and a reversed flow rate gradient (3 to 1 mL/min) were applied to avoid reaching the upper pressure limit of the pumping system (40 MPa) and to favor high chromatographic efficiency at every stage of the elution gradient. Detection was achieved with electrospray ionization-mass spectrometry (ESI(+)-MS). The method is then fast and straightforward as no derivatization step is necessary, and all isobaric species were chromatographically resolved.To demonstrate the applicability of the method, it was applied to the quantitation of amino acids in food supplements commonly consumed by sportsmen, containing taurine (a common natural amino acid) or branched-chain amino acids (BCAA), namely valine, and the isobaric leucine and isoleucine. A standard addition method was examined for sensitivity, linearity, repeatability and intermediate precision.

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.

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.

Racemization without deamidation: Effect of racemization conditions on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids

The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-13C15N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated.It was found that a 0.4 M borate buffer with a pH of 8.0 +/− 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-13C15N]-Met as well as its oxidation products, AQC-DL-[U-13C15N]-Met-O and AQC-DL-[U-13C15N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC–ESI–QTOF–MS analysis in positive ionization mode.

Derivatize, Racemize, and Analyze-an Easy and Simple Procedure for Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics.

A simple, controllable, and reproducible stereoisomerization (racemization and epimerization) protocol for the preparation of scalemic α-amino acid mixtures from stereoisomerically pure standards was developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 °C for defined time periods until the targeted d-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC), aminophenyl- N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl- N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly 13C and 15N labeled [U -13C15N]-amino acid standard with the desired d-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 °C will lead to a d-amino acid level of 1-5%, while 6 h at 95 °C provides 15-30% d-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as dl-Ile or dl-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.

A chiral unified chromatography-mass spectrometry method to analyze free amino acids.

In this project, we aimed at analyzing native (or free) amino acids with supercritical fluid chromatography coupled to mass spectrometric detection, with modern instruments and methods, and maintaining as simple a mobile phase as possible to ensure applicability of the method. The purpose was twofold: (i) a generic method allowing for satisfactory elution of a wide range of amino acids (acidic, basic, or neutral residue) and (ii) resolution of the enantiomeric pairs. The Chiralpak ZWIX (+) and (-) stationary phases were selected as they are well-known for the enantioresolution of amino acids in liquid chromatographic modes. A wide range elution gradient, starting with a large concentration of carbon dioxide (90%) and finishing at 100% solvent (methanol containing 70mM ammonium formate and 7% water) allowed the elution of 18 native proteinogenic amino acids out of 19 injected. In these conditions, enantioselectivity was achieved for 16 of them. The basic amino acids (arginine, histidine, and lysine) were the most difficult to elute in these conditions, resulting in rather poor peak shapes. Cysteine was never observed in any of the conditions tested. Sample application was attempted with two food supplements, tablets containing a mixture of 17 proteinogenic amino acids and capsules containing taurine and theanine that were not present in the standards used for the method development. The sample preparation method was very simple, involving dissolution of the tablets and capsules in acidified water, filtration, and dilution with methanol. Mass spectrometric detection (electrospray ionization with single-quadrupole mass detection) allowed for unambiguous identification of most amino acids, except for the leucine and isoleucine isomers that were not separated by the generic gradient. The observation of taurine and theanine also suggests that the method should be generally applicable to other native amino acids than the proteinogenic amino acids selected for the development of this method. As peak shapes and signal-to-noise ratios could still be improved, further developments are wanted to upgrade this method. Due to the wide gradient (10 to 100% co-solvent in carbon dioxide), the method cannot truly be called either supercritical fluid chromatography (SFC) or enhanced-fluidity liquid chromatography (EFLC), but should be related to "unified chromatography" (UC), joining SFC and HPLC. Graphical abstract.

Stereoselective separation of underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatized amino acids using zwitterionic quinine and quinidine type stationary phases by liquid chromatography–High resolution mass spectrometry

Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC).In addition, throughout this study a u-13C15N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u-12C14N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.

Effects of N-methylation and amidination of cyclic β-amino acids on enantioselectivity and retention characteristics using Cinchona alkaloid- and sulfonic acid-based chiral zwitterionic stationary phases

In the present work the effects of N-methylation and N-amidination of ampholytic cyclic β-amino acids on their retention and enantioseparation characteristics on Cinchona alkaloid- and sulfonic acid-based zwitterionic chiral selectors, namely Chiralpak ZWIX(+)™ and ZWIX(−)™ columns are described. In a polar-ionic mobile phase, a double ion-pairing interaction mechanism takes place between the charged sites of the chiral analytes (the selectands) and the chiral selector moieties. As a support to correlate the chromatographic results with the structural details of the analytes, pKa values and van der Waals volumes of the substituted amino groups were calculated.In order to ensure better understanding of the mechanistic details of the chromatographic system the composition of the bulk solvent, the role of acid base additives, the concentration of the counter-ions, temperature and the structures of the ampholytic analytes have been investigated. Applying N-Fmoc protection, the ampholytic character of the analytes diminished, leading to a marked loss of retention. In the temperature range studied (5–40 °C) thermodynamic parameters, such as the difference in the standard enthalpy change Δ(ΔH°), entropy Δ(ΔS) and Gibbs energy Δ(ΔG°) were calculated from linear van’t Hoff plots derived from the ln α vs. 1/T curves. The values of the thermodynamic parameters depended on the structures of the chiral selectors applied and the analytes studied.

Exploring the enantiorecognition mechanism of Cinchona alkaloid-based zwitterionic chiral stationary phases and the basic trans-paroxetine enantiomers.

The enantiomers of trans-paroxetine (the selectand) were separated on four chiral stationary phases incorporating either quinine [ZWIX(+), ZWIX(+A)] or quinidine [ZWIX(-), ZWIX(-A)] and (R,R)-aminocyclohexanesulfonic acid [in ZWIX(-), and ZWIX(+A)] or (S,S)-aminocyclohexanesulfonic acid [in ZWIX(+), and ZWIX(-A)] chiral selectors. The zwitterion nature of the phases is due to the presence of either (R,R)- or (S,S)-aminocyclohexanesulfonic acid in the selector structure bearing the quinuclidine moiety. ZWIX(+) and ZWIX(-) phases are available on the market with the commercial names CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-), respectively. With the aim of rationalizing the enantiomer elution order with the above chiral stationary phases, a molecular dynamic protocol was applied and two energetic parameters were initially measured: selectand conformational energy and selectand interaction energy. In the search for other descriptors allowing a better fitting with the experimental evidences, in the present work we consider an energetic parameter, defined as the selector conformational energy, which resulted to be relevant in the explanation of the experimental elution order in most of the cases. Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analytes.

Direct Separation of Pregabalin Enantiomers Using a Zwitterionic Chiral Selector by High Performance Liquid Chromatography Coupled to Mass Spectrometry and Ultraviolet Detection.

The chromatographic resolution of pregabalin enantiomers has been often achieved by derivatization of the molecule, in order to reach enough sensitivity at low concentrations of the minor enantiomer present in the active principle. In the present article, the development and optimization of two liquid chromatographic methods are presented for the direct resolution of pregabalin enantiomers on a chiral stationary phase (CSP) containing a zwitterionic selector derived from cinchona alkaloid and sulfonic acid (CHIRALPAK ZWIX). The key parameters for the separation as well as the compatibility of chromatographic conditions with different detection modes (ultraviolet and mass spectrometry) were investigated. The resulting methods were found to be selective, of high performance and low limits of detection (2 µg/mL by UV and 1 ng/mL by MS, respectively) and quantification (6 µg/mL by UV and 5 ng/mL by MS, respectively) for the minor enantiomer which is considered as a chiral impurity.

Chiral separation of 2-hydroxyglutaric acid on cinchonan carbamate based weak chiral anion exchangers by high-performance liquid chromatography

d- and l-2-Hydroxyglutaric acid (d- and l-2-HG, respectively) are metabolites related to some diseases (2-hydroxyglutaric aciduria, cancer), which make their identification and analysis crucially important for diagnostic purposes. Chiral stationary phases (CSP) based on tert-butylcarbamoyl-quinine and −quinidine (Chiralpak QN-AX and QD-AX), and the corresponding zwitterionic derivatives (Chiralpak ZWIX(+) and Chiralpak ZWIX(−)) were employed in a weak anion-exchange mechanism to perform the enantiomer separation of d- and l-2-HG without derivatization.QD-AX CSP showed the most promising separation and therefore optimization of eluent, additives, and temperature, required for the baseline separation of solutes was carried out. Depending on experimental conditions resolution values ranged up to 2.0 with run times <20min and MS-compatible conditions. Inversion on the elution order of d- and l-2-HG was possible by using the pseudo-enantiomeric QN-AX CSP.

High-performance liquid chromatographic enantioseparation of cyclic β-aminohydroxamic acids on zwitterionic chiral stationary phases based on Cinchona alkaloids

Cyclic β-aminohydroxamic acid enantiomer pairs were stereoselectively separated by high-performance liquid chromatography on the recently developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+)™, ZWIX(−)™, ZWIX(+A) and ZWIX(−A). The results of variation of the applied chromatographic conditions, such as the bulk solvent composition, the concentrations and ratio of the acid and base additives, the presence of water as mobile phase additive and the counter-ion concentration furnished a better understanding of the retention mechanism. A thermodynamic study in the temperature range 5–50 °C revealed enthalpy-controlled enantiodiscrimination in all cases. The structure–selectivity relationships clearly indicated the importance of the strereochemistry of the functional groups. From an enantiorecognition aspect, the diexo position of the functional groups always proved more favorable than the diendo position. The elution sequence was determined in all cases and was found to reversed when ZWIX(+)™ was changed to ZWIX(−)™ or ZWIX(+A) to ZWIX(−A).

Quinine-Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio- and Stereoselectivity of Underivatized Oligopeptides.

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 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5-16, 2016. © 2015 Wiley Periodicals, Inc.

Application of Cinchona alkaloid-based zwitterionic chiral stationary phases in supercritical fluid chromatography for the enantioseparation of Nα-protected proteinogenic amino acids

Stereoselective supercritical fluid chromatographic separations of the enantiomers of a large set of Nα-Fmoc proteinogenic amino acids were carried out on the recently developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+)™ and ZWIX(−)™ with protic solvents as co-solvents. The effects of the mobile phase composition, the natures and concentrations of the acid or base additives, the co- and counter-ions and temperature on the separations were investigated. The retention time in most cases slightly increased, while the separation factor decreased with increasing temperature. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van’t Hoff plots derived from the lnα vs 1/T curves in the studied temperature range (20–60°C). The values of the thermodynamic parameters depended on the natures of the selectors and the structures of the analytes. On both ZWIX(+)™ and ZWIX(−)™ columns, enthalpically-driven separations were observed. The elution sequence was determined in all cases and was observed to be opposite on ZWIX(+)™ and ZWIX(−)™ which acted for the presented applications as chiral anion exchanger.

Simultaneous analysis of d-alanine, d-aspartic acid, and d-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues

A highly sensitive and selective chiral LC-MS/MS method for d-alanine, d-aspartic acid and d-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1–500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.