Whelk-O1 Chiral Stationary Phase Research Articles

Reverse-phase chiral high-performance liquid chromatography for separation of a diastereomer in alalevonadifloxacin: A novel antibacterial agent.

Alalevonadifloxacin (ALA) is a novel antibacterial drug, recently launched in India to treat infections caused by Gram-positive bacteria. In present work, a chiral high-performance liquid chromatographic method was developed and validated for the quantification of a diastereomeric impurity (DI) in ALA. The separation was achieved on Pirkle type (R,R) Whelk-O1 chiral stationary phase, using ammonium formate buffer and acetonitrile in gradient fashion at a flow rate of 1.5 ml/min. The method was extensively validated for the quantification of DI in ALA. The detector response for DI was linear over the concentration range of 0.24-4.78 μg/ml. Limit of quantitation and limit of detection for DI were 0.24 and 0.07 μg/ml respectively. The mean recovery of the DI was 103.47 ± 5.14%. The impact of column temperature on the chiral separation was evaluated. The method was employed for controlling diastereomeric impurity in the batches of ALA used in preclinical studies.

Mass transfer kinetics on modern Whelk-O1 chiral stationary phases made on fully- and superficially-porous particles

In this work, a detailed study of mass transfer properties of trans-stilbene oxide (TSO) enantiomers on two Whelk-O1 chiral stationary phases (CSPs) has been performed. The CSPs were prepared by using both fully-porous silica particles of 2.5 μm particle diameter and superficially-porous ones of 2.6 μm particle diameter as base materials. By combining stop-flow and dynamic measurements in normal-phase conditions, the different contributions to mass transfer have been estimated. The study of intraparticle diffusion has revealed that the adsorption of both enantiomers is localized (i.e., characterized by absence of surface diffusion). The determination of thermodynamic binding constants (measured through adsorption isotherms) supports this finding.

Improved Achiral and Chiral HPLC-UV Analysis of Ruxolitinib in Two Different Drug Formulations

In this paper, two new reversed-phase (RP) HPLC-UV methods enabling the quantitative achiral and chiral analysis of ruxolitinib in commercial tablets (containing 20 mg of active pharmaceutical ingredient, API) and not commercially available galenic capsules (with 5 mg of API) are described. For the achiral method based on the use of a water/acetonitrile [70:30, v/v; with 0.1% (v) formic acid] eluent, a “research validation” study was performed mostly following the “International Council for Harmonization” guidelines. All the system suitability parameters were within the acceptance criteria: tailing factor, between 1.7 and 2.0; retention factor, 2.2; number of theoretical plates, >9000. The linearity curve showed R2 = 0.99 (Rxv2 = 0.99), while trueness (expressed as recovery) was between 96.3 and 106.3%. Coefficient of variations (CVs) (repeatability: CVw and intermediate precision: CVIP) did not exceed 1.3% and 2.9%, respectively. Moreover, the use of the enantiomeric Whelk-O1 chiral stationary phases operated under similar RP eluent conditions as for the achiral analyses, and the “inverted chirality columns approach (ICCA)” allowed us to establish that the enantiomeric purity of ruxolitinib is retained upon reformulation from tablets to capsules. The two developed methods can allow accurate determinations of ruxolitinib in drug formulations for medical use.

Integrating experimental and computational techniques to study chromatographic enantioresolutions of chiral tetrahydroindazole derivatives

With the selection of partially saturated 2H-indazoles as model compounds, we demonstrate the possibility to use Whelk-O1 chiral stationary phases (CSPs) to succeed in efficient small-scale preparative enantioseparations. Runs of three consecutive liquid chromatography injections (about 300 μg of racemate repeatedly injected in a 100 μL loop) produced groups of peaks without band contamination (α = 1.2 and RS = 2.57). With this procedure approximately 3.0 mg of each enantiomer, with enantiomeric excess ≥ 97% were obtained. Very profitably, the high volatility of n-hexane used as the sole eluent facilitated the solvent evaporation after the enantiomer recovery. High resolution mass spectrometry analysis confirmed that the chemical identity of the two enantiomers was preserved along the entire process.The ability of Whelk-O1 phases in enantioseparating structurally similar compounds was confirmed with the analysis of other two racemates. Moreover, the relevant chemoselectivity exhibited by the CSP towards the three racemates should allow to simultaneously optimizing the enantioselectivity of different analytes and perform small-scale enantioresolutions of different compounds during the same run. In this study, the integration of experimental off-line electronic circular dichroism analysis with ab initio time-dependent density-functional theory simulations facilitated the assignment of the absolute configuration of the single enantiomers, while a molecular dynamics protocol can be useful to make a priori predictions of the enantioseparation ability of CSP towards selected compounds.

Application of the “inverted chirality columns approach” for the monitoring of asymmetric synthesis protocols

In the present study, the “Inverted Chirality Columns Approach (ICCA)” was applied to follow an asymmetric synthetic reaction, namely, the addition of butan-2-one to trans-β-nitrostyrene, catalysed by (S)-proline, leading to the formation of 3-methyl-4-phenyl-5-nitro-2-pentan-2-one. The ICCA method was applied to overcome the lack of pure enantiomeric standards.The two widely employed (R,R)- and (S,S)-Whelk-O1 chiral stationary phases (CSPs), incorporating fully synthesized enantiomeric chiral selectors, were profitably used for this purpose. The enantioselective analysis with the two CSPs was performed under optimized reversed-phase conditions with a water/acetonitrile (60/40, v/v) eluent.In the probe reaction under investigation, a diastereomeric excess >90% was found according to a well-established reaction mechanism, thus affording the enantiomer couple (3S,4R)-3-methyl-4-phenyl-5-nitropentan-2-one and (3R,4S)-3-methyl-4-phenyl-5-nitropentan-2-one as the main product. Therefore, the attention was exclusively focused on this enantiomers pair.Rather similar retention and separation factor [1.12 with (R,R)-Whelk-O1 and 1.13 with (S,S)-Whelk-O1] values as well as resolutions [2.06 with (R,R)-Whelk-O1 and 2.30 with (S,S)-Whelk-O1] were produced by the two enantiomeric CSPs.Applying the ICCA concept allowed to identify the two enantiomers-related peaks in the chromatograms, ultimately indicating a 65-to-35 enantiomeric per cent ratio. Electronic circular dichroism (ECD) and high-resolution mass spectrometry analyses of the two peaks collected during the enantioselective analyses further confirmed the enantiomeric nature of the identified compounds. The (3S,4R) < (3R,4S) enantiomer elution order with the (R,R)-Whelk-O1 was fully disclosed thanks to ECD studies coupled with in silico quantum mechanical simulations. As expected, reversed elution order turned out with (S,S)-Whelk-O1.

On the effect of chiral selector loading and mobile phase composition on adsorption properties of latest generation fully- and superficially-porous Whelk-O1 particles for high-efficient ultrafast enantioseparations

The adsorption isotherms of trans-stilbene oxide (TSO) enantiomers have been measured under a variety of normal phase (NP) mobile phases (MPs) on three Whelk-O1 chiral stationary phases (CSPs), prepared respectively on 1.8 μm and 2.5 μm fully porous particles (FPPs) and 2.6 μm superficially porous particles (SPPs). Specific loading of chiral selector (moles per square meter) of the two FPPs was about 20% smaller than that of SPPs (even if they were prepared under exactly the same experimental conditions).Regardless of particle size or format, adsorption was described by means of a Bilangmuir model with ethanol/hexane MPs. On the other hand, in pure hexane, the Tóth isotherm was employed. Interestingly, it was found that selective and nonselective Henry's constants vary in opposite directions by increasing the percentage of strong MP modifier (between 3 and 10%, v/v). Saturation capacity of SPPs (referred only to the porous zone of the particle) was remarkably smaller than those of FPPs. On the other hand, binding constants on both selective and nonselective sites were significantly larger on SPPs. Finally, a correlation between the specific loading of chiral selector and the binding constants of enantiomers was suggested by data, which can be important also to understand the kinetic behavior of these particles in chiral ultrafast applications.

Unmatched Kinetic Performance in Enantioselective Supercritical Fluid Chromatography by Combining Latest Generation Whelk-O1 Chiral Stationary Phases with a Low-Dispersion in-House Modified Equipment.

This proof-of-concept work investigates the ultimate kinetic limits reachable in chiral supercritical fluid chromatography (SFC) with modern columns and advanced technological solutions. A commercial equipment (Waters Acquity UPC2) has been in-house modified to minimize its overall extra-column variance through a series of technical adjustments including low-volume connecting tubings, reduced-volume flow cell, an in-house made external column oven, external low-dispersion injection system, and electronic temperature controller. Compared to the original (as-shipped) configuration, the extra-column variance on the low-dispersion equipment was reduced by more than 97%, from about 85 to slightly more than 2 μL2 (measured at 2.0 mL/min). This was mainly achieved thanks to the occurrence of fully developed turbulent regime with a proper selection of capillary i.d. at significantly smaller flow rates (1.5-4 mL/min; CO2/methanol 80/20, v/v; 35 °C; back pressure regulator (BPR), 105 bar) than in entry-1 configuration. Ultrahigh efficiency columns of different geometries in-house packed with latest generation sub-2 μm UHPC-FPP-Whelk-O1 Chiral Stationary Phase (CSP) have been employed under sub- and supercritical fluid conditions. By carefully modulating the length and the internal diameter of connecting tubings in the function of column geometry, state of the art efficiencies (estimated in roughly 300 000 theoretical plates/m with reduced HETP of roughly 1.85) have been obtained on 4.6 mm i.d. chiral columns. Remarkably, for 3.0 mm × 100 mm (i.d. × length) columns, the efficiency gain on the fully modified SFC system (compared to an instrumental configuration where only the standard injector was replaced by the low-dispersion one) was greater than 90% for compounds with a retention factor of 1 and as large as 25% for retention factors of 2.5.

Resolution, determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)-whelk-O1 stationary phase.

The enantioresolution and determination of the enantiomeric purity of 32 new xanthone derivatives, synthesized in enantiomerically pure form, were investigated on (S,S)-Whelk-O1 chiral stationary phase (CSP). Enantioselectivity and resolution (α and RS ) with values ranging from 1.41-6.25 and from 1.29-17.20, respectively, were achieved. The elution was in polar organic mode with acetonitrile/methanol (50:50v/v) as mobile phase and, generally, the (R)-enantiomer was the first to elute. The enantiomeric excess (ee) for all synthesized xanthone derivatives was higher than 99%. All the enantiomeric pairs were enantioseparated, even those without an aromatic moiety linked to the stereogenic center. Computational studies for molecular docking were carried out to perform a qualitative analysis of the enantioresolution and to explore the chiral recognition mechanisms. The in silico results were consistent with the chromatographic parameters and elution orders. The interactions between the CSP and the xanthone derivatives involved in the chromatographic enantioseparation were elucidated.

Pirkle-type chiral stationary phase on core–shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations?

Pirkle-type Whelk-O1 chiral stationary phase (CSP) was prepared on 2.6μm superficially porous particles (SPPs). The chromatographic behavior of columns packed with this new CSP was compared with that of columns packed respectively with 1.8 and 2.5μm Whelk-O1 fully porous particles (FPPs). In the comparison, both thermodynamic and kinetic aspects were considered. Contrary to initial expectations, chiral columns packed with 2.6μm SPPs were quasi-comparable to those packed with 2.5μm FPPs, apparently due to larger contributions to band broadening from both eddy dispersion and, especially for the second eluted enantiomer, adsorption–desorption kinetics. These findings raise the question if SPPs, in spite of the undeniable advantages of their morphology to speed up mass transfer, are always the best choice for high-efficient ultrafast chiral separations. The last part of the work focuses on the use of short columns (10mm long) and very high flow rates to realize the separation of the enantiomers of trans-stilbene oxide (TSO) in normal phase mode in less than 1s.

Dynamic Behavior of Clobazam on High-Performance Liquid Chromatography Chiral Stationary Phases.

Clobazam, a 1,5-benzodiazepin-2,4-dione, is a chiral molecule because its ground state conformation features a nonplanar seven-membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring-flipping process. Variable temperature HPLC on the Pirkle type (R)-N-(3,5-dinitronenzoyl)phenylglycine and (R,R)-Whelk-O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence-decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high-performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on-column enantiomer interconversion. (enantiomerization) in the column temperature range between Tcol = 10°C and Tcol = 30°C, whereas on-column interconversion was absent at temperature close to or lower than Tcol = 5°C. Computer simulation of exchange-deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At Tcol = 20°C the averaged enantiomerization barriers, ΔG(‡), for clobazam were found in the range 21.08-21.53 kcal mol(-1) on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents.

Toward enantioselective nano ultrahigh-performance liquid chromatography with Whelk-O1 chiral stationary phase.

In this study, a Whelk-O1 chiral stationary phase immobilized on 2.5μm silica particles was employed in nanoLC. Two nanocolumns (180 and 250mm long, 75μm id) with a single polymeric organic monolithic outlet frit were packed under high-pressure ultrasonic-assisted packing procedure. The monolithic outlet frit was prepared by thermal polymerization of methacrylate-based monomers affording high-mechanical stability and high-pressure resistance. Very efficient enantioseparations with more than 70000 plates/m were achieved in normal phase mode by eluting (+/-) acenaphthenol. Nanocolumns were also tested in RP mode by using on-line MS detection with nano-spray ESI ion source. Kinetic performances of columns in RP mode were comparable to those in normal phase-conditions.

Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between −10°C and −35°C, whereas for flunitrazepam on-column interconversion was observed at temperatures between −40°C and −66°C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At −20°C the enantiomerization barriers, ΔG≠, for diazepam, prazepam and tetrazepam were determined to be in the range 17.6–18.7kcal/mol. At −55°C ΔG≠ for flunitrazepam was determined to be in the 15.6–15.7kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.

Enantioresolution of Chiral Derivatives of Xanthones on (S,S)‐Whelk‐O1 and l‐Phenylglycine Stationary Phases and Chiral Recognition Mechanism by Docking Approach for (S,S)‐Whelk‐O1

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)-Whelk-O1 and L-phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal-phase, polar-organic, and reversed-phase). The (S,S)-Whelk-O1 CSP, under polar-organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)-Whelk-O1 CSP by molecular docking technique. Data regarding the CSP-CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP-CDX interactions.

Drug synthesis methods and manufacturing technology preparative chromatographic separation of ibuprofen enantiomers on Whelk-O1 chiral stationary phase

The influence of experimental conditions (temperature, sample volume, mobile-phase composition) on the preparative separation of ibuprofen enantiomers on chiral stationary phase (S,S)-Whelk-O1 with hexane:EtOH eluents modified with acetic acid was studied. It was established that the effects of these factors on the elution profiles were complex with the patterns disturbed by overlap with the systemic peak. The importance of the last factor decreased with increasing alcohol concentration in the mobile phase. The possibility of optimizing the preparative separation with respect to both purity and yield of the target enantiomers was discussed.

Introducing Enantioselective Ultrahigh-Pressure Liquid Chromatography (eUHPLC): Theoretical Inspections and Ultrafast Separations on a New Sub-2-μm Whelk-O1 Stationary Phase

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.

Assessment of chiral stationary phases for suitability for combined enantiomeric impurity/related substances assays

Chiral stationary phases (CSP) for LC had a major impact on pharmaceutical R&D when they first became commercially available in the 1980s. Even although the use of CSP in pharmaceutical R&D is now very much a mature area, there is still scope for using CSP more effectively to bring about efficiencies. One such instance is the possibility of combining the chiral LC test for the level of a trace enantiomeric impurity in a chiral drug substance and the LC test for related substances into one test. It was envisaged that this could be achieved by carrying out reversed-phase LC on an ODS silica/CSP coupled column system. In evaluating Chiralpak QD-AX, Cyclobond I 2000 DNP and Whelk-O1 CSP using a polar organic - aqueous mobile phase it was found that the Whelk-O1 CSP had good achiral selectivity, the required match of retentivity with the ODS silica material, ACE 5 C18 and also exhibited an encouraging degree of enantioselectivity in the reversed-phase mode. Following consideration of the selectivity of the ACE 5 C18 and Whelk-O1 phases it became apparent that it might be possible to achieve the desired goal of achieving both the enantiomeric impurity and related substances separations in one system by using the Whelk-O1 CSP on its own. This was subsequently demonstrated to be the case using S-naproxen, laevokalim and S-flurbiprofen as illustrative examples.

The adsorption of Naproxen enantiomers on the chiral stationary phase ( [formula omitted])-Whelk-O1 under reversed-phase conditions: The effect of mobile phase composition

The adsorption of the Naproxen enantiomers on the Whelk-O1 chiral stationary phase from acidic (0.01 M CH 3COOH) methanol–water solutions was studied using the frontal analysis method and band profile calculations. The adsorption model that best accounted for these data is one assuming the coexistence of three types of adsorption sites, two of them being enantioselective, associated with the chiral selectors, the third type being the nonselective type of sites associated with residual surface silanol groups. The adsorption equilibrium is influenced by secondary processes that result in variations of the model parameters with the mobile phase composition. Several qualitative correlations between the model parameters and the methanol–water ratio were observed and are discussed.

Retention of Naproxen enantiomers on the chiral stationary phase Whelk-O1 under reversed-phase conditions. A reconsideration of the adsorption mechanism in the light of new experimental data

A model of adsorption of the Naproxen enantiomers on the ( S, S)-Whelk-O1 chiral stationary phase that we recently proposed was found to be inconsistent with new experimental data. A new model taking into account the variation of the dissociation coefficient of the analyte during elution of the band is discussed.

Extending the use of “Inverted Chirality Columns Approach” for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative

The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs.

Rational optimization of the Whelk-O1 chiral stationary phase using molecular dynamics simulations

Rational in silico optimization of the Whelk-O1 chiral stationary phase (CSP) has been carried out based on the chiral recognition mechanism extracted from previous molecular dynamics simulations [C.F. Zhao, N.M. Cann, Anal. Chem. 80 (2008) 2426] of this CSP. Three modified CSPs have been examined. The first two are designed to increase selectivity by reducing the docking probability of the less retained analyte. The third modified selector reverses the amide bridge to introduce a structural motif found in the popular carbamate-derivatized polysaccharide CSPs [Y. Okamoto, M. Kawashima, K. Hatada, J. Am. Chem. Soc. 106 (1984) 5357]. For each modified selector, an atomistic model has been obtained through extensive ab initio calculations. The effect of selector modification is then evaluated via simulations of the modified interface in the presence of target analytes. Simulation results show that the separation factors are increased for the modified CSPs but in some cases elution orders are reversed. The Whelk-O1 CSP was originally designed to separate naproxen [W.H. Pirkle, C.J. Welch, B. LAmm, J. Org. Chem. 57 (1992) 3854]. With this in mind, molecular dynamics simulations of naproxen are compared for the original, and the modified, selectors.