Anne-Catherine Servais

Separation of propranolol enantiomers by CE using sulfated beta-CD derivatives in aqueous and non-aqueous electrolytes: Comparative CE and NMR study.

Separations using CE employing non‐aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with non‐aqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non‐aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non‐aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non‐aqueous methanolic BGEs with two single isomer sulfated derivatives of β‐CD, namely heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD and heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non‐aqueous BGE in the case of heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD but remained the same in the case of heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non‐aqueous BGEs.

Enantioresolution of basic pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral stationary phase and polar organic mobile phases

A polysaccharide-based chiral stationary phase (Sepapak-4), with cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector, has been investigated in liquid chromatography (LC). Its enantioresolution power was evaluated towards 13 basic amino-drugs with widely different structures and polarities, using polar organic mobile phases. After preliminary experiments, acetonitrile was selected as the main mobile phase component, to which a low concentration of diethylamine (0.1%) was systematically added in order to obtain efficient and symmetrical peaks. Different organic solvents were first added in small proportions (5-10%) to acetonitrile to modulate analyte retention. Polar organic modifiers were found to decrease retention and enantioresolution while hexane had the opposite effect, indicating normal-phase behaviour under these conditions. The addition of an organic acid (formic, acetic or trifluoroacetic acid) was found to strongly influence the retention of the basic amino drugs in these nonaqueous systems. The nature and proportion of the acidic additive in the mobile phase had also deep impact on enantioresolution. Therefore, the studied compounds could be subdivided in three groups in respect to the acidic additive used. All analytes could be enantioseparated in relatively short analysis times (10-20 min) using these LC conditions.

Capillary electrophoretic and nuclear magnetic resonance studies on the opposite affinity pattern of propranolol enantiomers towards various cyclodextrins

In the present study the migration order of the propranolol enantiomers with various native CDs and neutral and charged CD derivatives was examined in capillary electrophoresis (CE). The reversal of the enantiomer migration order was observed due to sulfation of beta-CD on its primary hydroxy groups. The structures of intermolecular selector-select and temporary diastereomeric associates in solution were elucidated based on 1D rotating frame nuclear Overhauser effect spectroscopy (1D ROESY) experiments. Major structural differences were observed between the propranolol complexes with native beta-CD and heptakis(6-O-sulfo)-beta-CD.

Influence of the BGE composition on analyte response in CD‐mediated NACE‐MS

The influence of the BGE composition, including the addition of a single‐isomer sulfated β‐CD derivative, on the ionization performance of the model compound carvedilol in NACE‐ESI‐MS was studied using an alternative infusion method. This approach employs voltage‐induced infusion of the BGE containing the analyte, and takes into account the effects of variations in EOF and effective analyte mobility on the ESI‐MS intensity. First, the optimal composition of the sheath liquid for CE‐MS in terms of signal abundance and stability was determined. The BGE ammonium formate, acetate, and camphorsulfonate were found to have similar effects on analyte ionization. Addition of single‐isomer sulfated β‐CD derivatives (available as sodium salt) to the BGE revealed that the anionic CD derivatives did not give rise to the same ionization suppression effect. This result can be attributed to differences in the dissociation state of these sodium salts. Finally, it is shown that information about chiral selectivity can also be obtained with the applied infusion method.

Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography–electrospray ionization tandem mass spectrometry

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.

Determination of enantiomeric purity of S‐amlodipine by chiral LC with emphasis on reversal of enantiomer elution order

An LC method was developed and prevalidated for the enantiomeric purity determination of S-amlodipine in polar organic solvent chromatography using a chlorine-containing cellulose-based chiral stationary phase (CSP). The concentration of formic acid (FA) (0.01-0.2%) in the mobile phase containing acetonitrile as the main solvent was found to influence the elution order of amlodipine enantiomers as well as the enantioresolution. A reversal of the enantiomer elution order of amlodipine was only observed with chiral stationary phases with both electron-withdrawing (chloro) and electron-donating groups (methyl) on the phenyl moieties of the chiral selector, namely cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate). The highest enantioresolution (Rs : 4.1) value was obtained at the lowest FA concentration (0.01%) using cellulose tris(4-chloro-3-methylphenylcarbamate) as the chiral selector and the enantiomeric impurity, R-amlodipine, eluted first under these conditions. Therefore, the mobile phase selected for the prevalidation of the method consisted of ACN/0.1% DEA/0.01% FA and the temperature was set at 25°C. The method was prevalidated by means of the strategy based on the total measurement error and the accuracy profile. The method was found to be selective and the limit of quantification was found to be about 0.05% for R-amlodipine, while the limit of detection was close to 0.02%.

Combination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged β-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-β-CD or HDAS-β-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained.

Comparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selector–selectand interactions by nuclear magnetic resonance spectroscopy

The enantiomers of the chiral β-blocker drug talinolol were separated with two single component sulfated β-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-β-CD) (HDMS-β-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-β-CD) (HDAS-β-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol toward these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-β-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-β-CD was of the external type. The complex of the talinolol enantiomers with HDAS-β-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE.

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar non-aqueous mobile phases.

The resolving power of a new commercial polysaccharide-based chiral stationary phase, Sepapak-4, with cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n-hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face-centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n-hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.

Optimization of the separation of β‐blockers by ion‐pair capillary electrophoresis in non‐aqueous media using univariate and multivariate approaches

The separation of four β-Blocking drug substances (atenolol, sotalol, betaxolol, and metoprolol) selected as model basic analytes has been investigated in non-aqueous capillary electrophoresis (NACE) using the principle of ion-pair formation. Camphorsulphonate was selected as the counter-ion in a background electrolyte (BGE) made up of formate buffer in a mixture of acetonitrile/methanol or acetonitrile/ethanol. The influence on resolution of the concentration of the counter-ion, the nature and proportion of the organic solvents, as well as the concentration of the ionic components of the BGE was first studied by a univariate approach. An experimental design was then applied to estimate possible quadratic effects and first-order interactions. To identify the most important factors affecting the separation of the four β-blockers, a two-level fractional factorial design with 16 experimental points was applied as a screening test. A three factor Box-Behnken design with 12 experimental points was used to predict and optimize the selectivity. Finally the optimal conditions obtained by the univariate and the multivariate approaches were compared.