Katina Sourou Sylvestre Dossou

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.

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%.

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.

Implementation of a design space approach for enantiomeric separations in polar organic solvent chromatography

This paper focuses on implementing a design space approach and on the critical process parameters (CPPs) to consider when applying the Quality by Design (QbD) concepts outlined in ICH Q8(R2), Q9 and Q10 to analytical method development and optimization for three chiral compounds developed as modulators of small conductance calcium-activated potassium (SK) channels. In this sense, an HPLC method using a polysaccharide-based stationary phase containing a cellulose tris (4-chloro-3-methylphenylcarbamate) chiral selector in polar organic solvent chromatography mode was considered. The effects of trifluoroacetic acid (TFA) and n-hexane concentration in an acetonitrile (MeCN) mobile phase were investigated under a wide range of column temperatures. Good correlations were found between the observed data obtained after using a central composite design and the expected chromatographic behaviours predicted by applying the design of experiments-design space (DoE-DS) methodology. The critical quality attribute represented here by the separation criterion (S(crit)) allowed assessing the quality of the enantioseparation. Baseline separation for the compounds of interest in an analysis time of less than 20 min was possible due to the original and powerful tools applied which facilitated an enhanced method comprehension. Finally, the advantage of the DoE-DS approach resides in granting the possibility to concurrently assess robustness and identify the optimal conditions which are compound dependent.

Optimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases.

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25°C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1-0.3%) and the concentration of DEA (0.01-0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed.

Development and validation of a LC method for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation using a recently commercialized cellulose-based chiral stationary phase and polar non-aqueous mobile phase.

Ropivacaine is the first enantiomerically pure long-acting local anaesthetic used for surgical anaesthesia and post-operative pain relief. A liquid chromatographic (LC) method using acetonitrile as the main solvent and cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica as chiral stationary phase was successfully developed and applied for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation (Naropin(®)). The key role played by the acidic additive (trifluoroacetic acid or formic acid) in the enantioseparation of basic drugs in these LC systems was demonstrated by the reversal of ropivacaine enantiomers elution order observed when both acids were compared. In order to elute the enantiomeric impurity (R-ropivacaine) before S-ropivacaine, formic acid (FA) was selected. The temperature and the percentages of acidic additive and hexane in the mobile phase were found to significantly influence the retention and resolution of these enantiomers. The optimized mobile phase consisted of ACN/0.1% DEA/0.2% FA/5% hexane (v/v/v/v). The temperature was set at 35°C to avoid the interference from a peak system related to the presence of water in the sample on ropivacaine enantiomers. The LC method was then fully validated applying the strategy based on total measurement error and accuracy profiles. The accuracy profile obtained by linear regression after square root transformation was selected, the acceptance limits being settled at ±10% for the intended use of this analytical method. The relative bias was lower than 1.5%, while the RSD values for repeatability and intermediate precision were both below 1.0%. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be about 0.2 and 1.0 μg/mL, respectively, corresponding to 0.02 and 0.1% of the enantiomeric impurity in S-ropivacaine.

Evaluation of chlorine containing cellulose-based chiral stationary phases for the LC enantioseparation of basic pharmaceuticals using polar non-aqueous mobile phases.

The discrimination ability of three cellulose-based chiral stationary phases (CSPs) was evaluated towards the enantiomers of basic drugs, using ACN as the main solvent in polar organic mobile phases. The study was focused on CSPs containing cellulose tris(3-chloro-4-methylphenylcarbamate) (3-Cl-4-MePC), cellulose tris(4-chloro-3-methylphenylcarbamate) (4-Cl-3-MePC) or cellulose tris(3,5-dichlorophenylcarbamate) (3,5-diClPC) as the chiral selector. The behaviour of these CSPs was studied systematically in order to investigate the influence of the presence and position of the chlorine substituents on the phenylcarbamate moieties on the retention and resolution of the enantiomers. The evaluation was made with three different generic mobile phases, namely ACN/0.1%DEA/0.1% TFA (DEA, diethylamine), ACN/0.1%DEA/0.2% FA and ACN/0.1%DEA/0.2%AcA, deduced from the previous study. The nature of the acidic additive and of the chiral selector was found to be particularly important for the retention and enantioresolution of these basic compounds. High-resolution values could be obtained for most studied enantiomers with these CSPs, clearly demonstrating the interest of using them in combination with polar organic mobile phases. However, significant differences in enantioresolution between the CSPs have been observed for many compounds, indicating that these phases seem to be quite complementary.