Polysaccharide-based Stationary Phases Research Articles

Chiral HPLC-MS/MS determination of hyoscyamine enantiomers in baby herbal infusions after preconcentration with sulfonic HMS mesostructured silica synthesized by co-condensation

Tropane alkaloids are considered a major food safety issue in recent years, due to their worrying appearance in many foods, such as herbal infusions, usually due to cross-contamination with weeds during harvest. (−)-Hyoscyamine is synthesized by tropane alkaloids-producing plants and its ingestion can cause all types of disorders in humans. Its enantiomerization occurs easily over time, so the inactive (+)-hyoscyamine can be found in foods in variable proportions. Therefore, the goal of the current work was to develop a chiral high performance liquid chromatography tandem mass spectrometry method for the enantiomeric determination of (−)- and (+)-hyoscyamine. Chromatographic separation was optimized, and the best chiral resolution (Rs = 1.59) was achieved in 6.5 min with a Chiralpak® AY-3 polysaccharide-based stationary phase column using ethanol with 0.05 % diethylamine as mobile phase. For the preconcentration of the sample, two types of mesostructured silicas with sulfonic groups were synthesized, by grafting and co-condensation methods, and characterized. The materials were evaluated as sorbents for strong-cation exchange solid-phase extraction of hyoscyamine in baby herbal infusions. Based on the findings gathered, silica denoted as HMS-SO3−(co) was the best sorbent due to the high number of functional groups attached to silica (2.63 mmol/g) and its textural properties. Under optimal conditions, the preconcentration factor obtained was 167, using only 50 mg of the material, and a very low racemization of the (−)-hyoscyamine to the (+) enantiomer was found (<20 %). The method was validated with recoveries of 94–99 %, good linearity (R2 > 0.99), precision (RSD < 20 %), no matrix effect for both analytes and low method quantification limit (0.089 µg/L and 0.092 µg/L for (+)-hyoscyamine and (−)-hyoscyamine, respectively) to meet its applicability in monitoring real samples. Two types of herbal teas for babies (instant and teabags) were analyzed with the developed methodology. Two of the instantaneous infusions analysed were contaminated, one of them with 0.21 µg/L of (−)-hyoscyamine and 0.09 µg/L of (+)-hyoscyamine (ratio 70(−)/30(+)) and the other with 0.075 µg/L of (−)-hyoscyamine (the (+)- enantiomer was below the limit of quantification of the method). Taking these results into account, it is important to evaluate the content of hyoscyamine enantiomers in the infusions to avoid overestimation of (−)-hyoscyamine exposure.

Resolving phenylephrine HCl and guaifenesin enantiomers on cellulose-based chiral stationary phases: Separation of four enantiomers on 50-mm column.

Chiral high performance liquid chromatographic technique usually employs polysaccharide-based stationary phases in a normal phase mode. This frequently generates large waste of organic solvents. Using shorter columns of 50 mm length as well as a mobile phase with a high water percentage are common approaches for greening this analytical technique. In this context, a new chiral chromatographic technique was developed for simultaneous enantio-separation of phenylephrine HCl and guaifenesin racemates. Four 50 mm cellulose-based columns were experimented to separate the four enantiomers in a reversed phase mode. A face centered design was then employed to optimize the mobile phase acetonitrile% and flow rate on Lux Cellulose-1 (50 × 4.6 mm, 5 μm). The simultaneous resolution of the cited drugs enantiomers was achieved using acetonitrile-water (30:70, by volume), with a flow rate of 0.5 ml min-1 . These optimized chromatographic conditions separate the enantiomers in 7 min running time, generating about 1.0 ml acetonitrile per run. The proposed method was favorably compared with other reported chiral ones in terms of waste volume generated and analysis time required.

Modelling the enantiorecognition of structurally diverse pharmaceuticals on O-substituted polysaccharide-based stationary phases

This study aims to develop models to predict the retention, separation and elution sequence of the enantiomers of structurally diverse pharmaceuticals. More specifically, Quantitative Structure Retention Relationships (QSRR) models are built that describe the relationship between molecular descriptors and retention. Eighteen structurally diverse chiral mixtures, each consisting of a pair of enantiomers, were analyzed on two polysaccharide chiral stationary phases, Chiralcel OD-RH (cellulose tris(3,5-dimethylphenylcarbamate)) and Lux amylose-2 (amylose tris(5-chloro-2-methylphenylcarbamate)), applying either a basic or an acidic mobile phase, and their retention factor and elution sequence were determined. Both achiral and, in-house defined, chiral descriptors were used as descriptive variables to build the models. Linear regression techniques, i.e. stepwise multiple linear regression (sMLR) and partial least squares (PLS) regression, were applied to model the retention or separation as a function of the descriptors. In a first step, models were built with only achiral descriptors to model the global retention of both enantiomers of a chiral molecule. Subsequently, models were built with only chiral descriptors to predict the enantioseparation and elution sequence, and finally, models were considered with both descriptor types to predict the retention, the separation and the elution sequence of the enantiomers. The global retention was predicted well by the sMLR models with only achiral descriptors. The models with only chiral descriptors were not found suitable to predict the enantioseparation and elution sequence. Finally, the models containing both chiral and achiral descriptors allowed predicting the retention well, but their ability to predict the elution sequence and separation of the enantiomers differed widely for the chromatographic systems considered.

Determination of Chiral Impurity of Naproxen in Different Pharmaceutical Formulations Using Polysaccharide-Based Stationary Phases in Reversed-Phased Mode.

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (Rs) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a Rs = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.

Reversed phase liquid chromatography for the enantioseparation of local anaesthetics in polysaccharide-based stationary phases. Application to biodegradability studies

A comprehensive study on the chiral separation of bupivacaine, mepivacaine, prilocaine and propanocaine with eight commercial polysaccharide-based chiral stationary phases (CSPs) in reversed phase conditions compatible with MS detection is performed. Methanol and acetonitrile are used as organic modifiers. Retention and resolution values obtained for each compound in the different CSPs and mobile phases are compared. The polysaccharide-based CSPs tested present different enantioselectivity towards the analytes. From the results, the experimental conditions for determining the enantiomers of bupivacaine, mepivacaine, prilocaine and propanocaine in saline aqueous samples using MS detection are used, for the first time, to perform an enantioselective biodegradability study.

Effects of the Sorbent Backbone and Side Chain on Retention Mechanisms Using Immobilized Polysaccharide-Based Stationary Phases in Normal Phase Mode

The effects of the side chain and polymer backbone on the retention mechanisms of four polysaccharide-based sorbents, namely amylose 3,5-dimethylphenylcarbamate (Chiralpak IA), cellulose 3,5-dimethylphenylcarbamate (Chiralpak IB), amylose 3,5-dichlorophenylcarbamate (Chiralpak IE), and cellulose 3,5-dichlorophenylcarbamate (Chiralpak IC), were investigated using simple achiral molecules with distinct functional groups as probes. The thermodynamic properties of solute retention behaviors were investigated using retention factor data, van’t Hoff plots, adsorption isotherms, and retention models. Frontal analysis was used to obtain the adsorption isotherms of acetone (AC) and isopropanol (IPA). The results revealed that the adsorptions of AC on the four sorbents followed Langmuir isotherms, whereas heterogeneous adsorption mechanisms more accurately fit the adsorptions of IPA. For solutes associated with hydrogen-bonding groups, the retention factors and enthalpy changes suggest that cellulose sorbents may have more binding sites available than the amylose sorbents. The van’t Hoff analysis indicated that sorbents with Cl-substituted phenyl groups enhanced the binding strength of sorbent NH groups with the solutes. For aromatic solutes, larger retention factors and stronger adsorption energies were found for the cellulose sorbents. The results suggested that side chains with Cl-substituted phenyl groups weakened the π interactions. The inferences obtained from these sorbents were used for the interpretation of the chiral recognition mechanisms of pantolactone enantiomers.

Productivity and solvent waste in supercritical fluid chromatography for preparative chiral separations: a guide for a convenient strategy

The advent of supercritical fluid chromatography (SFC) in the 90s has changed preparative liquid chromatography. SFC is an improved way for separating chiral compounds during drug discovery processes yielding upwards of one hundred grams of pure enantiomers or during clinical trials requiring higher quantities.The need to purify approximately 45 mg of racemic mixture raises concerns regarding processing parameters, including injection volumes and frequency, column size, chromatographic method, and feed composition. In this study, Chiralpak® AD-H amylose tris(3,5-dimethylphenylcarbamate) polysaccharide-based stationary phase columns of various dimensions were investigated for the purification of propranolol using EtOH (+0.3% triethylamine)/CO2 15/85 v/v as the mobile phase. Production rate (mg/h), productivity (kilograms of racemate separated per kilogram of chiral stationary phase per day; kkd), solvent usage (L/g) and environmental factor (E Factor) were calculated for four column sizes for sequence and stacked modes of injection. The parameters were optimized to determine a method yielding high productivity or reduced environmental impact.In the stacked mode of injection, which allows for rapid processing compared with the sequential mode, the shortest column presents the best productivity of 0.176 kkd. A semi-preparative column (30 mm i.d.) yielded the best production rate of 467 mg purified per hour but had the worst environmental impact with an E Factor of approximately 56,414 (due to the solvent volume used during column equilibration). At Column Dilution (ACD) and mixed stream injection mode were also compared to separate 495 mg of propranolol. With ACD injection, 915 mL of ethanol and approximately 48 min were required, whereas with mixed stream injection, 1200 mL of ethanol and 63 min were required.

Simultaneous quantification of acidic and basic flupirtine metabolites by supercritical fluid chromatography according to European Medicines Agency validation

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

Enantiomers separation by capillary electrochromatography using polysaccharide-based stationary phases.

The separation of chiral compounds is an interesting and important topic of research because these compounds are involved in some biological processes, fundamentally in human health. Among the various application fields where enantiomers are remarkable, drug analysis has to be considered. Most of the drugs contain enantiomers and very often one of the two isomers could be pharmacologically more active or even dangerous. Therefore, the separation of these compounds is very important. Among the different analytical techniques usually employed, capillary electrochromatography has demonstrated great capability in enantiomers resolution. The great potential of this electromigration technique stands mainly in its high efficiency due to the use of an electrosmotic flow (flat flow profile) and on the high selectivity because of the use of a stationary phase. Chiral separation can be obtained utilizing several chiral stationary phases including a polysaccharide derivative. The aim of this review paper is to summarize the main features of capillary electrochromatography and polysaccharide derivatives of chiral stationary phase. It also report examples of practical applications utilizing this approach.

Chiral separation of novel diazenes on a polysaccharide-based stationary phase in the reversed-phase mode.

Chiral high-performance liquid chromatography separation of two recently synthesized liquid crystalline materials C1 and C2 was studied in the reversed-phase mode. Both materials have an azo-moiety and one chiral center in their molecular structures. They were available in racemic and pure S forms. For the enantiomeric separations, a Chiralpak AY-RH stationary phase based on amylose tris(5-chloro-2-methylphenylcarbamate) coated on 5μm silica was used. The compounds were analyzed in both of their possible forms, the more thermodynamically stable E form and the labile Z form. The conditions and time scale of the UV-induced E to Z transition were briefly evaluated. Under the optimized conditions, we were able to baseline separate S and R enantiomers of both of the studied materials not only in their E forms, but also in their Z forms. In comparison to the separation in the normal-phase mode, which we have reported recently, the resolution in the reversed-phase mode is significantly better. Interestingly, peak reversal was noticed for the S and R enantiomers when the separation was carried out with E versus Z forms of both compounds.

A new approach to the chiral separation of novel diazenes.

Two new types of potential liquid-crystalline azo compounds were synthesized in the form of racemic mixtures and as the individual S enantiomers. Both materials consisting of two substituted aromatic rings in the molecular core and one chiral center at the aliphatic moiety showed mesomorphic behavior. The separation of the R and S enantiomers by chiral high-performance liquid chromatography was unsuccessful when the azo compounds were in their natural E state. However, the irradiation of the compounds in the solution by UV light led to an almost quantitative transition to their Z forms. The chromatographic behavior of the compounds in their Z forms was significantly different, and partial separation of the individual enantiomers on chiral polysaccharide-based stationary phases was obtained. Thus, the proposed procedure represents a novel approach to the enantioseparation of chiral diazenes.

Immobilized Polysaccharide-Based Stationary Phases for Enantioseparation in Normal Versus Reversed Phase HPLC

A set of 31 structurally different chiral pharmaceutical compounds was used as model analytes for investigation of the enantioselective potential of two immobilized polysaccharide-based chiral stationary phases under normal and reversed phase separation conditions. These chiral stationary phases differed in the polymeric backbone, amylose or cellulose, but possessed the same derivatization functionality. The results showed that the tris(3,5-dimethylphenylcarbamate) of amylose and cellulose have very broad, and often complementary, enantiorecognition abilities. In general, normal phase separation mode seemed to be more advantageous for separation of the majority of studied pharmaceuticals no matter if amylose- or cellulose-based columns were used. However, in certain cases the reversed phase separation system yielded better results. The combination of these two immobilized chiral stationary phases offers a powerful tool for enantioseparation of different types of pharmaceuticals in the normal and/or reversed phase mode.

Enantioresolution of Chiral Derivatives of Xanthones on Different Types of Liquid Chromatography Stationary Phases: A Comparative Study

Since the development of chiral stationary phases (CSPs), liquid chromatography has emerged as one of the most useful methods for analyses and preparation of enantiomerically pure bioactive compounds. However, the key for success of enantioseparation lies in the first place in the choice of a suitable CSP having enantioselectivity for the target enantiomers. Thus, this work describes a comparative study of resolution of seven chiral derivatives of xanthones (CDXs) on nine CSPs of different nature (four macrocyclic antibiotics, two Pirkle-type and three polysaccharide-based), under multimodal elution conditions. The polysaccharide-based CSPs have proved to be most suitable to separate this group of compounds, since all the CDXs were enantioseparated with excellent enantioselectivity and resolution. Six of the seven CDXs were separated on macrocyclic antibiotic-based CSPs, however with low enantioresolution. The Pirkle-type CSPs exhibited specificity on chiral discrimination behavior, presenting very high enantioselectivity for CDXs with aromatic group linked to the chiral moiety. This comparative study provides valuable information in the field of enantioresolution, and constitutes a useful background for subsequent studies of this important class of compounds. Keywords: Amylose tris-3, 5-dimethylphenylcarbamate, amylose tris-3, 5-dimethoxyphenylcarbamate, cellulose tris-3, 5- dimethylphenylcarbamate, chiral derivatives of xanthones, chiral stationary phases, chirality, chirobiotic R, chirobiotic T, chirobiotic TAG, chirobiotic V, enantiomerically pure, enantioresolution, enantioselectivity, liquid chromatography, Lphenylglycine, macrocyclic antibiotic, multimodal elution, Pirkle-type stationary phases, polysaccharide-based stationary phases, (S, S)-Whelk-O1.

Pharmaceutical-enantiomers resolution using immobilized polysaccharide-based chiral stationary phases in supercritical fluid chromatography

Since their introduction on the market the applicability of immobilized polysaccharide-based chiral stationary phases in high-performance liquid chromatography has been thoroughly investigated. These immobilized phases have the benefit to be applicable with a wide range of modifiers, potentially extending the application range of the polysaccharide-based stationary phases. Because an increasing number of stationary phases are being introduced in the field of chiral chromatography it is important to evaluate their enantioselectivity in different techniques in order to get an idea about their applicability. In this study, three immobilized chiral polysaccharide-based stationary phases (Chiralpak IA, IB, and IC) are evaluated in supercritical fluid chromatography (SFC) with a test set of pharmaceutical racemates. This is done in a three-fold manner: their performance is evaluated (1) using traditional modifiers, (2) using mixtures of atypical modifiers, and (3) the results were compared to those on coated stationary phases with an equivalent chiral selector. To get a visual overview of the enantioselective patterns of the different chromatographic systems (mobile and stationary phase combinations), a Principal Component Analysis is performed, which allows determining the (dis)similarity between individual systems. To assess the complementarity cumulative success rates are determined. The immobilized chiral stationary phases prove to yield high cumulative success rates.

Updating a generic screening approach in sub- or supercritical fluid chromatography for the enantioresolution of pharmaceuticals

This work focusses on the update of a generic chiral screening approach in sub- or supercritical fluid chromatography (SFC). Newly generated data with 2-propanol-containing mobile phases on 12 polysaccharide-based stationary phases was combined with previous data obtained using methanol-containing mobile phases. As modifiers 2-propanol and methanol were selected for their earlier performance. An evaluation of the most appropriate solvent strength is made and the enantioselective behaviour of the chromatographic systems is discussed. A comparison of the systems is made and their complementarity investigated by analyzing the data by different means, e.g. principal component analysis. The generic screening sequence is proposed by selecting the most enantioselective and complementary systems. This allows updating an existing screening strategy. With the novel screening, all compounds of a 57-compounds test set were separated (48 baseline), on at least one of four selected systems, within an analysis time of 30min. The applicability and performance of the updated screening was demonstrated with a compound from the test set, i.e. alprenolol.

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 (Scrit) allowed assessing the quality of the enantioseparation. Baseline separation for the compounds of interest in an analysis time of less than 20min 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.

Resolution and determination of enantiomeric purity of new chiral derivatives of xanthones using polysaccharide-based stationary phases

The enantioresolution and determination of the enantiomeric purity of 24 new chiral derivatives of xanthones (CDXs) were investigated on polysaccharide-based chiral stationary phases (CSPs). The tris-3,5-dimethylphenylcarbamates of cellulose and amylose and tris-3,5-dimethoxyphenylcarbamate of amylose were selected as CSPs for this work. The separation of enantiomeric mixtures of CDXs was explored under multimodal elution conditions. All the enantiomeric mixtures of CDXs were enantioseparated with very high enantioselectivity and resolution with α and RS ranging from 1.43 to 12.41 and from 1.48 to 10.29, respectively. The best performances were achieved on amylose tris-3,5-dimethylphenylcarbamate stationary phase under polar organic elution conditions. Furthermore the enantiomeric purity for all the CDXs was measured, achieving values higher than 99%. Based on the obtained results, the influence of the mobile phases and structural features of the CSPs and CDXs on chiral discrimination are also discussed.

Synthesis and chromatographic enantioresolution of anti-HIV quinolone derivatives

The successful enantioseparation of five 6-desfluoroquinolones with three polysaccharide-based stationary phases (namely, the cellulose-based Chiralpak IB and the two amylose-based Chiralpak AD-H and Lux Amylose-2) is herein described. The investigated species differ for the nature of substituents and/or the position of the stereogenic centre on the quinolone scaffold.The effect on the enantioseparation performance exerted by the different morphology of the cellulose-based and amylose-based polymers, was systematically evaluated for all compounds. In this frame, the impact of alternative alcoholic (ethanol, 2-ethoxyethanol, methanol, 2-propanol) and acidic (acetic, methanesulfonic and trifluoroacetic acid) modifiers as well as of a “non-standard” solvent (chloroform), was investigated in normal phase conditions along with the stereo-electronic peculiarities of the selected polymers. While 7-[4-(1,3-benzothiazol-2-yl)-2-methyl-1-piperazinyl]-1-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (1) was enantioresolved with conventional normal-phase conditions by means of the largely employed amylose-based Chiralpak AD-H column, the recruitment of a bulky alcohol (2-ethoxyethanol) succeeded in the enantioresolution of 6-amino-1-methyl-7-[2-methyl-4-(2-pyridinyl)-1-piperazinyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (2) and 6-amino-1-[1-(hydroxymethyl)propyl]-4-oxo-7-(4-pyridin-2-ylpiperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (3) with the same column. The use of the amylose-based Lux Amylose-2 column, carrying both an electro-withdrawing (chlorine) and an electro-donating (methyl) group on the carbamate residue, allowed to get 6-amino-1-methyl-4-oxo-7-[3-(2-pyridinyl)-1-pyrrolidinyl]-1,4-dihydro-3-quinolinecarboxylic acid hydrochloride (4) enanantioresolved, and 6-amino-1-methyl-4-oxo-7-(3-pyridin-2-ylpiperidin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (5) enantioseparated.

Comparative enantioseparations of pharmaceuticals in capillary electrochromatography on polysaccharide‐based chiral stationary phases containing selectors with or without chlorinated derivatives

The screening conditions of an existing chiral strategy in CEC were tested for their applicability on four chlorine-containing polysaccharide-based stationary phases. The selectors of these phases are cellulose tris(3-chloro-4-methylphenylcarbamate), amylose tris(5-chloro-2-methylphenylcarbamate), cellulose tris(4-chloro-3-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate). The enantioselectivity of these phases was compared with those of the four phases without chlorine (Chiralpak® AD-RH, Chiralcel® OD-RH, Chiralpak® AS-RH and Chiralcel® OJ-RH) used in the earlier defined strategy. A test set of 48 structurally diverse drug compounds was analyzed using the screening conditions of the strategy. These results led to possibilities to upgrade the current screening strategy so that improved success rates are obtained. The chlorine-containing chiral stationary phases demonstrated an added value to the screening process since they showed enantioresolution for compounds not resolved by the chiral stationary phases not containing chlorine in their structure.

Supercritical fluid chromatography comparison of the poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1′-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.