Polysaccharide-type Chiral Stationary Phases Research Articles

New Levan-Based Chiral Stationary Phases: Synthesis and Comparative HPLC Enantioseparation of (±)-trans-β-Lactam Ureas in the Polar Organic Mode.

In this paper, the preparation of three new polysaccharide-type chiral stationary phases (CSPs) based on levan carbamates (3,5-dimethylphenyl, 4-methylphenyl, and 1-naphthyl) is described. The enantioseparation of (±)-trans-β-lactam ureas 1a-h was investigated by high-performance liquid chromatography (HPLC) on six different chiral columns (Chiralpak AD-3, Chiralcel OD-3, Chirallica PST-7, Chirallica PST-8, Chirallica PST-9, and Chirallica PST-10) in the polar organic mode, using pure methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN). Apart from the Chirallica PST-9 column (based on levan tris(1-naphthylcarbamate), the columns exhibited a satisfactory chiral recognition ability for the tested trans-β-lactam ureas 1a-h.

Comparative studies of immobilized polysaccharide derivatives chiral stationary phases for enantioseparation of furanocoumarins and dihydroflavones and discussion on chiral recognition mechanism.

Enantiomeric separation of furanocoumarins and dihydroflavones compounds were systematically studied in the normal-phase mode using four different polysaccharide-type chiral stationary phases, namely, Chiralpak IA, Chiralpak IC, Chiralpak IG, and Chiralpak IK-3 by high-performance liquid chromatography. The effect of alcohol modifiers and alcohol content on enantiomeric separation was evaluated for the separation of furanocoumarins and dihydroflavones. All the eight compounds have achieved baseline separation with the resolutions ranging between 1.52 and 23.11. For a better insight into the enantiorecognition mechanisms, thermodynamic analysis was carried out. The mechanisms of chiral recognition have been discussed. Among four chiral columns, Chiralpak IG exhibited the most universal and the best enantioseparation ability toward furanocoumarins and dihydroflavones when used n-hexane-isopropanol and n-hexane-ethanol as mobile phase, respectively. The steric hindrance, hydrogen bonding, and π-π interaction played major roles in chiral recognition on Chiralpak IG. By comparing four chiral columns, this work systematically analyzed the separation methods of furanocoumarins and dihydroflavones for the first time and reported some active chiral ingredients of traditional Chinese medicine that have never been separated, which provided a further insight into the enantioseparation of furanocoumarins and dihydroflavones on chiral stationary phases.

Simultaneous determination of enantiomeric and organic impurities of vildagliptin on a cellulose tris(3-chloro-4-methylphenylcarbamate) column under revered-phase conditions

A new, reversed-phase HPLC (RP-HPLC) method was developed for the simultaneous determination of the dipeptidyl-peptidase-IV-inhibitor antidiabetic drug vildagliptin (VIL) enantiomeric impurity and four other achiral related impurities. An initial screening was performed on five polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux Amylose-2, Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3) in polar organic mode with methanol, ethanol, 2-propanol, or acetonitrile containing 0,1% diethylamine as mobile phase to identify the best conditions for the separation of VIL enantiomers. Lux-Cellulose-2 column was found to provide the best chiral resolution for VIL enantiomers. Further experiments were conducted using different aqueous-organic mobile phases to achieve the simultaneous chiral-achiral separation of the selected compounds. Experimental design-based optimization was performed by using a face-centered central composite design. The optimal separation conditions (Lux Cellulose-2 stationary phase, 45 °C, mobile phase consisting of methanol/water/diethylamine 80:20:0.2 (v/v/v), and 0.45 mL/min flow rate) provided baseline separation for all 6 compounds. The optimized method was validated according to the ICH guideline and proved to be reliable, specific, linear, precise, and accurate for the determination of at least 0.1% for all impurities in VIL samples. The validated method was applied for determinations from a commercially available drug formulation and proved to be suitable for routine quality control of both enantiomeric and organic impurities of VIL.

Application of Green Chiral Chromatography in Enantioseparation of Newly Synthesized Racemic Marinoepoxides.

Enantioseparation of the newly synthesized series of novel quinoline-2(1H)-one epoxide structures rac-6a–c and rac-8a–c, named marinoepoxides, is described. Marinoepoxide rac-6a, the key intermediate in the total synthesis of natural products marinoaziridines A and B, as well as their structural analogues, was synthesized by addition of the achiral ylide generated in situ from the sulfonium salt 5 or 7, to the carbon-oxygen double bond of the corresponding quinoline-2(1H)-one-4-carbaldehyde 4a–c in good yield. Separation of enantiomers of (±)-2,3,3-trisubstituted marinoepoxides rac-6a–c and (±)-trans-2,3-disubstituted marinoepoxides rac-8a–c was studied using two immobilized polysaccharide type chiral stationary phases (CSPs); tris-(3,5-dichlorophenylcarbamoyl)cellulose stationary phase (CHIRAL ART Cellulose-SC) and tris-(3,5-dimethylphenylcarbamoyl)amylose stationary phase (CHIRAL ART Amylose-SA). Enantioseparation conditions were explored by high-performance liquid chromatography (HPLC) using dimethyl carbonate/alcohol mixtures and n-hexane/ethanol (80/20, v/v) as mobile phase, and by supercritical fluid chromatography (SFC) using CO2/alcohol mixtures as mobile phase. In all examined racemates, enantioseparation was successfully achieved, but its efficiency largely depended on the structure of chiral selector and type/composition of the mobile phase.

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.

Chiral separation of oxazolidinone analogues by liquid chromatography on polysaccharide stationary phases using polar organic mode

The enantioseparation of four oxazolidinone and one biosimilar thiazolidine derivatives was performed on seven different polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4) differing in backbone (cellulose or amylose), substituent or the immobilization technologies (coated or immobilized). Polar organic mode was employed using neat methanol (MeOH), ethanol (EtOH), 2-propanol (IPA) and acetonitrile (ACN) either alone or in combinations as mobile phases. Amylose-based columns with ACN provided the highest enantioselectivities for the studied compounds. The replacement of an oxygen with a sulfur atom in the backbone of the studied analytes significantly alters the enantiomer recognition mechanism. Chiral selector-, mobile-phase-, and interestingly immobilization-dependent enantiomer elution order reversal was also observed. Reversal of elution order and hysteresis of retention and enantioselectivity was further investigated using different mixtures of IPA:MeOH and ACN:MeOH on amylose-type chiral stationary phases. Hysteresis of retention and enantioselectivity was observed on all investigated amylose-type columns and binary eluent mixtures, which can be further utilized for fine-tuning chiral separation performance of the studied columns.

Liquid chromatographic method for the simultaneous determination of achiral and chiral impurities of dapoxetine in approved and counterfeit products.

A reversed-phase high performance liquid chromatographic method was developed and validated for the simultaneous determination of the related substances of S-dapoxetine, including R-dapoxetine, (3S)-3-(dimethylamino-3-phenyl-1-propanol), S-3-amino-3-phenyl-1-propanol, 1-naphtol, 4-phenyl-2H,3H,4H-naphtho[1,2-b]pyran and 1-(2E)-Cinnamyloxynaphthalene. During the screening experiments seven different polysaccharide-type chiral stationary phases (amylose-based Lux-Amylose-1, Lux-i-Amylose-1 and Lux-Amylose-2, as well as cellulose-based Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3 and Lux-Cellulose-4) were tested in polar organic mode using a mobile phase consisting of 0.1% diethylamine in methanol, ethanol, 2-propanol and acetonitrile with 0.5mL min-1 flow rate at 20 °C. Best results were obtained on Lux Cellulose-3 column with the ethanol-based mobile phase. To increase the retention factor of two, early-eluting impurities, water was added to the mobile phase. In order to counterbalance the increased total analysis time, higher column temperature (40°C) and gradient elution, combined with flow-programming` was applied. Using the optimized conditions baseline separations were achieved for all compounds within 30min. The method was validated according to the International Council on Harmonization guideline Q2(R1) and applied to the analysis of an approved, tablet formulation and dapoxetine-containing products sold on the internet. As expected, in the case of the pharmacy-acquired product, all of the monitored impurities were below 0.1%. However, interesting results were obtained when internet-acquired samples were analyzed. These tablets contained racemic dapoxetine and/or high concentration of R-dapoxetine impurity. Based on this work polysaccharide-based chiral stationary phases can be successfully applied for the simultaneous determination of achiral and chiral impurities in reversed-phase mode applying gradient elution and flow-rate programs. The study further underlines the importance of not only achiral, but also enantiomeric quality control, whenever counterfeiting of a single enantiomeric agent is suspected.

Determination of the Enantiomeric Purity of Solriamfetol by High-Performance Liquid Chromatography in Polar Organic Mode Using Polysaccharide-Type Chiral Stationary Phases

Solriamfetol is a novel FDA approved enantiopure drug used for the treatment of excessive sleepiness associated with narcolepsy or obstructive sleep apnea. Only the active R-enantiomer is marketed, therefore, a method to determine the chiral purity is essential. Chiral separation of solriamfetol was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode and described for the first time. Seven different polysaccharide-based chiral columns (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3 and Lux Cellulose-4) were employed for the separation of enantiomers using polar organic mode, with mobile phases consisting of 0.15% diethylamine in methanol (MeOH), 2-propanol (IPA) or acetonitrile (ACN). During the screening phase, the best results (Rs> 5, in less than 10 min) was obtained on a Lux Amylose-1 column with 0.15% diethylamine in methanol with an ideal elution order (the distomer eluted before the R-enantiomer). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN and MeOH:IPA. Using optimized parameters (Lux Amylose-1 column with 0.05% diethylamine in MeOH with 0.6 mL/min flow rate at 20 °C) high enantioresolution (Rs = 5.3) was achieved within 6 min. Thermodynamic analysis was performed and revealed an enthalpy-driven enantioseparation. The developed, isocratic HPLC method was validated according to current ICH guidelines and proved to be reliable, linear, precise and accurate for the determination of 0.1% S-enantiomer as a chiral impurity in R-solriamfetol.

Chiral Separation of 3,4-dihydropyrimidin-2(1H)-ones and 3,4-di -hydropyrimidin- 2(1H)-thiones Enantiomers by HPLC on Chiralcel®OD-H

Objective: Ten racemic 3,4-dihydropyrimidin-2(1H)-ones and 3,4-dihydropyrimidin- 2(1H)-thiones were separated by liquid chromatography on Chiralcel®OD-H column containing cellulose tris(3, 5-dimethylphenylcarbamate). Methods: The enantioseparation was carried out using Chiralcel®OD-H polysaccharide-type chiral stationary phase to resolve such enantiomers under normal-phase mode. Results: Complete separations of the 3,4-dihydropyrimidin-2(1H)-ones and 3,4-dihydropyrimidin- 2(1H)-thiones derivatives with good resolution (RS= 1.04-2.80) were achieved within a short time (10-15 min). Conclusion: An optimal baseline separation (Rs> 1.5) was achieved using Chiralcel®OD-H under normal-phase mode. Structure-retention relationships have also been discussed.

Enantioselective separation of twelve pairs of enantiomers on polysaccharide-based chiral stationary phases and thermodynamic analysis of separation mechanism.

The enantioseparation of twelve pairs of structurally related 1-aryl-1-indanone derivatives was studied in the normal-phase mode using three different polysaccharide-type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n-Hexane/2-propanol and n-hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n-hexane/2-propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25-40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1-indanone derivatives on enantioseparation was also discussed.

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (Rs = 5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min−1 flow rate at 25 °C (Rs = 5.4, t2 = 7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

Enantioseparation of napropamide by supercritical fluid chromatography: Effects of the chromatographic conditions and separation mechanism.

Supercritical fluid chromatography (SFC) is already used for enantioseparation in the pharmaceutical industry, but it is rarely used for the separation of chiral pesticides. Comparing with high performence liquid chromatography, SFC uses much more environmnetal friendly and economic mobile phase, supercritical CO2 . In our work, the enantioseparation of an amide herbicide, napropamide, using three different polysaccharide-type chiral stationary phases (CSPs) in SFC was investigated. By studying the effect of different CSPs, organic modifiers, temperature, back-pressure regulator pressures, and flow rates for the enantioseparation of napropamide, we established a rapid and green method for enantioseparation that takes less than 2minutes: The column was CEL2, the mobile phase was CO2 with 20% 2-propanol, and the flow rate was 2.0mL/min. We found that CEL2 demonstrated the strongest resolution capability. Acetonitrile was favored over alcoholic solvents when the CSP was amylose and 2-propanol was the best choice when using cellulose. When the concentration of the modifiers or the flow rate was decreased, resolutions and analysis times increased concurrently. The temperature and back-pressure regulator pressure exhibited only minor influences on the resolution and analysis time of the napropamide enantioseparations with these chiral columns. The molecular docking analysis provided a deeper insight into the interactions between the enantiomers and the CSPs at the atomic level and partly explained the reason for the different elution orders using the different chiral columns.

Chiral separation of lenalidomide by liquid chromatography on polysaccharide-type stationary phases and by capillary electrophoresis using cyclodextrin selectors.

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high-performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide-type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time-dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile-phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether-β-cyclodextrin, where quasi-equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high-performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6mL/min flow rate, 40°C) and capillary electrophoresis methods (30mM sulfobutylether-β-cyclodextrin, 30mM phosphate pH6.5, 12kV applied voltage, 10°C) were validated for the determination of 0.1% (R)-lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.

Enantioseparation of racecadotril using polysaccharide-type chiral stationary phases in polar organic mode.

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide-type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1-propanol, 2-propanol, and acetonitrile) at 5 different temperatures (5-40°C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector-and mobile-phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10°C, 100% methanol, flow rate: 0.6mL/min) baseline separation of racecadotril enantiomers (resolution=3.00±0.02) was achieved, with the R-enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.

Simultaneous enantioselective determination of triazole fungicide difenoconazole and its main chiral metabolite in vegetables and soil by normal-phase high-performance liquid chromatography

Herein is reported, for the first time, a simple and highly sensitive chiral high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of difenoconazole stereoisomers and their hydroxylated metabolite difenoconazole alcohol (CGA-205375) enantiomers in vegetables and soil matrix. The separation of difenoconazole and CGA-205375 including their simultaneous enantioseparation was studied using four different polysaccharide-type chiral stationary phases (CSPs) in combination with n-hexane-polar organic alcohols mobile phase. Chiralcel OJ consisting of 25 % of cellulose tris(4-methylbenzoate) coated on wide-pore polysaccharide silica gel exhibited higher resolving ability compared to cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) as well as to its similar amylose derivative (Chiralpak AD) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of difenoconazole and its metabolite CGA-205375 could be achieved under optimized separation conditions. Based on the established HPLC method, enantioselective analysis method for this fungicide and its main chiral metabolite in vegetables and soil matrix were developed and validated. Parameters including the matrix effect, linearity, precision, accuracy, and stability were evaluated. Under the optimal conditions, the mean recoveries from cucumber, tomato, and soil matrix ranged from 81.65 to 94.52 %, with relative standard deviations in the range of 1.05-8.32 % for all stereoisomers. Coefficients of determination R(2) ≥ 0.998 were achieved for each enantiomer in the cucumber, tomato and soil matrix calibration curves within the range of 0.5-50 μg mL(-1). The limits of quantification for all enantiomers in three matrices were all below 0.1 μg mL(-1). The methodology was successfully applied for simultaneous enantioselective analysis of difenoconazole stereoisomers and their metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of difenoconazole in food and environmental matrix.

High-performance liquid chromatography enantioseparation of atropisomeric 4,4′-bipyridines on polysaccharide-type chiral stationary phases: Impact of substituents and electronic properties

The high performance liquid chromatography (HPLC) enantioseparation of eleven atropisomeric 4,4′-bipyridines was performed in the normal and polar organic phase mode using three cellulose-based chiral stationary phases (CSPs), namely Lux® Cellulose-1, Lux® Cellulose-2, Lux® Cellulose-4, and two amylose-based CSPs, Chiralpak® AD-H and Lux® Amylose-2. n-Hexane/2-propanol mixtures and pure ethanol were employed as mobile phases. The combined use of Chiralpak® AD-H and Lux® Cellulose-2 allowed to enantioseparate all the considered bipyridines. Ten bipyridines were enantioseparated at the multimilligram level allowing the elution sequence determination of the enantiomers as well as their future use for the preparation of homochiral metal organic frameworks (MOFs). Moreover, the performance of the CSPs regarding the same bipyridine was different and dependent on the backbone as well as on the side chain of the polymer. The impact of substitution pattern, shape and electronic properties of the molecules on the separation behavior was investigated through the evaluation of retention factors (k), separation factors (α), resolution (Rs) and molecular properties determined using density functional theory (DFT) calculations. In this regard, the substituents at the 3,3′,5,5′ positions of the 4,4′-bipyridyl rings exhibited a pivotal role on the enantioseparation.

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R(S) = 0.81) were well resolved (R(S) > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26-40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation.

Enantiomer separation and indirect chromatographic absolute configuration prediction of chiral pirinixic acid derivatives: Limitations of polysaccharide-type chiral stationary phases in comparison to chiral anion-exchangers

Chiral α-arylthiocarboxylic acids with different substitution patterns, representing new pirinixic acid derivatives with dual PPARα/γ agonistic activities, have been separated into enantiomers on tert-butylcarbamoylquinine and quinidine based chiral anion-exchangers and amylose tris(3,5-dimethylphenylcarbamate) coated silica on analytical and preparative scale. Absolute configurations of individual enantiomers were assigned chromatographically via elution orders on the chiral anion-exchangers and were confirmed by stereoselective syntheses via Ewans auxiliaries that have lead to enantiomeric products with known absolute configurations. The results of both methods were in full agreement. Moreover, the receptor stereoselectivity in PPARα transactivation activities was consistent within the test set of structurally related compounds. Limited correlation (between elution order and substitution) was observed within the set of α-arylthiocarboxylic acids on the amylose tris(3,5-dimethylphenylcarbamate) based chiral stationary phase (CSP), in particular the elution order changed with remote substitution. This clearly demonstrates the risks of chromatographic absolute configuration assignments by prediction from one structural analog to another one, especially with CSPs such as polysaccharide CSPs that are recognized for their broad applicability due to multiple binding and chiral recognition modes. It is therefore of utmost importance that such chromatographic absolute configuration predictions by extrapolation to structural analogs are combined with orthogonal methods for verification of the results.

Determination of enantiomerization barriers of hypericin and pseudohypericin by dynamic high‐performance liquid chromatography on immobilized polysaccharide‐type chiral stationary phases and off‐column racemization experiments

Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high-performance liquid chromatography (HPLC) using immobilized polysaccharide-type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar-organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5-dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5-dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on-line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (DeltaG(enant)(#)) of 97.6-99.6 kJ/mol for both compounds (investigated temperature range 25-45 degrees C). Complementary variable temperature off-column (i.e., in solution) racemization experiments delivered DeltaG(enant)(#) = 97.1-98.0 kJ/mol (27-45 degrees C) for hypericin and DeltaG(enant)(#) = 98.9-101.4 kJ/mol (25-55 degrees C) for pseudohypericin. An activation enthalpy of DeltaH(#) = 86.0 kJ/mol and an activation entropy of DeltaS(#) = -37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and -82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions--at least as long as stereoselective intermolecular interactions with other chiral entities are absent.

Direct Chiral Resolution and its Application to the Determination of the Pesticide Tetramethrin in Soil by High-Performance Liquid Chromatography Using Polysaccharide-Type Chiral Stationary Phase

A high-performance liquid chromatography method is presented for the enantioseparation and quantitation of tetramethrin. The separation is achieved on amylose 3,5-dimethylphenyl-carbamate CSP (Chiralpak AD-H column) using a mobile phase consisting of a mixture of n-hexane-ethanol-2-propanol (99:0.9:0.1, v/v/v). Baseline chiral separation for the four isomers of tetramethrin is obtained within 20 min. Each of the resolutions of the two pairs of enantiomers is more than 2.0. The absolute configurations of each isomer are determined. The accuracy, precision, linearity, and limits of detection and quantitation of the method are investigated. For the determination of isomers in soil, the soil sample is extracted with acetone in an ultrasonic bath. The percentage recoveries from soil are in the range of 73.5% to 87.9%.