Enantioselective Extraction System Containing Binary Chiral Selectors and Chromatographic Enantioseparation Method for Determination of the Absolute Configuration of Enantiomers of Cyclopentolate

The distribution coefficients and enantioseparation of cyclopentolate were studied in an extraction system containing d-tartaric acid ditertbutyl ester in organic phase and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in aqueous phase. Various parameters involved in the enantioseparation such as the type and the concentration of chiral selectors, pH value and a wide range of organic solvents were investigated. The maximum enantioselectivity (α = 2.13) and optimum distribution coefficients (K _R = 0.85, K _S = 0.40) were obtained under the following conditions: 0.10 mol/L HP-β-CD in aqueous phase and 0.20 mol/L d-tartaric acid ditertbutyl ester in decanol as organic phase. Cyclopentolate is present as a racemic mixture to the aqueous phase. The potentially different biological activities of cyclopentolate enantiomers have not been examined yet. Two chiral liquid chromatography methods have been developed for the direct separation of the enantiomers of cyclopentolate. First method was used for the quantification analysis of cyclopentolate enantiomers in aqueous phase. Second method used two chiroptical detectors: electronic circular dichroism (ECD) and optical rotation (OR) for the identification of individual cyclopentolate enantiomers from the organic phase enriched with (R)-enantiomer. The absolute stereochemistry was determined by means of the comparison of the experimental and computed ECD spectra and signs of OR. The ECD spectra of chiral analytes were measured on-line using HPLC-ECD technique.     

Enantioselective extraction of fenvaleric acid enantiomers by two-phase (W/O) recognition chiral extraction

To establish an extraction method for fenvaleric acid (FA) enantiomers using l-iso-butyl-l-tartaric esters and hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector, the distribution of FA enantiomers was examined in methanol aqueous solution containing HP-β-CD and 1,2-dichloroethane organic solution containing l-iso-butyl-l-tartaric esters. The influences of the concentration of l-iso-butyl-l-tartaric esters and HP-β-CD, organic diluent, pH, extraction temperature and the concentration of methanol aqueous solution on the partition coefficient (k) and separation factor (α) of FA were investigated. The experiment results showed that the complex formed by l-iso-butyl-l-tartaric esters with S-enantiomer is stabler than that with R-enantiomer. With the increase of the concentration of l-iso-butyl-l-tartaric ester, k and α increased; With the increase of the concentration of HP-β-CD, k increased firstly, and then decreased, but α increased all the while, k was the highest when the concentration of HP-β-CD was 4 mmol L^?1. 1,2-dichloroethane organic diluent was better than the others. With the increase of pH, k and α decreased; with further increasing concentration of methanol aqueous solution, k and α decreased, k and α were the highest when the concentration of methanol aqueous solution was 10%. The extraction temperature had a great influence on k and α, too.     

Kinetic study on reactive extraction for chiral separation of phenylsuccinic acid enantiomers

The kinetics of the extraction of phenylsuccinic acid (PSA) enantiomers by hydroxypropyl-β-cyclodextrin (HP-β-CD) in a modified Lewis cell was studied, in which HP-β-CD dissolved in 0.1 mol L^?1 NaH₂PO₄/H₃PO₄ buffer solution (pH = 2.5) was selected as the chiral extractant. PSA enantiomers were extracted from organic phase to aqueous phase in the extraction module. The theory of extraction accompanied by a chemical reaction has been used to obtain the intrinsic kinetics of this extraction module. The different parameters affecting the extraction rate such as agitation speed, interfacial area, initial concentration of PSA enantiomers in organic phase as well as HP-β-CD concentration in aqueous phase were separately studied. The experimental results demonstrate that the extraction reactions are fast. The reactions were found to be first order with respect to PSA and second order with respect to HP-β-CD with forward rate constants of 3.4 × 10^?2 m⁶ mol^?2 s^?1 for R-PSA and 9.96 × 10^?3 m⁶ mol^?2 s^?1 for S-PSA. These data will be useful in the design of extraction processes.     

Enantioseparation of Phenylsuccinic Acid Enantiomers by Solvent Sublation with Collaborative Selectors

A new solvent sublation (SS) system for chiral separation is introduced by using phenylsuccinic acid (H₂A) as the model enantiomers. The experiments were carried out in a traditional SS apparatus but with collaborative chiral selectors: dibenzoyl-L-tartaric acid (L-DBTA) in the organic phase and hydroxypropyl-β-cyclodextrin (HP-β-CD) in the aqueous phase. The chiral recognition abilities of the two selectors are opposite for the H₂A enantiomers. Several important parameters were investigated. The results demonstrate that enantioselective sublation and partitioning behavior are mainly dependent on the pH of the solution, the concentrations of chiral selectors and H₂A. Furthermore, the flow rate of air and flotation time also have some effects on the enantioseparation. Under the optimized conditions, the enantioselectivity expressed by the separation factor (β) and enantiomer excess (e.e.%) are 2.47 and 29.50%, and the yields of R-H₂A and S-H₂A are 0.23 and 0.13 g·L^?1, respectively. Compared with the SS system with the single selector HP-β-CD in the aqueous phase (or L-DBTA in the organic phase), the increased values of β and e.e.% in the new SS system with collaborative selectors are 1.31 (or 1.38) and 5.90% (or 13.82%), respectively.     

Enantioselective resolution of chiral aromatic acids by biphasic recognition chiral extraction

This paper reports a new chiral separation technology—biphasic recognition chiral extraction for the separation of aromatic acid enantiomers such as α-cyclohexyl-mandelic acid (CHMA) and naproxen (NAP). The biphasic recognition chiral extraction system was established by adding hydrophobic d(l)-isobutyl tartrate in 1,2-dichloroethane organic phase and hydrophilic β-cyclodextrin (β-CD) derivative in aqueous phase, which preferentially recognize the (R)-enantiomer and (S)-enantiomer, respectively. These studies involve an enantioselective extraction in a biphasic system, where aromatic acid enantiomers form complexes with the β-cyclodextrin derivative in the aqueous phase and d(l)-isobutyl tartrate in the organic phase, respectively. Factors affecting the extraction mechanism are analyzed, namely the influence of the concentrations of the extractants and aromatic acid enantiomers, the types of the extractants, pH, and temperature. The experimental results show that the biphasic recognition chiral extraction is of much stronger chiral separation ability than the monophasic recognition chiral extraction, which utilizes the cooperations of the forces of the tartrate and the β-CD derivative. Hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and methyl-β-cyclodextrin (ME-β-CD) have stronger recognition abilities for the (S)-aromatic acid enantiomers than those for (R)-aromatic acid enantiomers, among which HP-β-CD has the strongest ability. d-Isobutyl tartrate preferentially recognizes (R)-CHMA and (S)-NAP, while l-isobutyl tartrate preferentially recognizes (S)-CHMA and (R)-NAP. The maximum enantioselectivities of CHMA and NAP are 2.49 and 1.65, under conditions at which the pH values of the aqueous phases are 2.7 and 2.5, at the ratio of 2:1 of [isobutyl tartrate] to [HP-β-CD].     

Equilibrium studies on reactive extraction of naproxen enantiomers using hydrophilic ��-cyclodetrin derivatives extractants

A simple and low-cost method using liquid?Cliquid extraction coupled with complexation reactive technique has been developed for enantioselective separation of naproxen enantiomers. Three kinds of modified ??-cyclodextrins including methyl-??-cyclodextrin (Me-??-CD), hydroxyethyl-??-cyclodextrin (HE-??-CD) and hydroxypropyl-??-cyclodextrin (HP-??-CD), were selected as hydrophilic chiral selectors for extraction naproxen from organic phase to aqueous phase. A systematic study of the factors affecting chiral separation performance were investigated. The experiment results obtained show that, HP-??-CD, HE-??-CD and Me-??-CD has stronger recognition abilities for S-naproxen than those for R-naproxen. Among the ??-CD derivatives studied, HP-??-CD has the strongest ability for chiral recognition and separation. Excellent enantioselectivity (a) of 1.59 is obtained under the optimal conditions of pH of 2.5 and temperature of 5 °C.     

Enantioseparation of mandelic acid enantiomers in ionic liquid aqueous two-phase extraction systems

In the past decade, ionic liquids have received great attention owing to their potential as green solvent alternatives to conventional organic solvents. In this work, hydrophobic achiral ionic liquids (1-butyl-3-methylimidazolium-hexafluorophosphate([bmim][PF₆]), 1-octyl-3-methylimidazolium tetrafluoroborate([omim][BF₄])) were used as solvents in chiral liquid-liquid extraction separation of mandelic acid (MA) enantiomers with β-cyclodextrin (β-CD) derivatives as hydrophilic chiral selectors preferentially forming complexes with (R)-enantiomers. Factors affecting the separation efficiency were optimised, namely the type of the extraction solvents and β-CD derivatives, concentrations of the β-CD derivatives and MA enantiomers, pH, and temperature. Excellent enantioseparation of MA enantiomers was achieved in the ionic liquid aqueous two-phase extraction systems under the optimal conditions of pH 2.5 and temperature of 5°C with the maximum enantioselectivity (α) of 1.74. The experimental results demonstrated that the ionic liquid aqueous two-phase extraction systems with a β-CD derivative as the chiral selector have a strong chiral recognition ability, which might extend the application of ionic liquids in chiral separation.     

Chiral separation of α-cyclohexylmandelic acid enantiomers using ionic liquid/salt aqueous two-phase system

In this paper, an ionic liquid-based aqueous two-phase system (ILATPS) was applied to the chiral separation of α-cyclohexylmandelic acid (α-CHMA) enantiomers with hydroxypropyl-β-cyclodextrin (HP-β-CD) as the chiral selector. Several influencing parameters were investigated including the types and concentration of ionic liquids, the amount of phase-forming salt, temperature, mixing time, pH, and the content of HP-β-CD. The results showed that not all ILATPS had the ability to chirally recognise the selected enantiomers and that [C₄mim]BF₄/(NH₄)₂SO₄-based ATPS possessed the best enantioseparation ability of the investigated ILATPSs. Under optimal conditions, the separation factor (α) attained 1.59 in a single-step extraction. ILATPS is much “greener” than other liquid-liquid extraction systems, showing its potential for application to the chiral separation.     

Use of polystyrene nanoparticles to enhance enantiomeric separation of propranolol by capillary electrophoresis with Hp-beta-CD as chiral selector

We describe the use of polystyrene (PS) nanoparticles to manipulate chiral selectivity of propranolol analysis by capillary electrophoresis, by dispersing PS nanoparticles into the run buffer employing hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. Distinct separational differences are observed between the buffer containing PS nanoparticles and buffer without, when changing separating conditions including PS nanoparticles concentration, pH, buffer concentration, HP-β-CD concentration and when adding an organic additive. Selectivity improvements are reflected by changes in the observed mobility as a result of interactions between the propranolol enantiomers and HP-β-CD governing the absorption process on the PS particles surface. The presence of PS nanoparticles increases the enantioseparation at low particle concentration in the presence of HP-β-CD as a chiral selector.     

反应萃取手性分离苯基琥珀酸对映体动力学研究

通过恒界面池研究了羟丙基-β-环糊精(HP-β-CD)萃取苯基琥珀酸对映体(PSA)动力学.采用伴随化学反应的萃取理论获得萃取动力学.实验分别考察了搅拌速率、界面面积、对映体浓度和萃取剂浓度等条件对PSA对映体萃取动力学的影响.实验结果表明:HP-β-CD萃取PSA对映体的反应为快反应;对对映体反应是一级反应,对萃取剂反应是二级反应;R-PSA,S-PSA反应速率常数分别为3.4×10-2m6mol-2s-1,9.96×103m6mol-2s-1.这些数据对萃取过程的设计是很重要的.     

Enantiospecific HPLC and CE Methods for Separation and Determination of S-Darifenacin in Pharmaceutical Formulations

Two separation techniques were developed for the determination of S-(−)darifenacin (DAR) in the presence of its R-(+) isomer: The first method is high performance liquid chromatography (HPLC) and the second is capillary electrophoresis (CE). Chiral separation for chromatographic HPLC method development was carried out for S-DAR on Daicel CROWNPAK CR (+) (5 μm, 4.0 × 150 mm) column which contains (3,3-diphenyl-1,1-binaphthyl)-crown-6 coated onto a 5.5 μm silica support. The mobile phase system was aqueous acidic 70 % HClO₄ (pH 2.5): methanol in the proportion of 90:10 v/v. This current mobile phase was delivered at flow rate 0.8 mL min⁻¹ using UV detector adjusted at 286 nm. In CE method, the enantiomers were separated using 50 μm inner diameter fused-silica capillary cut to total lengths of 31.2 cm using 50 mM phosphate buffer as background electrolyte adjusted to pH 2.5 by triethanolamine. A wide range of cyclodextrins (CDs) were used such as highly sulfated α, γ CDs, hydroxyl propyl-β-CD and sulfobutyl ether-β-CD as chiral selectors. The effects of chiral additives regarding its concentration and content of organic modifier on the enantioseparation were investigated. Linear concentration ranges were from 2.5 to 50 and 40 to 300 μg mL⁻¹ with detection limits 0.67 and 12.28 μg mL⁻¹ for chromatographic HPLC and electrophoretic CE methods, respectively. The two methods were validated according to ICH guidelines with respect to linearity, accuracy, precision, LOQ, LOD and robustness. The suggested methods are suitable for separation and quantitation of S-DAR in tablets.

     

Enantioseparation of Three β-Agonists Using Di-n-butyl d-Tartrate-Boric Acid Complex as Chiral Selector by Means of MEEKC

In this study, a self-prepared complex chiral selector, di-n-butyl d-tartrate-boric acid complex, by the reaction of di-n-butyl d-tartrate with boric acid in a running buffer was used as a chiral selector for the enantioseparation of three β-agonists including clenbuterol, cycloclenbuterol and tulobuterol by means of microemulsion electrokinetic chromatography (MEEKC). Three β-agonists were successfully enantioseparated using the chiral system, indicating that the di-n-butyl d-tartrate-boric acid complex was a useful chiral selector. The effects of di-n-butyl d-tartrate and sodium tetraborate concentration, surfactant concentration, cosurfactant, phosphate, buffer pH and composition, as well as applied voltage were extensively investigated to achieve a good enantioseparation. The di-n-butyl d-tartrate and sodium tetraborate concentration in the running buffer had great influence on the chiral resolution (R _s). Three β-agonists which could not be separated with only di-n-butyl d-tartrate, obtained good chiral separation using the complex chiral selector; among them, two pairs including clenbuterol and cycloclenbuterol could be baseline resolved in 7 min under optimized experimental conditions of 0.8% (w/v) di-n-butyl d-tartrate, 40 mM sodium tetraborate, 3.0% (w/v) Tween-20 and 60 mM sodium dihydrogen phosphate with 25 kV as running voltage. The results indicated that the method could be used for the enantioseparation of three β-agonists.

     

Computer-aided molecular modeling study of enantioseparation of iodiconazole and structurally related triadimenol analogues by capillary electrophoresis: Chiral recognition mechanism and mathematical model for predicting chiral separation

Chiral separation of iodiconazole, a new antifungal drug, and 12 new structurally related triadimenol analogues had been developed by capillary electrophoresis (CE) using hydroxypropyl-γ-cyclodextrin (HP-γ-CD) as the chiral selector. The effect of structural features of analytes on Δt and R_s was studied under the optimum separation conditions. Using molecular docking technique and binding energy calculations, the inclusion process between HP-γ-CD and enantiomers was investigated and chiral recognition mechanisms were discussed. The results suggest that hydrogen bonding between fluorine at position 4 of the phenyl group beside the chiral carbon and the hydroxyl group on the HP-γ-CD rim and face to face π–π interactions between two phenyl rings highly contributed to the enantiorecognition process between HP-γ-CD and iodiconazole. The N-methyl group beside chiral carbon also played an important role in enantiomeric separation. Additionally, the big difference in binding energy (ΔΔE) highly contributed to good separation in the presence of HP-γ-CD chiral selector, which may be a helpful initial guide for chiral selector selection and predicting the result of enantioseparation. Furthermore, the new mathematical equation established based on the results of molecular mechanics calculations exhibited good capability in predicting chiral separation of these triadimenol analogues using HP-γ-CD mediated CE.     

Enantiomeric separation of (R, S)-naproxen by recycling high speed counter-current chromatography with hydroxypropyl-β-cyclodextrin as chiral selector

Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of (R, S)-naproxen (NAP) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol L(-1) phosphate buffer solution with pH=2.67 (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated, including concentration of HP-β-CD, equilibrium temperature and pH of aqueous phase. Suitable elution mode was selected for HSCCC enantioseparation of (R, S)-NAP. Under optimum separation conditions, 29 mg of (R, S)-NAP was separated using preparative recycling HSCCC with the molar ratio HP-β-CD/NAP racemate 83:1. Technical details for recycling elution mode were discussed as for chiral HSCCC separation. The purities of both (S)-NAP and (R)-NAP were over 99.5% as determined by HPLC. Enantiomeric excess of (S)-NAP and (R)-NAP reached 99.4%. Recovery for NAP enantiomers from HSCCC fractions was 82-89%, yielding 13 mg of (S)-NAP and 12 mg of (R)-NAP.     

Monitoring the Supercritical Fluid Extraction of Pyrethroid Pesticides using Capillary Electrochromatography

Capillary electrochromatography (CEC) is reported for monitoring the extraction of the pyrethroid pesticides fenpropathrin, fenvalerate and fluvalinate by SFE using supercritical CO₂. The optimum SFE conditions obtained for the pyrethroid pesticides from spiked cellulose matrix, were for fenpropathrin 300 atm and 70°C, fenvalerate 300 atm and 60°C and for fluvalinate 200 atm and 75°C. Extracts collected in methanol were subjected to analysis by CEC on a 30 cm × 75 μm i.d. fused silica capillary packed with 5 μm Hypersil ODS (21 cm packed length). Electrochromatograms of the three pyrethroid pesticides were obtained in order of elution thiourea (as the EOF marker), fenpropathrin, fenvalerate and fluvalinate, with mobile phase ACN-25 mM NaH₂PO₄ pH 8.3 (85 : 15), voltage 25 kV, electrokinetic injection 5 kV, 3 sec and detection at 200 nm. The SFE recoveries were > 80% for all three solutes. In addition, enantioseparation of the pyrethroid pesticides was investigated using Me-β-CD and HP-β-CD as chiral additives. The enantioseparation of fenpropathrin was optimised to a methanol-25 mM Tris pH 8.3 mobile phase (75 : 25) containing 70 mM Me-β-CD.     

Direct TLC Resolution of (±)-Ketamine and (±)-Lisinopril by Use of (+)-Tartaric Acid or (−)-Mandelic Acid as Impregnating Reagents or Mobile Phase Additives. Isolation of the Enantiomers

Direct resolution of the enantiomers of the racemic drugs ketamine and lisinopril has been achieved by TLC. Enantiomerically pure tartaric acid and mandelic acid were used as chiral impregnating reagents and as mobile phase additives. When (−)-mandelic acid was used as chiral impregnating reagent use of ethyl acetate–methanol–water 3:1:1 (v/v) as mobile phase enabled successful resolution of the enantiomers of both compounds. For lisinopril, the mobile phase acetonitrile–methanol–water–dichloromethane 7:1:1:0.5 (v/v) was successful when (+)-tartaric acid was used as impregnating agent. When (+)-tartaric acid was used as mobile phase additive the mobile phase acetonitrile–methanol–(+)-tartaric acid (0.5% in water, pH 5)–glacial acetic acid 7:1:1.1:0.7 (v/v) enabled successful resolution of the enantiomers of lisinopril. The effects on resolution of temperature, pH, and the amount of chiral selector were also studied. The separated enantiomers were isolated and identified. Spots were detected with iodine vapour. LODs were 0.25 and 0.27 μg for each enantiomer of ketamine with (+)-tartaric acid and (−)-mandelic acid, respectively, whereas for lisinopril LODs were 0.14 and 0.16 μg for each enantiomer with (+)-tartaric acid (both conditions) and (−)-mandelic acid, respectively.

     

Inclusion complexes of cyclodextrins with 4-amino-1,8-naphthalimides (part 2)

Fluorescence spectroscopy was used to characterize inclusion compounds between 4-amino-1,8-naphthalimides (ANI) derivatives and different cyclodextrins (CDs). The ANI derivatives employed were N-(12-aminododecyl)-4-amino-1,8-naphthalimide (mono-C₁₂ANI) and N,N′-(1,12-dodecanediyl)bis-4-amino-1,8-naphthalimide (bis-C₁₂ANI). The CDs used here were α-CD, β-CD, γ-CD, HP-α-CD, HP-β-CD and HP-γ-CD. The presence of CDs resulted in pronounced blue-shifts in the emission spectra of the ANI derivatives, with increases in emission intensity. This behavior was parallel to that observed for the dyes in apolar solvents, indicating that inclusion complexes were formed between the ANI and the CDs. Mono-C₁₂ANI formed inclusion complexes of 1:1 stoichiometry with all the CDs studied. Complexes with the larger CDs (HP-β-CD, HP-γ-CD and γ-CD) were formed by inclusion of the chromophoric ANI ring system, whereas the smaller CDs (α-CD, HP-α-CD and β-CD) formed complexes with mono-C₁₂ANI by inclusion of the dodecyl chain. Bis-C₁₂ANI formed inclusion complexes of 1:2 stoichiometry with HP-β-CD, HP-γ-CD and γ-CD, but did not form inclusion complexes with α-CD, HP-α-CD and β-CD. The data were treated in the case of the large CDs using a Benesi-Hildebrand like equation, giving the following equilibrium constants: mono-C₁₂ANI:HP-β-CD (K ₁₁ = 50 M^?1), mono-C₁₂ANI:HP-γ-CD (K ₁₁ = 180 M^?1), bis-C₁₂ANI:HP-β-CD (K ₁₂ = 146 M^?2), bis-C₁₂ANI:HP-γ-CD (K ₁₂ = 280 M^?2).     

Biphasic recognition chiral extraction — novel way of separating pantoprazole enantiomers

This paper presents a biphasic recognition chiral extraction system developed as a new chiral separation technology for the separation of pantoprazole enantiomers, combining a hydrophilic β-CD derivative in the aqueous phase and a hydrophobic tartaric acid in the organic phase which preferentially recognise the (R)-enantiomer and (S)-enantiomer, respectively. In this study, a number of factors which influence the efficiency of the extraction were investigated including types of organic solvents, β-CD and tartaric acid esters and their concentrations, pH and temperature. As a result, enantioselectivity for pantoprazole enantiomers can be improved up to 1.42 under optimised conditions; in addition, it is clear that the combined action of β-CD and tartaric acid esters leads to formation of the biphasic chiral extraction system with a stronger separation capacity than a monophasic chiral extraction system.     

Resolution of α-cyclohexyl-mandelic acid enantiomers by two-phase (O/W) recognition chiral extraction

This paper presents a new chiral separation technology: two-phase (O/W) recognition chiral extraction. Distribution behavior of α-cyclohexyl-mandelic acid enantiomers was studied in the extraction system with D(L)-isobutyl tartrate in 1,2-dichloroethane organic phase and β-CD derivatives in aqueous phase, and the influence of the kind and concentration of extractant and pH on extraction performance was investigated. The experimental results indicate that two-phase (O/W) recognition chiral extraction is of strong chiral separation ability. HP-β-CD, HE-β-CD and Me-β-CD have higher recognition ability for S-CHMA than that for R-CHMA, among which HP-β-CD has the strongest ability; whereas, D-isobutyl tartrate has reversed recognition ability for them. In the extraction system containing HP-β-CD and D-isobutyl tartrate, e.e.% of S-CHMA in aqueous phase reached 27.6% by one stage extraction, and the distribution ratio for R-CHMA(k _R ) and for S-CHMA(k _s ) and separation factor (α) are 2.44, 0.89 and 2.49, respectively. Meanwhile, pH and concentration of extractant have great effects on chiral separation ability. Two-phase (O/W) recognition chiral extraction has great significance for preparative separation of racemic compounds.     

NMR studies of interactions of new CB2 cannabinoid receptor ligands with cyclodextrins hosts. Correlation with micellar electrokinetic chromatography and reversed phase high performance liquid chromatography

Three selective CB₂ cannabinoid receptor ligands have recently been discovered to be promising anti-inflammatory agents but their low water solubility hinder their per os administration. The popularity of the cyclodextrins, from a pharmaceutical standpoint lies on their ability to interact with poorly water-soluble drugs and improve their solubility. Herein, three experimental approaches for calculating the stability constant of complexes between the selective CB₂ ligands and either the β-CD or the HP-β-CD, were tested: nuclear magnetic resonance, micellar electrokinetic chromatography and high performance liquid chromatography in reversed phase. In NMR studies the calculated K values were relatively high and were between 1486 and 3571 M^?1 with β-CD. With HP-β-CD they were between 1203 and 2650 M^?1. Concerning the two others techniques the K values were found lower. In MECK studies with β-CD they were between 308 and 792 M^?1 and with HP-β-CD between 124 and 764 M^?1. Finally in RP-HPLC studies with β-CD, they were between 539 and 1144 M^?1 and with HP-β-CD between 196 and 396 M^?1. These calculated constants suggest that a complexation phenomenon occurs. A model for inclusion of one of the CB₂ ligands in the β-CD was then proposed from molecular modeling studies.