Enantioseparation of phenylsuccinic acid by high speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector

High speed counter-current chromatography (HSCCC) was successfully applied to resolution of phenylsuccinic acid (PSA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector (CS). The two-phase solvent system composed of n-hexane–methyl tert-butyl ether–0.1 mol L⁻¹ phosphate buffer solution with pH = 2.51 (0.5:1.5:2, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of chiral selector, pH value of the aqueous phase, the separation temperature, and the thermodynamic parameters of inclusion complex were calculated. The complex formation constants were determined using analytical instrument. Two HSCCC elution modes were studied and peak resolution equation was discussed. Under optimum separation conditions, 712 mg of PSA racemate was separated using preparative apparatus. The purities of both of the fractions including (+)-PSA and (−)-PSA from the preparative CCC separation were over 98.5% determined by HPLC and enantiomeric excess of (+)-PSA and (−)-PSA reached 97.6% and 98.6%, respectively. Recovery for the target compounds from the CCC fractions reached 80–82% yielding 285 mg of (+)-PSA and 292 mg of (−)-PSA.     

Chiral separation of pheniramine by capillary electrophoresis partial-filling technique using hydroxypropyl-β-cyclodextrin as chiral selector

A simple capillary electrophoresis partial-filling technique for the enantioseparation of pheniramine is presented. Phosphate buffer and hydroxypropyl-β-cyclodextrin (HP-β-CD) were used as the electrolyte and chiral selector, respectively. Several experimental parameters such as HP-β-CD concentration, HP-β-CD plug length, CE temperature and applied voltage were studied. Under the selected conditions, pheniramine enantiomers can be separated within less than 14 min. The assay was validated for linearity (5.0 × 10⁻⁶–5.0 × 10⁻⁴ M; R ² = 0.9987), limit of detection (5.0 × 10⁻⁷ M), limit of quantitation (5.0 × 10⁻⁶ M), analytical precision (%RSD ≤ 9.8) and accuracy (%recovery = 101 ± 3). The proposed methodology was then applied to the analysis of a commercially available pharmaceutical eye drop preparation. The results are in good agreement with that declared by the manufacturer. The proposed methodology provides adequate results in terms of simplicity, cost, sample throughput, repeatability and accuracy for quality control of pheniramine enantiomers in pharmaceutical preparations.     

Enantiomeric separation and determination of the enantiomeric impurity of armodafinil by capillary electrophoresis with sulfobutyl ether-β-cyclodextrin as chiral selector

A selective capillary electrophoresis method using sulfobutyl ether-β-cyclodextrin as a chiral selector was developed and validated for the determination of the enantiomeric impurity of (R)-modafinil, i.e., armodafinil. Several parameters were optimized for a satisfactory enantioresolution, including the type and concentration of chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature. The finally adopted condition was: 20 mmol/L phosphate buffer at pH 7.5, containing 20 mmol/L sulfobutyl ether-β-cyclodextrin and 20% methanol, at temperature of 25 °C. A good resolution of 3.3 for the two enantiomers of modafinil was achieved by applying the optimal conditions. The limit of detection (LOD) and limit of quantification (LOQ) of (S)-modafinil were 1.25 μg/mL and 2.50 μg/mL, respectively. The established method was also proven to display good selectivity, repeatability, linearity and accuracy. Finally, the method was used to investigate the enantiomeric purity of armodafinil in bulk samples.     

Determination of pheniramine enantiomers in eye drop by capillary electrophoresis using hydroxypropyl-β-cyclodextrin as chiral selector

A capillary zone electrophoresis procedure has been developed for the chiral determination of pheniramine in eye drop. Native and derivative cyclodextrins (CDs) including γ-CD, β-CD, hydroxypropyl-β-CD and dimethyl-β-CD were tested as chiral selectors. Using 30 mM hydroxypropyl-β-CD in 50 mM phosphate buffer (pH 3.0), the acceptable resolution value (R = 1.55) was obtained. The assay was validated for linearity (3.3 × 10⁻⁶–5.0 × 10⁻⁴ M; R ² = 0.9996), limit of detection (3.3 × 10⁻⁶ M), limit of quantification (8.5 × 10⁻⁶ M), analytical precision by terms of intra- and inter-day variability (RSD ≤ 2.57%), and accuracy (recovery ≥ 89.3%). The content of pheniramine in eye drop obtained by the proposed method was in good agreement with the declared value. The results indicated that pheniramine in the eye drop was present as the racemate.     

Enantiomeric separation in high-performance liquid chromatography using novel β-cyclodextrin derivatives modified by R-configuration groups as chiral stationary phases

Two new chiral stationary phases (CSP) were successfully prepared through bonding β-cyclodextrin (CD) derivatives modified by R-configuration groups (R-CPGCD, R-HMPGCD) to silica gel. Nineteen chiral nitro aromatic alcohol derivatives were separated under the polar organic and the reversed phase modes. Better enantioseparation was obtained in the reversed phase mode. The resolution values of the analytes ranged from 1.98 to 7.57 and from 2.19 to 8.14 on R-CPGCD and R-HMPGCD CSPs, respectively, using a mobile phase composed of methanol/water (v/v, 40/60). Better enantioseparation was obtained on R-HMPGCD CSP than on R-CPGCD CSP because of stronger hydrogen bonding and π-π interactions between the substituents on the cyclodextrin derivatives and the analytes. For different analytes, the increasing electronic density of the benzene ring was found to be favorable to the enantioseparation of the test analytes. The thermodynamic parameters showed that the enantioseparation of analytes was enthalpy-controlled and a lower temperature aided the enantiomeric separation of the solutes on the two CSPs. MD simulations were used to investigate the recognition mechanism between the chiral selectors and the analyte using R-, S-2-naphthalenemethanol and R-CPGCD and R-HMPGCD complexes as examples. S-2-naphthalenemethanol had the stronger interactions with R-CPGCD and R-HMPGCD than the R-isomer. The substituent derivatized on R-CPGCD and the cyclodextrin cavity contributed to the discrimination of the S-isomer, but only the derivatized group on R-HMPGCD was found to play a major role in separating prosess. In addition, the larger free energy deviation of the R- and S-isomers in the R-HMPGCD system brought about a higher resolution value (R_s = 8.14).     

Enantiomer separation of phenyllactic acid by HPLC with Hp-β-cyclodextrin as chiral mobile phase additive

In our current research work, a sensitive, specific, and economic HPLC method has been developed for the separation of phenyllactic acid (PLA) enantiomers. The effects of cyclodextrin (CD) type, column temperature, buffer pH, methanol content and CD concentration on enantioselective separation were investigated. Baseline chromatographic separation was achieved on an Inertsil ODS-SP (150 × 4.6 mm, i.d. 5 μm) column with HP-β-CD as chiral mobile phase additive. The LOD and LOQ for d-phenyllactic acid were 0.3 and 0.7 μg/mL, respectively. A good linear relationship was obtained in the concentration range of 0.71–5.13 μg/mL with r² >0.999 for d-PLA. The percentage recovery of the d-PLA ranged from 94.1 to 103.2 in l-PLA. This method is simple and cost-saving, and could be easily applied for chiral discrimination and determination of the racemic PLA in an enantioselective study in quality control laboratory.     

The 3-amino-derivative of γ-cyclodextrin as chiral selector of Dns-amino acids in electrokinetic chromatography

The enantioseparation of the enantiomeric pairs of 10 Dns derivatives of α-amino acids was successfully carried out by using for the first time the 3-amino derivative of the γ-cyclodextrin. The effects of pH and selector concentration on the migration times and the resolutions of analytes were studied in detail. 3-Deoxy-3-amino-2(S),3(R)-γ-cyclodextrin (GCD3AM) shows very good chiral recognition ability even at very low concentrations at all the three investigated values of pH, as shown by the very large values of selectivity and resolution towards several pairs of amino acids. The role played by the cavity, the substitution site and the protonation equilibria on the observed properties of chiral selectivity, on varying the specific amino acid involved, is discussed.     

A simple method for the separation of (6R)- and (6S)-5,6,7,8-tetrahydrofolic acid by reversed-phase HPLC with hydroxypropyl-β-cyclodextrin as the mobile phase additive

(6R)- and (6S)-5,6,7,8-tetrahydrofolic acid were resolved by reversed-phase HPLC on a C-18 column with hydroxypropyl-??-cyclodextrin (HP-??-CD) in the mobile phase. As the concentration of HP-??-CD increased from 0 to 45?mM, resolution increased and retention times decreased dramatically. The influence of different mobile phase pH, column temperature, and some other factors were evaluated, and we obtained the optimal HPLC separation conditions.     

Analysis of repaglinide enantiomers in pharmaceutical formulations by capillary electrophoresis using 2,6-di-o-methyl-β-cyclodextrin as a chiral selector

This study used the general applicability of 2,6-didi-o-methyl-β-cyclodextrin (DM-β-CD) as the chiral selector in capillary electrophoresis for fast and efficient chiral separation of repaglinide enantiomers. A systematic study of the parameters affecting separation was performed with UV detection at 243 nm. The optimum conditions were determined to be 1.25% (w/v) DM-β-CD in 20 mM sodium phosphate (pH 2.5) as the running buffer and separation voltage at 20 kV. DM-β-CD had the best enantiomer resolution properties under the tested conditions, whereas other β-cyclodextrins showed inferior performances or no performance. The proposed method had a linear calibration curve in the concentration range of 12.5-400 μg/mL. The limit of detection was 100 ng/mL. The intra-day and inter-day precisions were 2.8 and 3.2%, respectively. Recoveries of 97.9-100.9% were obtained. The proposed method was fast and convenient, and was determined to be efficient for separating enantiomers and applicable for analyzing repaglinide enantiomers in quality control of pharmaceutical production.     

Direct enantioseparation of lipoic acid in dietary supplements by capillary electrophoresis using trimethyl-β-cyclodextrin as a chiral selector

Lipoic acid, an antioxidant, naturally occurs as the (R)-enantiomer, while synthetic lipoic acid is racemic. It is thus of interest to know the (R)-enantiomer content of lipoic acid supplements. Here, we used capillary electrophoresis to directly enantioseparate lipoic acid in dietary supplements by using a sulfonated capillary with an effective voltage of +18 kV and direct detection at 200 nm. Factors affecting migration time and resolution of lipoic acid were investigated. The optimum background electrolyte was found to be 100 mM phosphate buffer (pH 7.0) containing 8 mM trimethyl-β-cyclodextrin as a chiral selector at 20°C. Under the proposed conditions, direct chiral resolution of lipoic acid in dietary supplements was conducted successfully.     

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase

In this paper a phenyl-carbamate-propyl-β-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2′-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H₂O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H₂O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2′-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.     

Enantiomeric separation of dansylamino acids by capillary zone electrophoresis with selectively methylated γ-cyclodextrin derivatives

The chiral separation ability of octakis2-, 3- and 6-mono-O-methyl, 2,3-, 2,6- and 3,6-di-O-methyl, and 2,3,6-tri-O-methyl)-γ-cyclodextrins as chiral selectors in capillary zone electrophoresis was investigated using twelve dansylamino acids. Unmodified and 6-monomethylated -γ-cyclodextrins (γ-CDs) exhibited similar high enantioselectivities. γ-CD still exhibited a chiral separation ability after 2-monomethylation or 2,6-dimethylation. 3-Monomethylated -γ-CD could only separate the enantiomers of two dansylamino acids, but further methylation of the hydroxyl groups at the 6-positions of 3-monomethylated γ-CD resulted in the highest chiral separation ability. γ-CD completely lost its high enantioselectivity after methylation of both the 2- and 3-positions, regardless of 6-methylation.     

Complex formation of some nonionic surfactants with hydroxypropyl-β-cyclodextrin and with heptakis (2,6-di-O-methyl)-β-cyclodextrin

The interaction of six nonionic surfactants -[4-(1,1,3,3-tetra-methylbutyl)phenyl]--hydroxypoly(oxy-1,2-ethanediyl) with hydroxypropyl--cyclodextrin (HPCD) and dimethyl--cyclodextrin (DIMEB) was studied by reversed-phase thin-layer chromatography in the presence and absence of sodium chloride. Each surfactant formed complexes with both cyclodextrin derivatives; however, the strength of interaction varied considerably. DIMEB formed more stable inclusion complexes with the surfactants than did HPCD. A longer ethyleneoxide chain decreased the strength of interaction, whereas sodium chloride exerted a negligible impact. Principal component analysis indicated that both the hydrophobicity and the specific hydrophobic surface are of the surfactant influenced the complex formation indicating the hydrophobic character of the interaction.Dedicated to Professor József Szejtli.     

Enantioseparation of aspartyl dipeptides by CE: Comparison between 18-crown-6-tetracarboxylic acid and carboxymethyl-β-cyclodextrin as chiral selector

Summary The simultaneous separation of isomeric α-and β-aspartyl peptides as well as their enantiomers by capillary electrophoresis was investigated. Resolution of the enantiomers of the peptides α/β-AspPhe-NH₂ and α/β-AspPhe-OMe was achieved with 18-crown-6-tetracarboxylic acid (18C6H₄) in untreated fused silica capillaries in the acidic pH range. Baseline resolution for the dipeptides was obtained using polyacrylamide (PAA)-coated capillaries. The selectivity of the crown ether is compared with the selectivity of another chiral selector, namely the negatively charged CD derivative carboxymethyl-β-CD (CM-β-CD).     

Enhancement of aqueous solubility of tricyclic acyclovir derivatives by their complexation with hydroxypropyl-β-cyclodextrin

Solubilities of tricyclic acyclovir derivatives in buffered aqueous solutions of hydroxypropyl-β-cyclodextrin (HP-β-CD) at pH 5.5 and 7.0 were determined at 25 and 37 °C. Complexation of these compounds with HP-β-CD resulted in a noticeable increase of their solubility; nevertheless it was limited to tricyclic derivatives of acyclovir carrying an aryl substituent. Combination of ¹H NMR and DSC techniques demonstrated the existence of inclusion complexes between acyclovir derivatives and HP-β-CD. The stability constants, estimated using the Higuchi–Connors method, were found in the range of 10–100 M⁻¹. Additionally, the pK _a values at 25 °C and molar extinction coefficients in aqueous buffered solutions were also determined for all studied compounds.     

Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20 mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365 nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.     

Synthesis and application of a novel single-isomer mono-6-deoxy-6-(3R,4R-dihydroxypyrrolidine)-β-cyclodextrin chloride as a chiral selector in capillary electrophoresis

A novel positively charged single-isomer of β-cyclodextrin, mono-6-deoxy-6-(3R,4R-dihydroxypyrrolidine)-β-CD chloride (dhypy-CDCl), was synthesized and employed as a chiral selector for the first time in capillary electrophoresis (CE) for the enantioseparation of anionic and ampholytic acids. The effects of the running buffer pH, chiral selector concentration, analyte structure and organic modifier on the enantioseparation were studied in detail. The chiral selectivity and resolution for most of the studied analytes decreased as the buffer pH increased in the range of 6.0–9.0. Increasing selector concentration led to decreased effective mobility, increased chiral selectivity and resolution for most of the studied analytes. Moreover, the hydroxyl groups located on the dihydroxypyrrolidine substituent of the dhypy-CDCl could have influence on the chiral separation.     

Enantioselective extraction of mandelic acid enantiomers by aqueous two-phase systems of polyethylene glycol and ammonium sulfate containing ��-cyclodextrin as chiral selector

A new chiral separation technology, aqueous two-phase extraction, was proposed for the separation of racemic mandelic acid. The distribution behavior of mandelic acid enantiomers was investigated in aqueous two-phase systems composed of polyethylene glycol and ammonium sulfate containing ??-cyclodextrin as chiral selector. The influences of the pH, the mass fraction of polyethylene glycol and ammonium sulfate, the polymerization degree of polyethylene glycol, the initial concentration of ??-cyclodextrin, mandelic acid enantiomers and extractive temperature on the distribution behavior were studied respectively. The results show that ??-cyclodextrin is inclined to recognize L-enantiomer; under the optimized conditions, the separation factor reaches 2.46 and the enantiomeric excess is 42.13% in the top phase and 40.43% in the bottom phase, respectively. Aqueous two phase chiral-extraction with strong chiral separation ability, plays great role in preparative separation of racemic compounds and is important for the development of aqueous two phase extraction technique.