Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l -methadone and d -methadone and their major metabolites, l - and d -2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d -methadone concentrations were lower than those of l -methadone and the d -EDDP levels were lower than those of L-EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two-compartment pharmacokinetic model. l -methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady-state volume of distribution than d -methadone. d -methadone and d -EDDP were eliminated faster than their respective l -enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.     

Chiral analysis of helquats by capillary electrophoresis: resolution of helical N-heteroaromatic dications using randomly sulfated cyclodextrins

Enantiomers of helical N-heteroaromatic dications, helquats, were separated by CE. An acidic 22/35 mM sodium/phosphate background electrolyte, pH 2.4, with addition of randomly sulfated α-, β- and γ- cyclodextrins allowed enantioresolution of a series of helquats, which comprised 5, 6 and 7 fused rings participating in the helical backbone. In general, at least one of the chiral selectors was found to provide baseline separation for 22 out of 24 helquats and partial separation for the remaining two. Individually, the sulfated γ-cyclodextrin turned out to separate 79% of the helquats, followed by the β- and α-congeners with 54 and 42% of the resolved compounds, respectively. Migration order of enantiomers was inspected for selected helquats and a relation of molecular size of the analytes to a cavity of the cyclodextrin selectors is discussed.     

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.     

Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis

Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H₃PO₄ buffer with 5.0 % CSD or CSE and 20 kV applied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs.

Separation of Tic-hydantoin enantiomers,potent sigma-1 agonists,by high performance liquid chromatography and capillary electrophoresis

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R_s > 3.3 with analysis times < 25 min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated β-cyclodextrins at pH 2.5 (R_s > 1.5 with analysis times <11 min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.     

Enantioseparation of solriamfetol and its major impurity phenylalaninol by capillary electrophoresis using sulfated gamma cyclodextrin

R-solriamfetol is a recently approved drug used for the treatment of excessive sleepiness associated with narcolepsy and sleep apnea. Herein, a capillary electrophoretic method was developed, enabling the simultaneous analysis of the API and its S-enantiomer in addition to the enantiomers of its major impurity phenylalaninol. Twenty-nine different cyclodextrins (CDs), including native, neutral, and charged ones were screened as potential chiral selectors, and the best results were obtained with sulfated CDs. Randomly sulfated-β-CD exhibited outstanding enantioresolution, the peaks of phenylalaninol enantiomers inserted between the two peaks of solriamfetol enantiomers, while sulfated-γ-CD (S-γ-CD) showed remarkable resolution values in a much shorter analysis time with the optimal enantiomer migration order. Among the single isomer sulfated CD derivatives, substituent dependent enantiomer migration order reversal could also be observed in the case of heptakis(6-O-sulfo)-β-CD (HS-β-CD) or heptakis(2,3-O-dimethyl-6-O-sulfo)-β-CD (HDMS-β-CD) with R-,S-solriamfetol, and heptakis(2,3-O-diacetyl-6-O-sulfo)-β-CD (HDAS-β-CD) resulting S-,R-solriamfetol migration order. The sulfated-γ-CD system was chosen for method optimization applying orthogonal experimental design. The optimized method (45 mM Tris-acetate buffer, pH 4.5, 4 mM S-γ-CD, 21°C, +19.5 kV) was capable for the baseline separation of solriamfetol and phenylalaninol enantiomers within 7 min. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of pharmaceutical preparation (Sunosi^® 75 mg tablet), thus it may serve as a routine procedure for the laboratories of regulatory authorities as well as in Pharmacopoeias.     

Development of a capillary electrophoretic method for determination of ketorolac enantiomers in human plasma using cationic β-cyclodextrin derivative as a chiral selector

A novel approach for the separation of ketorolac enantiomers by capillary electrophoresis is presented. A cationic β-cyclodextrin derivative based on imidazole was synthesized and used as a chiral selector in the background electrolyte. The influence of pH and ionic strength of background electrolyte, as well as cationic β-cyclodextrin derivative concentration on the resolution of ketorolac enantiomers, was investigated. The highest value of the resolution for ketorolac enantiomers was 1.46 when the background electrolyte consisted of 25 mM NaH₂PO₄ (pH 6.4) with 1 mM 1-butyl-3-β-cyclodextrinimidazolium tosylate. Additionally, the possibilities of cationic derivatives for the separation of ketoprofen enantiomers were shown (peak resolution 1.06). The two-step preconcentration mode was developed to reduce the limit of detection of individual enantiomers. The proposed approach was successfully applied to determine ketorolac enantiomers in tablet “Ketorol express” and human plasma. The calibration range of ketorolac enantiomers for plasma samples was 0.25–2.50 μg/ml with coefficients of determination ≥ 0.99. The relative standard deviation both of the peak area and migration time was less than 15%, as well as the accuracy ranged from 90.1% to 110.2% for both analytes. The limits of detection were 44 and 55 ng/ml for R- and S-ketorolac. The quantity of ketorolac in plasma was verified with high-performance liquid chromatography.     

γ(δ)-Thionolactones – Enantioselective Capillary GC and Sensory Characteristics of Enantiomers

The even numbered γ(δ)-thionolactones (C₆–C₁₂) were investigated, using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)- and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as chiral stationary phases in capillary gas chromatography. The odor characteristics of γ(δ)-thionolactone enantiomers were investigated by enantioselective gas chromatography/olfactometry.     

Chiral separation of lansoprazole and rabeprazole by capillary electrophoresis using dual cyclodextrin systems

Novel capillary electrophoresis methods using CDs as chiral selectors were developed and validated for the chiral separation of lansoprazole and rabeprazole, two proton pump inhibitors. Fourteen different neutral and anionic CDs were screened at pH 4 and 7 in the preliminary analysis. Sulfobutyl‐ether‐β‐CD with a degree of substitution of 6.5 and 10 at neutral pH proved to be the most suitable chiral selector for both compounds. Various dual CD systems were also compared, and the possible mechanisms of enantiomer separation were investigated. A dual selector system containing sulfobutyl‐ether‐β‐CD degree of substitution 6.5 and native γ‐CD proved to be the most adequate system for the separations. Method optimization was carried out using an experimental design approach, performing an initial fractional factorial screening design, followed by a central composite design to establish the optimal analytical conditions. The optimized methods (25 mM phosphate buffer, pH 7, 10 mM sulfobutyl‐ether‐β‐CD/20 mM γ‐CD, +20 kV voltage; 17°C temperature; 50 mbar/3 s injection, detection at 210 nm for lansoprazole; 25 mM phosphate buffer, pH 7, 15 mM sulfobutyl‐ether‐β‐CD/30 mM γ‐CD, +20 kV voltage; 18°C temperature; 50 mbar/3 s injection, detection at 210 nm for rabeprazole) provided baseline separation for lansoprazole (R_s = 2.91) and rabeprazole (R_s = 2.53) enantiomers with favorable migration order (in both cases the S‐enantiomers migrates first). The optimized methods were validated according to current guidelines and proved to be reliable, linear, precise, and accurate for the determination of 0.15% distomer as chiral impurity in dexlansoprazole and dexrabeprazole samples.     

Investigation of isomerization of dexibuprofen in a ball mill using chiral capillary electrophoresis

Besides the racemate, the S-enantiomer of ibuprofen (Ibu) is used for the treatment of inflammation and pain. Since the configurational stability of S-Ibu in solid state is of interest, it was studied by means of ball milling experiments. For the evaluation of the enantiomeric composition, a chiral CE method was developed and validated according to the ICH guideline Q2(R1). The addition of Mg²⁺, Ca²⁺, or Zn²⁺ ions to the background electrolyte (BGE) was found to improve Ibu enantioresolution. Chiral separation of Ibu enantiomers was achieved on a 60.2 cm (50.0 cm effective length) x 75 μm fused-silica capillary using a background electrolyte (BGE) composed of 50 mM sodium acetate, 10 mM magnesium acetate tetrahydrate, and 35 mM heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) as chiral selector. The quantification of R-Ibu in the mixture was performed using the normalization procedure. Linearity was evaluated in the range of 0.68–5.49% R-Ibu (R² = 0.999), recovery was found to range between 97 and 103%, the RSD of intra- and interday precision below 2.5%, and the limit of quantification for R- in S-Ibu was calculated to be 0.21% (extrapolated) and 0.15% (dilution of racemic ibuprofen), respectively. Isomerization of S-Ibu was observed under basic conditions by applying long milling times and high milling frequencies.     

New,selectively substituted cyclodextrins as stationary phases for the analysis of chiral constituents of essential oils

New β- and γ-cyclodextrin derivatives, selectively substituted with n-pentyl and methyl groups, e.g. heptakis(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin, octakis(2-O-methyl-3,6-di-O-pentyl)-γ-cyclodextrin, and octakis(2,6-di-O-methyl-3-O-pentyl)-γ-cyclodextrin, have been prepared from specifically protected intermediates. The new cyclodextrin derivatives exhibit unique enantioselectivity towards important chiral constituents of essential oils. The enantiomers of lavandulol, α-bisabolol, nerolidol, and other terpenoid alcohols could be resolved and their presence in different essential oils could be proved. Methyl jasmonate and epi-methyl jasmonate could, in addition, be detected in jasmine concrete by two-dimensional gas chromatography. The enantiomers of the macrocyclic ketone muscone have been separated for the first time.     

Separation of terbutaline enantiomers in capillary electrophoresis with neutral cyclodextrin-type chiral selectors and investigation of the structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy

The major goal of this study was to determine the affinity pattern of the terbutaline (TB) enantiomers toward α-, β-, γ-, and heptakis(2,3-di-O-acetyl)-β-cyclodextrins and using NMR spectroscopy for the understanding of the fine mechanisms of interaction between the cyclodextrins (CD) and TB enantiomers. It was shown once again that CE in combination with NMR spectroscopy represents a sensitive tool to study the affinity patterns and structure of CD complexes with chiral guests. Opposite affinity patterns of TB enantiomers toward native α- and β-CDs were associated with significant differences between the structure of the related complexes in solution. In particular, the complex between TB enantiomers and α-CD was of the external type, whereas an inclusion complex was formed between TB enantiomers and β-CD. One of the possible structures of the complex between TB and heptakis(2,3-di-O-acetyl)-β-CD (HDA-β-CD) was quite similar to that of TB and β-CD, although the chiral recognition pattern and enantioselectivity of TB complexation with these two CDs were very different.     

Gas chromatographic enantiomer separation of agrochemicals using modified cyclodextrins

The enantiomers of phenoxypropionic acid type herbicides have been resolved by capillary gas chromatography employing modified cyclodextrins as chiral stationary phases. Excellent separations were obtained with columns containing a 1:1 mixture of per-O-pentylated and per-O-methylated γ-cyclodextrin. The enantiomers of the methyl esters of mecoprop and dichlorprop were also resolved on octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. On this phase the order of elution of the enantiomers was temperature-dependent, the elution order being reversed as the temperature passed through the isoenantioselective temperature. This is the first time such behavior has been observed with cyclodextrin derivatives. The enantiomers of the polychlorinated polycyclic pesticides cis- and trans-chlordane, oxychlordane, heptachlor, heptachlorepoxide, and three chiral organophosphorus pesticides could be resolved using selectively derivatized cyclodextrin derivatives.     

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.     

Steroselective determination of trihexyphenidyl using carboxylmethyl-β-cyclodextrin by capillary electrophoresis with field-amplified sample stacking

A simple, sensitive and low-cost method using capillary electrophoresis coupled with field-amplified sample stacking (FASS) technique has been developed for enantioselective separation and quantification of trihexyphenidyl (THP) enantiomers in human serum. In this work, three kinds of modified β-cyclodextrin were tested as chiral selectors in CE. Among the CDs studied, THP enantiomers could only be separated by carboxylmethyl-β-cyclodextrin (CM-β-CD). A systematic study of the parameters (CD concentration and pH value in CE buffer, separation voltage and temperature, composition of sample solvent, injection voltage and time) affecting chiral separation and on-line concentration of THP enantiomers were investigated and optimized. The optimum FASS method provided a sensitivity enhancement of about 490-fold compared with usual hydrodynamic injection. Limits of detection for each enantiomer were in the low ng ml^− 1 concentration range (0.92 ng ml^− 1 or 3.06 nM). The quantification of each THP enantiomer in human serum was performed after serum sample extraction. To validate this CE-FASS method, linear regression analysis, intra and inter-day precision and recovery were determined with satisfying results.     

Glycogen: A novel branched polysaccharide chiral selector in CE

Various chiral selectors have been employed in CE and among them linear polysaccharides exhibited powerful enantioselective properties. Different from linear polysaccharides, the use of branched polysaccharides as chiral selectors in CE has not been reported previously. In this study glycogen belonging to the class of branched polysaccharides was used as a novel chiral selector for the enantiomeric separations for the first time. Since glycogen is electrically neutral, the method is applicable to ionic compounds. Eighteen chiral compounds including 12 basic drugs and six acidic drugs have been tested to demonstrate the potential of this chiral selector. BGE and selector concentrations and buffer pH were systematically optimized in order to obtain successful chiral separations. Among the tested compounds, the enantiomers of ibuprofen, which is an acidic drug, were successfully recognized by 3.0% w/v glycogen with 90 mM Tris‐H₃PO₄ buffer (pH 7.0). The enantiomers of basic drugs such as citalopram, cetirizine and nefopam were also baseline‐resolved with 50 mM Tris‐H₃PO₄ buffer (pH 3.0) containing 3.0% glycogen. Amlodipine belonging to basic compound only gave partial enantioseparation under the above‐mentioned condition.     

Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).     

Sulfated cyclodextrins as water-soluble chiral NMR solvating agents for cationic compounds

The utility of a series of sulfated cyclodextrins as water-soluble chiral NMR solvating agents for cationic substrates is described. Sulfated α-, β- and γ-cyclodextrin with degrees of substitution of 12, 13 and 14, respectively, a sulfated β-cyclodextrin with a degree of substitution of 9 and a sulfobutyl ether β-cyclodextrin with a degree of substitution of 6.3 are examined. Results with 33 water-soluble cationic organic salts are reported. Chiral differentiation with the sulfated cyclodextrins is compared to prior results obtained with anionic carboxymethylated and phosphated cyclodextrins. The highly sulfated cyclodextrins are often more effective at causing enantiomeric differentiation in ¹H NMR spectra than the sulfobutyl ether, carboxymethylated and phosphated cyclodextrins, and are recommended as the first choice of a chiral solvating agent for the analysis of chiral cationic organic salts in aqueous solution.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

Determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycines by chiral capillary electrophoresis

Summary A fast and practical chiral capillary electrophoretic method has been developed for determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG) compounds. In particular, the isomer PCCG-13, a potent selective and competitive antagonist at phospholipaseD-coupled metabotropic glutamate receptors (mGluRs), was completely resolved from its enantiomer PCCG-15 by use of dimethyl-β-cyclodextrin (DMCD) as chiral selector. pH 9.0 running buffer containing 2-amino-2-methyl-1,3-propanediol (AMPD; 100mM) was a suitable medium enabling resolution of the enantiomers in a short (32.5 cm total length) poly(vinyl alcohol) (PVA)-coated capillary. Because of the suppression of the electroosmotic flow and the good peak shape, baseline resolution of the enantiomers was obtained by use of the optimum concentration of chiral selector. For quantitative purposes at impurity levels, high sample loading was required and adequate separation was obtained in the presence of 80 mM DMCD. This CE method enabled quantification of 0.3% m/m of undesired enantiomer in PCCG-13; the samples analyzed, obtained from enantioselective synthesis, proved to be of high enantiomeric purity.