Analytical methodologies for the enantiodetermination of citalopram and its metabolites

Citalopram (CIT) is a highly selective serotonin reuptake inhibitor (SSRI) frequently used in the treatment of major depressive disorders. It has a chiral centre in its structure and is used in therapy both as a racemic mixture (R,S-CIT) and a pure enantiomer (S-CIT). The differences between the pharmacokinetic and pharmacological profiles of the two enantiomers are well established. Consequently, the development of new efficient chiral analysis methods for their enantiomeric separation is a topic of great actuality. CIT metabolism is stereoselective as it is metabolized in chiral active metabolites, which retain considerable SSRI activity and contribute to the pharmacological effect. Chiral analytical methods are employed for the determination of enantiomeric ratio in pharmaceutical preparations and for monitoring the enantiomer levels in biological samples for therapeutic and toxicologic purposes. The current study reviews the published literature for the chiral analysis of CIT and its metabolites based on chromatographic and electrophoretic methods coupled with UV, fluorescence and mass spectrometry detectors.     

Assignment of absolute configuration to an improved enantioselective naproxen selector

Assignment of absolute configuration to a recently developed chiral selector useful in the separation of the underivatized enantiomers of naproxen and other nonsteroidal anti-inflammatory drugs (NSAIDs) is described. Circular dichroism, ¹H NMR, and X-ray diffraction have been used to confirm the original assignment which was based solely upon elution orders from HPLC chiral stationary phases. All of these techniques agree in the assignment of the (S,S) absolute configuration to the enantiomer of the chiral selector which associates preferentially with (S)-naproxen. © 1994 Wiley-Liss, Inc.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Chiral analysis of methylphenidate and dextromoramide by capillary electrophoresis

Capillary electrophoretic methods have been developed to separate the enantiomers of methylphenidate (MPH) and dextromoramide. For MPH separation was achieved with heptakis (2,6-di-O-methyl)-β-cyclodextrin (DMCD) as chiral selector in a 100 mM phosphoric acid buffer adjusted to pH 3.0 with triethanolamine. Commercial samples of d,l-erytho-MPH HCl and d,l-threo-MPH HCl were analysed using the method. There was no evidence of the presence of d,l-threo-MPH HCl in d,l-erytho-MPH HCl and vice versa. The ratio of the enantiomers was determined for each diastereoisomer. Hydroxypropyl-β-cyclodextrin was the chiral selector of choice for the chiral separation of the enantiomers of moramide. The separation which gave a resolution of about 3.5 was achieved in 4 min using only a 6 cm of length of capillary. In a sample of dextro-R-moramide tartrate only a small quantity (4.9% w/w) of levo-S-moramide was detected with this method.     

Indirect synchronous fluorescence spectroscopy and direct high‐performance thin‐layer chromatographic methods for enantioseperation of zopiclone and determination of chiral‐switching eszopiclone: Evaluation of thermodynamic quantities of chromatographic separation

Economic and enantioselective synchronous fluorescence spectroscopy and high‐performance thin‐layer chromatography methods have been developed and validated as per ICH guidelines for the separation of zopiclone enantiomers using L‐(+)‐tartaric acid as a chiral selector, followed by determination of the chiral‐switching eszopiclone. Synchronous fluorescence spectroscopy was successfully applied for chiral recognition of R & S enantiomers of zopiclone at ?λ = 110 nm based on creating of diastereomeric complexes with 0.06M tartaric acid in an aqueous medium containing 0.2M disodium hydrogen orthophosphate. Synchronous fluorescence intensities of eszopiclone were recorded at 296 nm in concentration range 0.2‐ to 4‐μg/mL eszopiclone. High‐performance thin‐layer chromatography method depends on resolution of zopiclone enantiomers on achiral HPTLC silica‐gel plates using acetonitrile:methanol:water (8:2:0.25, v/v/v) containing L‐(+)‐tartaric acid as a chiral mobile‐phase additive followed by densitometric measurements at 304 nm in concentration range of 1 to 10 μg/band of eszopiclone. The effect of chiral‐selector concentration, pH, and temperature on the resolution have been studied and optimized for the proposed methods. The cited procedures were successfully applied to determine eszopiclone in commercial tablets of pure and racemic forms. Enantiomeric excess was evaluated using optical purity test and integrated peak area to describe the enantiomeric ratio. Thermodynamics of chromatographic separation, enthalpy, and entropy were evaluated using the Van't Hoff equation. The proposed methods were found to be selective for identification and determination of the eutomer in drug substances and products.     

Maltodextrins as Chiral Selectors in CE: Molecular Structure Effect of Basic Chiral Compounds on the Enantioseparation

Prediction of chiral separation for a compound using a chiral selector is an interesting and debatable work. For this purpose, in this study 23 chiral basic drugs with different chemical structures were selected as model solutes and the influence of their chemical structures on the enantioseparation in the presence of maltodextrin (MD) as chiral selector was investigated. For chiral separation, a 100‐mM phosphate buffer solution (pH 3.0) containing 10% (w/v) MD with dextrose equivalent (DE) of 4‐7 as chiral selector at the temperature of 25°C and voltage of 20 kV was used. Under this condition, baseline separation was achieved for nine chiral compounds and partial separation was obtained for another six chiral compounds while no enantioseparation was obtained for the remaining eight compounds. The results showed that the existence of at least two aromatic rings or cycloalkanes and an oxygen or nitrogen atom or –CN group directly bonded to the chiral center are necessary for baseline separation. With the obtained results in this study, chiral separation of a chiral compound can be estimated with MD‐modified capillary electrophoresis before analysis. This prediction will minimize the number of preliminary experiments required to resolve enantiomers and will save time and cost. Chirality 26:620–628, 2014. © 2014 Wiley Periodicals, Inc.     

Enantioselective determination of metoprolol and major metabolites in human urine by capillary electrophoresis

The enantiomeric separation of metoprolol and its metabolites in human urine was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using carboxymethyl-β-cyclodextrin (CM-β-CD) as the chiral selector. A 100-mM acetate buffer (pH 4.0) containing 5% 2-propanol and 10 mM CM-β-CD resulted in the optimum separation of the metoprolol enantiomers and its acidic metabolite in human urine. Following a single metoprolol oral administration of 100 mg racemic metoprolol tartrate, stereoselective pharmacokinetic analysis showed that urinary acidic metabolite 3 of metoprolol accounted for 62.3% of the dose with an R/S ratio of 1.23 and urinary unchanged metoprolol 1 accounted for 6.3% of the dose with an R/S ratio of 0.72.     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.     

Bioanalytical chiral chromatographic technique and docking studies for enantioselective separation of meclizine hydrochloride: Application to pharmacokinetic study in rabbits

Enantiomeric resolution and molecular docking studies of meclizine hydrochloride on polysaccharide-based chiral stationary phase comprising cellulose tris(4-methylbenzoate) chiral selector (150 × 4.6 mm, 3.0 μm) were presented. The mobile phase used was acetonitrile:10mM ammonium bicarbonate (95:05, v/v). The developed technique was used to perform the enantioselective assay of meclizine hydrochloride in its marketed formulation. The elution order of meclizine hydrochloride enantiomers was determined by docking studies. Target compound was extracted from rabbit plasma using protein precipitation technique, followed by development of bioanalytical chiral separation method using the same matrix. Application of the method to determine pharmacokinetic parameters of meclizine hydrochloride enantiomers was performed using Phoenix WinNonlin 8.1 software. The results demonstrated stereoselective disposition of meclizine hydrochloride enantiomers in rabbits.     

Enantiomeric separation of bupropion enantiomers by electrokinetic chromatography: quantitative analysis in pharmaceutical formulations

The first CE method enabling the quantitation of the two enantiomers of bupropion was developed in this work. Electrokinetic chromatography (EKC) mode using cyclodextrins as chiral selectors was employed. A study on the enantiomeric separation ability of different neutral and anionic CDs was carried out. Sulfated-beta-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, temperature, and separation voltage on the enantiomeric separation of bupropion was also studied. The use of 10 mM sulfated-beta-CD in 50 mM borate buffer (pH 9.0) with an applied voltage of 30 kV and a temperature of 30 degrees C enabled the separation of the enantiomers of bupropion with high resolution (Rs > 7) and short analysis time (approximately 3.5 min). Finally, the method was successfully applied to the quantitation of bupropion in two pharmaceutical formulations.     

Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5‐s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 27:223–227, 2015. © 2014 Wiley Periodicals, Inc.     

Separation and determination of warfarin enantiomers in human plasma samples by capillary zone electrophoresis using a methylated β-cyclodextrin-containing electrolyte

A methyl β-cyclodextrin with a degree of substitution of 1.8 proved to be an effective chiral selector, among other cyclodextrins tested, for the separation of warfarin enantiomers by capillary electrophoresis. The operating conditions were optimized with respect to electrolyte composition (buffer pH, ionic strength, cyclodextrin concentration, methanol content) and applied voltage. The influence of a high ionic strength on the resolution was clearly shown. A baseline separation can be obtained in less than 15 min with an efficiency of ca. 250 000 theoretical plates. These conditions were applied to the determination of warfarin enantiomers in the plasma of patients under warfarin therapy. The limit of detection for the whole procedure (dichloromethane extraction followed by evaporation to dryness and capillary electrophoresis) was of the order of 0.2 mg/l (6.5 · 10⁻⁷M) of each enantiomer.     

Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-beta-cyclodextrin as chiral selector in partial filling mode

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Novel Stereoselective High‐Performance Liquid Chromatographic Method for Simultaneous Determination of Guaifenesin and Ketorolac Enantiomers in Human Plasma

A novel method was developed for the simultaneous determination of guaifenesin (GUA) and ketorolac tromethamine (KET) enantiomers in plasma samples. Since GUA probably increases the absorption of coadministered drugs (e.g., KET), it would be extremely important to monitor KET plasma levels for the purpose of dose adjustment with a subsequent decrease in the side effects. Enantiomeric resolution was achieved on a polysaccharide‐based chiral stationary phase, amylose‐2, as a chiral selector under the normal phase (NP) mode and using ornidazole (ORN) as internal standard. This innovative method has the advantage of the ease and reliability of sample preparation for plasma samples. Sample clean‐up was based on simply using methanol for protein precipitation followed by direct extraction of drug residues using ethanol. Both GUA and KET enantiomers were separated using an isocratic mobile phase composed of hexane/isopropanol/trifluoroacetic acid, 85:15:0.05 v/v/v. Peak area ratios were linear over the range 0.05–20 µg/mL for the four enantiomers S (+) GUA, R (–) GUA, R (+) KET, and S (–) KET. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines in terms of system suitability, specificity, accuracy, precision, robustness, and solution stability. Finally, this procedure was innovative to apply the rationale of developing a chiral high‐performance liquid chromatography (HPLC) procedure for the simultaneous quantitative analysis of drug isomers in clinical samples. Chirality 26:629–639, 2014. © 2014 Wiley Periodicals, Inc.     

Optimum operational conditions for chiral separation of tryptophan enatiomers using ligand exchange liquid chromatography

A simple and precise method for chiral separation of tryptophan enantiomers using high performance liquid chromatography with aligand exchange mobile phase was developed. Chiral separation was performed on a conventional C₁₈ column, using a mobile phase that consisted of a water-methanol solution (88∶12, v/v) containing 10 mmol/Ll-leucine and 5 mmol/L copper sulfate as a chiral ligand additive at a flow rate of 1.0 mL/min. This method allowed baseline separation of two enantiomers with a resolution of 1.84 in less than 30 min. The effect of various conditions, including concentration, type of ligand, organic modifier, pH, flow rate, and temperature, on enantioseparation were evaluated and chiral recognition mechanisms were investigated. Thermodynamic data (ΔΔH and ΔΔS) obtained by van't Hoff plots revealed that enantioseparation is an enthalpy-controlled process.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Lambda-carrageenan: A novel chiral selector for capillary electrophoresis

Lambda-carrageenan, a linear high molecular weight sulfated polysaccharide, has been employed as a chiral selector in capillary electrophoresis for the separation of enantiomers of weakly basic pharmaceutical compounds. The racemic compounds that were enantioresolved included propranolol, pindolol, tryptophanol, laudanosine and laudanosoline. In addition, the diastereomeric pair of cinchonine and cinchonidine were also resolved. Method conditions such as buffer pH, electrolyte concentration, column temperature, and chiral selector concentration were found to be important for improvement of enantioselectivity. © 1996 Wiley-Liss, Inc.     

Chiral separation of salbutamol and bupivacaine by capillary electrophoresis using dual neutral cyclodextrins as selectors and its application to pharmaceutical preparations and rat blood samples assay

An attempt for the simultaneous separation of salbutamol (Sal) and bupivacaine (Bup) enantiomers was performed by capillary elecytrophoresis with a dual mixture of neutral cyclodextrins as chiral selector. The influence on the separation of several parameters such as buffer composition, pH, the concentration ratio of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to dimethyl-beta-cyclodextrin (DM-beta-CD) was investigated. A better separation was obtained for Sal and Bup with the CD mixtures compared to the use of HP-beta-CD or DM-beta-CD alone. The best simultaneous separation of Sal and Bup enantiomers was achieved with a 20 mM HP-beta-CD and 20 mM DM-beta-CD at pH 2.5 in a triethanolamine (TEA)/phosphate buffer. This method-utilized chlorphenamine (Chl) as an internal standard was found to be linear in the range 0.5-100 microg/mL and 0.5-150 microg/mL for Sal and Bup enantiomers, respectively. The limits of detection for both enantiomers of Sal and Bup were 0.18 and 0.24 microg/mL, respectively. The proposed method was applied to monitor Sal and Bup enantiomers concentration change in rat blood samples obtained from a male rat after celiac doses administration 0-30 min of Sal and Bup racemate. The method could also be used to determine Sal enantiomers in a pharmaceutical aerosol.     

Enantiomer separation of 7-des-methyl-ormeloxifene using sulfated beta-cyclodextrin in countercurrent chromatography

The enantiomers of 7-des-methyl-ormeloxifene were separated by countercurrent chromatography (CCC) using sulfated beta-cyclodextrin as chiral selector, representing the first reported successful application of a cyclodextrin derivative in CCC-based resolutions. The choice of chiral selector relies on extreme separation factors observed in chiral capillary electrophoresis, and suitable CCC conditions were developed employing an analytical toroidal coil countercurrent chromatograph. Preparative separation of the enantiomers was performed using a conventional, preparative CCC-instrument. Copyright 1999 Wiley-Liss, Inc.