High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high‐performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate‐based chiral derivatizing reagents, (S)‐1‐(1‐naphthyl) ethyl isothiocyanate, (S)‐NEIT, and (R)‐α‐methyl benzyl isothiocyanate, (R)‐MBIT. The diastereomers synthesized with (S)‐NEIT were enantioseparated by reversed‐phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)‐MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040–0.043 µg/mL for each of the diastereomers prepared with (S)‐NEIT. Copyright © 2013 John Wiley & Sons, Ltd.     

(S)‐Naproxen as a platform to develop chiral derivatizing reagent for reversed‐phase high‐performance liquid chromatographic enantioseparation of analytes having a carbonyl functional group

(S)‐Naproxen was used to synthesize a chiral reagent, (S)‐2‐(6‐methoxynaphthalen‐2‐yl)propanehydrazide, by itsreaction with hydrazine hydrate in the presence of dicyclohexylcarbodiimide as coupling agent. The reagent was characterized and its chiral purity was established. It was used as a chiral derivatizing reagent for the synthesis of hydrazone diastereomers, under microwave irradiation, of certain chiral aldehydes and ketones. The respective diastereomers were separated by reversed‐phase high‐performance liquid chromatography using a binary solvent combination containing trifluoroacetic acid. The diastereomers were detected at 231 nm. The method was validated for accuracy, precision, and limit of detection (LOD). For a series of hydrazones the LOD was found to be in the range 1.62–1.65 pmol/mL. Copyright © 2012 John Wiley & Sons, Ltd.     

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

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Application of optically pure amines as chiral auxiliaries to develop trichloro‐s‐triazine‐based new chiral derivatizing reagents for reversed‐phase high‐performance liquid chromatographic enantioseparation of dl‐selenomethionine

(R)‐(+)‐naphthylethyl amine and (S)‐(+)‐1‐benzyl‐3‐aminopyrrolidine were incorporated as chiral auxiliaries, by nucleophilic substitution of chlorine atoms, in cyanuric chloride (CC) or its 6‐butoxy derivative. There were obtained four new chiral derivatizing reagents (CDRs) as two dichloro and two monochloro triazine reagents. The CDRs so obtained were characterized and their optical purity was ascertained. Diastereomers of dl ‐selenomethionine were synthesized under microwave irradiation for 60 or 90 s (at 80% power of 800 W). Reversed‐phase high‐performance liquid chromatographic separation of diastereomers was carried out on a C₁₈ column using mixtures of acetonitrile with aqueous trifluoroacetic acid as mobile phase. The detection was made at 230 nm using a photodiode array detector. The separation behaviors in terms of retention times and resolutions were compared. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative study on the liquid chromatographic enantioseparation of cyclic β‐amino acids and the related cyclic β‐aminohydroxamic acids on Cinchona alkaloid‐based zwitterionic chiral stationary phases

The enantiomeric pairs of cis and trans stereoisomers of cyclic β‐aminohydroxamic acids and their related cis and trans cyclic β‐amino acids containing two chiral centers were directly separated on four structurally related chiral stationary phases derived from quinine and quinidine modified with (R,R)‐ and (S,S)‐aminocyclohexanesulfonic acids. Applying these zwitterionic ion‐exchangers as chiral selectors, the effects of the composition of the bulk solvent, the acid and base additives, the structures of the analytes, and temperature on the enantioresolution were investigated. To study the effects of temperature and obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range 5–50°C. The differences in the changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) were calculated from the linear van't Hoff plots derived from the ln α versus 1/T curves in the studied temperature range. Results thus obtained indicated enthalpy‐driven separations in all cases. The sequence of elution of the enantiomers was determined and found to be reversed when ZWIX(–)™ was changed to ZWIX(+)™ or ZWIX(–A) to ZWIX(+A).     

Chiral separation of isoxanthohumol and 8‐prenylnaringenin in beer,hop pellets and hops by HPLC with chiral columns

Xanthohumol, isoxanthohumol, and 8‐prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2‐propanol. Fractions of isoxanthohumol and 8‐prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8‐prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD‐H column with a mobile phase composed of hexane–ethanol (90:10, v/v) and a Chiralpak AD‐RH column with a mobile phase composed of methanol–2‐propanol–water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8‐prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2−10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5−5.5) during boiling.     

A novel C2 symmetric chiral stationary phase with N‐[(4‐Methylphenyl)sulfonyl]‐l‐leucine as chiral side chains

In this study, a series of chiral stationary phases based on N‐[(4‐methylphenyl)sulfonyl]‐l ‐leucine amide, whose enantiorecognition property has never been studied, were synthesized. Their enantioseparation abilities were chromatographically evaluated by 67 enantiomers. The chiral stationary phase derived from N‐[(4‐methylphenyl)sulfonyl]‐l ‐leucine showed much better enantioselectivities than that based on N‐(4‐methylbenzoyl)‐l ‐leucine amide. The construction of C₂ symmetric chiral structure greatly improved the enantiorecognition performance of the stationary phase. The C₂ symmetric chiral stationary phase exhibited superior enantioresolutions to other chiral stationary phases for most of the chiral analytes, especially for the chiral analytes with C₂ symmetric structures. By comparing the enantioseparations of the enantiomers with similar structures, the importance of hydrogen bond interaction, π–π interaction, and steric hindrance on enantiorecognition was elucidated. The enantiorecognition mechanism of trans‐N,N′‐(1,2‐diphenyl‐1,2‐ethanediyl)bis‐acetamide, which had an excellent separation factor on the C₂ symmetric chiral stationary phase, was investigated by ¹H‐NMR spectroscopy and 2D ¹H‐¹H nuclear overhauser enhancement spectroscopy.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

Rapid quantification of amlodipine enantiomers in human plasma by LC‐MS/MS: application to a clinical pharmacokinetic study

A rapid, simple, specific and sensitive LC‐MS/MS method has been developed and validated for the enantiomeric quantification of amlodipine (AML) isomers [R‐amlodipine (R‐AML) and S‐amlodipine (S‐AML)] with 200 μL of human plasma using R‐AML‐d₄ and S‐AML‐d₄ as corresponding internal standards as per regulatory guidelines. A simple liquid–liquid extraction process was used to extract these analytes from human plasma. The total run time was 3.5 min and the elution of R‐AML, S‐AML, R‐AML‐d₄ and S‐AML‐d₄ occurred at 1.62, 2.51, 1.63 and 2.53 min, respectively. This was achieved with a mobile phase consisting of 0.2% ammonia–acetonitrile (20:80, v/v) at a flow rate of 1 mL/min on a Chiralcel OJ RH column. A linear response function was established for the range of concentrations 0.1–10 ng/mL (r >0.998) for each enantiomer. The intra‐ and inter‐day precision values for both enantiomers met the acceptance criteria. Both enantiomers were stable in a set of stability studies, viz. bench‐top, auto‐sampler, freeze–thaw cycles and long‐term. The current assay was successfully applied to a pharmacokinetic study to quantitate AML enantiomers following oral administration of 10 mg AML tablet to humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Enantioseparation of 3‐phenyllactic acid by chiral ligand exchange countercurrent chromatography

3‐Phenyllactic acid is an antimicrobial compound with broad‐spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3‐phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3‐phenyllactic acid with a synthesized chiral ligand. A two‐phase solvent system was composed of n‐butanol/hexane/water (0.4:0.6:1, v/v/v) to which N‐n‐dodecyl‐l ‐hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid–liquid extraction. Baseline enantioseparation of racemic 3‐phenyllactic acid by analytical high‐speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3‐phenyllactic acid reached 99.0%, as determined by chiral high‐performance liquid chromatography.     

Determination of enantiomeric purity of S‐amlodipine by chiral LC with emphasis on reversal of enantiomer elution order

An LC method was developed and prevalidated for the enantiomeric purity determination of S‐amlodipine in polar organic solvent chromatography using a chlorine‐containing cellulose‐based chiral stationary phase (CSP). The concentration of formic acid (FA) (0.01–0.2%) in the mobile phase containing acetonitrile as the main solvent was found to influence the elution order of amlodipine enantiomers as well as the enantioresolution. A reversal of the enantiomer elution order of amlodipine was only observed with chiral stationary phases with both electron‐withdrawing (chloro) and electron‐donating groups (methyl) on the phenyl moieties of the chiral selector, namely cellulose tris(3‐chloro‐4‐methylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate). The highest enantioresolution (R_s: 4.1) value was obtained at the lowest FA concentration (0.01%) using cellulose tris(4‐chloro‐3‐methylphenylcarbamate) as the chiral selector and the enantiomeric impurity, R‐amlodipine, eluted first under these conditions. Therefore, the mobile phase selected for the prevalidation of the method consisted of ACN/0.1% DEA/0.01% FA and the temperature was set at 25°C. The method was prevalidated by means of the strategy based on the total measurement error and the accuracy profile. The method was found to be selective and the limit of quantification was found to be about 0.05% for R‐amlodipine, while the limit of detection was close to 0.02%.     

Enantiomeric separation of precursors of rho-kinase inhibitors by HPLC on polysaccharide-based chiral stationary phases

Summary The separation of enantiomers of substituted cyclohexanecarboxamides, benzamides and chemical precursors of Rho-kinase inhibitors was achieved using derivatized polysaccharide-based chiral stationary phases. Separations were by normal phase HPLC with a mobile phase ofn-hexane-alcohol (methanol, ethanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), tris-methylbenzoate (Chiralcel OJ), a silica-based amylose tris-(S)-1-phenylethylcarbamate (Chiralpak AS), or tris-3,5-dimethylphenylcarbamate (Chiralpak AD). The effects of cencentration of various aliphatic alcohols in the mobile phase were investigated. The effect of structural features on the discrimination between the enantiomers was examined. The isolation of milligram amounts of enantiomers of two derivatives was performed on an analytical column by multiple repetitive injections under overload conditions.     

Binding modes identification through molecular dynamic simulations: A case study with carnosine enantiomers and the Teicoplanin A2‐2‐based chiral stationary phase

In the present study, an in silico methodology able to define the binding modes adopted by carnosine enantiomers in the setting of the chiral recognition process is described. The inter‐ and intramolecular forces involved in the enantioseparation process with the Teicoplanin A2‐2 chiral selector and carnosine as model compound are successfully identified. This approach fully rationalizes, at a molecular level, the (S) < (R) enantiomeric elution order obtained under reversed‐phase conditions. Consistent explanations were achieved by managing molecular dynamics results with advanced techniques of data analysis. As a result, the time‐dependent identification of all the interactions simultaneously occurring in the chiral selector‐enantiomeric analyte binding process was obtained. Accordingly, it was found that only (R)‐carnosine is able to engage a stabilizing charge–charge interaction through its ionized imidazole ring with the carboxylate counter‐part on the chiral selector. Instead, (S)‐carnosine establishes intramolecular contacts between its ionized functional groups, that limit its conformational freedom and impair the association with the chiral selector unit.     

Synergetic mechanism and enantioseparation of aromatic β‐amino acids by biphasic chiral high‐speed counter‐current chromatography

A biphasic chiral recognition system based on chiral ligand exchange with Cu(II)‐N‐n‐dodecyl‐L‐proline and hydroxypropyl‐β‐cyclodextrin as an additive was developed to enantioseparate aromatic β‐amino acids by high‐speed counter‐current chromatography. The biphasic chiral recognition system was established with an n‐butanol/water (1:1, v/v) solvent system by adding N‐n‐dodecyl‐L‐proline and Cu(II) ions to the organic phase and hydroxypropyl‐β‐cyclodextrin to the aqueous phase. Several separation parameters, such as temperature, pH value, and chiral selector concentration, were systematically investigated by enantioselective liquid–liquid extraction. Under the optimal separation conditions, 54.5 mg of (R,S)‐β‐phenylalanine and 74.3 mg of (R,S)‐β‐3,4‐dimethoxyphenylalanine were baseline enantioseparated. More importantly, the synergistic enantiorecognition mechanism, based on the Cu(II)‐N‐n‐dodecyl‐L‐proline and hydroxypropyl‐β‐cyclodextrin, was discussed for the first time.     

Development and validation of an enantioselective LC‐MS/MS method to quantify enantiomers of (±)‐TAK‐700 in rat plasma: lack of in vivo inversion of (+)‐TAK‐700 (Orteronel) to its antipode

A highly sensitive, specific and enantioselective assay has been developed and validated for the estimation of TAK‐700 enantiomers [(+)‐TAK‐700 and (?)‐TAK‐700] in rat plasma on LC‐MS/MS‐ESI in the positive‐ion mode. Liquid–liquid extraction was used to extract (±)‐TAK‐700 enantiomers and IS (phenacetin) from rat plasma. TAK‐700 enantiomers were separated using methanol and 5 mm ammonium acetate (80:20, v/v) at a flow rate of 0.7 mL/min on a Chiralcel OJ‐RH column. The total run time was 7.0 min and the elution of (+)‐TAK‐700, (?)‐TAK‐700 and IS occurred at 3.71, 4.45 and 4.33 min, respectively. The MS/MS ion transitions monitored were m/z 308.2 → 95.0 for TAK‐700 and m/z 180.2 → 110.1 for IS. The standard curves for TAK‐700 enantiomers were linear (r² > 0.998) in the concentration range 2.01–2015 ng/mL for each enantiomer. The inter‐ and intra‐day precisions were in the ranges 3.74–7.61 and 2.06–8.71% and 3.59–9.00 and 2.32–11.0% for (+)‐TAK‐700 and (?)‐TAK‐700, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method was applied to the study of stereoselective oral pharmacokinetics of (+)‐TAK‐700 and it was unequivocally demonstrated that (+)‐TAK‐700 does not undergo chiral inversion to its antipode in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

High‐performance liquid chromatographic enantioseparation of isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids on a chiral ligand‐exchange stationary phase

The application of a chiral ligand‐exchange column for the direct high‐performance liquid chromatographic enantioseparation of unusual β‐amino acids with a sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate‐Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline‐fused 2‐aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5–45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from –2.3 to 2.2 kJ/mol, Δ(ΔS°) from –3.0 to 7.8 J mol^?1 K^?1 and –Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy‐ and entropy‐controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.     

Enantioselective HPLC analysis of escitalopram oxalate and its impurities using a cellulose-based chiral stationary phase under normal- and green reversed-phase conditions

Normal-phase and reversed-phase high-performance liquid chromatography methods for the separation of the active pharmaceutical ingredient escitalopram from its (R)-enantiomer impurity have been developed on the cellulose-based Chiralcel OJ-H chiral stationary phase. Both methods share two features: they use ethanol as a cosolvent and are able to give a complete enantioseparation without interference from other associated chiral impurities. With the green eluent mixture ethanol–water–diethylammine 70:30:0.1 (v/v/v), the resolution between escitalopram and (R)-enantiomer was 2.09 at 30°C. The limits of quantification for the (S) and (R) enantiomers were 4.5 and 3.8 μg mL⁻¹, respectively.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.     

Synthesis and application of a chiral ionic liquid functionalized β‐cyclodextrin as a chiral selector in capillary electrophoresis

A novel chiral ionic liquid functionalized β‐cyclodextrin, 6‐O‐2‐hydroxpropyltrimethylammonium‐β‐cyclodextrin tetrafluoroborate ([HPTMA‐β‐CD][BF₄]), was synthesized and used as a chiral selector in capillary electrophoresis. [HPTMA‐β‐CD][BF₄] not only increased the solubility in aqueous buffer in comparison with the parent compound, but also provided a stable reversal electroosmotic flow, and the enantioseparation of eight chiral drugs was examined in phosphate buffer containing [HPTMA‐β‐CD][BF₄] as the chiral selector. The effects of the [HPTMA‐β‐CD][BF₄] concentration and the background electrolyte pH were studied. Moreover, the chiral separation abilities of β‐CD and [HPTMA‐β‐CD][BF₄] were compared and possible mechanisms for the chiral recognition of [HPTMA‐β‐CD][BF₄] are discussed. Copyright © 2013 John Wiley & Sons, Ltd.