Chiral HPLC Separation of Rosiglitazone and its Main Metabolites and Studies on Their Racemization

Rosiglitazone (RSG) is marketed as a racemic mixture although the antidiabetic activity is essentially related to the (S)-enantiomer. The chiral center has an adjacent carbonyl group; therefore, the (R)-enantiomer could be transformed to the (S)-enantiomer or vice versa by keto-enolic tautomerism. The literature indicates that this racemization is slow enough to allow the evaluation of the properties of the isolated enantiomers. However, there is no information about the enantioselective kinetic disposition and metabolism of RSG. Additionally, there are no studies on the racemization of its metabolites. Considering these facts, a chiral HPLC method was developed and used for the first time to study the racemization of RSG and its main metabolites. Different conditions, including those used to evaluate the in vitro enantioselective metabolism, were employed. The simultaneous chiral separation of RSG and metabolites was achieved on a Chiralcel OJ-H column by employing methanol/ethanol (90:10, v/v) as mobile phase. The racemization studies showed that the half-life of RSG decreased more than 30 times when the temperature increased from 4 to 37 °C. It was also observed that the half-life of RSG changed from approximately 20 h at pH 3.5 to approximately 2 h at pH 7.4. The same profile was observed for its metabolites. Organic solvents and UV light did not present influence on the racemization process. In addition, a Complete Factorial Design was conducted to evaluate the influence of some parameters that can be changed during an in vitro metabolism study. The results obtained showed that the racemization occurs under in vitro metabolism conditions.     

A Validated LC Method for Determination of the Enantiomeric Purity of Darifenacin in Bulk Drug and Extended Release Tablets

A new and accurate chiral liquid chromatographic method has been developed for the determination of enantiomeric purity of darifenacin [(S)-enantiomer] in bulk drugs and extended release tablets. Normal phase chromatographic separation was performed on an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with n-hexane:ethanol:diethylamine (50:50:0.3, v/v/v) as mobile phase at a flow rate of 1.0 mL min⁻¹. The elution time was ~15 min. The resolution (R _s ) between the enantiomers was greater than four and interestingly the (R)-enantiomer was eluted prior to darifenacin in the developed method. The limit of detection (LOD) and limit of quantification (LOQ) for the (R)-enantiomer were 0.02 μg and 0.07 μg, respectively, for a 10 μL injection volume. The method was extensively validated in terms of linearity, precision and accuracy and satisfactory results were obtained. Robustness studies were also conducted. The sample solution stability of darifenacin was determined and the compound was found to be stable for a study period of 48 h.

     

Validated Chiral LC Method for the Enantiomeric Separation of Palonosetron Hydrochloride

A new and accurate chiral liquid chromatographic method has been developed for the separation of palonosetron hydrochloride (PALO) and its (R,R)-enantiomer in bulk drug samples with an elution time of about 20 min. The chromatographic separation was carried out by normal phase chromatography using an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with a mobile phase composed of n-hexane:ethanol:1,4 dioxane:trifluoroacetic acid:diethylamine (65:30:5:0.3:0.3, v/v) pumped at a flow rate of 1.0 mL min⁻¹. The resolution (R _s ) between the enantiomers was found to be greater than 3.0 and interestingly the (R,R)-enantiomer was eluted prior to the (S,S)-enantiomer (PALO) in the developed method. Mobile phase additives, trifluoroacetic acid and diethylamine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. The limit of detection (LOD) and limit of quantification (LOQ) of the (R,R)-enantiomer were found to be 0.03 and 0.1 μg respectively for 10 μL injection volume. The developed method shows excellent linearity (r ² > 0.999) over a range of LOQ to 0.3% for the (R,R)-enantiomer. The percentage recovery of the (R,R)-enantiomer in bulk drug samples ranged from 97.2 to 102.3 revealing good sensitivity of the developed method. Robustness studies were also carried out on the developed method.

     

A Validated LC Method for Determination of the Enantiomeric Purity of Darifenacin in Bulk Drug and Extended Release Tablets

A new and accurate chiral liquid chromatographic method has been developed for the determination of enantiomeric purity of darifenacin [(S)-enantiomer] in bulk drugs and extended release tablets. Normal phase chromatographic separation was performed on an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with n-hexane:ethanol:diethylamine (50:50:0.3, v/v/v) as mobile phase at a flow rate of 1.0 mL min^?1. The elution time was ~15 min. The resolution (R _s ) between the enantiomers was greater than four and interestingly the (R)-enantiomer was eluted prior to darifenacin in the developed method. The limit of detection (LOD) and limit of quantification (LOQ) for the (R)-enantiomer were 0.02 μg and 0.07 μg, respectively, for a 10 μL injection volume. The method was extensively validated in terms of linearity, precision and accuracy and satisfactory results were obtained. Robustness studies were also conducted. The sample solution stability of darifenacin was determined and the compound was found to be stable for a study period of 48 h.     

Rhodium-Catalyzed Enantioselective Synthesis,Structures, and Properties of Single and Double Azahelicene-Like Molecules

The enantioselective synthesis of aza[6] and [7]helicene-like molecules have been achieved by the cationic rhodium(I)/axially chiral biaryl bisphosphine complex-catalyzed intramolecular [2+2+2] cycloaddition of cyanodiynes. This protocol was successfully applied to the diastereo- and enantioselective synthesis of an S-shaped double aza[6]helicene-like molecule with a high ee value of 89 %. Although no epimerization and racemization were observed in the double carbo[6]helicene-like molecule at 80 °C, epimerization and racemization of the double aza[6]helicene-like molecule proceeded at 80 °C. This double aza[6]helicene-like molecule showed good fluorescent quantum yields and chiroptical responses under both neutral and acidic conditions.     

Enantiomeric Separation of Moxifloxacin and Its (R,R)-Isomer by Ligand-Exchange Chiral Chromatography

A new stereospecific LC method for the separation and quantification of moxifloxacin and its (R,R)-enantiomer in bulk drug was developed and validated by ligand-exchange liquid chromatography on a reversed phase column using aqueous mobile phase containing the chiral reagent l-isoleucine-Cu(II). The UV detector was operated at 293 nm. The flow rate of mobile phase was set at 0.9 mL min^?1. The achiral ODS column offers good separation of the two enantiomers in less than 20 min. The test concentration was 1,000 μg mL^?1 in the mobile phase. This method was capable of detecting the (R,R)-enantiomer of moxifloxacin up to 0.1 μg mL^?1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with the (R,R)-enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the (R,R)-enantiomer in bulk drug samples ranged from 98.1 to 104.4%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

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.     

Study on the Enantioselective Degradation of Imazethapyr in Soil by CE

A method was developed for the separation of imazethapyr enantiomers using capillary electrophoresis and large volume injection. Optimized buffer conditions were found using 6% hydroxypropyl-β-cyclodextrin as the chiral selector at pH 11 with an injection time of 20 s (0.5 psi). The limits of detection (LOD, S/N = 3) were 0.22 and 0.23 mg L⁻¹ for the two imazethapyr enantiomers (imazethapyr-I and imazethapyr-II) respectively. The relative standard deviation was less than 5%. This method was successfully used in the study of enantioselective degradation of this herbicide in field soil and the results suggested that imazethapyr-I degraded at a higher rate when compared with imazethapyr-II.

     

Characterization of racemization of chiral pesticides in organic solvents and water

Eight chiral pesticides, which were selected to cover different pesticide species and origins of chirality, were investigated to explore their chiral stability in organic solvents and water. Profenophos, fenamiphos, quizalofop-ethyl, dichlorprop-methyl (DCPP-methyl) and acetochlor were showed stable under all test conditions. However, significant racemization was observed for malathion, phenthoate and fenpropathrin in methanol, ethanol and water, but not in n-hexane, isopropanol, acetone or methylene chloride. The kinetic parameters (rate constant k and half-life T_1/2) of the abiotic racemization were calculated through a mathematical model of the first-order reaction. Furthermore, the extent of racemization varied among the solvents and was also affected by temperature dependence. The racemization of malathion, phenthoate and fenpropathrin in water was documented to be pH-dependent and took place more rapidly at pH 7.0 than at pH 5.8. The observed racemization was deduced to occur via a proton exchange process at the chiral center, and the relationship between the abiotic racemization and pesticide structure was further explored. Findings from this study are useful for better understanding enantioselectivity of chiral pesticides in environment and also for proper analysis, formulating or handling of enantiopure products.     

Separation of α-cyclohexylmandelic acid enantiomers using biphasic chiral recognition high-speed counter-current chromatography

This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether–water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5–22.0 mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (−)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (±)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85–88% yielding 186 mg of (+)-enantiomer and 190 mg of (−)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.     

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

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

     

Enantioselective noncovalent synthesis of hydrogen-bonded double-rosette assemblies

The noncovalent synthesis of enantiomerically pure hydrogen-bonded assemblies (M)- and (P)-1(3).(CA)(6) is described. These dynamic assemblies are of one single handedness (M or P), but do not contain any chiral components. They are prepared by using the "chiral memory" concept: the induction of supramolecular chirality is achieved through initial assembly with chiral barbiturates, which are subsequently replaced by achiral cyanurates. This exchange process occurs quantitatively and without loss of the M or P handedness of the assemblies. Racemization studies have been used to determine an activation energy for racemization of 105.9+/-6.4 kJ mol(-1) and a half-life time to racemization of 4.5 days in benzene at 18 degrees C. Kinetic studies have provided strong evidence that the rate-determining step in the racemization process is the dissociation of the first dimelamine component 1 from the assembly 1(3).(CA)(6). In addition to this, it was found that the expelled chiral barbiturate (RBAR or SBAR) acts as a catalyst in the racemization process. Blocking the dissociation process of dimelamines 1 from assembly 1(3).(CA)(6) by covalent capture through a ring-closing metathesis (RCM) reaction produces an increase of more than two orders of magnitude in the half-life time to racemization.     

A Validated LC Method for the Determination of Chiral Purity of (1S)-6,11-Dioxo-1,2,3,4,6,11-Hexahydropyridazino[1,2-b]Phthalazine-1-Carboxylic Acid: A Key Intermediate of Cilazapril

A simple and accurate normal phase liquid chromatographic method was developed for the determination of chiral purity of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, S-enantiomer used as key intermediate in the manufacturing of cilazapril bulk drug. Chromatographic separation between (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, and its opposite enantiomer (1R)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, R-enantiomer was achieved using a Chiralpak AD-H column using a mobile phase containing hexane, isopropyl alcohol and tri-fluoro acetic acid (80:20:0.1 v/v/v). The resolution between the two enantiomers was found to be more than 3.2. The limit of detection (LOD) and limit of quantitation (LOQ) of the R-enantiomer was 0.15 and 0.5 μg mL^?1, respectively, for 10 μL injection volume. The percentage recoveries of the R-enantiomer ranged from 96.5 to 105.3 in the bulk samples of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid. The test solution and mobile phase was observed to be stable up to 24 h after the preparation. The developed method was validated as per International Conference on Harmonization guidelines in terms of LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

A Validated Chiral LC Method for the Separation and Quantification of (S,R,S)-Enantiomer and (R,R,R)-Isomer of Aprepitant

A new and accurate chiral liquid chromatographic method has been developed for the separation and quantification of (S,R,S)-enantiomer (unwanted enantiomer) and (R,R,R)-isomer (key intermediate) of aprepitant in bulk drug and formulation samples of apprepitant. The elution time was approximately 20 min using an immobilized amylose-based chiral stationary phase (Chiralpak-IA). The mobile phase was n-hexane and ethanol (90:10, v/v) and was delivered at a flow rate of 1.0 mL min^?1. Detection was carried out with a wavelength set to 220 nm. The resolution factor between enantiomers was found to be greater than five. Limit of detection for both (S,R,S) enantiomer and (R,R,R) isomer of aprepitant was 0.035 µg, and limit of quantification for both (S,R,S) enantiomer and (R,R,R) isomers of aprepitant was 0.1 µg, for a 10 µL injection. The developed method showed excellent linearity (r > 0.999) for both isomers. When the method was applied to bulk drug samples and in pharmaceutical formulations recoveries were obtained ranging from 97.2 to 103.1%. Aprepitant sample solutions were found to be stable when characterized over a period of 48 h.     

In vivo metabolism for the hydroxylation of FK778 to the metabolite M3 in humans studied by enantioselective liquid chromatography/tandem mass spectrometry

A major active metabolite of malononitrilamide FK778 (an immunosuppressant under development) is labeled M3. Due to a chiral center created during in vivo metabolism, the exploration of enantiomer profiles in clinical samples is critical to the characterization of the immunosuppressive activity of M3. An enantioselective liquid chromatography method with detection by tandem mass spectrometry (LC/MS/MS) was developed for the resolution of M3 enantiomers. It was experimentally confirmed that no interconversion between the two enantiomers occurred during sample preparation. This new approach was applied to measure the enantioselectivity of the M3 metabolite in human plasma samples from kidney transplanted patients. The assay results of 91 in vivo human samples from three subjects showed a ratio of 57:43 for the (-)-enantiomer (the 2nd eluter) vs. the (+)-enantiomer (1st eluter), indicating that the enantiometabolism of FK778 through human enzymes is essentially non-specific.     

Enantioselective synthesis of the bisabolane sesquiterpene (+)-1-hydroxy-1,3,5-bisabolatrien-10-one and revision of its absolute configuration

The enantioselective synthesis of (S)-1-hydroxy-1,3,5-bisabolatrien-10-one 1 is here described. This sesquiterpene was prepared using (S)-3-(2-methoxy-4-methyl-phenyl)butan-1-ol as a chiral building block. Two different pathways were employed and both turned out to be high yielding, affording 1 in good chemical purity and without any racemization of the existing stereocenter. The spectroscopic data of the synthetic (S)-1 were in very good agreement with those reported for the natural compound, which was extracted from Juniperus formosana heartwood and from the leaves of J. chinensis. The positive sign of the measured optical rotation value of synthetic (S)-1 allows the unambiguous assignment of the absolute configuration of (+)-1 as the (S)-enantiomer. This finding corrects the previous configuration determination which indicated the opposite result. At last, since even (R)-3-(2-methoxy-4-methyl-phenyl)butan-1-ol is preparable in high enantiomer purity by mean of a different biocatalytic process, the formal synthesis of natural (R)-1 was also accomplished.     

Enantioselective synthesis of fatty acid amide hydrolase inhibitors with 1,3-disubstituted butan-2-one scaffold

Fatty acid amide hydrolase is a key enzyme in the inactivation of the analgesic and anti-inflammatory endocannabinoid anandamide. Previously, the chiral compound 1-(1H-benzotriazol-1-yl)-3-(4-phenylphenoxy)butan-2-one was identified as a potent inhibitor of fatty acid amide hydrolase and is therefore of interest as a potential agent against pain and inflammation. Two different approaches for the enantioselective synthesis of fatty acid amide hydrolase inhibitors with a 1,3-disubstituted butan-2-one scaffold were carried out. The first one uses the chiral epoxide 2-[1-(4-phenylphenoxy)ethyl]oxirane with an (R)- or (S)-configuration at the exocyclic stereocenter as central intermediates. These substances were obtained by separation of the non-stereoselectively synthesized epoxide into its racemic diastereomers by reversed phase chromatography followed by Jacobsen’s hydrolytic kinetic resolution of each enantiomer with the (S)-configured oxirane ring. Furthermore, a chiral pool based enantioselective synthesis was developed. In that case, the starting compound for both target enantiomers was methyl 3,4-O-isopropylidene-l-threonate. In comparison to the first approach, the chiral pool synthesis consisted of more steps, but generated the enantiomers with much better enantiomeric excess. Biological evaluation showed that the (R)-enantiomer inhibits isolated fatty acid amide hydrolase with a 200-fold higher activity than the (S)-enantiomer.     

A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R _s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min^?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL^?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

In Vitro and In Vivo Primary Metabolic Characterization of F18, a Novel Histone Deacetylase-6 (HDAC6) Inhibitor,Using UHPLC–QqQ–MS/MS and Q-TOF–MS Methods

F18, N-hydroxy-4-(2-methoxy-5-(methyl (2-methylquinazolin-4-yl) amino) phenoxy) butanamide, is a novel selective HDAC6 inhibitor with good antitumor activity. In the early drug development, drug-metabolism studies are a crucial and indispensable part. In this study, we proposed to evaluate the in vitro primary metabolism of F18 in phase Ι in liver microsomes from human, rat, dog, monkey and mouse and investigate the metabolite profile both in vitro and in vivo using LC–MS/MS methods. F18 showed high metabolic stability in human, rat, dog, monkey and mouse liver microsomes over 120 min, with t _1/2 >8 h in human, rat, and dog, and t _1/2 <3.5 h in monkey, with almost no clearance in mouse. Human cytochrome P450 (P450) phenotyping showed that F18 was predominantly metabolized by CYP2C9, CYP2E1, CYP2D6 and CYP3A4. The investigation of the effect of F18 on CYP enzymes in HLM demonstrated that this compound did not significantly inhibit CYP 1A2 (IC₅₀ >100 μM), was a moderate inhibitor of CYP3A4 (IC₅₀ = 1.63 μM) and had negligible effects on CYP3A1/2 activity in rats. The results will be valuable in understanding drug–drug interactions (DDI) when F18 is co-administered with other drugs. The metabolites of F18 were investigated in rat plasma, urine, feces and different liver microsomes in NADPH samples, yielding at least 11 metabolites in these biological samples. The prominent metabolic pathways were de-methylation, de-amination, de-oxidation and O-glucuronidation. In summary, this work provides the first clues regarding F18 metabolism, providing important information for comprehensive understanding of F18 metabolites.

     

Thermal racemization of biaryl atropisomers

Many biaryl compounds possess atropisomerism due to the steric hindrance of substituents at the ortho-position of the two aromatic moieties. Upon heating, atropisomers may have enough energy to surpass the rotational energy barrier and racemize. The thermal stability of five atropisomers was studied using chiral chromatography by following the change in enantiomeric excess ratio at different temperatures. The first order racemization reaction rate was obtained at a given temperature as the slope of the change in enantiomeric excess ratio versus time. For each atropisomer, the racemization rates at different temperatures led to the value of the rotational energy barrier for racemization, ΔG³, and to the racemization half lifetime, t_1/2, indicating the atropisomer thermal stability. Binaphthol started to racemize significantly at temperature of 190 °C and above while binaphthyldiamine was much more stable showing little or very minor racemization up to 210 °C. A chloro-substituted phenylamino-naphthol was very sensitive to thermal racemization starting at a low 40 °C.