Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.     

Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.     

2D Ising Model for Adsorption-induced Enantiopurification of Racemates

Mechanisms for the spontaneous transformation of achiral chemical systems into states of enantiomeric purity have important ramifications in modern pharmacology and potential relevance to the origins of homochirality in life on Earth. Such mechanisms for enantiopurification are needed for production of chiral pharmaceuticals and other bioactive compounds. Previously proposed chemical mechanisms leading from achiral systems to near homochirality are initiated by a symmetry-breaking step resulting in a minor excess of one enantiomer via statistical fluctuations in enantiomer concentrations. Subsequent irreversible processes then amplify the majority enantiomer concentration while simultaneously suppressing minority enantiomer production. Herein, equilibrium adsorption of amino acid enantiomer mixtures onto chiral and achiral surfaces reveals amplification of surface enantiomeric excess relative to the gas phase; i. e. enantiopurification of chiral adsorbates by adsorption. This adsorption-induced amplification of enantiomeric excess is shown to be well-describe by the 2D Ising model. More importantly, the 2D-Ising model predicts formation of homochiral monolayers from adsorption of racemic mixtures or prochiral molecules on achiral surfaces; i. e. enantiopurification with no apparent chiral driving force.     

Validation of a method using an achiral liquid chromatography sorbent and a circular dichroism detector. Analysis of the efaroxan enantiomers

The known HPLC method using an achiral C8 silica sorbent and a circular dichroism (CD) detector for the determination of efaroxan enantiomeric excess has been validated. After optimization of the mobile phase, the enantiomers were detected at 278 nm offering maximum ellipticity between two optically active forms. The calibration curve of the anisotropy factor (g) versus the enantiomeric excess was linear with a correlation coefficient (r2) of 0.9985. The accuracy of the method was assessed by comparing the enantiomeric excess obtained by measuring the g factor (C8 column, CD and UV detections) with those determined by enantioselective HPLC (Chiralpak AD-H column, UV detection). Statistical tests (level of confidence of 95%) were assessed to compare the two orthogonal methods. The straight line gave a correlation coefficient of 0.9995, an intercept not significantly different from zero (0.0549) and a slope of 1.026. The precision evaluated on retention time (RSD<0.6%), g factor (RSD<8.3%) and CD peak area (RSD<7.5%) was suitable both in term of intra- and inter-day precisions. The proposed method has the advantages of being fast and precise without using expensive chiral column. Non-enantioselective HPLC-CD was suitable for the simultaneous determination of the optical and chemical purity of efaroxan.     

Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions

High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol–water–DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76–26.28 μg mL⁻¹ for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL⁻¹, respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol–DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of ≥99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.     

Simultaneous determination of optical and chemical purities of a drug with two chiral centers by liquid chromatography-circular dichroism detection on a non-chiral stationary phase

The need for a rapid and cheap chromatographic technique for the simultaneous determination of diastereoisomeric and chemical purities of a drug has led to develop a non-enantioselective HPLC method on a porous graphitic carbon (PGC) sorbent and by using circular dichroism (CD) detection. Among a lot of non-chiral chromatographic supports tested, PGC was the only one allowing the separation of all benzoxathiepin diastereoisomers. After optimization of the chromatographic conditions, a mobile phase composed of chloroform, acetonitrile and methanol was suitable to elute the diastereoisomers with their related chemical impurities in less than 20 min by gradient mode. CD detection was found to be compatible with elution gradient and both the UV, CD and g anisotropy factor signals were recorded without disturbance. The determination of chemical purity was achieved with UV detection (254 nm) whereas the diastereoisomeric purity was determined using CD detection (258 nm) by plotting the anisotropy factors (R,S-S,R and R,R-S,S) versus the enantiomeric excess of each couple of enantiomers. A mathematic model was developed in order to express the diastereoisomeric excess versus the enantiomeric excess of each couple of enantiomers. Hence, the HPLC-UV/CD method was convenient to give access simultaneously to the optical and chemical purities of a chiral drug.     

Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.     

High-performance liquid chromatographic and capillary electrophoretic methods for the quantitation of the (S)-terbutaline in (R)-terbutaline

Summary The resolution of chiral drugs, metabolites and related substances continues to be an important area in pharmaceutical analysis. Two methods for the optical purity testing of (R)-(-)-terbutaline were developed, namely capillary electrophoresis using hydroxypropyl-β-cyclodextrin and high-performance liquid chromatography using a chiral stationary phase. Validation data such as linearity, recovery, detection limit, and precision of the two methods are presented. The detection limit of (S)-terbutaline in (R)-terbutaline was 0.05% by the HPLC method and 0.03% by the CE method. The was generally good agreement between the HPLC and CE results. These methods were found to be applicable as a practical quality control method for the enantiomeric purity determination of(R)-terbutaline.     

Chiral capillary electrophoretic determination of the enantiomeric purity of tetrahydronaphthalenic derivatives, melatoninergic ligands, using highly sulfated beta-cyclodextrins

Using cyclodextrin capillary zone electrophoresis (CD-CZE), baseline separation of synthetic tetrahydronaphthalenic derivatives, potential melatoninergic compounds, was achieved. A method for the enantioresolution of these tetralins and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters such as the nature and concentration of the chiral selectors, buffer pH, organic modifiers, temperature and applied voltage were investigated. The use of charged CDs provides a driving force for our neutral compounds in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-beta-CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution of three tetralin compounds was obtained using 25 mM phosphate buffer at pH 2.5 containing 2.5% w/v of highly S-beta-CD at 25 degrees C with an applied field of 0.25 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of linearity, sensitivity, accuracy and recovery. The enantiomeric purity for the three molecules was determined and the detection limit of enantiomer impurities is about 0.3-0.6%.     

高效液相色谱-圆二色检测法分析甲霜灵的对映体纯度

以手性农药甲霜灵为研究对象,使用非手性高效液相色谱在不拆分对映体的条件下,利用圆二色检测器所测的各向异性系数(g)测定手性对映体纯度。实验结果表明,g与对映体过剩率(ee)具有良好的线性关系;通过比较g所测ee与手性色谱所测的ee,二者所测ee相对平均偏差小于3.0%,说明该方法具有较高的准确性,可应用于手性化合物对映体纯度的测定。     

Supercritical fluid extraction: a novel method for the resolution of tetramisole

A new resolution method, based on the selective distribution of enantiomers between a chiral solid and an achiral supercritical fluid phase, is reported. The chiral solid phase is formed from the optically active dicarboxylic acid derivative, (2R,3R)-O,O′-dibenzoyltartaric acid, and the racemic base (tetramisole). A new method is also described for the enrichment of enantiomeric mixtures which have an enantiomeric ratio other than 1:1. This is based on the partial salt formation of the enantiomeric mixture with an achiral substance, which is then followed by supercritical fluid extraction of the free enantiomer. The extract has an enantiomeric composition which is different from the starting mixture. The method is applied to an enantiomeric mixture of tetramisole with hydrochloric acid.     

Simultaneous chiral discrimination of multiple profens by cyclodextrin-modified capillary electrophoresis in normal and reversed polarity modes

Simultaneous enantioseparations of nine profens for their accurate chiral discrimination were achieved by capillary electrophoresis (CE) in the normal polarity (NP) mode with a single cyclodextrin (CD) system and in the reversed polarity (RP) mode with a dual CD system. The single CD system in the NP mode employed heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) added at 75 mM-100 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) as the optimum run buffer. The dual CD system operated in the RP mode used 30 mM TMbetaCD and 1.0% anionic carboxymethyl-beta-cyclodextrin dissolved in pH 3.0, 100 mM phosphoric acid-triethanolamine buffer containing 0.01% hexadimethrine bromide added to reverse the electroosmotic flow. Fairly good enantiomeric resolutions and the opposite enantiomer migration orders were achieved in the two modes. Relative migration times to internal standard under respective optimum conditions were characteristic of each enantiomer with good precision (< 2% relative standard deviation, RSD), thereby enabling to crosscheck the chemical identification of profens and also their accurate chiralities. The method linearity in the two modes was found to be adequate (r > or = 0.9991) for the chiral assay of the profens investigated. Simultaneous enantiomeric purity test of ibuprofen, ketoprofen and flurbiprofen in a mixture was feasible in a single analysis by the present method.     

手性溶剂-NMR法测定2,4-滴丙酸对映体纯度的定量分析

研究了手性溶剂法测定2,4-滴丙酸的^1H、^13C谱,在满足NMR准确定量所要求的分离度和信噪比的条件下,能准确测定手性化合物的对映体纯度。比较了以对映体百分含量（R％）和对映体过量（ee）表示手性农药的对映体纯度的差别,发现以对映体百分含量代替对映体过量来表示手性农药的对映体纯度更为准确。     

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859–4.524 μg/mL was found to be R² = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.     

Packed column supercritical fluid chromatography of a peroxysome proliferator-activating receptor agonist drug. Achiral and chiral purity of substance, formulation assay and its enantiomeric purity

This paper describes packed column supercritical fluid chromatography (SFC) for the analysis of a peroxysome proliferator-activating gamma-receptor agonist that is a carboxylic acid. Evaluation of conditions for the separation of this candidate drug and related compounds in bulk substance is described. A Chiralcel OD column was used for this purpose due to its high selective retention of related substances and relative inertness, though the enantioselectivity was negligible, with methanol as polar modifier. A high enantioselectivity was obtained on Chiralpak AD and it was possible to determine the enantiomeric purity within 10 min on a 5 cm short column. Both the achiral and the chiral systems were run without acid additive in the mobile phase and the level of detection of impurities by area was about 0.1%. For the analysis of samples dissolved in water, without any isolation step, 2-propanol was used as modifier. Due to the column surface activity, evidently generated by injected water, citric acid 1 mM was included as additive in the 2-propanol in order to maintain symmetric and undistorted peak shape. The detection limit for the assay was 21 microg mL(-1) (50 nmol mL(-1)) for 5 microL injected (R.S.D. 6.4%, n = 8). A 5 cm short Chiralcel OD column was used. Determination of enantiomeric purity of the drug in aqueous samples required increased sensitivity. The sample was acidified and extracted into a small volume of 1-pentanol, out of which 25 microl was analyzed by SFC. The minor enantiomer at the 3% (w/w) level added could be confirmed. Its ratio remained constant during the procedure as measured relative to a reference solution in organic media.     

Enantio- and chemo-selective HPLC separations by chiral-achiral tandem-columns approach: the combination of CHIROBIOTIC TAG and SCX columns for the analysis of propionyl carnitine and related impurities

We describe a new tandem-columns chiral-achiral HPLC arrangement by using a chiral column (CHIROBIOTIC TAG) connected in series with an achiral column (Spherisorb S5 SCX), based on a strong cationic exchange mechanism; this approach is very useful for the analysis of chiral molecules, containing cationic groups in their structures. We used this special combination to develop an easy and convenient procedure for the enantio- and chemo-selective dosage of propionyl L-carnitine (1) and relative impurities (2-6), which allowed for the simultaneous separation and quantitation within 30 min. Under the best chromatographic conditions (acetonitrile-10 mM sodium dihydrogen phosphate 65:35, v/v (pHa 6.80) as the mobile phase and UV detection at 205 nm], all the individual peaks were well separated. The applicability of the method, fully validated, was demonstrated by the analysis of a pharmaceutical batch of propionyl L-carnitine, where we found the following contents: 98.5% for 1 (drug substance); 0.15% for 3; 0.1% for 5 and 0.2% for 6. The enantiomeric excess (e.e.%) measured for the drug substance was 98.9%. Finally, a single mixed-bed column, packed with a binary mixture of the chiral and achiral phases, in a 1:1 ratio, gave similar chromatographic results as the tandem-columns approach, and thus, offered an easy alternative solution to the separation of the considered mixture.     

Analytical and semipreparative enantioseparation of 9-hydroxyrisperidone, the main metabolite of risperidone, using high-performance liquid chromatography and capillary electrophoresis. Validation and determination of enantiomeric purity

The HPLC semipreparative enantioseparation of 9-hydroxyrisperidone (9-OHRisp) was studied by optimizing various experimental conditions: the nature of the chiral stationary phase (CSP), mobile phase composition, temperature and analyte loading. This semipreparative enantioseparation was successfully completed using the polysaccharide Chiralcel OJ chiral stationary phase and a n-hexane/ethanol/methanol (50/35/15, v/v/v) ternary mobile phase. To assess the enantiomeric purity of both isolated isomers, three analytical methods using UV detection were developed and validated: one CE method using dual cyclodextrin mode and two HPLC methods using either the Chiralcel OJ CSP in normal-phase mode or the alpha-acid glycoprotein (alpha-AGP) CSP in reversed-phase mode. The three methods make it possible to obtain excellent enantioseparations (R(s) >3) with analysis times lower than 15 min, and the calculated limits of detection allow for the determination of minor enantiomeric impurities (0.1%). Enantiomeric purity obtained for dextrorotatory and levorotatory enantiomers was superior to 99.9% and equal to 98.9%, respectively, which proved the success of the semipreparative enantioseparation. A brief comparison of the performances of the analytical methods completes this work.     

Quantitative analysis of three chiral pesticide enantiomers by high-performance column liquid chromatography

Methods for the enantiomeric quantitative determination of 3 chiral pesticides, paclobutrazol, myclobutanil, and uniconazole, and their residues in soil and water are reported. An effective chiral high-performance liquid chromatographic (HPLC)-UV method using an amylose-tris(3,5-dimethylphenylcarbamate; AD) column was developed for resolving the enantiomers and quantitative determination. The enantiomers were identified by a circular dichroism detector. Validation involved complete resolution of each of the 2 enantiomers, plus determination of linearity, precision, and limit of detection (LOD). The pesticide enantiomers were isolated by solvent extraction from soil and C18 solid-phase extraction from water. The 2 enantiomers of the 3 pesticides could be completely separated on the AD column using n-hexane isopropanol mobile phase. The linearity and precision results indicated that the method was reliable for the quantitative analysis of the enantiomers. LODs were 0.025, 0.05, and 0.05 mg/kg for each enantiomer of paclobutrazol, myclobutanil, and uniconazole, respectively. Recovery and precision data showed that the pretreatment procedures were satisfactory for enantiomer extraction and cleanup. This method can be used for optical purity determination of technical material and analysis of environmental residues.     

Direct determination of the enantiomeric purity of oxyphenonium using chiral HPLC with post-column extraction detection

Summary A method is described for the determination of the enantiomeric purity (enantiomeric excess) of the anticholinergic drug oxyphenonium. The method for this quaternary ammonium compound is based on the direct HPLC analysis with a chiral stationary phase. Two kinds of 1-acid glycoprotein-bonded phases were used.For the detection a post-column extraction with fluorescence detection of the ion-pair counter ion dimethoxyantracene sulphonate was used.     

Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated beta-cyclodextrins

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S-alpha-CD, S-beta-CD or S-gamma-CD at 30 degrees C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng.mL(-1) for the first enantiomer.