High-performance liquid chromatographic enantioseparation of (R,S)-fluoxetine using Marfey's reagent and (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as chiral derivatizing reagents along with direct thin-layer chromatographic resolution and isolation of enantiomers using L-tartaric acid as mobile phase additive

Chiral assay of enantiomers of fluoxetine was achieved in pharmaceutical formulations using direct and indirect methods. L-tartaric acid was used as a mobile phase additive in thin-layer chromatography; the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (R,S)-flouxetine was derivatized with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE], Marfey's reagent and 1-fluoro-2,4-dinitrophenyl-L-methionine amide (FDNP-L-Met-NH?. The diastereomers were separated using RP-HPLC. The effect of flow rate and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP-L-Met-NH? were also separated by RP-TLC.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Chiral separation of methadone, 2-ethylidene- 1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) by capillary electrophoresis using cyclodextrin derivatives

A stereoselective method was developed for the simultaneous determination of methadone and its two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) by capillary electrophoresis. Five beta-cyclodextrin (betaCD) background electrolyte (BGE) additives were evaluated for resolution efficiency. The conditions for baseline resolution of each of the three enantiomer pairs was determined to be 1 mM heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DMbetaCD) in 100 mM phosphate at pH 2.6. This method represents the first successful method for the resolution of the six enantiomers associated with the metabolism of methadone. The utilisation of doubly coated capillaries in conjunction with betaCD derivatives for a faster separation of the methadone-related enantiomers is also reported. The coated capillaries were prepared using a polycation of poly(diallyldimethylammonium chloride) (PDDAC) and a polyanion of dextran sulfate. Baseline resolution of the methadone enantiomers was achieved with a BGE of 8 mM (2-hydroxy)propyl-beta-cyclodextrin (HPbetaCD) in 100 mM phosphate at pH 2.6. The migration times for the stereoselective methadone separation were approximately 4 min, which represented a reduction by a factor of approximately three, compared to that attained using analogous conditions with the uncoated capillary.     

Selectivity tuning of serially coupled (S,S) Whelk-O 1 and (R,R) Whelk-O 1 columns in HPLC

The selectivity tuning of two columns coupled in series is investigated in chiral high-performance liquid chromatography. Two columns with reversal enantioselectivities [(R,R) Whelk-O 1 and (S,S) Whelk-O 1] are coupled in series via a T connector. Selectivity of such a column series is tuned by varying the mobile phase flows in the individual columns. The flow ratio necessary for the required selectivity is calculated on the basis of retention factors measured on the individual columns. The performance of this method for adjusting the required selectivity is studied by the separation of enantiomers of alkoxy substituted esters of phenylcarbamic acid. It is demonstrated that the change of the mobile phase flows in the individual columns enables change in the elution order of enantiomers.     

Illustration of a simple and versatile scheme for reversing enantiomeric elution order and facilitating enantiomeric impurity determination in capillary electrophoresis

Determination of enantiomeric purity is most often done under overload conditions, which leads to deformed peaks. In general, the best resolutions are obtained when the small peak appears before the large peak in the electropherogram. To be able to determine the R(+)-impurity in the S(-)-form as well as the S(-)-impurity in the R(+)-form the elution orders have to be reversed. The present paper describes reversal of enantiomeric elution order for the basic analyte propranolol and the acidic analyte ibuprofen. For propranolol, a charged heptakis-(6-sulfo)-beta-cyclodextrin (CD) is used in the background electrolyte. For ibuprofen, a mix of the charged heptakis-(6-sulfo)-beta-CD and the uncharged heptakis-(2,3,6-tri-O-methyl)-beta-CD is used in the background electrolyte. The use of a coated capillary and reversal of the polarity shift the elution order, buffer composition is unchanged in both cases. The enantiomers of propranolol and ibuprofen are well separated on both the coated and uncoated capillaries. Detection limits of enantiomer impurities are investigated using spiked samples of both propranolol and ibuprofen.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and quantification of (polymeric) hindered-amine light stabilizers in polymers using pyrolysis-gas chromatography-mass spectrometry and liquid chromatography-ultraviolet absorbance detection-evaporative light scattering detection

Direct analysis of polymers containing polymeric hindered amine light stabilizers (HALS) by using pyrolysis coupled to GC-MS is applied successfully for fast and straightforward identification of these HALS additives. Each of the HALS additives shows different pyrolysis gas chromatograms containing characteristic pyrolysis products. As a result, HALS additives with very similar chemical structures, e.g. Chimassorb 944 and Chimassorb 2020, can be distinguished. A HPLC method with both ultraviolet (UV) and evaporative light scattering detection (ELSD) is developed to quantify the various HALS additives in extracts of polymers. The critical factor of the HPLC method is the use of a basic amine, like n-hexylamine, as a solvent additive to facilitate the elution of HALS additives. The various HALS additives can be distinguished according to retention time and peak shape and by using different detection methods. The suitability of the developed methods is demonstrated by the analytical performance of the HPLC method and the identification and determination of the actual content of HALS additives in polyolefines using pyrolysis GC-MS and HPLC. The HPLC method can also be used for the determination of the specific migration of HALS additives from food contact materials.     

A fast CE method for the achiral separation of methadone and its major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline

The utilization of dynamic doubly coated capillaries for a fast separation of methadone and its two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) was investigated. The coated capillaries were prepared using a polycation of poly(diallyldimethylammonium chloride) and a polyanion of dextran sulfate. A fast achiral separation was developed using the coated capillaries with a BGE of 100 mM phosphate buffer at pH 2.6. Complete achiral separation of methadone, EDDP and EMDP was achieved, with migration times of approximately 4 min. The method offers considerable advantages with respect to BGE simplicity and analysis time compared to previously published CE methods for methadone and its related analytes.     

Enantiomeric separation of some pharmaceutical intermediates and reversal of elution orders by high-performance liquid chromatography using cellulose and amylose tris(3,5-dimethylphenylcarbamate) derivatives as stationary phases

Several pairs of enantiomers of pharmaceutical intermediates were separated by HPLC directly on cellulose and amylose tris(3,5-dimethylphenylcarbamate) derivatives (Chiralcel OD and Chiralpak AD) using hexane as mobile phase with 2-propanol or ethanol as modifier. The separation and elution order of the enantiomers on the two columns using different alcohol modifiers were compared. Reversal of the elution order of some enantiomeric pairs associated with increased retention of many of these solutes upon changing the mobile phase modifier from 2-propanol to ethanol was observed. The effect of structural variation of two pairs of enantiomers on their k' and separation factor alpha was noted. Chiralcel OD and Chiralpak AD columns provided different retention, separation and elution order of some of the enantiomeric pairs.     

Chiral discrimination studies of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid by high-performance liquid chromatography and NMR spectroscopy

Chiral discrimination studies using (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) as a chiral selector were performed by high-performance liquid chromatography (HPLC) and NMR spectroscopy. The enantiomers of alanine (Ala) or alanine methyl ester (Ala-ME) were well separated on the chiral stationary phases (CSPs) derived from (+)-18-C-6-TA by HPLC. The chiral selector, (+)-18-C-6-TA, used in the CSP was also applied for the chiral discrimination of the Ala and Ala-ME enantiomers, and it discriminated these enantiomers successfully by NMR spectroscopy. The chemical shift differences (Delta Delta delta) of the alpha-proton of these enantiomers in the presence of an equimolecular solution of 18-C-6-TA were observed to be 0.10 ppm for Ala in methanol-d4 containing 10 mM H2SO4 and 0.11 ppm for Ala-ME in methanol-d4. The observed NMR results agreed with the chromatographic data on the (+)-18-C-6-TA-derived CSP by HPLC in terms of both the elution order and solvents effects.     

直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相拆分布地奈德对映体及其制剂含量的测定

22R-布地奈德的药物活性比22S-布地奈德的强2~3倍,开发布地奈德对映体拆分和定量分析方法,可为其药物研发及质量控制提供重要依据。目前,主要以反相C₁₈固定相对布地奈德对映体进行拆分,而采用手性固定相对其进行拆分少有报道。通过考察固定相、流动相和柱温对布地奈德对映体拆分的影响,建立了基于直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相快速拆分和检测布地奈德对映体的高效液相色谱方法,其色谱条件如下:色谱柱为Chiralpak AS-RH色谱柱(150 mm×4.6 mm, 5.0 μm),流动相为乙腈-水(45∶55, v/v),柱温40 ℃,流速1.0 mL/min,二极管阵列检测器(DAD),检测波长246 nm,进样量10 μL。在该色谱条件下,布地奈德的两个对映体得到较好拆分,22R-布地奈德和22S-布地奈德的保留时间分别6.40 min和7.77 min,分离度为4.64; 22R-布地奈德和22S-布地奈德分别在各自范围内线性关系良好,相关系数(R²)均为0.9999,检出限分别为0.05 μg/mL和0.07 μg/mL,定量限分别为0.16 μg/mL和0.20 μg/mL; 4个添加水平的样品加标回收率为102.63%~104.17%,相对标准偏差(RSD)为0.08%~0.57%(n=6)。将该方法应用于1批次4个吸入用布地奈德混悬液实际样品进行检测,22R-布地奈德和22S-布地奈德的含量分别为283.15~284.63 μg/mL和259.86~261.51 μg/mL。该方法操作简便,分析时间短,重复性好,准确度高,可用于布地奈德对映体的拆分及其制剂的质量控制。     

High-performance liquid chromatography and thin-layer chromatography assays for Devil's Club (Oplopanax horridus)

Devil's root, Oplopanax horridus, is a widely used folk medicine in Alaska and British Columbia. The inner bark of the root and stem has been used to treat colds, cough, fever, and diabetes. The present study involves the development of high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods to detect the presence of trans-nerolidol and sterols in the root bark. The HPLC and TLC analytical methods presented are suitable for the characterization and identification of Oplopanax horridus.     

Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (1-22) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (n-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines 1-22 and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC.     

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.     

Noncovalent interactions between ([18]crown-6)-tetracarboxylic acid and amino acids: electrospray-ionization mass spectrometry investigation of the chiral-recognition processes

Chiral recognition of enantiomers by host compounds is one of the most challenging topics in modern host-guest chemistry. Amongst the well-established methods, mass spectrometry (MS) is increasingly used nowadays, due to its low detection limit, short analysis time, and suitability for analyzing mixtures and for studying chiral effects in the gas phase. The development of electrospray-ionization (ESI) techniques provides an invaluable tool to study, in the gas phase, diastereoisomeric complex ions prepared from enantiomer ions and a chiral selector. This paper reports on an ESIMS and ESIMSMS study of the molecular mechanisms that intervene in the chiral-recognition phenomena observed between amino acids and a chiral crown ether. The modified crown ether, namely (+)-([18]crown-6)-2,3,11,12-tetracarboxylic acid, is used as the chiral selector when covalently bound on a stationary phase in liquid chromatography. This study was stimulated by the fact that, except with threonine and proline, consistent elution orders were observed, which indicates that the D enantiomers interact more strongly with the chiral selector than the L enantiomers. For proline, the lack of a primary amino group is likely to be responsible for the nonresolution of the two forms, whereas the second stereogenic center on threonine could explain the reversed elution order. In light of those observations, we performed mass spectrometry experiments to understand more deeply the enantiomeric recognition phenomena, both in solution by the enantiomer-labeled guest method and in the gas phase by gas-phase ligand-exchange ion/molecule reactions. The results have been further supported by quantum chemical calculations. One of the most interesting features of this work is the identification of a nonspecific interaction between proline and the crown ether upon ESIMS analysis.     

Comparative study on the enantiomer separation of 1,1'-binaphthyl-2,2'diyl hydrogenphosphate and 1,1'-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems

The chiral recognition ability of single and dual selectors, that were used as additives, have been investigated by HPLC and CE. Native beta- and gamma-cyclodextrins, permethylated beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, cholic acid and taurodeoxycholic acid sodium salts were applied as chiral selectors, whereas the atropisomers of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate, and 1,1'-bi-2-naphthol served as model compounds. It was found that all investigated selectors, except for gamma-cyclodextrin, display the same affinity pattern for binaphthyl enantiomers, i.e., binding the S more strongly than the R enantiomer. However, the differences in the phase distribution of chiral selectors led to the opposit elution order of enantiomers: with cyclodextrins, the first eluted is S enantiomer, while R is the first eluted for bile salts. Under the conditions studied, cyclodextrins (except gamma-cyclodextrin), as well as cholic acid sodium salts acting singly, enable the separation of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate enantiomers both by HPLC and CE methods, while 1,1'-bi-2-naphthol enantiomers were resolved only under CE conditions with permethylated cyclodextrin or bile salts. In both techniques the application of dual systems could improve resolution or make it worse (oreven cancel), depending on the sign of enantioselectivity of particular selectors, their concentrations and localization: mobile or stationary phase. It has been found that the mechanism of separation as well as interactions occurring between two selectors may be followed by using combined HPLC and CE methods. The obtained results proved that, as well as beta-CD, TM-beta-D and gamma-CD also form inclusion complexes with cholic acid sodium salts. The reversal of elution order may be realized by two procedures: changing a single selector, i.e., cyclodextrin on cholic acid sodium salt or vice versa, and by changing the proportion of selectors in the combined bile salt-cyclodextrin system.     

超临界流体色谱与高效液相色谱分离手性化合物的比较

超临界流体色谱(SFC)分离具有速度快、分离效率高、溶剂消耗少等优点,近年来在手性化合物的分离分析中得到诸多应用。本文对比研究了涂覆型多糖手性色谱柱在SFC和高效液相色谱(HPLC)上拆分24种手性化合物的差异。通过比较这些化合物在色谱柱上的保留时间和选择因子等发现多数化合物在SFC上的分离效率要高于其在HPLC上的分离效率,但HPLC对轴手性化合物的分离效率要优于SFC。SFC和HPLC的分离表现出一定的互补性,随着苯环侧链烷基的碳数增加,化合物在SFC上的保留逐渐增强,而在HPLC的保留却逐渐减弱。叶菌唑在使用SFC和HPLC分析时出现了洗脱顺序反转的现象。这些结果为SFC手性拆分提供了参考。     

Use of evaporative light scattering detector in the detection and quantification of enantiomeric mixtures by HPLC

Routinely used in our laboratories at analytical scale, an evaporative light scattering detector (ELSD) has proved to be versatile in the detection of enantiomeric resolution using chiral stationary phases by HPLC. Though this kind of detector has been widely used in various domains, its application in enantiomeric resolution has not been discussed in the literature and is found to have very specific features especially in the quantitative perspective. In contrast with the UV detection, the peak area from ELSD for both enantiomers of a racemic mixture may not be the same. This complicates the assessment of the enantiomeric purity of unknown samples. This current work deals with some practical aspects in the detection of enantiomers and in accurate quantitative determination of enantiomeric purity by ELSD. Effects of analyte nature (more precisely molecular weight and volatility), peak shape and peak shape difference between enantiomers on the quantitative integration by ELSD are discussed in connection with the UV-detection results. The calibration for quantitative enantiomeric analysis and its effectiveness are demonstrated.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Chromatographic Chiral Recognition and Computer Aided Deconvolution of the Enantiomeric Secondary Alcohol with a Self‐Packed Chiral Stationary Phase HPLC Column

The γ‐aminopropyl‐derivatized silica was treated with the (R)‐(‐)‐N‐(3,5‐dinitrobenzoyl)‐α‐phenylglycine and was packed into a standard 250 mm × 4.5 mm HPLC column to perform the chiral separation of secondary alcohol. Enantiomers of four homologous series of aryl alkyl substituted alcohol were resolved in different degrees. The chiral recognition mechanism was proposed that could be used to rationalize well the elution order and the resolution between the R‐ and S‐configuration of a secondary alcohol. The unresolved peak of the enantiomers of 1‐phenylethanol and the partially resolved peak of the enantiomers of 1‐(1‐naphthyl)ethanol were deconvoluted by the assistance of computer and a program written in BASIC computer language. Two mathematical models were used for the deconvolution and were based on modified Gaussian functions with a fixed baseline that can take into account the skew of a chromatographic peak. Parameters in the mathematical model were estimated by using a nonlinear regression algorithm. The selected mathematical models were suitable for deconvolving the less skewed overlapping peaks of 1‐phenylethanol.