Enantiomeric separation and discrimination of 2-hydroxy acids as O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl esters by achiral dual-capillary column gas chromatography

An efficient method is described for the simultaneous enantiomeric separation of 18 different racemic 2-hydroxy acids for the determination of their absolute configurations. It involves the conversion of each enantiomer into a diastereomeric O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl ester for the direct separation by achiral dual-capillary column gas chromatography with subsequent identification and determination of its chirality by retention index (I) library matching. The enantiomers of each acid were well separated with high resolution values (R > or = 1.4) on DB-5 and DB-17 columns of different polarity. When temperature-programmed I values of 2-hydroxy acid enantiomers as their diastereomeric derivatives were measured on both columns, the I values were characteristic of each enantiomer. Simple I matching with the reference values was thus useful in cross-checking each acid enantiomer for the identification and chiral discrimination. When applied to urine samples, the present method allowed positive identification of most of the spiked 2-hydroxy acids from normal urine and for endogenous (S)-lactic acid and (S)-2-hydroxybutyric acid from a clinical urine specimen.     

Enantioseparation of chiral amino acids as the N(O,S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography

The enantioseparation of 30 racemic amino acids in a single analysis is described for the determination of their absolute configurations. Two-phase extractive ethoxycarbonyl (EOC) reaction with ethyl chloroformate present in the dichloromethane phase was performed to recover amino acids from alkaline aqueous solutions. The resulting N(O,S)-EOC amino acids extracted into an organic solvent after acidification were reacted with a chiral alcohol such as (S)-(+)-3-methylbutan-2-ol, (S)-(+)-butan-2-ol and (S)-(+)-octan-2-ol for gas chromatographic analysis on achiral dual-capillary DB-5 and DB-17 columns of different polarities. Among the chiral reagents examined, (S)-(+)-3-methylbutan-2-ol provided the best diastereomeric structures in resolving all the racemic amino acids into their enantiomeric pairs with high resolution factors (1.2-8.0). Moreover, the temperature-programmed retention index (I) values measured on the two columns were characteristic of each enantiomer. Hence simple I matching with the reference values was useful in cross-checking for chemical identification and also chiral discrimination. When the present method was applied to a fermented dairy product (Yakult), D-alanine, D-aspartic acid, D-glutamic acid and D-proline were positively detected along with their respective L-forms in addition to glycine.     

Liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl alpha-amino acids and their ester derivatives on polysaccharide-derived chiral stationary phases

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) protected alpha-amino acids and their ethyl ester derivatives was performed on polysaccharide-derived chiral stationary phases, Chiralcel OD, Chiralpak AD, and Chiralpak AS. In general, Chiralcel OD and Chiralpak AD showed good performance for resolution of N-FMOC alpha-amino acids and their ethyl esters, respectively. All investigated N-FMOC alpha-amino acid enantiomers were baseline separated on Chiralcel OD or Chiralpak AD, whereas N-FMOC alpha-amino acid ethyl ester enantiomers were baseline resolved (alpha = 1.15-3.03) on Chiralpak AD, except for two analytes. The L-enantiomers of all examined FMOC alpha-amino acid ethyl ester derivatives are preferentially retained on Chiralpak AD, while the elution orders of the other enantiomer separations are not consistent.     

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.     

N-Menthoxycarbonylation combined with trimethylsilylation for enantioseparation of beta-blockers by achiral dual-column gas chromatography

Solvent extractive two-phase menthoxycarbonyl (MnOC) derivatization was combined with trimethylsilyl (TMS) reaction for enantioseparation of beta-blockers by gas chromatography employing achiral DB-5 and DB-17 dual-columns of different polarity. beta-Blockers in alkaline solution were vortex-mixed with menthyl chloroformate present in dichloromethane to be extracted as diastereomeric N-MnOC derivatives. The subsequent O(N)-TMS reaction allowed complete enantioseparations of two beta-blockers and partial separations of five as N-MnOC/O(N)-TMS derivatives in a single analysis. The temperature-programmed retention index sets were characteristic of each derivative, facilitating chiral discrimination of each enantiomer.     

Extending the scope of enantiomer separation on diluted methylated β-cyclodextrin derivatives by high-resolution gas chromatography

High-resolution open-tubular columns coated with solutions of heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (Phase I) or heptakis(2,6-di-O-methyl-3-O-trifluoroacetyl)-β-cyclodextrin (Phase II) in moderately polar polysiloxanes such as OV-1701 (5% cyanopropyl/7% phenyl/88% methylpolysiloxane) and OV-225 (25% cyanopropyl/50% phenyl/25% methylpolysiloxane) are used for the gas chromatographic enantiomer separation of volatiles belonging to different classes of compounds. No derivatization procedures are necessary for most of the resolved chiral molecules. The chiral stationary phases can be operated between 25 and 190°C for extended periods of time. The enantiomer separation of saturated, unfunctionalized hydrocarbons clearly demonstrates the importance of molecular inclusion in chiral recognition using cyclodextrins for this class of compounds. The different, and in some cases complementary, selectivity of the Phases I and II is demonstrated.     

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.     

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of d-amino acids typically found in biological fluids and tissues. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Enantiomeric separation of chiral phenoxy acid herbicides by electrokinetic chromatography. Application to the determination of analyte-selector apparent binding constants for enantiomers

The enantiomeric resolution of chiral phenoxy acid herbicides was performed by electrokinetic chromatography using a cyclodextrin as chiral pseudophase (CD-EKC). A systematic evaluation of several neutral and charged cyclodextrins was made. Among the cyclodextrins tested, (2-hydroxy)propyl beta-cyclodextrin (HP-beta-CD) was found to be the most appropriate for the enantioseparation of phenoxy acids. The influence of some experimental conditions, such as nature and pH of the background electrolyte, chiral selector concentration, and temperature, on the enantiomeric separation of phenoxy acids was also studied. The use of a 50 mM electrolyte solution in ammonium formate at pH 5 and a temperature of 40 degrees C enabled the enantiomeric resolution of four of the six phenoxy acids investigated (2-phenoxypropionic acid, 2(3-chlorophenoxy)propionic acid, 2-(4-chlorophenoxy)propionic acid, and 2-(2,4-dichlorophenoxy)propionic acid) obtaining migration times ranging from 9 to 15 min. Mixtures of the two phenoxy acids not enantiomerically resolved (2-(4-chlorophenoxy)-2-methylpropionic acid and 2-(2,4,5-trichlorophenoxy)propionic acid) and up to three of the phenoxy acids enantiomerically resolved were separated in about 15 min. Finally, the apparent binding constants for each enantiomer-HP-beta-CD pair were calculated at two temperature values (20 and 40 degrees C).     

Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-6-2,3,11,12-tetracarboxylic acid of the same chiral selector

Liquid chromatographic comparisons for enantiomer resolution of α-amino acids and chiral primary amino compounds were made using chiral stationary phases (CSPs) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) of the same chiral selector. The resolution of all α-amino acids on CSP 1 developed in our group was found to be better than that on CSP 2 reported by Machida et al. All α-amino acids examined in this study were well enantioseparated on CSP 1 (α=1.22–2.47), while four analytes were not resolved or all the other analytes were poorly resolved on CSP 2 than on CSP 1. However, in resolving the primary amino compounds without a carbonyl group, CSP 1 was comparable with CSP 2. Although (+)-18-C-6-TA of the same chiral selector was used to prepare CSP 1 and CSP 2, this study showed that different connecting methods for the CSPs might influence their ability to resolve the analytes depending on their structures related to the chiral recognition mechanism.     

Liquid chromatographic resolution of proline and pipecolic acid derivatives on chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

Two liquid chromatographic chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid were applied to the resolution of the amide derivatives of cyclic α‐amino acids including proline and pipecolic acid. Among the five amide derivatives of proline, aniline amide was resolved best on the first chiral stationary phase, which contains two N–H tethering amide groups, with the separation factor of 1.31 and the resolution of 2.60, and on the second chiral stationary phase, which contains two N–CH₃ tethering amide groups, with the separation factor of 1.57 and the resolution of 5.50. Among the five amide derivatives of pipecolic acid, 2‐naphthyl amide was resolved best on the first chiral stationary phase with the separation factor of 1.30 and the resolution of 1.75, but 1‐naphthylmethyl amide was resolved best on the second chiral stationary phase with the separation factor of 1.30 and the resolution of 2.26. In general, the second chiral stationary phase was found to be better than the first chiral stationary phase in the resolution of the amide derivatives of cyclic α‐amino acids. In this study, the second chiral stationary phase was first demonstrated to be useful for the resolution of secondary amino compounds.     

Resolution of enantiomers and isotopic compositions by selective complexation gas chromatography on metal complexes

Summary The high selectivity of complexation gas chromatography has been employed for enantiomer resolution and isotope separation. Thus, a chiral olefin, 3-methylcyclopentene, has been resolved analytically on optically active dicarbonyl-rhodium(I)-3-trifluoroacetyl-1R-camphorate in squalane. The deuterated ethylenes C₂H_4–nD_n have been separated on the same rhodium(I)-containing stationary phase. The chiral aliphatic oxiranes epoxypropane andtrans-2,3-epoxybutane have been resolved on optically active nickel(II)-bis-3-trifluoroacetyl-1R-camphorate in squalane.     

Comparison Between Methyl and Trimethylsilyl Ester Derivatives in the Separation and GC Quantification of Triterpene Acids in Eugenia brasiliensis Leaf Extract

This study details a method to characterize the triterpene acid-rich extract obtained from the defatted leaves of Eugenia brasiliensis (Myrtaceae) via extraction with 2 % NaOH in ethanol at room temperature. The crude extract (yield 2.35 %) was submitted to analysis by gas chromatography coupled to mass spectrometry (GC–MS) confirming ursolic acid as its major compound. The optimal conditions for the separation of oleanolic, betulinic and ursolic acids were assayed by GC with flame ionization detection (GC–FID) using two different columns (DB-5 and DB-17HT) and by applying two distinct derivatizing protocols. The use of a DB-17HT column led to the best results, with a shorter runtime and a better resolution (Rs) between the oleanolic and betulinic signals for both the bis-trimethylsilyl (Rs 2.84) and methyl ester derivatives (Rs 2.47). A DB-5 column also gave satisfactory results for the TMS ester, with a runtime of 30 min and Rs 2.14. Ursolic acid in the crude extract was quantified by comparison to two individual standard curves determined using commercial ursolic as its TMS derivative on the DB-5 column and its methyl ester on the DB-17HT column. Good linearity was achieved in both cases (r ² = 0.9776 and 0.9953, respectively), and the amounts of ursolic acid in the extracts were calculated to be 144.7 and 147.9 mg·g^?1, respectively. These results showed no significant differences when compared using Tukey’s HSD test. Total triterpene acids amounted to 0.52 % in E. brasiliensis dry leaves.     

The CGC enantiomer separation of 2-arylcarboxylic acid esters by using β-cyclodextrin derivatives as chiral stationary phases

Chiral 2-arylcarboxylic acid esters are important intermediates in preparation of enantioenriched 2-arylpropionic acids type Non-steroidal anti-inflammatory drugs (NSAIDs). Enantiomer separation of 2-arylcarboxylic acid esters is crucial for evaluation of the asymmetric synthesis efficiency and the enantiomer excess of chiral 2-arylcarboxylic acid derivatives. The capillary gas chromatography (CGC) enantiomer separation of 17 pairs of 2-arylcarboxylic acid esters enantiomers was conducted by using seven different β-cyclodextrin derivatives (CDs) as chiral stationary phases. It was found that for the 7 pairs of 2-phenylpropionates enantiomers, CDs with both alkyl and acyl substituents especially 2,6-di-O-pentyl-3-O-butyryl-β-cyclodextrin exhibited better enantiomer separation abilities than the other CDs examined. For the 7 pairs of 2-(4-substituted phenyl)propionates enantiomers, 2,3,6-tri-O-methyl-β-cyclodextrin possessed better enantiomer separation abilities than the other CDs. Among the 3 pairs of 2-phenylbutyrates enantiomers examined, only methyl 2-phenylbutyrate enantiomers could be separated by three CDs among the 7 CDs tested, while enantiomers of ethyl 2-phenylbutyrate and isopropyl 2-phenylbutyrate couldn't be separated by any of the 7 CDs tested. Besides the structures of CDs, the structures of 2-arylcarboxylic acid esters including different ester moieties, substituents of phenyl, and different carboxylic acids moieties in 2-arylcarboxylic acid esters also affected the enantiomer separation results greatly. The CGC enantiomer separation results of 2-arylcarboxylic acid esters on different CDs are useful for solving the enantiomer separation problem of 2-arylcarboxylic acid esters.     

3-Hydroxyglutarate and β,γ-Epoxy Esters as Substrates for Pig Liver Esterase (PLE) and α-Chymotrypsin

The pH dependence of the α-chymotrypsin-catalyzed hydrolysis of dimethyl 3-hydroxyglutarate ( 3 ) has been studied. The e.e. was determined by HPLC analysis of diastereoisomeric camphanoic-acid derivatives. Kinetic resolution of the β,α-epoxy esters 10 and 24 by pig liver esterase has been shown to provide an alternative access to chiral β-hydroxy esters and acids of high optical purity. By this latter method, the unnatural enantiomer of γ-amino-β-hydroxybutyric acid (GABOB) has been synthesized. Finally, dimethyl meso-3,4-epoxyadipate ( 19 ) was hydrolyzed by pig liver esterase with almost 100% selectivity.     

Direct enantiomeric TLC resolution of dl-penicillamine using (R)-mandelic acid and l-tartaric acid as chiral impregnating reagents and as chiral mobile phase additive

DL-Penicillamine has been resolved into its enantiomers by normal-phase TLC using L-tartaric acid as chiral impregnating reagent as well as chiral mobile phase additive, while (R)-mandelic acid has been found to be successful as a chiral impregnating reagent. The solvent system acetonitrile-methanol-water (5:1:1, v/v) was found to be successful when L-tartaric acid was used as impregnating agent while the solvent combination acetonitrile-methanol-(0.5% l-tartaric acid in water, pH 5)-glacial acetic acid (7:1:1.1:0.7, v/v) was successful as mobile phase as it contained L-tartaric acid as the chiral additive. (R)-mandelic acid was successful as chiral impregnating reagent with ethyl acetate-methanol-water (3:1:1, v/v), as the mobile phase. The effects of concentration of chiral selectors, temperature and pH were examined on enantiomeric resolution. The spots were detected with iodine vapors and the detection limits were found to be 0.12 microg for each enantiomer of penicillamine with L-tartaric acid, under both the conditions, and 0.11 microg with (R)-mandelic acid.     

Liquid chromatographic resolution of 2-hydroxycarboxylic acids on a new chiral stationary phase derived from (S)-leucine

Enantiomers of racemic 2-hydroxycarboxylic acids have been resolved as their O-ethoxycarbonyl pi-basic anilide derivatives on a new chiral stationary phase (CSP) derived from N-(3,5-dinitrobenzoyl)leucine N-phenyl N-alkylamide and the resolution results have been compared with those on various commercial pi-acidic CSPs. The resolution results demonstrate that the new CSP derived from N-(3,5-dinitrobenzoyl)leucine N-phenyl N-alkylamide is most effective among the five CSPs tested for the resolution of 2-hydroxycarboxylic acid derivatives. In order to elucidate the chiral recognition mechanism exerted by the new CSP, the resolution of slightly differently modified derivatives of 2-hydroxycarboxylic acids on the new CSP has been investigated. Based on the resolution results, a chiral recognition mechanism utilizing three simultaneous interactions such as the face to face pi-pi interaction and the two hydrogen bonding interactions between the CSP and the more retained enantiomer of the analyte has been proposed.     

Simultaneous determination of dl-lactic acid and dl-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7?min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12?fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.     

分子印迹聚合物在毛细管电色谱拆分手性药物中的研究进展

手性药物通过与生物体内生物大分子之间的手性匹配与分子识别来发挥药理作用。两个对映体与体内手性环境相互作用的不同导致每个对映体表现出不同的药理活性、代谢过程、代谢速率及毒性等药代动力学特征。因此发展手性药物的拆分方法,对于手性药物的开发和生产过程的质量监控具有重要意义。分子印迹聚合物（MIPs）是以目标分子作为模板而制备的高分子聚合物,它具有特定的空间分子结构和官能团,对目标分子具有高度的特异性识别能力。基于该特点,MIPs非常适合于手性药物的拆分和纯化。毛细管电色谱（CEC）可同时基于毛细管电泳和液相色谱的分离机理对目标物进行分离,因此具有高分离效率和高选择性的特点。将MIPs材料作为CEC的固定相,可将这两种技术的优势结合,从而实现对手性药物的高效拆分。MIPs材料在1994年首次应用于CEC手性拆分,此后该研究领域开始获得关注和发展。MIPs材料主要通过4种模式在CEC中实现手性拆分,分别是作为开管柱、填充柱和整体柱的固定相以及分离介质中的准固定相。该综述以这4种模式作为分类基准,根据MIPs制备所需的材料和分离对象对其在CEC手性拆分中的应用进行了总结,揭示了MIPs在CEC手性拆分中的潜力,同时评述了这4种模式各自的优势与不足,并对将来MIPs在CEC手性拆分中的发展进行了展望。     

Evaluation of the separation characteristics of application-specific (volatile organic compounds) open-tubular columns for gas chromatography

The solvation parameter model is used to characterize the separation characteristics of two application-specific open-tubular columns (Rtx-Volatiles and Rtx-VGC) and a general purpose column for the separation of volatile organic compounds (DB-WAXetr) at five equally spaced temperatures over the range 60-140 degrees C. System constant differences and retention factor correlation plots are then used to determine selectivity differences between the above columns and their closest neighbors in a large database of system constants and retention factors for forty-four open-tubular columns. The Rtx-Volatiles column is shown to have separation characteristics predicted for a poly(dimethyldiphenylsiloxane) stationary phase containing about 16% diphenylsiloxane monomer. The Rtx-VGC column has separation properties similar to the poly(cyanopropylphenyldimethylsiloxane) stationary phase containing 14% cyanopropylphenylsiloxane monomer DB-1701 for non-polar and dipolar/polarizable compounds but significantly different characteristics for the separation of hydrogen-bond acids. For all practical purposes the DB-WAXetr column is shown to be selectivity equivalent to poly(ethylene glycol) columns prepared using different chemistries for bonding and immobilizing the stationary phase. Principal component analysis and cluster analysis are then used to classify the system constants for the above columns and a sub-database of eleven open-tubular columns (DB-1, HP-5, DB-VRX, Rtx-20, DB-35, Rtx-50, Rtx-65, DB-1301, DB-1701, DB-200, and DB-624) commonly used for the separation of volatile organic compounds. A rationale basis for column selection based on differences in intermolecular interactions is presented as an aid to method development for the separation of volatile organic compounds.