Enantioselective Column Research Articles

Enantiomeric analysis of amino acids by using comprehensive two‐dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.

Comprehensive analysis of branched aliphatic d-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic d-amino acids in the presence of large amounts of their l-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the d-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5min. In the rat, significant amounts of d-Leu were found in all tissues and physiological fluids tested (trace – 1.3nmol/g tissue), and in the urine, the presence of high amounts of d-allo-Ile (d-isomer of a non-proteinogenic amino acid, 22.2nmol/ml) was demonstrated. d-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual d-amino acid and the potential need to clarify its unique metabolism in mammals.

Determination of toxaphene enantiomers by comprehensive two-dimensional gas chromatography with electron-capture detection

Comprehensive two-dimensional gas chromatography with micro electron-capture detection (GC × GC–μECD) has been evaluated for the enantioseparation of five chiral toxaphenes typically found in real-life samples (Parlar 26, 32, 40, 44 and 50). From the two enantioselective β-cyclodextrin-based columns evaluated as first dimension column, BGB-176SE and BGB-172, the latter provided the best results and was further combined with three non-enantioselective columns in the second dimension: HT-8, BPX-50 and Supelcowax-10. The combination BGB-172 × BPX-50 was finally selected because it provided a complete separation among all enantiomers. A satisfactory repeatability and reproducibility of the retention times in both the first and the second dimension were observed for all target compounds (RSDs below 0.8%, n = 4). Linear responses in the tested range of 10–200 pg/μl and limits of detection in the range of 2–6 pg/μl were obtained. The repeatability and reproducibility at a concentration of 100 pg/μl, evaluated as the RSDs calculated for the enantiomeric fraction (EF), was better than 11% ( n = 4) in all instances. The feasibility of the method developed for real-life analyses was illustrated by the determination of the enantiomeric ratios and concentration levels of the test compounds in four commercial fish oil samples. These results were compared to those obtained by heart-cut multidimensional gas chromatography using the same enantioselective column.

Chiral comprehensive two-dimensional gas chromatography with electron-capture detection applied to the analysis of chiral polychlorinated biphenyls in food samples

The feasibility of comprehensive two-dimensional gas chromatography with electron-capture detection (GC × GC–ECD) for the enantioseparation of chiral PCBs from other possible interfering compounds has been evaluated. Three commercially available enantioselective β-cyclodextrin-based capillary columns (Chirasil-Dex, BGB-172 and BGB-176SE) have been tested as first-dimension columns. Three non-enantioselective stationary phases (HT-8, BPX-50 and Supelcowax-10) were combined with the enantioselective columns to allow the unambiguous determination of the enantiomers of the target chiral PCBs. Each enantioselective first-dimension column tested was able to separate into enantiomers different PCB congeners, but in all cases, the use of Supelcowax-10 as second-dimension column provided the most satisfactory results. The Chirasil-Dex × Supelcowax-10 column combination allowed the determination of the enantiomeric fraction (EF) of PCBs 84, 91, 95, 132, 136, 149, 174 and 176 in the working standard solution, while that of congener 135 was hindered. The BGB-172 × Supelcowax-10 column set allowed a proper EF determination of congeners 45, 84, 131, 132, 135, 171, 174 and 183, while that of PCB 91 was interfered with co-elutants. The column combination BGB-176SE × Supelcowax-10 allowed the determination of all congeners that this enantioselective stationary phase was able to separate into enantiomers, i.e. PCBs 45, 91, 95, 136, 149 and 176. These column combinations have also been evaluated for the simultaneous determination of the 12 congeners with a toxic equivalency factor assigned by the WHO (PCBs 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, 189) and the seven indicator congeners (PCBs 28, 52, 101, 118, 138, 153 and 180), and evaluated for the analysis of food samples.

Chirally-functionalized monolithic materials for stereoselective capillary electrochromatography

Despite the demonstrated high performance of stereoselective capillary electrochromatography (CEC) [1], the broader acceptance of this appealing separation technique has been strongly impeded by the weakness of the non-dedicated, conventional packed column technology normally employed, which is based on microparticulate materials (3–5 lm) and depends on sintered retaining frits. To alleviate the need for frits and related problems (such as bubble formation, barely reproducible manufacture, fragility) and in particular poor column longevity, researchers’ attempts to improve on the CEC column technology and thereby simplify the practical work have recently focused on monolithic type columns where the continuous porous solid is anchored to the capillary wall. Along this line, three distinct types of enantioselective monolithic CEC columns have evolved [2, 3]. (i) Enantioselective monolithic capillaries that make use of particulate CSPs have been prepared by either embedding the particles (25–30%) in a monolithic bed (such as a sol–gel matrix [4] or a polyacrylamide polymer [5]) or by stabilizing the densely-packed particulate bed via a sol–gel matrix [6]. (ii) Silica monolithbased enantioselective columns are prepared in situ in a capillary using sol–gel technology via steps of gelation and phase separation (macropore formation) and subsequent hydrothermal treatment or aging (mesopore formation), which allows independent control of mesopores and macropores and yields an inorganic material with a bimodal pore distribution and high active surface area [7, 8]. In a subsequent step, the chiral selector is immobilized through either covalent linkage [9] or via adsorptive coating of a polymeric selector [10, 11]. (iii) In contrast to the above approaches, enantioselective organic polymer monoliths may be prepared in a single step from a chiral monomer (a chiral selector with a polymerizable functionality), an ionizable co-monomer for EOF generation, another co-monomer that allows the surface polarity to be adjusted, a crosslinker, and porogens that induce phase separation during polymerization. This approach is very flexible and allows tuning of the polymer in terms of porosity, macropore diameter and surface chemistry, in order to meet the specific requirements of CEC and enantiomer separation. Chirally-functionalized polyacrylamide [12–15], brush-type chiral polymethacrylate [16], and molecularly-imprinted chiral polymer monoliths [17] have been successfully developed for enantioselective CEC by incorporating various different chiral recognition principles.

Determination of zolmitriptan enantiomers in rat liver microsomes by chiral high performance liquid chromatography with fl uorescence detection.

A selective chiral high performance liquid chromatographic method was developed and validated to separate and quantify the enantiomers of a new potent selective 5-HT(1B/1D) receptor partial agonist, S-zolmitriptan, and its antipode in rat liver microsomes induced with beta-naphtho flavone. S- and R-zolmitriptan were extracted from rat hepatic microsomal incubates with chloroform/isopropanol (75:25, v/v), and were separated on a narrow-bore enantioselective normal phase Chiralpak AD-H column (250 x 0.46 mm) with hexane-isopropanol-triethylamine (72/28/0.25, v/v/v) as mobile phase and fluorescence detection with emission at 350 nm and excitation at 291 nm. The calibration curves were linear for R- and S-zolmitriptan concentration over the range 0.1-5.0 microg/mL (r = 0.9996 and 0.9999), and the limits of quantitation were 0.1 microg/mL. The metabolism and interaction of the enantiomers of zolmitriptan in treated hepatic microsomes were investigated using chiral HPLC. There was significant difference between the disposition of the S- and R-zolmitriptan when racemic zolmitriptan or single enantiomers of zolmitriptan were incubated for 5, 10 and 20 min, suggesting that the metabolism of zolmitriptan in rat liver microsomes is enantioselective. In addition, there was also a significant difference between the IC(50) of R- to S-zolmitriptan and S- to R-zolmitriptan (IC(50S/R)/IC(50R/S) = 45.2). This indicated that the disposition process favored the S-form of zolmitriptan.

Contributions to the theory and practice of the chromatographic separation of enantiomers

The theory and practice of enantioselective capillary chromatography employing metal coordination compounds and modified cyclodextrins as chiral stationary phases are treated. A unified approach involving all contemporary chromatographic methods and a single enantioselective column is described. Reliable thermodynamic data of enantioselectivity are derived by the retention-increment method. The existence of an isoenantioselective temperature is demonstrated. Kinetic enantiomerization studies are presented. The preparative-scale separation of enantiomers by gas chromatography with enantioselective packed columns is achieved. Unusual phenomena and future aspects of enantioselective chromatography are discussed.

Analysis of strawberry volatiles using comprehensive two-dimensional gas chromatography with headspace solid-phase microextraction

The aims of the current study were to develop an enantioselective multi-dimensional gas chromatography (GC × GC) method for the examination of strawberry volatiles and to use this method to make comparisons between the volatile profiles of different cultivars and between fresh picked and post-harvest berries of the same cultivar. Strawberry volatiles were sampled using solid-phase microextraction (SPME), and the repeatability and reproducibility of this method was examined. Semi-quantitative analysis of the volatiles was conducted using the relatively new technique of comprehensive multi-dimensional gas chromatography, using enantioselective (chiral) columns for the differentiation of analyte enantiomers. Chiral GC × GC facilitated the detection of key enantiomers in strawberry flavour. The (−)-enantiomer of 2,5-dimethyl-4-hydroxy-(2 H)-furan-3-one (DMHF) and the S-enantiomer of linalool were tentatively identified as the predominant forms in both the cultivars Selva and Adina. The compounds benzaldehyde and methyl hexanoate were shown to decrease in post-harvest berries, whilst DMHF and nerolidol increased upon storage.

Enantioselective gas chromatographic analysis of monoterpenes in essential oils of the family Myrtaceae

AbstractThe elution profiles of several essential oils of the Myrtaceae family were acquired using a single enantioselective stationary phase column. These chromatograms were compared with those obtained by using a two‐column coupled system, which comprised a low‐polarity stationary phase column connected to the terminal end of the enantioselective column. The enantiomeric distributions of β‐pinene, sabinene, α‐phellandrene, limonene, trans‐ and cis‐sabinene hydrate, linalool, terpinen‐4‐ol and α‐terpineol were determined. The use of a two‐column coupled system is demonstrated to be useful for improving the resolution of target (chiral) compounds from otherwise interfering matrix components. Copyright © 2004 John Wiley & Sons, Ltd.

Separation and analysis of diastereomers and enantiomers of cypermethrin and cyfluthrin by gas chromatography.

Synthetic pyrethroid (SP) insecticides are of environmental significance because of their high aquatic toxicity. Due to their chirality, SP compounds contain multiple diastereomers and enantiomers. However, due to great structural similarities and lack of isomer standards, gas chromatographic (GC) analysis of SP diastereomers or enantiomers is poorly developed. In this study, we used a HP-5 column to separate the diastereomers and a beta-cyclodextrin-based enantioselective column (BGB-172) to separate the enantiomers of cypermethrin (CP) and cyfluthrin (CF). Resolved peaks were identified by comparing chromatograms of isomer-enriched CP products. Diastereomers of both CP and CF were separated on the HP-5 column. On the BGB-172 column, enantiomers of all cis diastereomers were separated, while those of trans diastereomers were not separated. The elution order appears to be regulated by configuration, a finding which may allow peak identification in the absence of isomer standards. When coupled with electron capture detection, the developed methods had low detection limits and may be used for analysis of SP diastereomers and enantiomers in environmental samples.

Determination of enantiomers of synthetic pyrethroids in water by solid phase microextraction - enantioselective gas chromatography.

Solid phase microextraction (SPME) is an ideal sample preparation technique because of its speed and solvent-free features. Sampling by SPME is selective and only the dissolved concentration is measured, which allows measurement of the bioavailable fraction of a contaminant in aqueous media. One potential application of SPME is for analysis of enantiomers of chiral contaminants in environmental samples. In this study, a method was developed for determining enantiomers of (Z)-cis-bifenthrin and cis-permethrin in water using coupled SPME and enantioselective gas chromatography (GC). Following SPME sampling, enantiomers of (Z)-cis-bifenthrin and cis-permethrin were separated at the baseline on a beta-cyclodextrin-based enantioselective column, and analyte enrichment onto the SPME fiber was not enantioselective. The GC response increased as sampling time was increased from 0 to 240 min, and as sampling temperature was increased from 20 to 40 degrees C. Organic solvents such as methanol, acetone, and acetonitrile enhanced, while soil extracts slightly decreased, the GC response. The integrated SPME-enantioselective GC method was used to analyze surface runoff samples. The analysis showed preferential degradation of the 1S-3S enantiomer over the 1R-3R enantiomer for both (Z)-cis-bifenthrin and cis-permethrin. The concentrations detected by SPME-GC were substantially smaller than those determined following solvent extraction, suggesting that SPME-enantioselective GC analysis selectively measured the dissolved fraction.

Recent progress in enantiomeric separation by capillary electrochromatography.

Recent progress in enantiomeric separations by capillary electrochromatography (CEC) is reviewed. The development of simple and robust CEC column technologies plays an important role for popularization of CEC. During the last several years, various approaches for the preparation of enantioselective columns have been reported. Currently, the monolithic column technology (continuous beds) represents the most advanced approach for the preparation of CEC columns. The development of new chiral stationary phase used for CEC is another important issue in this field. Fundamental investigations on electrochromatographic behaviors of various CSPs are necessary in order to understand the separation mechanism and thus improve the separation performance. Some chiral stationary phases performed better under nonaqueous CEC conditions than reversed-phase conditions. Coupling CEC with mass spectrometry (MS) provides a powerful tool for enantiomeric separation. Finally, some applications of enantiomeric separation by CEC are summarized.

Analytical and semipreparative enantiomeric separation of azole antifungal agents by high-performance liquid chromatography on polysaccharide-based chiral stationary phases: Application to in vitro biological studies

High-performance liquid chromatography (HPLC) was used for the enantiomeric separation of chiral imidazole derivatives endowed with antimycotic activity. Enantioselective columns, containing carbamates of cellulose and amylose, were used. The influence of the nature and content of an alcoholic modifier in the mobile phase was studied. The isolated enantiomers, separated on semipreparative columns, were submitted to in vitro biological investigations.

Comparison of Dynamic HPLC and Dynamic NMR in the Study of Conformational Stereodynamics: Case of the Enantiomers of a Hindered Secondary Phosphine Oxide1

Static and dynamic stereochemistry of HP(O)ButAr (Ar = 2-methyl-1-naphthyl) has been studied by a combination of variable-temperature NMR (1H and 13 P), HPLC, and CD measurements as well as by MM calculations. Two unequally populated stereolabile isomers for each configurational enantiomer have been detected and their anticlinal and synclinal structures assigned. All the four species have been physically separated and identified on a cryogenic HPLC enantioselective column at −83 °C. The interconversion barrier measured by dynamic NMR yields essentially the same value as that measured by dynamic HPLC (14.75 and 14.95 kcal mol-1, respectively).

Enantioselective analysis of ( R)- and ( S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of ( R, S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of ( R)- and ( S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.

Determination of Enantiomer Ratios of Bornane Congeners in Biological Samples Using Heart-Cut Multidimensional Gas Chromatography

The enantiomeric ratios of the chlorinated bornanes, 2-exo, 5,5,9,9,10, 10-heptachlorobornane (B[20030]-(022)), 2-endo,3-exo,5-endo,6-exo,8, 8,10,10-octachlorobornane (B[12012]-(202), Parlar No. 26), 2-exo,3-endo,6-endo,8,9,10,10-heptachlorobornane (B[21001]-(112)), 2,2,5,5,9,9, 10,10-octachlorobornane (B[30030]-(022), Parlar No. 38), 2-endo,3-exo,5-endo6-exo,8,8,9,10,10-nonachlorobornane (B[12012]-(212), Parlar No. 50), and 2,2,5,5,8,9,9,10,10-nonachlorobornane (B[30030]-(122), Parlar No. 62) were determined in a hake liver (Merluccius merluccius) and in two whitebeaked dolphin blubber (Lagenorynchus albirostris) samples. The analysis was performed by heart-cut multidimensional gas chromatography using an electron capture detector (ECD). Ultra 2 (5%-phenyl-95%-methylsilicone) was used as stationary phase in the first column and enantioselectivity was obtained in the second column with a phase consisting of a mixture of OV-1701 and heptakis(2,3,6-O-tert- butyldimethylsilyl)-β-cyclodextrin (10 : 1). All reference standards were also tested on the enantioselective column alone. The comparison of the two systems showed that deviations of ±0.12 from the racemic enantiomer ratio can be observed for the single column due to co-eluting impurities which are present in the reference standards. In most of the heart-cut chromatograms of the biota samples about 15 peaks could be found, showing the importance of a multidimensional separation system for an interference-free quantification by ECD. In all biological samples a significant deviation from the racemic enantiomer ratio was found for B[30030]-(022) (Parlar No. 38) and for B[30030]-(122) (Parlar No. 62). This indicates that an enantioselective disposition of the congeners occurs. In addition, considerations are presented concerning the relationship between congener structure and enantiomeric disposition.

Quantification of ketoprofen enantiomers in human plasma based on solid-phase extraction and enantioselective column chromatography.

An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C18 solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; (R)-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves (n = 14) were linear over the concentration range of 0.16 to 5.00 micrograms/ml per enantiomer [mean r2 of 0.999 for both enantiomers, root mean square error were 0.015 for R(-) and 0.013 for S(+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the over the concentration range of 0.2 to 4.0 micrograms/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.

Optimisation of chiral separation of omeprazole and one of its metabolites on immobilized α1-acid glycoprotein using chemometricsglycoprotein using chemometrics

A strategy for the optimisation of direct chiral separation of omeprazole and a metabolite, hydroxi-omeprazole, in reversed phase liquid chromatography is described. A factorial design was used, where mobile phase pH, concentration of a mobile phase modifier, ionic strength and column temperature were tested as the variables and enantioselective retention, column efficiency and asymmetry factor as the responses. The experimental results were evaluated with multivariate analyses, which demonstrated that the column temperature and content of mobile phase acetonitrile were by far the most important variables. The enantiomers of omeprazole and one of its metabolites were baseline resolved within 15 minutes. The optimised chromatographic system was used for a separation of the enantiomers of omeprazole and its main metabolite in a patient plasma sample.