Use of derivatized cyclodextrins as chiral selectors for the separation of enantiomers by gas chromatography.

The state-of-the-art of enantioseparations of various chiral compounds on derivatized cyclodextrins, employed as chiral stationary phases (CSPs) by gas chromatography, is reviewed. Heptakis(2,3,6-O-trimethyl)-beta-cyclodextrin and octakis(3-O-butanoyl-2,6-di-O-pentyl)-gamma-cyclodextrin (Lipodex E), either dissolved in a semipolar polysiloxane or bonded to poly(dimethylsiloxane) (Chirasil-Dex), proved to be the most popular chiral selectors. Special applications refer to the use of heptakis (2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin. Enantioselectivity is generally low with enantioseparation factors in the range of 1.02

A mixed stationary phase containing two versatile cyclodextrin-based selectors for the simultaneous gas chromatographic enantioseparation of racemic alkanes and racemic alpha-amino acid derivatives.

In an effort to simultaneously enantioseparate racemic unfunctionalized alkanes and racemic alpha-amino acid derivatives by gas chromatography (GC) in forthcoming experiments related to the search for extraterrestrial homochirality, the two versatile modified cyclodextrin (CD) selectors octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin (Lipodex G) and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin were dissolved in a polysiloxane and the mixed binary chiral selector system was coated onto a 50m x 0.25 mm i.d. fused silica capillary column. Whereas the former CD selector enantioseparates racemic unfunctionalized alkanes the latter CD selector preferentially resolves N-(O,S)-trifluoroacetyl-alpha-amino acid alkyl esters. With both CD selectors employed as mixed binary chiral selector system present in one chiral stationary phase (CSP), the simultaneous gas chromatographic enantioseparation of racemic alkanes and of racemic derivatized alpha-amino acids is achieved in a single temperature-programmed run. Also for other classes of racemic compounds, the scope of enantioseparation could be extended as compared to the conventional use of the single CD selectors in GC.

Chiral discrimination using an immunosensor.

Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized alpha-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.

Stereoselective pharmacokinetic analysis of valnoctamide in healthy subjects and in patients with epilepsy.

OBJECTIVE: To investigate the pharmacokinetics of the four stereoisomers of valnoctamide, a mild tranquilizer endowed with anticonvulsant properties. METHODS: Racemic valnoctamide, 400 mg, was administered orally to seven healthy subjects and to six patients with epilepsy stabilized with long-term carbamazepine therapy. In the patients with epilepsy, valnoctamide kinetics was also reassessed after 8-day oral dosing at a dosage of 600 mg daily. Plasma samples were assayed by gas chromatography-mass spectrometry with use of a capillary column coated with chiral stationary phase that enabled baseline resolution of the four stereoisomers, designated hereafter as A, B, C, and D (where A and C, together with B and D, represent enantiomeric pairs). RESULTS: In healthy subjects, stereoisomers A, C, and D showed similar kinetics, with an apparent oral clearance (CL/F) of about 4 1/2 L/hr, a half-life (t1/2) of about 10 hours, and an apparent volume of distribution (VSS/F) of about 65 L. However, stereoisomer B showed a much higher clearance (8.7 +/- 0.9 L/hr) and a shorter t1/2 (5.8 hours). For all stereoisomers, CL/F values in patients with epilepsy were about tenfold higher than those found in healthy subjects. Compared with healthy subjects, patients with epilepsy also showed shorter t1/2 values and higher VSS/F values for each of the stereoisomers. After 7-day dosing, CL/F values at steady state were lower than those determined in the same patients after a single dose. CONCLUSIONS: Valnoctamide exhibits enantioselectivity and diastereoselectivity, an observation that may have important practical implications if pharmacodynamic differences between stereoisomers are also found. The observed pharmacokinetic differences between healthy subjects and patients with epilepsy are likely to be related to induction of metabolism of valnoctamide stereoisomers by carbamazepine.

Direct binding of low molecular weight haptens to ELISA plates.

Immobilization of low molecular weight haptens in ELISA usually involves their coupling to protein molecules or covalent binding to the solid phase. In this study we demonstrate that it is possible to directly bind the hapten p-aminophenylalanine to gamma-irradiated polystyrene microtiter plates for the detection of antibodies that stereospecifically recognize the chiral center of alpha-amino acids. Simple incubation of the hapten in aqueous buffer, without additional activation, results in a stable coating that is suited for use in ELISA.

Nonprogressive cerebellar disorder with mental retardation and autosomal recessive inheritance in Hutterites.

We report a nonprogressive neurological disorder in at least 11 Hutterites with healthy but consanguineous parents. In several of the affected, hypotonia was noted at birth. Retarded motor and mental development became apparent during the first year of life. The age of unsupported walking varied from 5-21 years. Consistent signs were unsteady, broadly based gait and stance, exaggerated deep tendon reflexes mainly in the lower limbs, and mild to moderate mental retardation. Variable signs were extensor plantar reflexes (9/11), short stature (-2SD in 8/11), strabismus (7/11), small muscle mass (6/11), mild intention tremor (3/11), cataracts (1/11), and epilepsy (1/11). CAT scans in two affected sisters showed slight enlargement of the fourth ventricle in one and hypoplasia of the cerebellum in both. The disorder is probably the same as that described earlier under the heading, dysequilibrium syndrome.

The Meckel syndrome in the Hutterites.

At least three cases of the Meckel syndrome have been identified in the Hutterites. Two of these were sibs and were studied during life; the other, a close relative, was diagnosed retrospectively by a review of hospital records. All parents were consanguineous. The phenotype ranged in severity from the association of occipital meningocele, cystic kidneys, postaxial polydactyly, and microphthalmia to a milder expression consisting of cystic kidneys, ocular defects apparent only on funduscopic examination, and a brain abnormality demonstrated by computer tomography. Survival ranged from 5 to 13 months. In one patient, the renal lesion was manifested as a tubular rather than a glomerular defect, and was probably not the primary cause of death.

Biological activation of 1,3-butadiene to vinyl oxirane by rat liver microsomes and expiration of the reactive metabolite by exposed rats.

When 1,3-butadiene is incubated with rat liver microsomes and NADPH both enantiomers of vinyl oxirane are formed, the amount of epoxide being dependent on incubation time, microsomal protein, and substrate concentration. Inhibition by SKF 525 A or dithiocarb as well as induction by pretreatment with phenobarbital or 20-methylcholanthrene suggest participation of cytochrome P-450 in this reaction. The amount of epoxide is enhanced by addition of 1,1,1-trichloropropene oxide and reduced by glutathione, especially in the presence of hepatic cytosol. When rats are exposed to 1,3-butadiene in a closed chamber (conditions of maximal metabolism) vinyl oxirane is exhaled and can be quantitatively determined from the gas phase. The concurrent exhalation of acetone is consistent with the idea of biologic action of a reactive metabolite.

Separation of enantiomers by gas chromatography.

The separation of enantiomers by gas chromatography is performed on chiral stationary phases (CSPs) via hydrogen bonding, coordination and inclusion. Thus, typical chiral selectors are amino acid derivatives, terpene-derived metal coordination compounds and modified cyclodextrins. In Chirasil-type stationary phases the chiral selector is anchored to a polysiloxane backbone improving gas chromatographic performance. The present review article describes the state-of-the-art, scope and limitations, applications and mechanistic considerations at the advent of the millennium incorporating 16 figures and 168 references.

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography.

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.

Enantioselective determination of chiral organochlorine compounds in biota by gas chromatography on modified cyclodextrins.

Approaches to the gas chromatographic enantiomer separation of chiral organchlorines (alpha-hexachlorocyclohexane, cis- and trans-chlordane, heptachlor, heptachlorepoxide, oxychlordane, o,p'-DDT, compounds of technical toxaphene and stable atropisomeric polychlorinated biphenyls) are reviewed. Chiral stationary phases based on cyclodextrin derivatives and used for the gas chromatographic enantiomer separation of the chiral organochlorines are described. Enantiomeric ratios of chiral organochlorines in technical mixtures and biological samples are reported and discussed.

Approach to the thermodynamics of enantiomer separation by gas chromatography. Enantioselectivity between the chiral inhalation anesthetics enflurane, isoflurane and desflurane and a diluted gamma-cyclodextrin derivative.

The thermodynamics of enantioselectivity, -delta D,L(delta G), -delta D,L(delta H), delta D,L(delta S) and Tiso, have been determined by gas chromatography employing the concept of the retention increment R' for the inhalation anesthetics enflurane (1), isoflurane (2) and desflurane (3) and the selector octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-gamma-cyclodextrin (4) in the polysiloxane SE-54. It is shown that the separation factor alpha is concentration-dependent. Therefore, the separation factor alpha should not be employed as a criterion for enantioselectivity in diluted systems. The -delta DL(delta G) data for 1 and 4 are corroborated by 1H NMR spectroscopic measurements.

Approximation function for the direct calculation of rate constants and Gibbs activation energies of enantiomerization of racemic mixtures from chromatographic parameters in dynamic chromatography.

An approximation function for enantioselective dynamic chromatography of racemic mixtures of interconverting enantiomers has been derived that allows the direct calculation of enantiomerization rate constants (k1 and k(-1)) and Gibbs activation energies of enantiomerization, deltaG++ , from chromatographic parameters, i.e., retention times of the enantiomers A and B ((t(A)R and t(B)R), peak widths at half height (WA and wB) and the relative plateau height (hplateau), without computer simulation. The reaction rate constants of enantiomerization, k(-1), obtained with this approximation function, have been validated by comparison with a simulated dataset of 15,625 chromatograms. The mean, standard deviation and confidence interval show a high correlation between the approximated and simulated rate constants. The average deviation from the Gibbs activation enthalpy of enantiomerization, deltaG++, has been estimated to be as small as about +/- 0.11 RT.

Preparative enantiomer separation of the inhalation anesthetics enflurane, isoflurane and desflurane by gas chromatography on a derivatized gamma-cyclodextrin stationary phase.

The preparative enantiomeric separation of the inhalation anesthetics enflurane (1) and isoflurane (2) in very high chemical (> 99.5%) and enantiomeric excess (ee > 99%) by gas chromatography (GC) on octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-gamma-cyclodextrin (4), dissolved in the apolar polysiloxane SE-54 and coated on Chromosorb P AW DMCS, is described. Up to 1 g of each enantiomer of 1-2 can been obtained per diem. The enantiomers of the highly volatile desflurane (3) can also be separated, albeit with diminished ee. The enantiomeric excess of 1-3 was checked by analytical GC on 4 and the absolute configuration of 2 and 3 has been determined via anomalous X-ray diffraction.

Enantioselective stopped-flow multidimensional gas chromatography. Determination of the inversion barrier of 1-chloro-2,2-dimethylaziridine.

Enantioselective stopped-flow multidimensional gas chromatography (stopped-flow MDGC) is a fast and simple technique to determine enantiomerization (inversion) barriers in the gas phase in a range of delta G#gas(T)=70-200 kJ mol(-1). After complete gas-chromatographic separation of the enantiomers in the first column, gas phase enantiomerization of the heart-cut fraction of one single enantiomer is performed in the second (reactor) column at increased temperature and afterwards this fraction is separated into the enantiomers in the third column. From the observed de novo enantiomeric peak areas a(j), the enantiomerization time t and the enantiomerization temperature T, the enantiomerization (inversion) barrier delta G#gas(T) is determined and from temperature-dependent experiments, the activation enthalpy delta H#gas and the activation entropy delta S#gas are obtained. Enantiomerization studies on chiral 1-chloro-2,2-dimethylaziridine by stopped-flow MDGC yielded activation parameters of nitrogen inversion in the gas phase, i.e., delta G#gas(353 K)=110.5+/-0.5 kJ mol(-1), delta H#gas=71.0+/-3.8 kJ mol(-1) and delta S#gas=-109+/-11 J mol(-1) K(-1). By the complementary method of dynamic gas chromatography (GC), the apparent enantiomerization (inversion) barrier of 1-chloro-2,2-dimethylaziridine in the gas-liquid biphase system was found delta G#app(353 K)=108 kJ mol(-1). The values obtained by stopped-flow MDGC in the gas phase were used to calculate the activation parameters of nitrogen inversion of 1-chloro-2,2-dimethylaziridine in the liquid phase in the presence of the chiral selector Chirasil-nickel(II), i.e.. deltaG#liq(353 K)=106.0+/-0.4 kJ mol(-1), delta H#liq=68.3+/-1.4 kJ mol(-1) and deltaS#liq=-106+/-3.0 J mol(-1) K(-1).

Enantiomer separation on a Chirasil-Dex-polymer-coated stationary phase by conventional and micro-packed high-performance liquid chromatography.

Polymer-coating of Chirasil-Dex (a polysiloxane-anchored permethyl-beta-cyclodextrin) on Nucleosil 300-5 followed by thermal immobilization furnishes a chiral stationary phase for enantiomer separation under reversed-phase conditions by conventional and micro-packed HPLC.

Enantiomer separation by pressure-supported electrochromatography using capillaries packed with a permethyl-beta-cyclodextrin stationary.

Efficient enantiomer separation by pressure-assisted, micro-packed capillary electrochromatography (CEC) has been carried out using a permethyl-beta-cyclodextrin-modified silica support (PM-beta-CD-silica). When comparing this method with micro-packed-high-performance liquid chromatography in the single-column-mode, CEC displays higher column efficiencies (about three times higher theoretical plate numbers at comparable elution times). The pressure support (about 10 bar), applied to avoid bubble formation, has a negligible influence on elution times in CEC. The influence of the type and composition of organic modifiers is described.

Time-resolved cryogenic modulation reveals isomer interconversion profiles in dynamic chromatography.

The dynamic chromatographic study of interconversion of E and Z forms of oximes has been investigated by using a novel cryogenic modulation method in a two-dimensional gas chromatographic array. The primary column is a conventional capillary GC column on which the molecular interconversion proceeds. In this case, the molecular dynamical process leads to a peak profile describing the kinetics and thermodynamics of the interconverting molecules during its chromatographic elution. Thus an interconversion region intercedes the elution of the individual stereoisomers of the reaction. Since the molecules are isomers, classical molecular identification methods such as gas chromatography-mass spectrometry are unable to study the individual instantaneous amounts of each of the compounds. Hence the infinitesimal profiles of interconversion along the entire column have never been experimentally observed; rather the total profile is normally subjected to mathematical modelling studies in order to match experiment with theory, and to gain the kinetic parameters of the process. In the present study, an instantaneous ratio of the individual isomers can be found during the chromatographic elution by direct measurement. This is achieved by using a cryogenic zone focussing process, with rapid longitudinal modulation of a cold trap and continual pulsing of collected zones into a fast-analysis high-resolution capillary column on which isomer interconversion is minimized. The data can be displayed as a two-dimensional contour plot to demonstrate the individual isomer profiles. The two-dimensional analysis also allows easy measurement of the peak ratios of the two isomers which is an indicator of the extent of interconversion that has taken place. Two model systems, acetaldoxime and butyraldoxime, were chosen to illustrate the use of the cryogenic modulation procedure. It is anticipated that the procedure could be applied to other molecules which exhibit gas-phase isomerizations or reactions.

Unified enantioselective capillary chromatography on a Chirasil-DEX stationary phase. Advantages of column miniaturization.

Immobilized Chirasil-DEX (mono-6-O-octamethylenepermethyl-beta-cyclodextrin chemically linked to dimethylpolysiloxane) can be employed as a versatile chiral stationary phase in chromatography. The chiral polymer has a long lifetime and is configurationally and thermally stable. The concept of unified enantioselective chromatography has been demonstrated for the enantiomer separation of hexobarbital by gas chromatography, supercritical fluid chromatography, liquid chromatography and capillary electrochromatography on a single open-tubular column (1 m x 50 microns I.D.) coated with Chirasil-DEX. The advantages of miniaturization in contemporary chromatographic enantiomer separation are demonstrated. Chirasil-DEX coated on porous silica is also useful for enantiomer separation in high-performance liquid chromatography.

Enantiomer separation by complexation SFC on immobilized Chirasil-nickel and Chirasil-zinc.

The use of complexation SFC for enantiomer separation of Lewis base selectands on chiral nickel(II)- and zinc(II)-bis[(3-heptafluorobutanoyl)-10-methylene-(1R)-camphora te] chemically bonded to poly(dimethylsiloxane) (Chirasil-nickel and Chirasil-zinc) and employed as Lewis acid selectors is described. The method is especially suited for less volatile and configurationally labile racemates. The variation of the experimental parameters temperature T, pressure p and density rho of the mobile phase carbon dioxide on the retention factor k, relative retention r and chiral separation factor alpha is studied, providing insights into the mechanisms of chiral recognition under supercritical conditions. For mecoprop methyl ester (methyl 2-(4-chloro-2-methylphenoxy)propanoate) an unusual increase of alpha at increased temperature is observed on Chirasil-nickel. Supercritical carbon dioxide does not inadvertently affect the complexation equilibria between Lewis donor selectands and the Lewis acid metal selectors during complexation SFC.