Chiral Stationary Phase High-performance Liquid Research Articles

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250mm, 5μm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50ml:50μL). Ultraviolet wavelength for detection was 220nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000ngmL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.

An Auxiliary Approach for the Stereoselective Synthesis of Topologically Chiral Catenanes

SummaryCatenanes, molecules in which two rings are threaded through one another like links in a chain, can form as two structures related like an object and its mirror image but otherwise identical if the individual rings lack bilateral symmetry. These structures are described as “topologically chiral” because, unlike most chiral molecules, it is not possible to convert one mirror-image form to the other under the rules of mathematical topology. Although intriguing and discussed as early as 1961, to date all methods of accessing molecules containing only this topological stereogenic element require the separation of the mirror-image forms via chiral stationary phase high-performance liquid chromatography, which has limited their investigation to date. Here, we present a simple method that uses a readily available source of chiral information to allow the stereoselective synthesis of topologically chiral catenanes.

Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors

Chiral stationary phase-high performance liquid chromatography coupled with various detectors has been one of most commonly used methods for analysis and separation of chiral compounds over the past decades. Various detectors exhibit different characteristics in qualitative and quantitative studies under different chromatographic conditions. Herein, a comparative evaluation of HPLC coupled with ultraviolet, optical rotation, refractive index, and evaporative light scattering detectors has been conducted for qualitative and quantitative analyses of metalaxyl racemate. Effects of separation conditions on the peak area ratio between two enantiomers, including sample concentration, column temperature, mobile phase composition, as well as flow rate, have been investigated in detail. In addition, the limits of detection, the limits of quantitation, quantitative range and precision for these two enantiomers by using four detectors have been also studied. As indicated, the chromatographic separation conditions have been slight effects on ultraviolet and refractive index detections and the peak area ratio between two enantiomers remains almost unchanged, but the evaporative light scattering detection has been significantly affected by the above-mentioned chromatographic conditions and the corresponding peak area ratios varied greatly. Moreover, the limits of detection, the limits of quantitation, and the quantitative ranges of two enantiomers with UV detection were remarkably lower by 1–2 magnitudes than the others.

Determination of trantinterol enantiomers in human plasma by high-performance liquid chromatography - tandem mass spectrometry using vancomycin chiral stationary phase and solid phase extraction and stereoselective pharmacokinetic application.

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human.

Direct separation of the diastereomers of phosphatidylcholine hydroperoxide bearing 13-hydroperoxy-9Z,11E-octadecadienoic acid using chiral stationary phase high-performance liquid chromatography

Increasing evidence suggests that phospholipid peroxidation plays important roles in the pathogenesis of various diseases, such as atherosclerosis. With regard to the biochemical processes that initiate phospholipid peroxidation in vivo, enzymatic conversion of phosphatidylcholine to phosphatidylcholine hydroperoxide (PCOOH) by lipoxygenase (LOX) may play a crucial role. This will become clear if we can analyze PCOOH bearing hydroperoxy fatty acids with S-stereoconfiguration. In this study, we therefore attempted such an analysis. Initially, we used LOX, linoleic acid and Lyso phosphatidylcholine, and synthesized PCOOH bearing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE). PCOOH bearing racemic 13-9Z,11E-HPODE was also prepared. We used liquid chromatography equipped with CHIRALPAK OP (+) (poly (o-pyridyl diphenylmethacrylate) coated on silica), a UV detector and a quadrupole-time-of-flight mass spectrometer, and achieved diastereomer separation of PCOOH stereoisomers with excellent resolution and peak shape. This is the first study reporting the diastereomer separation of PCOOH. The present method will be beneficial in developing a better understanding of the biochemical processes that initiate oxidative stress (PCOOH formation) in vivo, which may lead to further elucidation of the involvement of PCOOH in the development of diseases. In addition to clinical applications, the present method may also be effective in the evaluation of enzymatic oxidative food deterioration.

Racemization and stereoselective alcoholysis of temazepam

Enantiomers of temazepam (TMZ), an anxiolytic drug in clinical use, were resolved and stabilized by the use of aprotic solvents in chiral stationary phase high-performance liquid chromatography (CSP-HPLC). The enantiomers were used to study racemization and heteronucleophilic substitution (alcoholysis) reactions in anhydrous acidic methanol and ethanol. Kinetics of spontaneous racemization and stereoselective conversions to 3-O-methyltemazepam (MeTMZ) and 3-O-ethyltemazepam (EtTMZ) were determined by circular dichroism (CD) spectropolarimetry and CSP-HPLC. The rates of conversion of rac-TMZ to rac-MeTMZ (or rac-EtTMZ) were determined by ultraviolet-visible spectrophotometry. The results indicated that, for example, both N4-protonated and unprotonated forms of (S)-TMZ underwent spontaneous racemization and its 3S-hydroxyl group was highly stereoselectively substituted at C3 position by the ethoxy group of ethanol to form EtTMZ enriched in (S)-EtTMZ. Similar stereoselective reactions occurred for TMZ enantiomers in acidic methanol. Chirality 11:179–186, 1999. Published 1999 Wiley-Liss, Inc.

Enantiomeric Separation and Detection of 2-Arylpropionic Acids Derivatized with [(N,N-Dimethylamino)sulfonyl]benzofurazan Reagents on a Modified Cellulose Stationary Phase by High-Performance Liquid Chromatography

(RS)-2-Arylpropionic acids (2-APAs) were derivatized with the fluorogenic reagents, 4-[(N,N-dimethylamino)sulfonyl]-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) and 4-[[(N-hydrazinoformyl)methyl]-N-methyl]amino-7-[N,N-(dimethylamino)sulfonyl]-2,1,3-benzoxadiazole (DBD-COHz), and their enantiomeric separation by a chiral stationary phase high-performance liquid chromatography was investigated in the reversed-phase mode with H(2)O/CH(3)CN or H(2)O/MeOH as the mobile phase on a column of cellulose tris(3,5-dimethylphenyl carbamate) coated on a silica gel support (Chiralcel OD-R). The derivatives with DBD-PZ were enantiomerically separated well under the elution condition of H(2)O/MeOH, based on the π-π interaction between the derivatives and the stationary phase. The rigid and bulky structure of DBD-PZ was demonstrated to be more effective as compared to the less rigid ones. The derivatives with DBD-COHz were more efficiently separated into each enantiomer with H(2)O/CH(3)CN as the eluent. The effective separation was based on hydrogen-bonding interaction between the acid hydrazide of the derivatives and the carbamoyl moiety of the stationary phase. There was a reversal in the elution order of the enantiomers between the two fluorescent derivatives. The detection limits obtained for each enantiomer were approximately 10-30 fmol on column. The derivatization with the reagent and the concomitant use of the reversed-phase and chiral stationary-phase HPLC were demonstrated to be useful for the enantiomeric quantification in rat plasma after intravenous administration of flurbiprofen racemate, a representative of 2-APAs.

Stereoselective nucleophilic substitution of oxazepam and racemization in acidic methanol and ethanol.

Enantiomeric and racemic oxazepam (OX), 3-O-methyloxazepam (MeOX), and 3-O-ethyloxazepam (EtOX) were used to study racemization, heteronucleophilic, and homonucleophilic substitution reactions in anhydrous acidic methanol and ethanol. Kinetics of racemization and nucleophilic substitution reactions in nondeuterated and deuterated solvents were determined by circular dichroism spectropolarimetry, chiral stationary phase high-performance liquid chromatography (HPLC), reversed-phase HPLC, and mass spectrometry. Several reactions occurred when (S)-OX, for example, was dissolved in acidic methanol: (1) (S)-OX itself underwent spontaneous racemization, (2) the 3-hydroxyl group of (S)-OX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (S)-MeOX, and (3) the 3-methoxy group of (R)-MeOX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (S)-MeOX, and (4) the 3-methoxy group of (R)-MeOX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (R)-MeOX. Repetitive reactions 3 and 4 eventually resulted in a racemic MeOX. Similar reactions occurred for an enantiomeric OX in acidic ethanol.

Enantioselectivity suggests a cytosolic origin for a commercial pig liver esterase preparation

A widely utilized pig liver esterase preparation has been found to be derived essentially exclusively from the cytosolic fraction of pig livers. Esterases in cytosol and microsomes prepared from a fresh pig liver hydrolyzed the S- and R-enantiomers of racemic oxazepam 3-acetate (rac-OXA) with specific activity ratios of approximately 2.3:1 and 1:62, respectively. Product formations were analyzed by chiral stationary phase high-performance liquid chromatography. The commercial pig liver esterase preparation showed greater activity toward S-OXA than did the esterases in the cytosolic fraction prepared from fresh pig liver. The results established that (i) esterases contained in microsomes and cytosol of pig liver have opposite enantioselectivity in the hydrolysis of rac-OXA and (ii) the commercial pig liver esterase preparation has a cytosolic origin.

Acid-catalyzed stereoselective heteronucleophilic substitution and racemization of 3-O-methyloxazepam and 3-O-ethyloxazepam.

Enantiomers of 3-O-methyloxazepam (MeOX) and 3-O-ethyloxazepam (EtOX) were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Reaction kinetics and deuterium isotope effects of acid-catalyzed racemization of enantiomeric MeOX in ethanol and enantiomeric EtOX in methanol were studied by spectropolarimetry. The acid-catalyzed heteronucleophilic substitution reactions of racemic MeOX in ethanol and racemic EtOX in methanol were studied by reversed-phase HPLC. Thermodynamic parameters involved in the reactions were obtained by temperature-dependent reaction rates. The effects of solvent's dielectric constant on the heteronucleophilic substitution reactions were also determined. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer, derived from a 1,4-benzodiazepine either in M (minus) or P (plus) conformation, is proposed to be an intermediate and responsible for the acid-catalyzed stereoselective nucleophilic substitution and the resulting racemization.

Stereoselective homonucleophilic substitution of 3- O-methyl and 3- O-ethyloxazepam enantiomers by chiral stationary phase high-performance liquid chromatography

Enantiomers of 3- O-methyloxazepam and 3- O-ethyloxazepam were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Temperature-dependent and acid-catalysed racemization of 3- O-methyloxazepam enantiomers in methanol and 3- O-ethyloxazepam enantiomers in ethanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of reaction product were determined by CSP-HPLC. Thermodynamic parameters in the formation of the activated complex ( E act, Δ H‡, Δ S‡ and Δ G‡) were consistent with those determined by a spectropolarimetric method. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer is proposed to be an intermediate and responsible for the acid-catalysed stereoselective homonucleophilic substitution and the resulting racemization.

Resolution of enantiomeric triols, triol-hydroxyethylthioethers, and methoxy-triols derived from three benzo[ a]pyrene diol-epoxides by chiral stationary phase high-performance liquid chromatography

Benzo[ a]pyrene 7,8-diol- anti-9,10-epoxide, 7,8-diol- syn-9,10-epoxide, and 9,10-diol- anti-7,8-epoxide were converted to triol, triol-hydroxyethylthioether, and methoxy-triol derivatives. Enantiomeric pairs of these derivatives were resolved by high-performance liquid chromatography with Pirkle's π-electron acceptor chiral stationary phases. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analyses. Relative to those of tetrols, these derivatives are less polar and have shorter retention times and improved enantiomeric resolution on chiral stationary phases. Absolute stereochemistries of most enantiomeric derivatives were deduced by comparing their circular dichroism spectra to those of similar compounds derived from enantiomeric diol-epoxides of known absolute stereochemistry.

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols.

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.

Stereoselective formation and hydration of benzo[ c]phenanthrene 3,4-and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[ c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58–72%) in the 5 S,6 R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78–86%) in the 5 S,6 R enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3 S,4 R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5 S,6 R absolute stereochemistry. Absolute configurations of enantiomeric BcPh 5,6-epoxides were determined by CD spectral analyses of the enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5 S,6 R-epoxide was enzymatically hydrated exclusively at the C 6 position to form an optically pure BcPh 5 S,6 S-dihydrodiol. However, optically pure BcPh 5 R,6 S-epoxide was hydrated at both C 5 and C 6 positions to form a BcPh trans-5,6-dihydrodiol with a (5 S,6 S):(5 R,6 R) enantiomer ratio of 32:68.

Elution order—absolute configuration relationship of k-region dinhydrodiol enantiomers of benz[ a]anthracene derivatives in chiral stationary phase high-performance liquid chromatography

The direct resolution of K-region cis and trans-hydrodiol enantiomers of 14 unsubstituted and methyl- and bromo-substituted benz[ a]anthracene (BA) derivatives was investigated by high-performance liquid chromatography with commercially available columns, packed with γ-aminopropylsilanized silica to which eitehr ( R)-N-(3,5-dinitrobenzoyl)phenylglycine ( R-DNBPG) or ( S)-N-(3-5-dinitrobenzoyl)leucine ( S-DNBL) is either ionically or covalently bonded. BA derivatives used in this study include: BA, 1-methyl-BA, 4-methyl-BA, 7-methyl-Ba, 8-methyl-Ba, 10-methyl-BA, 11-methyl-BA, 12-methyl-Ba, 7,12-methyl-BA, 7-bromo-Ba, 7-bromo-1-methyl-Ba, 7-bromo-11-methyl-BA, 7-bromo-12-methyl-BA, and 3-methylcholanthrene. The enantiomers of BA trans-5,6-dihydrodiol were the only compounds not resolved by any of the four chirals stationary phases (CSPs) tested. The results indicate that conformational preference of the hydroxyl group is one of the most important factor in determining the elutionorder of dihyrodiol enantiomers. The presence and the location of a substituent and the molecular size and shape of the dihydrodiols can significantly affect the efficiency of enantiomeric resolution. In general, the ionically bonded R-DNBPG provides the best resolution of enantiomeric quasidiequatorial trans-dihydrodiols and the R,R enantiomers ar consistently more strongly retained. In contranst, the enantiomeric pairs of quasidiaxial trans-dihydrodiols are generaly better resolved by the covalently bonded R-DNBPG, and the S,S enantiomers are more strongly retained. The enantiomers of cis-dihydrodiols having hydroxyl groups that adopt quasiequatorial-quasiaxial and/or quasiaxial-quasiequatorial conformations are more consistently resolved by the ionically bonded S-DNBL and in all cases the S,R enantiomers are more strongly retained. Thus, it is possible to choose a CSP which resolves the K-region dihydrodiol enantiomers with a predictable elution order.

Two K-regions of 5-methylchrysene are sites of oxidative metabolism

Two K-region trans-dihydrodiols were identified as products formed in the metabolism of 5-methylchrysene by liver microsomes from phenobarbital-treated male Sprague-Dawley rats. These two dihydrodiols were isolated from a mixture of metabolites by reversed-phase and normal-phase high-performance liquid chromatographies. Both K-region dihydrodiols were characterized by ultra-violet, mass, and circular dichroism spectral analyses. Chiral stationary phase high-performance liquid chromatographic analyses indicated that 5-methylchrysene 5,6-dihydrodiol and 11,12-dihydrodiol contain (S,S): (R,R) enantiomer ratios of 2:98 and 12:88, respectively. Although it is a bay-region dihydrodiol, the hydroxyl groups of 5-methylchrysene trans-5,6-dihydrodiol adopt a quasidiequatorial conformation.

Stereoselective metabolism of chrysene by rat liver microsomes. Direct separation of diol enantiomers by chiral stationary phase h.p.l.c.

The direct enantiomeric resolution of non-K region trans-1,2-dihydrodiol, 1,2,3,4-tetrahydro-trans-1,2-diol, trans-3,4-dihydrodiol and 1,2,3,4-tetrahydro-trans-3,4-diol, K region trans- and cis-5,6-dihydrodiols and their monomethyl ethers of chrysene was studied by chiral stationary phase high-performance liquid chromatography (CSP-h.p.l.c.). The chiral stationary phase columns were packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)-phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine was bonded either ionically or covalently. Enantiomers of all dihydrodiol derivatives were resolved by one or more, but not all, of the chiral stationary phases utilized. Enantiomeric resolutions were substantially improved when the non-K region dihydrodiols were converted to tetrahydrodiols. The absolute configurations of the K region trans- and cis-5,6-dihydrodiols were established by the exciton chirality circular dichroism method. The (R,R):(S,S) enantiomer ratios, determined by CSP-h.p.l.c., of the 1,2-, 3,4- and 5,6-trans-dihydrodiols formed in the metabolism of chrysene by liver microsomes from untreated male rats of the Sprague--Dawley strain were found to be 51:49, 99:1 and 86:14, respectively; from phenobarbital-treated rats, 41:59, 99:1 and 87:13, respectively; from 3-methylcholanthrene-treated rats, 96:4, 99:1 and 92:8, respectively. The absolute configurations of chrysene 5,6-epoxide enantiomers, resolved by CSP-h.p.l.c., were elucidated by the determination of the structures and absolute configurations of their methoxylation products. Both enantiomers of chrysene 5,6-epoxide were hydrated by microsomal epoxide hydrolase to chrysene trans-5,6-dihydrodiol enriched (67-92%) in the 5R,6R enantiomer. Chrysene 5R,6S-epoxide was hydrated to trans-5,6-dihydrodiol at a rate approximately 6-fold faster than chrysene 5S,6R-epoxide.

Direct enantiomeric resolution of cyclic alcohol derivatives of polycyclic aromatic hydrocarbons by chiral stationary phase high-performance liquid chromatography

The enantiomers of some cyclic alcohol derivatives of phenanthrene, benz-[ a]anthracene, benzo[ a]pyrene, cholanthrene, and 3-methylcholanthrene were resolved by high-performance liquid chromatography using a commercially available preparative column packed with an ( R)-N-(3,5-dinitrobenzoyl)phenylglycine ionically bonded to γ-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet—visible absorption, mass and circular dichroism spectral analyses. This method has been applied to the determination of optical purity of 1-hydroxycholanthrene and 1-hydroxy-3-methylcholanthrene formed in the metabolism of cholanthrene and 3-methylcholanthrene, respectively, by rat liver microsomes.