Chiral Stationary Phase Column Research Articles

Direct Liquid Chromatographic Separation of 2,3-Dihydro-2-ethylbenzofuran-2-carboxylic Acid Enantiomers Using a Cellulose Carbamate-Based Chiral Stationary Phase Column

Abstract Direct liquid chromatographic separation of 2,3-dihydro-2-ethylbenzofuran-2-carboxylic acid enantiomers on a commercial cellulose carbamate-based chiral stationary phase column is described. The addition of a small amount of organic acid in the mobile phase is required for the elution and separation of both enantiomers. Better peak shapes are obtained when trifluoroacetic acid is used. The method allows the detection of the undesired (+)-enantiomer down to a level of 0.5 % and can be used to measure the enantiomeric purity of (-)-enantiomer after resolution.

Development of optically active fluorescent Edman-type reagents

The optically active fluorescent Edman-type reagents 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [(R)-(–)- and (S)-(+)-NBD-PyNCS] and 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [(R)-(–)- and (S)-(+)-DBD-PyNCS] were synthesized and characterized by their physico-chemical properties. High optical purities of all four enantiomers were identified by high-performance liquid chromatography (HPLC) with a chiral stationary phase (CSP) column. Good stability in both solid and liquid forms is one of the excellent properties of these novel Edman-type reagents. The applicability of these reagents to the resolution of mirror-image enantiomers of amines, including amino acids and some drugs (β-adrenergic blocking agents), by reversed-phase HPLC was demonstrated. The proposed reagents read quantitatively with primary and secondary amino functional groups under mild conditions (55 °C for 10 min) in the presence of triethylamine (TEA) to produce the corresponding fluorescent thiourea derivatives. The maximum excitation/emission wavelengths of the resulting diastereomers in acetonitrile–water (1 + 1) are around 490/530 nm for NBD-PyNCS derivatives and around 460/550 nm for DBD-PyNCS derivatives. The fluorescence wavelengths are independent of the amino compounds. The diastereomers that were derived from the amines were efficiently resolved on an achiral ODS column with water–acetonitrile containing 0.05% trifluoroacetic acid (TFA). The Rs values were in the range 0.83–4.12 for the diastereomers obtained with NBD-PyNCS and 1.35–6.06 for those with DBD-PyNCS. The diastereomer corresponding to the R configuration of the amines elute faster than those of the S configuration with the (R)-(–)-enantiomer of the Edman-type reagents used as the derivatization reagent. As expected, the opposite elution order is obtained with the use of the (S)-(+)-enantiomers.

Sub- and supercritical fluid chromatography on packed columns: a versatile tool for the enantioselective separation of basic and acidic drugs.

Various chiral stationary phases were investigated in sub- and supercritical fluid chromatography for the separation, without derivatization, of basic (beta-blockers, benzodiazepines) and acidic (nonsteroidal anti-inflammatory drugs, beta-agonists) pharmaca. For all racemates, baseline separation was achieved within short analysis times. For several solutes, the high resolution obtained allowed injection of milligram amounts and semipreparative collection of the enantiomers. The parameters affecting enantioselectivity (column efficiency, influence of modifiers and basic or acidic additives, and temperature) have been studied. Enantiomerization of 3-OH-benzodiazepines could be suppressed by working at low temperatures or by using acetonitrile as a comodifier. Serial coupling of different chiral stationary phase columns resulted in a column triplet (Chiralpak AD, an amylose derivative; Chiralcel OD, a cellulose derivative; and Chirex 3022, a Brush-type with pi-donor characteristics) on which all solutes investigated could be baseline separated.

Enantiomer resolution of camazepam and its derivatives and enantioselective metabolism of camazepam by human liver microsomes

Camazepam [3-(N,N-dimethyl)carbamoyloxy-7-chloro-1-methyl-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one, CMZ] possesses anxiolytic, anticonvulsant, muscle relaxant, and hypnotic properties. CMZ is clinically used as a racemate. Enantiomer resolution of CMZ and 11 of its derivatives was studied by high-performance liquid chromatography (HPLC) using 5 different chiral stationary phase (CSP) columns. Enantiomers of 10 compounds were resolved by at least one of the 5 CSP's tested. Enantiomers of two other compounds, which have either one or two hydroxymethyl groups at the carbamoyl nitrogen, were either not resolved or resolved with very low efficiency. However, enantiomers of the hydroxymethyl derivatives were resolved via base-catalyzed dehydroxymethylation. In vitro metabolism of racemic CMZ by human liver microsomes was found to be enantioselective. Major metabolites were isolated by normal-phase and reversed-phase HPLC and further characterized by ultraviolet absorption and circular dichroism spectral analyses, and by chiral stationary phase HPLC analysis. Following an in vitro incubation of rac-CMZ, the unmetabolized CMZ was found to be enriched in ( S)-CMZ, indicating that the R-enantiomer was enantioselectively metabolized. Metabolites were formed primarily by hydroxylation and demethylation of the methyl groups at the C3 side chain. All metabolites were found to be optically active, enriched in either the S-enantiomer or the R-enantiomer.

Resolution of the enantiomers of ibuprofen; comparison study of diastereomeric method and chiral stationary phase method

In this study, an indirect diastereomeric method and a direct method utilizing a chiral stationary phase (CSP) were investigated for the resolution of ibuprofen enantiomers. In the indirect method, ethylchloroformate (ECF) and 2-ethoxy-1-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) were utilized as first-step derivatizing reagents in acetonitrile or toluene. In the direct CSP method, ibuprofen enantiomers were derivatized to p-nitrobenzyl ureides and then resolved on an ( R)-(−)-(1-naphthyl)ethylurea CSP column. The derivatization procedure took place in 10 min with an overall inversion efficiency of 90.3%. Racemization was not observed under the derivatization conditions used. The HPLC-CSP method was utilized to study the pharmacokinetics of ibuprofen enantiomers in dog plasma after a single oral administration of 200 mg of ibuprofen racemate.

High-performance liquid chromatographic determination of the isomeric purity of a series of dioxolane nucleoside analogues

Racemic (±)- cis-2 hydroxymethyl-4-(cytosin-1′-yl)-1,3-dioxolane analogue (BCH-204) exhibited high levels of anti-HIV activity, but also showed cytotoxicity at the active concentration. To examine the possibility of enantiomerically separating the HIV activity from the cytotoxicity in the dioxolane nucleosides, high-performance liquid chromatography using chiral stationary phase columns was examined. The successful separation of dioxolane compounds was demonstrated utilizing optimum conditions of columns, solvents, flow-rates and temperature. In the reversed-phase mode, the cyclodextrin columns Cyclobond I SP and RSP were used to separate the enantiomers of cis- and trans-(±) dioxolane-C, and the protein column α-AGP was successful in separating enantiomerically cis-(±) dioxolane-G and cis- and trans-enantiomeric forms of (±)- dioxolane-A. In the normal-phase mode, one of the cellulose columns, Chiralcel OJ, successfully separated enantiomerically (±)- dioxolane-T nucleosidic analogue.

Enantiomeric Composition of the trans -Dihydrodiols Produced from Phenanthrene by Fungi

The trans-dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans-1,2-dihydrodiol produced by C. elegans was a mixture of the 1R,2R and 1S,2S enantiomers in variable proportions. The phenanthrene trans-3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3R,4R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3R,4R and 3S,4S enantiomers. The phenanthrene trans-9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9S,10S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9R,10R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.

Further studies on the resolution of carboxylic acid enantiomers by liquid chromatography with fluorescence and laser-induced fluorescence detection

The optical purities of chiral derivatization reagents 4-( N,N-dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APys), 4-(aminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (ABD-APys) and 4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APys) were determined by chromatographic separation with an Ultron ES-PHCD (β-cyclodextrin phenylcarbamate derivative) chiral stationary phase column. With the exception of (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole ( d-NBD-APy), which was found to have a purity of 99.5%, the enantiomers were determined to be more than 99.8% pure. The rate of the derivatization reaction for each enantiomer of DBD-APy at room temperature in the presence of the carboxylic acid reaction promoters 2,2′-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP), was essentially the same for each enantiomer of naproxen. The reaction rates of 50°C were comparable at room temperature. When the derivatizations with DBD-APy and NBD-APy were carried out in the presence of diethyl phosphorocyanidate (DEPC) instead of DPDS and TPP quantitative derivatizations were not obtained even after a prolonged reaction time. The diastereomers derived from anti-inflammatory drups and N-acetylamino acid enantiomers were efficiently resolved by both normal-phase ( R s = 2.58–7.60) and reversed-phase ( R s = 1.62–6.96) chromatography. The resolution of carboxylic acid enantiomers by liquid chromatography with laser-induced fluorescence (LIF) detection was also investigated. The minimum detectable levels by reversed-phase chromatography with an argon ion laser at 488 nm were 29 fmol of ABD-APy-Nap, 11 fmol of DBD-APy and 2.9 fmol of NBD-APy. Ibuprofen enantiomers added to rat plasma and human urine were completely separated by an ODS column with water-acetonitrile as eluent without interference from endogenous compounds. Sensitive detection was achieved with a commercially available LIF detector. Negligible mutagenicity of the reagents was observed by the Ames test using Salmonella typhimurium strains TA100 and TA98

Enantiomeric Separation of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

Abstract We have found that high-performance liquid chromatographic analysis of enantiomeric N-protected amino acid esters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) can be utilized as one of the procedures for determining the optical purities of non-protein amino acids. The methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of non-protein amino acids showed excellent to good enantiomeric separations using hexane - 2-propanol as a mobile phase. There was a regularity in the elution order of enantiomers: the L-isomer had a shorter retention time than the D-isomer. We have also investigated the effect of the N-protecting groups and the ester groups on the enantiomeric separation. The Z, 4-methoxybenzyloxycarbonyl (Z(OMe)), and 9-fluorenylmethoxycarbonyl (Fmoc) derivatives gave exceptionally good resolutions. By contrast, the formyl and t-butoxycarbonyl (Boc) groups impaired the enantiomeric separation. Almost all the alkyl esters e...

Synthesis of (2S,3S)-2-amino-3-methylpent-4-ynoic acid, a precursor amino acid for the preparation of tritium- or deuterium-labelled peptide in the isoleucine residue

The synthesis of (2S,3S)-2-amino-3-methylpent-4-ynoic acid (Amp)1, its incorporation into a peptide chain, and the preparation of a deuterium-labelled peptide in the isoleucine residue are reported. Amino acid 1 was synthesized in three steps from 3-chlorobut-1-yne. The optical purity of amino acid 1 was determined by HPLC with a chiral stationary-phase column and was found to be more than 99%(ee). Tyr-Amp-Leu was synthesized by solid-phase synthesis based on Fmoc strategy. The tripeptide was deuteriated catalytically to yield Tyr-[2H]lle-Leu. The distribution of deuterium was investigated by fast-atom-bombardment mass spectrometry (FAB-MS) and 13C NMR spectroscopy, which confirmed that the deuterium was located entirely at the isoleucine residue.

Determination of manidipine enantiomers in human serum using chiral chromatography and column-switching liquid chromatography

A stereoselective and highly sensitive method using chiral chromatography and successive column-switching liquid chromatography is described for the determination of manidipine enantiomers in human serum. A human serum sample obtained after ingestion of manidipine was extracted twice with a mixture of n-hexane—diethyl ether under alkaline conditions. The enantiomers in the extract were separated on a chiral stationary phase column (Chiralcel OJ), and the effluents containing the respective enantiomers were collected. Each fraction was then analysed by column-switching liquid chromatography. The proposed stereoselective method offered high sensitivity: detection limits for both isomers were 0.2 ng/ml in human serum, both at a signal-to-noise ratio of 3. The method is suitable for the pharmacokinetic studies of manidipine enantiomers.

Resolution of terfenadine enantiomers by beta-cyclodextrin chiral stationary phase high-performance liquid chromatography.

Enantiomers of terfenadine were resolved by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) column packed with beta-cyclodextrin (beta-CD) covalently bound to silica. Separation was achieved in both the reverse phase and normal phase modes. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analysis.

Direct enantiomeric separation of terfenadine and its major acid metabolite by high-performance liquid chromatography, and the lack of stereoselective terfenadine enantiomer biotransformation in man

Direct enantiomeric separation of terfenadine and its major acid metabolite was achieved by using two different chiral stationary phase columns with two different mobile phase systems. Further, the enantiomeric composition of the human urinary acid metabolite has been determined, indicating a non-stereoselective biotransformation in man.

N,N‐dimethylcarbamyl derivatives of oxazepam

Three N,N-dimethylcarbamyl derivatives of oxazepam (1-(N,N-dimethylcarbamyl)oxazepam, 3-O-(N,N-dimethylcarbamyl)oxazepam, and 1,3-O-bis(N,N-dimethylcarbamyl) oxazepam) and a 3-O-acyl-1-(N,N-dimethylcarbamyl)-oxazepam were synthesized from either oxazepam or demoxepam. Enantiomeric pairs of these derivatives and of camazepam were resolved by high-performance liquid chromatography on at least two of three commercially available chiral stationary phase columns employed. Absolute configurations of resolved enantiomers were established by comparing their circular dichroism spectra to those of enantiomeric oxazepams with known absolute stereochemistry. Similar to those of oxazepam, enantiomers of 1-(N,N-dimethylcarbamyl)oxazepam undergo rapid racemization (t1/2 1.9 min at 23 degrees C and 0.9 min at 37 degrees C) in an aqueous solution at pH 7.5. The (R)-enantiomer of rac-3-O-acyl-1-(N,N-dimethylcarbamyl)oxazepam was hydrolyzed approximately 4.6-fold faster than the (S)-enantiomer by esterases in rat liver microsomes, whereas the (S)-enantiomer was hydrolyzed approximately 43-fold faster than the (R)-enantiomer by esterases in rat brain S9 fraction.

Interactions of chiral molecules with an (r)‐n‐(3,5‐dinitrobenzoyl) phenylglycine HPLC stationary phase

AbstractForty different chiral molecules were studied by liquid chromatography with a Pirkle‐type, (R)‐N‐(3,5‐dinitrobenzoyl) phenylglycine (DNBPG), chiral stationary phase column. The dramatic effect of a small molecular change on chiral recognition was demonstrated using DL‐amino acid derivatives. The inductive effect on chiral recognition was also studied using trifluoro‐, trichloro‐, dichloro‐, monochloroacetyl, and acetyl derivatives of four different chiral amines. The study of the enantiomer separation of 11 different crown ethers of 2,2′‐binaphthyldiyl showed that the rigidity of the chiral center can be an additional parameter in chiral recognition for the DNBPG phase but not for a β‐cyclodextrin bonded chiral phase. It is apparent from this study that steric effects, inductive effects, and molecular rigidity play important roles in chiral recognition with DNBPG chiral stationary phases.

Simultaneous determination of disopyramide and its mono-N-dealkylated metabolite enantiomers in human plasma and urine by enantioselective high-performance liquid chromatography

Enantiomers of disopyramide (DP) and its mono-N-dealkylated metabolite (MND) were determined in human plasma and urine by enantioselective high-performance liquid chromatography using a chiral stationary-phase column. This method was precise and sensitive: the mean recoveries from plasma at a concentration of 0.5 μg/ml were 101.1% for (+)-DP, 98.0% for (−)-DP, 94.4% for (+)-MND and 82.9% for (−)-MND; the within- and between-day coefficients of variation at the same concentration were 4.4 and 3.3% for (+)-DP, 4.7 and 4.1% for (−)-DP, 6.5 and 4.1% for (+)-MND and 7.8 and 2.4% for (−)-MND for plasma; the lower detection limits were 40 ng/ml for (+)-DP, 80 ng/ml for (−)-DP, 100 ng/ml for (−)-MND and 200 ng/ml for (+)-MND, for 0.5 ml of plasma and 0.2 ml of urine. The ultrafiltration technique was used for determination of the unbound concentration of DP enantiomers in plasma. A preliminary study of the determination of DP and MND enantiomers in plasma and urine samples from a healthy subject given racemic DP demonstrated the clinical applicability of the present method for therapeutic monitoring and pharmacokinetic studies.

Direct separation and optimization of timolol enantiomers on a cellulose tris-3,5-dimethylphenylcarbamate high-performance liquid chromatographic chiral stationary phase

A high-performance liquid chroamtographic method was developed for the direct resolution and optimizationof the separationof timolol enantiomers. The method involves the use of a cellulose tris-3,5-dimethylphenylcarbamate chiral stationary phase (OD-Chiralcel) column. the effects of concentration of 2-propanol, various aliphatic alcohols and diethylamine in the mobile phase and column temperature on the retention and enantioselectivity of timolol enantiomers were studied. The maximum resolution factor obtained was 4.00 when using the solvent system hexane—2-propanol (95:5) containing 0.4% (v/v) diethylamine at 5°C.

Enantioselective aliphatic hydroxylations of racemic 1‐hydroxy‐3‐methylcholanthrene by rat liver microsomes

Enantiomeric pairs of 1-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-1,2-diols, and 1-hydroxy-3-methylcholanthrene (1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-1,2-diol was established by the exciton chirality CD method following conversion to a bis-p-N,N-dimethylaminobenzoate. Incubation of an enantiomeric 1-OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols; the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-1,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-1,2-diol. Absolute configurations of enantiomeric 1-OH-3-OHMC were determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1-OH-3-OHMC greater than 3MC cis-1,2-diol greater than 3MC trans-1,2-diol. Enzymatic hydroxylation at C2 of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (approximately 3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (approximately 58/42). Rat liver microsomal C2-hydroxylation of racemic 1-OH-3MC resulted in a 3MC trans-1,2-diol with a (1S,2S)/(1R,2R) ratio of 63/37 and a 3MC cis-1,2-diol with a (1S,2R)/(1R,2S) ratio of 12/88, respectively.

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3- b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [( S) and ( R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4 H-thieno[2,3- b]thiopyran-2-sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25–250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single ( S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5–100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.

Analysis of Enantiomers of a New Antidiabetic Agent in Plasma by High-Performance Liquid Chromatography

Abstract A new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), and its L-enantiomer were successfully separated and quantified by high-performanece liquid chromatograph. This direct resolution was accomplished using a chiral stationary phase column packed with 5 μm N-(tert-butylaminocarbonyl)-L-valylaminopropyl silica gel and a mobile phase consisting of n-hexane/n-propanol/trifluoroacetic acid. The method has been used for the analysis of plasma samples from beagle dogs.