Chiral Stationary Phase Column Research Articles

Direct separation of regio- and enantiomeric isomers of diacylglycerols by a tandem column high-performance liquid chromatography

A novel HPLC-based method for direct separation of the three isomers of mono-acid diacylglycerols (DAGs), i.e., 1,2-DAG, 2,3-DAG and 1,3-DAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and a chiral stationary phase column) are connected in series. Two isomeric mixtures of DAGs (i.e., dicapryloylglycerol and dioleoylglycerol) and lipase-catalyzed reaction mixtures were successfully resolved on the tandem column HPLC system without any derivatization prior to the analysis. According to the established analytical method, stereoselectivity of two lipases toward mono-acid triacylglycerols in ethanolysis reaction was investigated. The tested enzymes were immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). Analyses of the enantiomeric purity of 1,2-DAG and 2,3-DAG, generated as intermediates during the reaction, revealed that CALB and RML have sn-3 and sn-1 stereopreference, respectively.

Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

Analysis of benidipine enantiomers in human plasma by liquid chromatography–mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column

We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1 ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction-monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C max and AUC inf values of (+)-alpha benidipine were higher than those of (−)-alpha benidipine by 1.96- and 1.85-fold, respectively ( p < 0.001), whereas, the T max and t 1/2 for each of the benidipine stereoisomers were not significantly different.

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

The high-performance liquid chromatographic separation of enantiomers of N-protected non-protein amino acid esters was investigated by using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiracel OD). The effect of the N-protecting groups and the ester groups on chiral discrimination was examined. The benzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl derivatives gave good enantiomeric separations, while the formyl and t-butoxycarbonyl groups marred them. Almost all the alkyl esters examined and the benzyl ester gave enantiomeric separations better than or of the same order as the methyl ester. The N-Z-protected methyl esters of a number of non-protein α-amino acids were well resolved using hexane–2-propanol as a mobile phase. The resolution of β-amino acid derivatives was inferior to that of the isomeric α-amino acid derivatives.

Synthesis of (S)-(+)-2-methyl-4-octanol: male-specific compound released by sugarcane weevil Sphenophorus levis (coleoptera: curculionidae)

(S)-2-Methyl-4-octanol (1) is a male-specific compound released by the sugarcane weevil Sphenophorus levis, being its biological activity associated with the aggregation behaviour of the species. Continuing our studies on the chemical ecology of this species, this work describes an expeditious synthesis of alcohol (1), starting from the easily available D-mannitol (2), in a route that employ the known (R)-glyceraldehyde acetonide (4) as key intermediate. GC analysis of an acetyl derivative of the final product, using a chiral stationary phase column, revealed an enantiomeric excess of 99.5%. Compound (1) has been identified as component of the male-produced aggregation pheromone in several curculionid species.

Enantioselective HPLC determination of E-6087, a new COX-2 inhibitor, in human plasma: Validation and pharmacokinetic application.

E-6087 is a nonsteroidal anti-inflammatory compound that selectively inhibits cyclooxygenase-2. Because E-6087 has a chiral center, this compound is a racemic mixture of two stereoisomers, (+)-(R)-E-6087 (E-6231) and (-)-(S)-E-6087 (E-6232). A normal-phase liquid-chromatographic method for the enantioselective determination of E-6087 in human plasma was developed and validated. The samples were extracted using solid-phase extraction cartridges containing C(18) sorbent, and the extracts were redissolved in absolute ethanol and injected into the chromatographic system. The enantiomeric separation was achieved on a chiral stationary-phase column of derivatized amylose, and the enantiomers were quantified by fluorescence detection. The method was validated for drug concentrations ranging from 5 to 400 ng/ml for both enantiomers. No peaks interfering with the quantification of enantiomers were observed. The limit of quantification was 5 ng/ml, with precision expressed as a coefficient of variation lower than 10.6% and accuracy expressed as relative error lower than 12.2%. The utility of this method was demonstrated by analysis of plasma samples from healthy volunteers given an oral dose of rac-E-6087. Peak plasma levels of E-6231 were higher than levels obtained for E-6232. Results were consistent with those obtained with a conventional reversed-phase method used for determination of the racemic compound.

HPLC microdetermination of flurbiprofen enantiomers in plasma with a glycopeptide-type chiral stationary phase column.

A rapid and stereospecific HPLC micromethod to quantify flurbiprofen enantiomers was developed. Both flurbiprofen enantiomers and indomethacin, used as internal standard, were extracted with methylene chloride from 100 microL of acidified plasma. The resolution of the R- and S-forms was performed on a bonded vancomycin chiral stationary phase (Chirobiotic V) with 20% of tetrahydrofuran in ammonium nitrate (100 mM, pH 5) as mobile phase. Calibration curves were linear in the range 0.5-10 microg/mL for both enantiomers. A good accuracy (< or = 5%) was obtained for all quality controls, with intra-day and inter-day variation coefficients equal or less than 7.7%. Recovery of both enantiomers was found in the range 77.4-86.3%. The lower limit of quantitation was 0.25 microg/mL for both enantiomers, without interference of endogenous components. This validated micromethod has been successfully applied for quantifying R- flurbiprofen and S- flurbiprofen in rat plasma.

Separation, bioactivity, and dissipation of enantiomers of the organophosphorus insecticide fenamiphos

Most chiral pesticides are used as racemates despite the fact that the pesticidal activity of the given pesticide is usually the result of the preferential reactivity of only one enantiomer while the other enantiomers may have toxic effects against other nontarget organisms. Accordingly, the enantiomer of fenamiphos, an organophosphorus pesticide, was separated by high-performance liquid chromatography with reverse phase and normal phase on a Pirkle model chiral stationary phase column. It was found that n-hexane/isopropanol (95/5) was the best solvent system for enantiomer resolution of fenamiphos. Chromatographic data including capacity factor ( k′), separation factor ( α), and resolution (Rs) are presented. Inhibitory activity to enzyme butyrylcholinesterase and toxicity to Daphnia of enantiomers and racemic insecticide fenamiphos were also studied. In the toxicity tests of Daphnia, the lethal concentration (LC 50) of (+)-fenamiphos, (−)-fenamiphos, and racemate were 0.0016, 0.0061, and 0.0019 μg/mL, respectively. No significant difference of LC 50 values between (+)-fenamiphos and racemate were found, but (−)-fenamiphos showed significantly lower toxicity to Daphnia. The inhibitory concentration (IC 50) to the cholinesterase were 0.008, 0.15, and 0.46 μg/mL for (+)-fenamiphos, (−)-fenamiphos, and racemate, respectively. Both enantiomers and the racemate showed significant difference in inhibiting the cholinesterase. However, (+)-fenamiphos proved to be about 20 times more toxic to Daphnia and only about four times more inhibitory activity to butyrylcholinesterase than (−)-fenamiphos. The dissipation of (+)-fenamiphos, (−)-fenamiphos, and racemate in selected soils and natural water samples were also studied. The half-life ( t 1/2) of (+)-, (−)-, and racemate in soils showed no related to the soil texture, pH, or organic carbon content. By comparing the residues of (+)-, (−)-, and racemate in the water, it was found that (+)-fenamiphos was degraded faster than the others after 21 days. Briefly, (+)-fenamiphos is more toxic than (−)-fenamiphos to a nontarget organism ( Daphnia), but the environmental persistence of the two compounds showed no significant difference.

Detection of 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) carbamoyl amino acids generated by post-column desulfuration in the simultaneous determination of the sequence and d/ l-configuration of peptides using a fluorogenic Edman reagent, 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate

A modified Edman sequencing method using 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for amino acid sequence and d/ l-configuration determination has been developed. DBD-thiazolinone (TZ)-amino acids generated by Edman sequencing method were hydrolyzed and then oxidized to DBD-carbamoyl (CA)-amino acids, instead of the conversion reaction to thiohydantoin (TH)-amino acid in the conventional Edman method. The oxidative desulfuration step operated manually with formation of DBD-CA-amino acids was difficult to control because of the production of by-products by the side-chain oxidation of amino acid and inapplicability to the conventional automated protein sequencer. Therefore, post-column desulfuration method in which the reaction was carried out after the separation of analyte on a column was designed. DBD-thiocarbamoyl (TC)-amino acids generated by the modified sequencing method were separated on the reversed phase or Pirkle type chiral stationary phase column and then oxidized to DBD-CA-amino acids with hydrogen peroxide as a post-column reagent and detected fluorimetrically. Using the method, the determination of the amino acid sequence and d/ l-configuration of a d-amino acid containing peptide was achieved and gave us the recognition to the utility of the method.

CHIRAL HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF ENANTIOMERS OF α-METHYL α-AMINO ACIDS

The enantiomeric separation by chiral high performance liquid chromatography of derivatized and underivatized α-methyl α-amino acids was achieved by two different methods. A cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) was used to resolve N-benzyloxycarbonylated methyl esters. Excellent separations of enantiomers were achieved with all the α-methyl α-amino acids examined. A column packed with octadecylsilanized silica coated with N,S-dioctyl-d-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000) was also used to resolve the underivatized amino acids. Excellent to good separations of enantiomers were achieved with a variety of underivatized α-methyl α-amino acids carrying aliphatic or aromatic side-chains by optimizing the amount of the organic component and the concentration of the copper(II) ion in the hydro-organic eluent.

Simultaneous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral high-performance liquid chromatography

Chiral HPLC methods were developed and utilized for the simultaneous determination of plasma protein binding of enantiomers of two racemic aminohydantoin compounds. Reversed-phase HPLC with the use of a polysaccharide-type chiral stationary phase column was employed for the separation and quantitation of the enantiomers of the two compounds with detection limits in the range 5–10 ng/ml in the plasma matrix. The chiral HPLC methods were selective, sensitive and reproducible. The R and S enantiomers of both compounds were baseline-resolved under the chromatographic conditions employed. Ultrafiltration techniques were applied to determining the plasma protein binding for each enantiomer in rat, dog and human plasma. The results clearly show stereoselective binding of the two enantiomers of each compound with higher protein binding of the R enantiomer than the S enantiomer in rat, dog and human plasma. Binding association constants were also determined to be in the range 1.01–14.0·10 4 M −1 at 37°C. Both the protein binding percentage and binding association constant were enantioselective and species-dependent. Such information is important for a clear understanding of the differences in biological activity as well as in pharmacokinetic and pharmacodynamic properties between the two enantiomers of each compound in the drug discovery and development process.

Synthesis and Optical Resolution of Novel Chiral Silanols

Abstract Silanols bearing an asymmetric center on silicon, methyl(2-phenylethenyl)(3,3,3-trifluoropropyl)silanol and methyl(phenyl)(vinyl)silanol are synthesized by alkylative cleavages of cyclic siloxanes with an organolithium. Optical resolution of their enantiomers is performed by HPLC with a chiral stationary phase column. Optical characters of thus separated enantiomers are also examined.

Enantioselective high performance liquid chromatographic assay of acebutolol and its active metabolite diacetolol in human serum.

A stereoselective direct liquid chromatographic method for assay of the enantiomers of the beta-adrenergic blocker acebutolol (AC) and its active metabolite, diacetolol (DC), in human serum was developed. The assay is based on extraction with ethyl acetate and separation of enantiomers on an amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak AD) column. The method was validated and proved useful for the determination of the enantiomers in serum samples of patients suffering from hypertension and chronically treated with racemic AC. The results were compared and found similar with an indirect assay based on derivatization of the enantiomers with (+)-(S)-1-(1-naphthyl)ethyl isocyanate (NEIC).

OPTIMIZED ENANTIOSELECTIVE SEPARATION OF CLENBUTEROL ON MACROCYCLIC ANTIBIOTIC TEICOPLANIN CHIRAL STATIONARY PHASE

An isocratic and simple high performance liquid chromatographic method was developed for the direct resolution of clenbuterol enantiomers. The method involved the use of the macrocyclic antibiotic teicoplanin chiral stationary phase column known as Chirobiotic T. Several mobile phase compositions were studied to optimize the resolution of racemic clenbuterol. The role of using different solvents, e.g. methanol, ethanol, and acetonitrile as organic modifiers and the effect of triethylamine and acetic acid on the enantioseparation of clenbuterol were investigated. The best base-line separation was achieved using mobile phase consisting of ethanol containing 0.3% acetic acid and 0.2% triethylamine where the stereochemical separation factor (α) obtained was 1.58 and the stereochemical resolution factor (Rs) was 1.48. Under these chromatographic conditions, the (-)-R-clenbuterol eluted first with a capacity factor k′1 of 1.89 followed by the (+)-S-enantiomer with a capacity factor of k′2 of 2.99.

Isocratic HPLC Separation of Several Racemic Drugs with Two Stereogenic Centers on a Pirkle Urea-Type Chiral Stationary Phase

A simple and rapid isocratic enantiomeric separation of several racemic drugs with two stereogenic centers, namely for-moterol, labetalol, nadolol, indenolol, and U-54494A, was achieved under normal phase mode using a urea-type chiral stationary phase (CSP) column of (S)-indoline-2-carboxylic acid and (R)-1-(α-naphthyl) ethylamine, known as Chirex 3022 column. Labetalol and formoterol were resolved successfully into its four enantiomers. Furthermore, racemate mixture of U-54494A has been resolved into its individual enantiomers. However, nadolol and indenolol was partially separated.

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.

Solid-phase extraction–high-perfomance liquid chromatography determination of verapamil and norverapamil enantiomers in urine

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C 8 or C 18 bonded phase (C 8 in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5–300 ng/ml. Intra-day and inter-day reproducibilities were 10.5–14.2% at 3 ng/ml, 4.8–9.3% at 138.5 ng/ml and 7.8–10.1% at 280 ng/ml level, respectively, for all the enantiomers.

Enantiomeric separation of several cyclic imides on a macrocyclic antibiotic (vancomycin) chiral stationary phase under normal and reversed phase conditions

Several cyclic imidic compounds (barbiturates, piperidine-2,6-diones, and mephenytoin) are enantiomerically resolved via high-performance liquid chromatography (HPLC) on a macrocyclic antibiotic covalently bonded to a silica gel support. The Chirobiotic V chiral stationary phase (CSP) column contains the antibiotic vancomycin as the chiral selector. The results of the analysis show that the substituents at the chiral carbon position of the racemic drugs affect chiral resolution. In addition, ring size may also play a role when considering the formation of analyte-CSP inclusion complexes. Contrary to the piperidine-2,6-diones, the chromatographic parameters for the barbiturates are much the same under normal- or reversed-phase conditions. The details of these results are discussed. Chirality 10:358–361, 1998. © 1998 Wiley-Liss, Inc.

Chiral separations of β-blocking drug substances using the Pirkle-type α-Burke 1 chiral stationary phase

The Pirkle-type α-Burke 1 chiral stationary phase (CSP) column has been evaluated for the enantiomeric separation of β-blocking drug substances and suitability for the determination of enantiomeric purity of these substances. Eighteen β-blockers currently on sale in Denmark were investigated. By varying the amount of alcohol modifier in the mobile phase, a total of fifteen of the eighteen investigated β-blockers were successfully separated by straight-phase HPLC within acceptable analysis times (i.e. below 20 min). The separation factors ranged between 1.07 and 1.41, and the resolutions were in no case less than 0.9. The possible use of the column for a direct determination of enantiomeric purity of the drug substances was also investigated. Validation studies, in which the enantiomers of propranolol were used, were performed in accordance with the ICH guideline, Validation of Analytical Procedures. The limit of detection for R-propranolol in S-propranolol was estimated to be ca. 0.1% and the limit of quantification ca. 0.3%.

Stereoselective high-performance liquid chromatographic analysis of ketoprofen and its acyl glucuronides in chronic renal insufficiency

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S, S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standards were resolved using a C 18 reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S, S-Whelk-O1 chiral stationary phase column.