Enantiomeric resolution of new triazole compounds by high-performance liquid chromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was synthesized and coated on aminopropylsilica to prepare a chiral stationary phase (CSP). HPLC methods were developed for the direct enantioseparation of 12 chiral triazole compounds on the CSP. The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, 1-butanol, 2-propanol, and t-butanol) in various portions. The column temperatures were studied for the optimization of the resolutions. The effects of structural features of the solutes on the discrimination between the enantiomers were examined. Baseline separation was easily obtained in many cases.     

Enantiomeric resolution of bupivacaine by high-performance liquid chromatography and chiroptical detection

This work reports a new analytical procedure for the separation and determination of the enantiomers of bupivacaine and the determination of the enantiomeric purity. The isomers were separated using a Chirex 3020 (250 mm x 4.6 mm) with a mobile phase of n-hexane:dichloroethane:ethanol (82:9:9, v/v/v) at a flow-rate of 1 ml min(-1) and UV, polarimetric and circular dichroism (CD) detection. Obtained retention times were 5.93 and 7.53 min (R and S) with a resolution of Rs=2.36. Precisions (RSD) were 1.83 and 2.02% (CD detection) and 3.07 and 1.26% (UV detection) for R- and S-enantiomers, respectively (at 10 microg level). Detection limits were 0.5 and 0.5 microg (R and S) with CD detection, and 0.9 and 0.3 microg with UV detection. Polarimetric detection was inadequate to perform a quantitative method at similar concentration ranges as UV and CD because of poor sensitivity. A procedure for determination of enantiomeric purity using a conventional chromatographic column (RP18, Luna) coupled to a CD detector and anisotropy factor (CD/UV) as analytical signal was also developed. Obtained results show that RSDs of 6.7-1.6% were obtained in the range of 0-100% enantiomeric purity.     

Enantiomeric resolution of a series of chiral sulfoxides by high-performance liquid chromatography on polysaccharide-based columns with multimodal elution

The enantiomeric resolution of a series of 20 asymmetric sulfoxides was systematically investigated by HPLC using multimodal elution with amylose trisR(S)-1-phenylethylcarbamate], amylose tris(3,5-dimethoxyphenylcarbamate) and amylose and cellulose tris(3,5-dimethylphenylcarbamate) phases. The sulfoxide series was composed of aromatic, olefinic and ketosulfoxides, sulfinyl acids and esters. This work has shown that enantioselectivity and enantioresolution of the polysaccharide-based columns can be achieved by changing the type and composition of the mobile phase, widening the applicability of these chiral phases.     

Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column

In our previous studies, we employed recycle HPLC for the separation of triacylglycerol (TAG)-positional isomers (PIs). In this study, a recycle HPLC system equipped with a polysaccharide-based chiral column was applied to the enantiomeric separation of some asymmetric TAGs having straight-chain C16-C18 acyl residues. As a result, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (rac-PPO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (rac-OOP), and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol (rac-PPL) were resolved into their respective enantiomers. However, neither 1,2-dioleoyl-3-linoleoyl-rac-glycerol (rac-OOL), consisting of only unsaturated fatty acids, nor 1,2-dipalmitoyl-3-stearoyl-rac-glycerol (rac-PPS), consisting of only saturated fatty acids, was resolved. These results suggest that the asymmetric TAGs, used in this study, having both a palmitic acid moiety and an oleic acid (or a linoleic acid) moiety at the sn-1 or sn-3 positions are resolved by the chiral column. This new chiral separation method can be used in combination with atmospheric pressure chemical ionization mass spectrometry to determine the sn-OOP/sn-POO ratio in palm oil. This method is applicable for the chiral separation of asymmetric TAGs in palm oil.     

Optical resolution of asymmetric triacylglycerols by chiral-phase high-performance liquid chromatography

A simple method for direct optical resolution of some asymmetric triacylglycerols (TGs) has been established. The method employs chiral-phase high-performance liquid chromatography (HPLC). An enantiomeric pair of TGs comprising 1-eicosapentaenoyl-2,3-dicapryroyl-sn-glycerol (ECC) and 1,2-dicapryroyl-3-eicosapentaenoyl-sn-glycerol (CCE) was resolved on a CHIRALCEL OF or on a CHIRALCEL OD column. The separation of another pair of asymmetric TGs, 1-docosahexaenoyl-2,3-dicapryroyl-sn-glycerol (DCC) and 1,2-dicapryroyl-3-docosahexaenoyl-sn-glycerol (CCD), was achieved with the CHIRALCEL OD column. The chiral-phase HPLC method in combination with silver-ion HPLC and high-temperature gas chromatography was used for monitoring two interesterification reactions, whose products were chiral TGs. Interesterification of tricapryloylglycerol with ethyleicosapentaenoate or with ethyldocosahexaenoate was performed using Rhizomucor miehei lipase as the catalyst. The products targeted were the asymmetric pair of TGs, ECC and CCE or DCC and CCD. The amounts of sn-1-substituted products (ECC or DCC) were greater than their sn-3-substituted counterparts (CCE or CCD) throughout the reaction period, suggesting that R. miehei lipase had a stereopreference towards the sn-1 position over the sn-3 position.     

Enantiomeric resolution of bifonazole by supercritical fluid chromatography

The enantiomeric separation of bifonazole by supercritical fluid chromatography on Chiralpak AD has been studied. The effect of different modifiers (methanol, ethanol, 2-propanol, and acetonitrile) was examined. Enantioseparation was possible with all of them, but the best results were provided by the alcohol-type ones. The resolution was higher than 5 in all cases. The isoelution temperatures Tiso were calculated from the study of the temperature effect for the different organic modifiers. The value of Tiso was below the working temperature range on using methanol, but above it with ethanol or 2-propanol.     

Enantiomeric resolution of new aromatase inhibitors by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)]-benzothiazolinone and benzoxazolinone derivatives with one chiral center. Those analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom). The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined. Baseline separation (Rs > 1.5) was easily obtained in many cases.     

Enantiomeric separation of various lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters by high-performance liquid chromatography

Lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters were separated by chiral high-performance liquid chromatography (HPLC) with a Chiralcel OD-H column in the normal-phase mode. Major lipoxygenase derivatives of linoleic, -linolenic and arachidonic acids are well resolved by this column, provided they have been individually purified. Our method allows an easy and rapid determination of lipoxygenases enantioselectivity. In all cases tested the R enantiomer is eluted first.     

Preparation and rapid resolution of Xenopus phosvitins and phosvettes by high-performance liquid chromatography

The presence of acidic phosivitins phosvettes in Xenopus laevis yolk platelets and their purification by (NH4)2SO4 precipitation of associated lipovitellin were documented by polyacrylamide gel electrophoresis followed by staining with Stains-all. Procedures were further developed to resolve the various entities present in the crude phosvitin/phosvette fraction by size-exclusion, anion-exchange, and hydrophobic interaction chromatography, using a Pharmacia FPLC system, and their resolution was documented by both electrophoresis and two-dimensional chromatography. Four major entities (phosvitins 1 and 2; phosvettes 1 and 2) were observed, but microheterogeneity was also apparent, particularly by hydrophobic interaction chromatography. The new separation procedures require min/h rather than h days.     

Complementary mobile-phase optimisation for resolution enhancement in high-performance liquid chromatography

An optimisation methodology in high-performance liquid chromatography (HPLC) is presented for the selection of two or more mobile phases having an optimal complementary resolution. The complementary mobile phases (CMPs) are selected in such a way that each one resolves optimally only some compounds in the mixture, while the remainder, resolved by the other mobile phase(s), can overlap among them. The methodology is based on the computation of a peak purity measurement for each solute, using an asymmetrical peak model for peak simulation. Two global resolution criteria (product of elementary resolutions and worst elementary resolution) and two methods for solving the problem (a systematic examination of all possible solute arrangements, and the use of genetic algorithms to expedite the calculation time) were used to find the optimal CMPs. The CMP optimisation methodology was applied to the resolution of a mixture of 10 diuretics and beta-blockers, which could not be resolved using a single mobile phase; virtual baseline resolution was achieved, however, with two CMPs.     

Multicomponent self-modelling curve resolution in high-performance liquid chromatography by iterative target transformation analysis

Iterative target transformation factor analysis can provide a method for resolving elution profiles consisting of any number of compounds. The results obtained for 3-component resolution are consistent with the results obtained with conventional methods of curve resolution. The same restrictions with regard to overlap and relative signal heights of the compounds seem to apply to the conventional method of curve resolution and the proposed method. The method is tested on data from high-performance liquid chromatography with a diode-array detector obtained for polynuclear aromatic hydrocarbons and for proteins.     

Pirmenol determination by high-performance liquid chromatography

A method for the determination of pirmenol in serum is presented in this paper. The method consists of extraction of pirmenol and chlorodisopyramide (internal standard) from serum at an alkaline pH using methylene chloride. The organic extract was analysed using high-performance liquid chromatography. The mobile phase consisted of 0.01 M K2HPO4 (pH 2.4)-acetonitrile (94:6, v/v) delivered at ambient temperature and 2 ml/min through a 25 cm x 0.4 mm C18 reversed-phase column. Detection of the compounds of interest was achieved at 210 nm. The analytical method demonstrated low intra- and inter-assay variation. During the analysis of patient samples and a therapeutic drug mixture test serum, no substances that interfered with pirmenol detection were found. The method is shown to be stable, accurate, selective and sensitive enough to be utilized for the analysis of multiple samples such as may be encountered in clinical or research situations.     

On-line racemization by high-performance liquid chromatography

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (±)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((±)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.     

Sensitive derivatizatton reagents for the resolution of carboxylic acid enantiomers by high-performance liquid chromatography

Two highly sensitive, chiral derivatization reagents, D- and L-1-aminoethyl-4-dimethyl-aminonaphthalene, were synthesized from 1-dimethylaminonaphthalene. Condensation of carboxylic acids with the chiral reagent was readily effected in the presence of a watersoluble carbodiimide. The diastereoisomeric amides formed from N-acetylamino acid and α-arylpropionic acid enantiomers were efficiently resolved by normal phase chromatography (μPorasil column) -with hexane/ethyl acetate or hexane/tetrahydrofuran as a mobile phase. With a fluorescence detector (excitation 320 nm, emission 395 nm), the detection limit was 0.1 ng.     

Non-intuitive separation of vanilla compounds using rapid resolution high-performance liquid chromatography

A method is presented for modeling the retention peak migration in rapid resolution high-performance liquid chromatography (HPLC) depending on experimental parameter values. It allows time reduction on the determination of the experimental conditions for optimal resolution (especially for untrained chromatographers). Separation for 18 species present in a conventional vanilla formulation was not possible in a single chromatogram, due to a systematic error in defining single peak migration with the usual assumptions. This was achieved by the means of two runs under different experimental conditions. Prediction of the peak inversion for quantitation purposes in a given mixture is now possible and can help to avoid misidentification on set-ups with UV-ELSD or other non-specific detectors.     

Enantiomeric determination of L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography

Enantiomeric determination of L- and D-lactate in human cerebrospinal fluid (CSF) was achieved by HPLC on a chiral stationary phase with UV detection. Samples were submitted to a solid-phase extraction procedure using Oasis HLB Plus Extraction Cartridge and L- and D-lactate in the extract were separated by Shodex ORpac CRX-453 B column, a ligand exchange column for chiral separation, using a mobile phase containing copper (II) ion. L- and D-lactate were determined in 25 min. Intra-assay precision in CSF was 4.98% (mean 1.85 mmol/L) for L-lactate and 10.1% (mean 4.96 micromol/L) for D-lactate (n = 5). Detection limits were between 1.0 (L-lactate) and 1.5 (D-lactate) pmol. The mean values (n = 3) of analytical recovery for L- and D-lactate were 95% and 107%, respectively. The mean +/- SD of concentrations of L- and D-lactate in CSF (n = 20) were 1.52 +/- 0.54 mmol/L and 10.98 +/- 5.15 micromol/L, respectively.