Development of new isothiocyanate-based chiral derivatizing agent for amino acids

Summary (1S,2S)-1,3-Diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate [(S,S)-DANI] has been developed as a new chiral derivatizing agent for resolution of compounds containing an amino group. The reagent is readily available in both enantiomeric forms. Its applicability was demonstrated by the resolution of representative α-amino acids. The diastereomeric thiourea derivatives produced were separated by reversed-phase (C₁₈) high-performance liquid chromatography, with mixtures of 0.1% aqueous trifluoroacetic acid (pH∼2) and methanol as eluents.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.     

Determination of the enantiomeric composition of chiral carboxylic acids using chiral derivatization and HPLC

Summary The enantiomers of chiral carboxylic acids were separated as their diastereomeric amides with (1R,2R)-(−)-1-(4-nitrophenyl)-2-amino-1,3-propanediol (“levobase”) and with “dextrobase” (the enantiomer of levobase) by high-performance liquid chromatography using a conventional C-18 column and various solvent systems containing acetonitrile, methanol, water, and phosphoric acid.     

Synthesis and evaluation of new fluorescent derivatization reagents for resolution of chiral amines by RP-HPLC

New fluorescent chiral derivatization reagents (i.e., DBD-trans-4-hydroxy-l-proline, DBD-cis-4-hydroxy-l-proline, DBD-cis-4-hydroxy-d-proline, and DBD-trans-3-hydroxy-l-proline) were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole with corresponding hydroxy-prolines. These reagents reacted with chiral amine to produce a couple of diastereomers. The labeling efficiently proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine as the activation reagents. The reaction conditions are mild and no racemization occurred during both the reagent synthesis and the diastereomer formation (<0.4%). The resulting diastereomers fluoresce at around 560 nm (excitation at around 450 nm). Good linearity of the calibration curves was obtained in the range of 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was evaluated in terms of separation factor (α) and resolution value (Rs). DBD-trans-4-hydroxy-l-proline was efficient for the resolution of dl-phenylalanine methylester; while DBD-cis-4-hydroxy-d(or l)-proline was excellent for the separation of 1-(1-naphtyl)ethylamines, as comparing with trans-4-hydroxy isomer. The reagents of cis-isomer seemed to be predominant for the resolution of hydrophobic enantiomers. On the other hand, trans-isomers were suitable for the separation of the racemic amines containing ester in the structure. With respect to the position of OH group, the effect seems to be less, judging from the results of DBD-trans-4-hydroxy-l-proline and DBD-trans-3-hydroxy-l-proline toward phenylalanine methylester. The results suggest that the separation is dependent upon both structures of the amines and the reagent used. Thus, the stereostructure, hydrophobicity and hydrogen bonding of the diastereomer, etc. seem to be affecting the separation.     

Separation of the stereoisomers of racemic fluoroquinolone antibacterial agents on a crown-ether-based chiral HPLC stationary phase

Summary A chiral stationary phase derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid has been successfully used for the direct separation of the enantiomers of recemic fluoroquinolines containing a primary amino group being investigated as antibacterial agents. The chromatographic resolution behavior was found to depend on the content and the type of acidic and organic modifiers in the mobile phase and on the column temperature.     

Direct enantiomeric separation of racemic precursors to diltiazem hydrochloride and clentiazem maleate by HPLC on a chiral ovomucoid column

Summary A direct HPLC separation method was developed for the determination of the enantiomers of racemic precursors to diltiazem (I) and its 8-chloro derivatives (II). The enantiomers were successfully separated on a chiral ovomucoid column using an aqueous-organic mobile phase (reversed-phase HPLC). The influence of the organic modifier and buffer pH on the retention and enantioselectivity was investigated. The chromatographic conditions chosen for the separation permitted complete resolution of the enantiomers of both the acid (I_b and II_b) and methyl ester precursors (I_a and II_a) within 20 min. The influence of sample load on retention times, theoretical plates numbers, peak heights and peak areas was also investigated. The peak areas showed a good linearity over the concentration range examined, although all the others were influenced significantly by the sample size. An optical antipode of the intermediate to be determined could be detected by the area-percentage method down to ca. 0.1%, together with the determination of its precursor, including its optical purity.     

Reversed-Phase Liquid Chromatographic Separation, on Cellulose Chiral Stationary Phases, of the Stereoisomers of Methoxytetra-hydronaphthalene Derivatives, New Agonist and Antagonist Ligands for Melatonin Receptors

A stereoselective high-performance liquid chromatographic method has been established for chiral separation of melatoninergic derivatives with one or two chiral centers, new agonist and antagonist ligands for melatonin receptors. Reversed-phase separations were performed on cellulose-based chiral stationary phases—tris-3,5-dimethylphenylcarbamate (Chiralcel OD-RH) or tris-methylbenzoate (Chiralcel OJ-R). Water–modifier (methanol or acetonitrile) mixtures in different proportions were used as mobile phases. The effects of organic mobile-phase modifier concentration, temperature, and compound structure were examined. Baseline separation (R_S > 1.5) was readily obtained for many of the compounds.     

Separation of the enantiomers of pyrethroic acids and their esters by high-performance liquid chromatography on a polysaccharide-derived chiral stationary phase

Summary The liquid-chromatographic separation of the enantiomers of pyrethroic acids and their esters has been investigated on a polysaccharide-derived chiral stationary phase (CSP), Chiralpak AS. Good separation of the enantiomers of underivatized pyrethroic acids was achieved on the column, and the enantiomers of pyrethroic acid methyl and ethyl ester derivatives were also resolved.     

Chiral resolution of diphenylalanine by high-performance liquid chromatography on a crown-ether-based chiral stationary phase and by NMR spectroscopy

Summary The enantiomers of diphenylalanine (DPA) were well separated by chiral HPLC and NMR spectroscopy on the chiral stationary phase (CSP) derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA). The chromatographic parameters such as separation factors and retention times were greatly influenced by the mobile phase conditions. The (+)-18-C-6-TA used in the CSP was also employed as a chiral solvating agent for the enantiodiscrimination of the DPA enantiomers by NMR spectroscopy. The proton of the DPA analyte showing the chemical shift nonequivalences was used in determining the enantiomeric composition of the analyte.     

The preparative chromatographic enantioseparation of a racemic morphanthridine analog on a chiral stationary phase

Summary The preparative chromatographic enantioseparation of a chiral morphoanthridine analog has been performed on an analytical column using amylose-tris(3,5-dimethylphenylcarbamate) as chiral stationary phase. The racemate (100 mg) was resolved to baseline within 15 min. This paper describes the development of the method, estimation of the capacity of the chiral stationary phase and discussed the potential of the chromatography if performed under preparative conditions. From the results and calculations presented it seems likely that the resolution of 70 tons year⁻¹ could easily be achieved on 30 kg of stationary phase with a mobile-phase consumption of only 720 L day⁻¹.     

Interaction of cyclodextrins with drugs. Chromatographic investigation using a cyclodextrin bonded-phase and its application to chiral separations

Summary The interaction between cyclodextrin and the drug (1R,2S,3S,4S)-(5Z)-7-(3-((phenylsulfonyl)amino)bicyclo[2.2.1]hept-2-yl)hept-5-enoic acid ((+)-S-145), was studied using -, -, and -cyclodextrin bonded-phase columns. Retention behavior of (+)-S-145 on these columns revealed that the strength of inclusion was -cyclodextrin. Interaction between -cyclodextrin and (+)-S-145 was found to increase as the proportion of carboxylic ion in the (+)-S-145 molecule increased. Comparison of binding capacities of these bondedsilica gels obtained by frontal analysis and surface coverage indicated that availability of the immobilized - and -cyclodextrin was 20–25%. The synthesized -cyclodextrin bonded-phase column was superior to that of commercial columns in terms of chiral separation of (±)-S-145. A typical usage of the -cyclodextrin column is discussed for separation of (±)-S-145 in plasma samples.     

Design of enzyme reactors as chromatographic columns for racemic resolution of amino acid esters

Summary Racemic resolution of aromatic and aliphatic amino acid esters into L-amino acid and D-amino acid ester via LC and HPLC is achieved by using enzyme reactors as chromatographic columns. For this purpose α-chymotrypsin and trypsin are immobilized on Eupergit C, Sepharose 4B and Lichrosorb-Diol.     

2-Bromoacetyl-6-methoxynaphthalene: A useful fluorescent labelling reagent for HPLC analysis of carboxylic acids

Summary The use of 2-bromoacetyl-6-methoxynaphthalene as a fluorogenic labelling reagent in pre-column derivatization for the HPLC separation of biologically active carboxylic acids (fatty acids and bile acids) has been investigated. The compound reacts (30 min. at 70°C) with carboxylic acids to give fluorescent esters that can be separated by reversedphase HPLC and detected at ex. 300 nm, em. 460 nm. The experimental conditions for the derivatization and chromatographic separation are discussed. Applications to the determination of valproic acid and chenodeoxycholic acid in pharmaceutical formulations are described.     

Attempts on the semi-preparative resolution of racemic esters of aminoacids on a chiral liquid chromatographic column derived from N-formylisoleucine

Summary A semi-preparative (250mm×10mm i.d.) chiral liquid chromatographic column incorporating a chiral material derived from N-formylisoleucine has been found to be suitable for the resolution of milligram or larger quantities of two aminoacid esters. The method should be capable of being used for other racemic materals on an even larger scale.     

Separation of aliphatic and aromatic amines by the ion interaction reagent RP-HPLC technique

Summary Ion interaction reagent RP-HPLC has been employed for the separation of some typical aliphatic and aromatic amines. The effects on retention of the alkyl chain length of the eluent, and of the eluent flow-rate have been studied. The use of solutions of hexylaminium-, octylaminium-and decylaminium-salicylate has been tested and compared, employing both conductometric and spectrophotometric detection. On the basis of the results obtained, the optimal experimental conditions can be chosen for each separation. Aliphatic and aromatic mono and diamines can be separated and detected, with an average sensitivity of the order of 40 ng without any pretreatment or derivatization. The retention data obtained for amines, compared with those obtained with the same ion interaction reagents for anions, help in the interpretation of the mechanism involved in the technique.     

Direct chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using different cellulose based chiral stationary phases

Summary Two improved methods for the enantiomeric separation of racemic aminoglutethimide (±AG) and its acetylated metabolite (±AAG) have been developed. Direct liquid chromatographic resolution of the enantiomers of aminoglutethimide and its acetylated metabolite was accomplished using Chiralcel OD and Chiralcel OJ columns without any derivatization. Maximum resolution of 8.87 and 2.23 was obtained for the enantiomers of aminoglutethimide and its acetylated metabolite using a Chiralcel OD column, while maximum resolution of 10.34 and 7.01 was obtained for the enantiomers using a Chiralcel OJ column. Optimization of separation was obtained using different concentration of 2-propanol in hexane as a mobile phase.     

Chromatographic behaviour of homologous amines: Comparison of derivatizing agents,columns and mobile phases

Summary The liquid chromatographic behaviour of aliphatic mono- and polyamines belonging to different homologous series has been investigated using various pre-column derivatizing agents (OPA and CNB), mobile phases (water-MeOH and water-ACN), RP columns (different packing sizes and shapes) and detectors (UV and fluorescence) in order to characterize the different contributions to retention and the different selectivities under the conditions investigated. The parameters derived allowed computation of capacity factors in good agreement with experiments. The chromatographic method optimized with respect to OPA derivatization has been successfully utilized for the determination of putrescine, cadaverine and 2-phenyl-ethylamine in cheese samples under different ageing conditions.     

L-N-(3,5-Dimethoxybenzoyl)isoleucine chiral stationary phase for separation of enantiomers by HPLC

Summary L-N-(3,5-dimethoxyoxybenzoyl)isoleucine, ionically bonded to γ-aminopropyl silica, has been tested as a chiral stationary phase for the separation of racemates by HPLC. The phase shows good selectivity towards different types of racemates and in particular for those having an electron-poor aromatic group in their molecule. The separation of benzoin racemate can be achieved on the developed chiral phase with an α value of 1.10.     

Optimisation of chromatographic separations by use of a chromatographic response function, empirical modelling and multivariate analysis

Summary The chiral separation of the drug substance R,S-oxybutynin chloride on a reversed phase HPLC system has been optimised by use of empirical modelling and multivariate analysis. The separation was characterised by a new chromatographic response function developed to modulate both quality of separation and retention time. The study includes a comparison between three different multivariate techniques (multi-layer feed-for-ward neural networks, multiple linear regression and partial least squares regression) of their capabilities to model the new chromatographic response function and predict its value for new experiments. It was indicated that the most accurate models were achieved with neural networks, although partial least squares regression could also be used to solve the problem since it gives the major directions for the optimal settings of the variables.