Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis.

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R(s)>3.3 with analysis times<25min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated beta-cyclodextrins at pH 2.5 (R(s)>1.5 with analysis times <11min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts.

Separations of the diastereoisomers of three nucleoside 5'-phosphotriester derivatives of Ara-C (tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation (Rs>1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5'-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis.

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.

Chiral resolution of the enantiomers of new selective CB(2) receptor agonists by liquid chromatography on amylose stationary phases.

Analytical HPLC methods using derivatized amylose chiral stationary phases, Chiralpak AD-H and Chiralpak AS, were developed for the direct enantioseparation of eight substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with one stereogenic center. Baseline separation (Rs>1.5) was always achieved on amylose based Chiralpak AD-H column to the difference with Chiralpak AS. Using UV detection, a linear response was observed within a 180-420 micromol L(-1) concentration range (r2>0.991) for three racemic compounds 1, 3 and 4 with best pharmacological potentials; repeatability, limit of detection (LD) and quantification (LQ) were also determined: LD varied, for the solutes, from 0.36 to 2.56 micromol L(-1). Finally, the enantiopurity of these compounds was determined. Additionally, the effect of temperature variations upon isomer separations was investigated.

Determination of pKa values of benzoxa-, benzothia- and benzoselena-zolinone derivatives by capillary electrophoresis. Comparison with potentiometric titration and spectrometric data.

Acidity constants of benzoxa-, benzothia- and benzoselena-zolinone derivatives were determined by capillary electrophoresis, potentiometry and spectrophotometry experiments. These three analytical techniques gave pK(a) results that were in good agreement. A convenient, accurate and precise method for the determination of pK(a) was developed to measure changes in acidity constants induced by heteroatom or 6-benzoyl substituted derivatives. pK(a) values were determined simultaneously for two compounds characterized by different electrophoretic mobility (micro(e)) and pK(a) value and in the presence of an analogous neutral marker.

Enantioseparation of cis and trans nucleosides, aromatic analogues of stavudine, by capillary electrophoresis and high-performance liquid chromatography.

Compounds 1-4 are diastereoisomeric thymine derivatives of isochroman aromatic analogues of stavudine, an approved drug. Both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) techniques were used to separate these species with high resolution and thus permit the determination of enantiomeric excess. Chiral selectivity was developed using anionic (highly sulfated) cyclodextrins as chiral selectors in CE and amylose, cellulose and cyclodextrin chiral stationary phases by HPLC. The HPLC method was found to be more efficient than the CE method and was applied, after validation (repeatability, limit of detection, limit of quantification) to follow and quantify the kinetics of a stereoselective esterification.

Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.

A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.

Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs. Application to cellular extract analysis.

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12-0.20 and 0.40-0.67 microm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract.

Enantioseparation of four cis and trans diastereomers of 2',3'-didehydro-2',3'-dideoxythymidine analogs, by high-performance liquid chromatography and capillary electrophoresis.

Compounds 1-4 are the four stereoisomers of a synthetic new potential antiviral agent (d4T analog) containing two chiral centers and a base (uracil). Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) techniques were used to separate and quantify enantiomers with high resolution. The determination of enantiomeric purity of the compounds was developed using both amylose chiral stationary phase by HPLC and anionic cyclodextrins (highly S-CD) as chiral selectors in CE. The HPLC method was found to be superior in sensitivity to the CE method.

Determination of ionization constants of N-imidazole derivatives, aromatase inhibitors, using capillary electrophoresis and influence of substituents on pKa shifts.

Capillary electrophoresis (CE) was used as a method to determine the acidity constants of eight aromatase inhibitors. This method was validated by comparison of results obtained with a traditional method, UV spectroscopy, and additionally with computational calculations. We confirmed here, with our series of compounds, that capillary electrophoresis is an attractive method for pKa measurements which is based on migration time or mobilities of the ionic species over a range of pH values. The precision of pKa measurements of N-imidazole derivatives is useful to observe pKa shifts induced by chemical modifications introduced on adjacent aromatic rings such as heterocycle (benzoxa- or benzothiazolinone) or substituted benzyle. The knowledge of these pKa values is a great interest to predict migration of solutes and qualitative interactions with ionized cyclodextrines as chiral selectors in further enantioseparative CE studies.