Comparison of liquid and supercritical fluid chromatography for the separation of enantiomers on chiral stationary phases

Comparisons of liquid (LC) and supercritical fluid chromatography (SFC) were conducted using commercially available chiral stationary phases (CSPs) bearing three different types of chiral selectors. Chiral compounds of pharmaceutical and agricultural interest were used to probe advantages of limitations of SFC relative to LC for enantiomeric separations. Column equilibrium and parameter optimization were generally accomplished more rapidly in SFC than in LC. Although improved resolution was often observed in SFC, analysis times were not always lower in SFC than in LC. In some instances, SFC provided separation capabilities not readily accessible in LC.     

Recent applications of polysaccharide-type chiral stationary phases for the enantioselective analysis of chiral drugs with one and two stereogenic centers

Abrupt onset pulmonary edema, that rapidly resolves (flash edema) may be due to renal artery stenosis. We describe two patients with renal artery stenosis who experienced a life-threatening episode of flash edema. Relief of the stenosis prevented recurrence of the flash edema.     

Enantiomeric separation of pirlindole by liquid chromatography using different types of chiral stationary phases

The enantioseparation of pirlindole by liquid chromatography (LC) was investigated using three different chiral stationary phases (CSPs) containing either cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-R), ovomucoid (OVM) or beta-cyclodextrin (beta-CD). The effects of the mobile phase pH on retention, enantioselectivity and resolution were studied. Methanol and acetonitrile were tested as organic modifiers while the influence of the addition to the mobile phase of sodium alkanesulfonates or sodium perchlorate was also investigated. Sodium perchlorate was only used on the Chiralcel OD-R column while sodium alkanesulfonates were tested as mobile phase additives on the three kinds of CSPs. The enantioseparation of pirlindole could be obtained on all CSPs tested, the best results with respect to chiral resolution being achieved on the Chiralcel OD-R and the OVM columns. The use of sodium octanesulfonate (NaOS) was found to improve the enantioseparation of pirlindole on the OVM column while enantioselectivity was considerably enhanced by addition of sodium perchlorate on the Chiralcel OD-R column.     

Evaluation of biological effects of three neodymium compounds on silkworm,Bombyx mori

Neodymium (Nd) is one of the most famous members of rare earth elements family. With the increasing global demand for Nd, it can inevitably enter the environment and affect the ecological environment through the food chain and other ways. Therefore, it is very important to study the biological effects of Nd. In this study, the biological effects of 3 kinds of Nd compounds, i.e. neodymium oxide (Nd₂O₃), neodymium chloride (NdCl₃) and neodymium nitrate (Nd(NO₃)₃), were evaluated by using silkworm models through the method of intravascular injection. Each of the three Nd compounds was prepared into 6 groups at different concentrations (40, 50, 60, 70, 80, 90 μg/μL). The results show that within the certain concentration range, Nd compounds show great damage to the growth of silkworms since high death rate is observed: the death rate from Nd₂O₃ group is floating between 30% and 80%, the one from NdCl₃ group is between 17% and 33% and that from Nd(NO₃)₃ group is between 23% and 37%. It also reveals that Nd compounds have some negative effects on the body length and weight of silkworms. However, the histophysiological study shows that the three Nd compounds cause no obvious negative effects on the cell morphologies in midgut, fat body and silk gland. The XRD and FTIR spectra show that the secondary and the crystalline structures of silks are not destroyed, the TGA and DTG data exhibit that the thermal stability of silks is not damaged, and the tensile test shows that the mechanical properties of silks are not affected significantly by the intake of Nd compounds. In general, the intake of Nd compounds has significant inhibitory effects on silkworm growth but little negative impact on silk structures or cell morphology, the study can provide useful toxicological data for the safe application of Nd compounds in various fields.     

Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases

A method previously developed for the analysis of organohalogenated compounds in dairy products is now validated for polychlorinated biphenyls (PCBs) determination in soybean infant formulas. The results of this study are consistent with those found for PCBs in powdered full-fat milk. The methodology is based on a solid-liquid extraction step enabling a semi-selective extraction of the apolar lipids of the matrix without affecting the efficiency for the recovery of PCBs. Mean recoveries for the spiked coplanar congeners studied were in the 88-114% range, with relative standard deviations (R.S.D.s) lower than 9.8%. The R.S.D.s related to the determination of endogenous PCBs were in the 1.5-10.0% range. The validated methodology was applied to the PCB analysis in different trademarks of soybean infant formulas commercialised in Spain. Toxic tetraequivalents of tetrachlorodibenzo-p-dioxin and daily intake corresponding to each one were calculated and compared with values previously published and with those found in literature for human breast milk in different countries.     

Separation of chiral compounds by capillary electrophoresis

This review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.     

Determination of the antitumor agent depsipeptide in plasma by liquid chromatography on serial octadecyl stationary phases

We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5'-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides-793 and-720 that was greater than 97% A/G-rich. A repeat of the sequence 5'-AGGGAGGG-3' separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated approximately 3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased approximately 11 fold in both rat and human glial tumor cells. Four specific complexes (Cl-4) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 > C4) while Cl and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of Cl and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5'-gGGGAGGG-3' sequence previously implicated in the response of the atrial natriuretic factor promoter to the alpha-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (Cl-4) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5'-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.     

Differential susceptibility of L cells in the exponential and stationary phases to cadmium chloride

Comparative studies of the cellular toxicity and uptake of cadmium in L cells in the exponential and stationary phases were done. The LD50 of cadmium chloride to L cells in the exponential and stationary phases was 5.5 and 30.5 muM respectively and the cadmium content of L cells exposed to 5.4 muM cadmium chloride for 3.5 hr in the exponential and stationary phases was 0.123 and 0.065 mug/10(6) cells, respectively. These results suggest that the higher susceptibility of cells in the exponential phase to cadmium may be caused by an increased permeability of the cell membrane to cadmium.     

Effect of temperature on retention of enantiomers of beta-methyl amino acids on a teicoplanin chiral stationary phase

The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-methylphenylalanine, beta-methyl-tryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was studied on a teicoplanin-containing chiral stationary phase at different temperatures and at different mobile phase compositions, using the reversed-phase mode. With variation of both mobile phase composition and temperature, almost baseline separations could be achieved for all four enantiomers of sterically hindered amino acids. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van 't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (delta H degree), the apparent change in entropy (delta S degree) and the apparent change in Gibbs free energy (delta G degree) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic constants (delta H degree, delta S degree and delta G degree) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.     

Coupled achiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds

A multicolumn approach was developed to address the limited achiral selectivity of chiral stationary phases. Groups of structurally related compounds, including beta-blockers and 1,4-benzodiazepines, were separated using coupled achiral/chiral stationary phases under subcritical fluid conditions. The achiral selectivity of amino and cyano stationary phases was used to modify the resolution of compounds on a Chiralcel OD chiral stationary phase by combining the achiral and chiral columns in series. In the case of the benzodiazepines, separation of achiral compounds was performed concurrently with the enantioseparation of chiral molecules. The separation of components of a multidrug cough and cold medication was also demonstrated on a cyano column coupled with a Chiralpak AD chiral stationary phase. The use of modified carbon dioxide eluents eliminated the mobile phase incompatibility problems associated with column coupling in liquid chromatography and incorporated the high efficiency of sub- and supercritical fluid chromatography.     

Direct chiral resolution of tiaprofenic acid in pharmaceutical formulations by capillary zone electrophoresis using cyclodextrins as chiral selector

Capillary zone electrophoresis (CZE) was used for the enantiomeric separation of (R,S)-tiaprofenic acid ([R,S]-Tia) in a pharmaceutical formulation employing an acetate buffer at pH 4.5 and 2,3,6-tri-O-methyl-beta-cyclodextrin (tri-OMe-beta-CD) as the chiral selector. The effect of the concentration of trimethylated-beta-cyclodextrin in the presence of carboxymethylated-beta-cyclodextrin (CM-beta-CD) on enantiomeric resolution of (R,S)-Tia and (R,S)-5-benzoyl-alpha-methyl-3-thiopheneacetic acid (3-isomer of tiaprofenic acid, 3-Tia) was investigated at pH 4, 4.5, and 5.     

Capillary electrophoresis chiral separations of basic compounds using cationic cyclodextrin

Chiral separations of basic enantiomers were carried out by using a cationic cyclodextrin (CD), quaternary ammonium beta-cyclodextrin (QA-beta-CD), under counter-electroosmotic flow (counter-EOF) conditions. The special characteristics of using a cationic CD to separate cationic enantiomers is that the EOF can be reversed and the analyte-CD complexation is reduced. This is especially useful for chiral separation of cationic compounds, which strongly bind with neutral and anionic CDs (such as tricyclic amine compounds). The reduction in the binding constants between the CD and the cationic enantiomers makes it easier to control the optimum CD concentration. The application of the cationic CD also eliminated the peak tailing problem caused by electrodispersion. The effect of pH and the concentration of QA-betaCD on chiral separation has been studied. At pH 3.02, no separation for any of the enantiomeric amines was observed. At pH 8.20, chiral separation of some tricyclic compounds was achieved at very high resolution due to the counter-EOF setup. At pH 11.6, most enantiomers were neutral and chiral separation of some bicyclic compounds can be obtained.