Michael Schleimer

Enantiomer separation by high-performance liquid chromatography on polysiloxane-based chiral stationary phases

Abstract The synthesis of two polysiloxane-based chiral stationary phases (CSPs) derived from a π-acidic N-(3.5-dinitrobenzoyl)-β-amino acid (JEM-1) and a π-basic N-(1-naphthyl)-leucine selector is described as is their systematical comparison with the corresponding “brush”- type CSPs. The enantioselectivity of the polysiloxane-based CSPs is higher under both normal- and reversed-phase conditions. In the normal- phase mode, the greater enantioselectivity stems from smaller retention factors for the least retained enantiomers, presumably because of a reduction of analyte interactions with the support silanols owing to effective shielding of the surface by the polymer. The retention factors of the second-eluted enantiomers are shifted to higher values on the π-basic CSP and to lower values on the π-acidic CSP. The latter CSP shows but a small increase in enantioselectivity relative to the corresponding “brush”-type CSP having a comparable selector loading. The silanophilic interactions can be further reduced by end-capping with hexamethyldisilazane (HMDS). When lower amounts of polar modifier are used, the resolution of the polymeric CSPs approaches that of the corresponding brush-type CSP. Under reversed-phase conditions enantioselectivity is reduced but not to the extent generally found for brush-type CSPS. The presence of the non-polar polymeric backbone can introduce hydrophobic interactions which may alter enantioselectivity. It would seem advantageous to use dimethylpolysiloxanes having a high selector concentration in order to reduce the extent of any non-chiral contribution by the polysiloxane backbone to analyte retention while enhancing the favorable chiral recognition properties of the polymer.

Enantiomer separation by complexation gas and supercritical fluid chromatography on immobilized polysiloxane-bonded nickel(II) bis[(3-heptafluorobutanoyl)-10-methylene-(1R)-camphorate] (Chirasil-nickel)

Abstract The synthesis of a polysiloxane containing chemically bonded chiral metal complex derived from nickel(II) bis[(3-heptafluorobutanoyl)-10-methylene-(1 R )-camphorate] (Chirasil-nickel) and its effective immobilization on the inner surface of fused-silica capillaries by thermal treatment is described. As expected, the immobilization properties were found to be dependent on the content of residual reactive SiR groups in the polymer backbone (R = OCH 3 , H). The immobilized Chirasil-nickel stationary phase was employed for the analytical enantiomer separation of coordinating solutes by high-resolution capillary gas and supercritical fluid chromatography. The increase in the relative retention monitored for racemic test solutes after rinsing the columns indicates a slight increase in the effective complex concentration which does not affect the chiral separation factor α. The temperature limit of analysis was raised to 170–180°C for temperature-programmed runs and to 140–150°C under iso thermal conditions, thus extending the scope of complexation gas chromatography considerably, as demonstrated by various enantiomer separations performed at elevated temperatures. When Chirasil-nickel is immobilized on the inner surface of short, narrow-bore capillaries (50 μm I.D.), it can be employed in complexation supercritical fluid chromatography , combining the high solvation strength of supercritical carbon dioxide with the benefit of low operating temperatures, enhancing enantioselectivity.

Enantiomer Separation by Capillary Supercritical Fluid Chromatography

The unambiguous determination of enantiomeric compositions and absolute configurations is an important analytical task in the synthesis, characterization and use of chiral nonracemic compounds (optical isomers, enantiomers) such as chiral auxiliaries, catalysts, pharmaceuticals, pesticides, herbicides, pheromones, flavours and fragrants. As the insight into chirality-activity relationships steadily improve and, as a consequence, legislation of chiral drugs becomes more and more stringent, the development of precise methods for the determination of enantiomeric purities up to ee>99% is of great importance.

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry.

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-D-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.

Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-(14C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs

A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary.