Olle Gyllenhaal

Enantiomeric separations of amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector.

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.

Chiral supercritical fluid chromatography on porous graphitic carbon using commercial dimethyl β-cyclodextrins as mobile phase additive

Using dimethylated-beta-cyclodextrin mixtures (MeCD) as chiral selectors in CO2-polar modifier mobile phase and porous graphitic carbon as solid-phase, chiral supercritical (or subcritical) fluid chromatography was performed. The adsorbed quantity of MeCD onto the porous graphitic carbon (Hypercarb) was measured for various chiral selector concentrations using the breakthrough method with evaporative light scattering detector. The effects of MeCD concentration in the mobile phase, the nature of the polar modifier, the outlet pressure, the column temperature and the nature of the commercial MeCD mixture on the retention and the enantioselectivities were studied. For a given solute, the enantioselectivity is greatly dependent on the commercial MeCD mixture used. The retention mechanism was also studied. From the data, we find that the dominant mechanism for the chiral discrimination is the diastereoisomeric complexation in the mobile phase.

HPLC enantiomer separation of a chiral 1,4-dihydropyridine monocarboxylic acid

An enantioselective anion exchanger based on tert-butylcarbamoylquinine as chiral selector and thiol-modified silica as chromatographic support was applied for the enantiomer separation of the shortacting calcium antagonist clevidipine after its hydrolysis to methyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate. This hydrolyzed derivative of clevidipine is the primary metabolite and precursor of the parent drug. Method development included the steps of optimization of the composition of the mobile phase (pH, type and content of organic modifier, polar organic mode), screening of various structural analogs of above mentioned CSP, and evaluation of the effect of the flow rate. These detailed studies gave insight into the operational mode of the CSP, which is basically an enantioselective anion exchange mechanism. The polar organic mode turned out to be advantageous with regard to enantioselectivity and resolution. The optimized method makes use of an eluent composed of 0.125% acetic acid in acetonitrile with flow rate of 1 ml/min at a constant temperature of 25 degrees C, and allows the separation of the both enantiomers with enantioselectivity alpha of 1.25 and a resolution RS of 3.0 within 10 min. On the above mentioned quinine carbamate phase the (R)-enantiomer is stronger retained, while a change to the corresponding quinidine carbamate CSP allows the reversal of the elution order.

Chiral separations of metoprolol and some analogs with carbon dioxide on Chiralcel OD and Chiralpak AD stationary phases. Use of chemometrics

SummaryThe influence of methanol concentration, column temperature, and column back-pressure on the enantios-electivity of the separation of eighteen amino alcohols in supercritical-fluid chromatography has been investigated by use of statistical experimental design. The enantioselective retention of the amino alcohols was studied using Chiralcel OD and Chiralpak AD as the chiral stationary phases and the experimental responses obtained—retention factors (k) and selectivity factors (α)—were evaluated by use of the partial least squares algorithm. The performance of the columns was compared and the enantioselectivity of the Chiralcel OD column was found to be superior. Almost all the racemic amino alcohols tested were separated and separation factors as high as 4.5 were obtained by use of the Chiralcel OD column.Experimental results from a factorial design with three centerpoints resulted in a statistical model based on linear terms only. The first-eluted enantiomers, which, when identified, were found to be theR forms, had similar retention times, whereas the retention times of theS forms were more varied. The column temperature had a greater effect on enantioselectivity than methanol content. Changes in the system back-pressure had no significant influence on enantioselectivity.The results obtained from the factorial design were used to predictk and α. Differences between predicted and experimental data were less than 10%.The effect on enantioselectivity of protolytic mobile phase additives, e. g. dimethyloctylamine, acetic acid, and trifluoroacetic acid, and of mobile-phase flow-rate, were also studied, as was the effect of solute structure. The position of substituents on the aromatic ring, type of alkyl group attached to the nitrogen atom, and the number of methylene groups between the stereogenic center and the nitrogen atom all affected enantioselectivity.The chromatographic system developed could be used to determine enantiomeric purity even if the chiral impurity eluted after the main peak.

Packed-column supercritical fluid chromatography for the purity analysis of clevidipine, a new dihydropyridine drug.

In this paper we describe a packed column supercritical fluid chromatography method that can be used for the analysis of a new dihydropyridine substance. The method is based on methanol-modified carbon dioxide as the mobile phase and Hypersil bare silica as column support at a column temperature of 50 degrees C and 150 bar as back pressure. Using an adjusted methanol gradient the most likely by-products can be separated and detected (240 nm) within 13 min. Occasionally the column needed treatment with 4 mM citric acid in the methanol modifier in order to give a narrow peak of an acidic analogue. The present method can detect analogues at the 0.1% (w/w) level. The precision at this level for one of the analogues was 5.9% RSD. This method shows a higher selectivity than a corresponding reversed-phase liquid chromatographic method.

Enantioresolution of dihydropyridine substituted acid by supercritical fluid chromatography on hypercarb® with Z-(L)-arginine as chiral counter ion

SummaryA chromatographic method is described for separation of substituted dihydropyridines on Hypercarb, porous-graphitic carbon as support and methanol-modified carbon dioxide containing Tween 60® deactivation additive, as mobile phase.Enantiomers of a dihydropyridine substituted acid were resolved after addition of Z-(L)-arginine as ion-pairing counter ion. A 0.2 mM concentration was sufficient to achieve a resolution factor>2 or baseline resolution. The system was used to estimate the minor enantiomer below 0.1% level.

Packed-column supercritical fluid chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets.

We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 40 degrees C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related compounds. In order to elute NO(3)(-) with acceptable retention time a quarternary ammonium hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2, 5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0. 1 the precision was less than 10%.

Direct injection of aqueous samples in packed column supercritical fluid chromatography of isosorbide-5-mononitrate from drug release testing.

A method for the analysis of aqueous samples of isosorbide-5-mononitrate (5-ISMN) is presented. It is based on packed column supercritical fluid chromatography (SFC) using 20% of 2-propanol in carbon dioxide as the mobile phase and a diol silica column as the stationary phase. Using the described conditions it is possible to quantitate 5-ISMN released from Imdur tablets in gastric media. The precision upon repeated injections was 2% (RSD) at the 20 microg/ml level (n=8), using peak height measurements, when the solution was circulated through the sample loop of the injector. Samples from drug release testing that had been analyzed with reversed phase LC were analyzed with the present method and the results agreed well. It is also possible to monitor the drug released in a dissolution-testing vessel through direct on-line continuous loading (recirculation) of the sample loop of the SFC instrument.