Thomas Jira

Extracolumn band broadening in capillary liquid chromatography

A commercially available capillary LC instrument was modified to investigate and control the contribution of different instrument components on extracolumn band broadening. Quantitative estimations of dispersion induced by several equipmental parts were carried out. Injection parameters could be optimized to achieve the theoretical value of 12 for a profile factor describing a rectangular sample profile. Additionally, an additive injector flow channel dependent dispersion effect was found. A practical approach for minimizing instrumental effects in capillary LC is suggested. The results were compared with those obtained with an HPLC instrument designed for conventional size columns.

Chiral resolution of diols by capillary electrophoresis using borate-cyclodextrin complexation

In this study, the chiral separation of compounds with a cis-diol structure using an electrolyte containing borate and cyclodextrin (CD) is described. A dual mechanism involving both inclusion of the aromatic moiety into the cavity of the CD and the formation of mixed borate complexes is assumed to be responsible for chiral recognition. It has been shown that the separation is greatly influenced by the structure of the CD, the pH and the concentration of both the CD and borate. The method was optimized with some simple diol compounds and applied to quinazoline-(3H,1H)-4-on-2-thiones and tetrazol-5-thiones of potential pharmaceutical interest.

Chiral separation of unmodified amino acids with non-aqueous capillary electrophoresis based on the ligand-exchange principle.

A ligand exchange mechanism in non-aqueous capillary electrophoresis was employed for the separation of eight unmodified amino acids using chiral complexes of copper(II) with L-proline and L-isoleucine. The electrophoretic medium consisted of 25 mM ammonium acetate and 1 M acetic acid in methanol. We were able to completely separate the enantiomeric pairs of each of the investigated racemic amino acids. We also report the optimization of the separation parameters, such as pH*, composition of the complex, and concentration of the complexing agents.

Effect of chromatographic conditions on liquid chromatographic chiral separation of terbutaline and salbutamol on Chirobiotic V column

In this study, a method for enantioseparation of terbutaline and salbutamol was established using Chirobiotic V column as a stationary phase. Polar ionic mode applying mobile phase containing ammonium nitrate in 100% ethanol, pH 5.1 was found to give the best separation. The salt concentration in the mobile phase and pH value were found to be the most important chromatographic factors affecting separation. Separation of enantiomers of these two basic analytes was complete in less than 10 min without applying ammonium trifluoroacetate (ATFA) or triethylamine (TEA) salts.

NONAQUEOUS CAPILLARY ELECTROPHORESIS : APPLICATION POSSIBILITIES AND SUITABILITY OF VARIOUS SOLVENTS FOR THE SEPARATION OF BASIC ANALYTES

Eleven organic solvents were tested for their suitability in the use of capillary electrophoresis (CE). In all cases, 25 mM ammonium acetate and 1 M acetic acid were used as electrolytes. Three basic therapeutical agents, propranolol, carteolol and imipramine, were used as analytes. Four solvents (2‐propanol, ethylene glycol, propylene glycol, dichloromethane) were not suitable for use with CE under our conditions. Depending on the other solvents used, the analytes showed very different behavior. We observed that the ε/η quotient alone is not sufficient for a characterization of the solvents. Further investigations with all the solvents as 1:1 mixtures with methanol were carried out. As a result some principal changes occurred compared to the pure solvents. Working with mixtures of different solvents proved to be advantageous because of the possibility to influence properties like high viscosity or low ionizing abilities by the addition of a suitable second solvent.

Use of chiral and achiral ion-pairing reagents in combination with cyclodextrins in capillary electrophoresis

Abstract The suitability of chiral and achiral acidic ion-pairing reagents to effect chiral separation in capillary electrophoresis in the presence of cyclodextrins (CDs) has been demonstrated previously. This method was applied to a great variety of analytes. Besides alkylsulfonates, alkanoic acids are able to improve chiral resolution in combination with CD. The dependence of the separation on alkyl chain length is described. The use of the cationic ion-pairing reagent quinine, is a powerful tool in influencing enantiomeric separation of acidic and basic analytes. Methods of direct and indirect UV detection are used in quinine containing running buffers. The influences of pH value, quinine and CD concentration on the separation factors are reported. l -Hyoscyamine showed the best capability to improve the enantioseparation of propranolol.

Use of cationic cyclodextrin for enantioseparation by capillary electrophoresis

The usability of 2-hydroxy-3-trimethylammoniopropyl-β-cyclodextrin for chiral discrimination of various basic and acidic substances is described. The dependence of chiral separation on cyclodextrin (CD) concentration and pH value was investigated. Altering the pH value the migration order of the enantiomers of acidic analytes could be changed. Due to the quaternary ammonium structure of the CD molecule, a reversal of the electroosmotic flow (EOF) was observed. The direction of the migration of CD molecule was in opposite direction to the EOF because of its positive charge. The influence of CD concentration and pH on the EOF was determined.

Description of retention characteristics of calixarene-bonded stationary phases in dependence of the methanol content in the mobile phase.

Calixarene-bonded stationary phases in HPLC are known to support additional interactions compared to conventional alkyl-bonded phases (pi-pi interactions, complex-building interactions). Thus it cannot be presumed that the same mechanisms of retention apply and that retention can be predicted in similar ways. Here 31 solutes of highly various molecular structures have been analysed at different mobile phase compositions (0-98% (v/v) methanol) in order to characterise the chromatographic behaviour of the novel stationary phases and to test the applicability of established models predicting retention factors. The influence of a change of the methanol content is discussed for non-polar, polar and ionic solutes and differences of their behaviour on the differing column types are shown. Additionally estimates about underlying retention mechanisms are given.

Chiral separations of 1,3,4-thia- and 1,3,4-selenadiazine derivatives by use of non-aqueous capillary electrophoresis.

Our aim was to establish suitable conditions for the chiral separation of 12 1,3,4-thia- and 1,3,4-selenadiazine derivatives; some of them were identified in screening tests as potential antituberculotics. To overcome possible problems with the water insolubility of most analytes, we profited by the advantages of non-aqueous capillary electrophoresis. Methanol, formamide, and a mixture of formamide with acetonitrile (1:2, v/v) were used as separation media. Hydroxyethyl-, hydroxypropyl-, and methyl-beta-cyclodextrin were applied as chiral selectors in concentrations of 200 mM. Besides the effect of these different electrophoretic media and selectors, we also investigated the consequences of using different electrolytes (25 mM ammonium acetate/1 M acetic acid and 25 mM citric acid/12.5 mM TRIS). Distinct differences of the separation factors in the different separation media were observed. Depending on structure characteristics of the analytes, we established clear classifications to these cyclodextrins (CD), which were most appropriate for the separation of the enantiomers of the particular analytes.

Analytical power of LLE–HPLC–PDA–MS/MS in drug metabolism studies: Identification of new nabumetone metabolites

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date. In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE-HPLC-PDA and achiral LLE-UHPLC-MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC-MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite. To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE-HPLC-PDA-MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA).