Separation of cis- and trans-isomers of thioxanthene and dibenz[b,e]oxepin derivatives on calixarene- and resorcinarene-bonded high-performance liquid chromatography stationary phases

The chromatographic behavior of six calix[n]arene phases (n=4, 6, 8) and one calix[4]resorcinarene phase is described for the separation of cis- and trans-isomers of three thioxanthene (flupentixol, clopenthixol, chlorprothixene) and one benz[b,e]oxepin derivative (doxepin). The influences of two different organic modifiers (MeOH, MeCN) for the separation of the isomers on every column are described. Different selectivities of the stationary phases exist as a function of the ring size of the calixarenes and their substitution at the "upper rim" with p-tert.-butyl groups. Furthermore, the influence of free phenol groups on the resorcinarene phase is discussed. Relations between structural elements of the analytes and the retention behavior on the stationary phases are found. The selectivity of the calixarene and resorcinarene stationary phases is compared with a RP-C18 phase containing the same base silica. Advantages of the resorcinarene as well as of the calixarene columns exist for the separation of cis- and trans-isomers of three compounds dependent from the substitution in position 2 of the thioxanthenes, respectively the kind of the basic side chain of all substances.     

Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

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 epsilon/eta 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.     

Chiral separation of amino acid esters by micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was used for the chiral separation of uncharged analytes (C- and N-protected amino acids). Sodium dodecyl sulfate (SDS) was the micelle forming agent, and different cyclodextrin (CD) derivatives were added as chiral selectors. Suitable conditions for the enantioseparation were found by variation of the separation conditions. The influence of addition of organic solvents like acetonitrile or methanol, and other chiral additives (camphor-10-sulfonic acid, malic acid) was examined. The addition of an organic modifier resulted in different effects on micelle formation, and thereby on the separation. The used chiral additives did not improve the selectivity. Furthermore, dependence of the electroosmotic flow (EOF), and the capacity factors on the concentration of CDs was investigated. Increasing the CD concentration, both the EOF to a smaller extent as well as the capacity factors decrease. Nevertheless, the enantioseparation is improved with a CD-concentration up to 30 mM. Higher CD-concentrations reduce the separation of the analytes.     

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.     

Peak shape improvement of basic analytes in capillary liquid chromatography

The analysis of bases is of special interest in pharmaceutical research because numerous active substances contain basic functional groups. Capillary and conventional size LC separations of drug substances spiked with potential impurities were compared. In the case of the nonpolar drug levonorgestrel equal separation efficiency was readily attained by both techniques. The peaks of basic substances, however, showed extensive tailing when separated by capillary LC. The peak deformation was attributable to interactions of the basic substances with the polar inner surface of the fused silica capillaries employed in capillary LC and does not appear with the steel tubing generally used in conventional size LC. This drawback of capillary LC was overcome by use of deactivated fused silica capillaries for column hardware and transfer lines.     

Chromatographic Applications on Monolithic Columns: Determination of Triamcinolone, Prednisolone and Dexamethasone in Pharmaceutical Tablet Formulations Using a Solid Phase Extraction and a Monolithic Column

A method is described for extraction, separation and quantification of 3 corticosteroids in tablets (triamcinolone, prednisolone and dexamethasone). The extraction was achieved using a solid phase extraction (SPE) on RP-18 cartridges. The steroids eluates were examined by HPLC. The chromatographic separation was carried out on Chromolith^® Performance RP-18e column, a new packing material consisting of monolithic rods of highly porous silica, using isocratic binary mobile phase of MeOH and H₂O in the ratio 1: 1. A diode array detector was used at 254 nm for detection. The method was validated for system suitability, linearity, precision, limits of detection, specificity, stability and robustness. The limits of detection were 6.25, 6.25 and 12.50 ng per 25 L injected volumes for them respectively. The recovery values of this method were between 89 and 101% and the reproducibility was within 6.86.     

Fast-GC/HRMS to quantify the EU priority PAH

For the analysis of the 15 polycyclic aromatic hydrocarbons (PAH) classified as priority from the Scientific Committee on Food (SCF) and benzo[c]fluorene (BcL) assessed to be relevant by the Joint FAO/WHO Experts Committee on Food Additives (JECFA) in food, a sensitive analytical method is necessary. In the past, many methods were established for only the analysis of the 16 priority PAH recommended by the US Environmental Protection Agency (16 EPA-PAH) but not for the 15 SCF-PAH and BcL. Therefore, at the Max-Rubner-Institut, Federal Research Institute of Nutrition and Food in Kulmbach, Germany, an analytical method with a runtime of 72 min was developed. To shorten this runtime without a loss of separation, a Fast-GC/high resolution MS (HRMS) method with a runtime of only 25 min using a TR-50ms column (10 m x 0.1 mm x 0.1 microm) was created. The repeatability (n = 3) of spiked PAH concentrations in test-matrix tea ranged from 0.1 to 11%. The analytical parameters LOD (0.01-0.02 microg/kg) and LOQ (0.03-0.06 microg/kg) also in the test-matrix tea were determined. A good linearity for all PAH (R(2) >0.99) and averaged recoveries from 75 to 117% in the concentration range of 1-20 microg/kg were achieved.