Paul K. Owens

Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers

Polymers imprinted with clenbuterol were used to study the influence of various post-polymerization treatments [e.g., thermal annealing, microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid template desorption] on the bleeding of residual template. The aim of the study was to reduce the bleeding to levels that would allow the use of the materials as affinity phases for extraction of clenbuterol from bovine urine at concentrations below 1 ng ml-1. After treatment, the clenbuterol imprinted polymers were packed into solid-phase extraction columns and the bleeding was estimated by quantifying the amount of template released in 10 ml of methanol-acetic acid (9 + 1 v/v). This was followed by an assessment of selectivity and recovery in comparison with non-treated material. The lowest bleeding level was found after MAE using 100% trifluoroacetic acid for 3 x 20 min at 100 degrees C. The collected eluate contained in this case 3 ng ml-1 of clenbuterol. The same material was subsequently used for the extraction of clenbuterol from spiked bovine urine. The resulting selectivity and recovery were lower compared with those obtained using the untreated material. A milder but still efficient method to reduce the bleeding level was found to be MAE with formic acid. In this case a bleeding level of 14 ng ml-1 was found after only a 1 h extraction time. In a second model system, using a polymer imprinted with L-phenylalanine anilide, the bleeding was reduced to a similar level by extensive on-line washing in good swelling solvents containing acid or base additives and after thermal annealing of the polymers in the dry state.

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography.

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 microm LiChrospher 100 A diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100000 plates meter(-1), respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run (n=5, 0.74% and 1.5%) and column-to-column (n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 microm LiChrospher 1000 A diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 A material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80000 plates meter(-1), with acceptable repeatability (n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120000 plates meter(-1), with acceptable repeatability (n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four beta-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45000 plates meter(-1), respectively.

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 microm 100-A porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers (Rs = 2.3 and 53000 plates m(-1)). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor (Rs = 3.0 and 86000 plates m(-1)) and alprenolol (Rs = 3.3 and 135000 plates m(-1)) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography.

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous‐bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 νm ID) byin situ copolymerization of N‐(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2×10–5 cm2V–1 s–1) and the aqueous reversed‐phase modes of operation (8.5×10–5 cm2V–1 s–1). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120 000 plates meter –1) values.

Application of molecularly imprinted polymers in supercritical fluid chromatography.

Molecularly imprinted polymers (MIPs), for the templates free base racemic propranolol and the L-enantiomer of phenylalanine anilide (L-PA), were investigated as stationary phases in supercritical fluid chromatography (SFC). Large retention differences were observed on the propranolol MIP for both the template molecule and the structural analogue metoprolol compared to that observed on the corresponding blank polymer. Mobile phase composition and solute concentration were found to affect this retention behaviour. The phenylalanine anilide MIP (L-PA MIP) was found to be enantioselective in SFC with stronger retention observed for the template enantiomer. Throughout the study, characteristic imprinting peak shapes for the stronger retained template molecule were observed for both MIPs examined. After a number of days under supercritical fluid conditions, the performance of the photochemically initiated L-PA MIP was found to significantly deteriorate whereas the thermally initiated propranolol MIP revealed only small changes in its separation performance after a long term of operation. The separation behaviour of these two MIPs in SFC was compared with results obtained on the same columns in high-performance liquid chromatography (HPLC) both before and after their application in SFC.

Complexation of Voriconazole Stereoisomers with Neutral and Anionic Derivatised Cyclodextrins

A number of native, neutral derivatised and anionicderivatised cyclodextrins (CDs) were examined aschiral electrolyte additives in capillaryelectrophoresis (CE) to separate the fourstereoisomers of the new antifungal agent,voriconazole. A very large difference in interactionbetween each diastereoisomer and the CDs was observedin the CE study, where enantioselectivity was easilyobtained for one and extremely difficult to obtain forthe other. Nuclear magnetic resonance spectroscopy(1H-NMR) indicated a strong interaction betweenthe easily separated diastereoisomer and each of theCDs with enantiomeric shift nonequivalence values ofover 100 Hz obtained when using the anionicsulphobutylether-β-CD chiral solvating agent. Inaccordance with observations from the CE study, theopposite diastereoisomer indicated no shiftnonequivalence at all. The nature of the complexationbetween the easily separated diastereoisomer and theanionic sulphobutyletherβ-CD was also probedusing a two-dimensional nuclear Overhauser enhancementexperiment and a series of spin lattice relaxationtime measurements. It was found that theenantioselective interaction occurred through thepartial inclusion of a difluorophenyl group into theCD toroid which was also aided through a number ofadditional interactions between the drug molecule andthe sulphobutylether derivatives outside the CDtoroid.

Development and validation of a capillary electrophoresis method for ximelagatran assay and related substance determination in drug substance and tablets

The advantages of simplicity, selectivity, versatility and ease of use of free solution capillary electrophoresis (CE) present an orthogonal and complementary separation technique to the established methods of liquid chromatography (LC) for pharmaceutical analysis. This work presents the development and performance of a suitable CE method for ximelagatran (formerly H 376/95) assay and related substance determination in both drug substance and tablets. The method employed was a low pH phosphate buffer, to which acetonitrile and hydroxypropyl-beta-cyclodextrin were added, in order to facilitate the separation of ximelagatran and its related substances. An applied field of 350 V/cm was used and all compounds were resolved in approximately 20 min. Benzamidine hydrochloride was used as an internal standard in quantification. The data indicate that the performance of the validated method offers equivalent and complementary information, in terms of selectivity, sensitivity, accuracy, linearity and precision, to that of an established gradient LC method employed for similar purposes. Robustness of the method was investigated by experimental design and evaluated using multivariate calculations.

Development and validation of a chiral capillary electrophoresis method for melagatran and ximelagatran drug substances

This work presents the development and validation of an enantioselective capillary electrophoresis (CE) method for two new drug substances, the novel direct thrombin inhibitor melagatran and its oral prodrug ximelagatran. CE with a derivatised cyclodextrin as the chiral discriminant was examined as a viable alternative after failing to achieve sufficient selectivity and sensitivity using chiral liquid chromatography (LC). A robust and sensitive chiral method was developed which utilised a low pH phosphate buffer with heptakis(2,6-Di-O-methyl)-β-cyclodextrin to achieve chiral recognition for both drugs with additional organic modifier to fine tune selectivity. Quantitation at the 0.05% (w/w) level was achieved by employing sample stacking effects possible with a 200 mM phosphate buffer in combination with a narrow 50 μm ID capillary. A full validation was subsequently carried out according to the ICH guidelines, including selectivity, linearity, detection and quantitation limits, accuracy, precision, stability of analytical solutions, and robustness. Linearity, precision, and accuracy data were shown to be adequate over the entire range including at the 0.05% (w/w) quantitation limit. Several electrolyte parameters in addition to buffer depletion and cyclodextrin batch variations were amongst the parameters verified for method robustness.

Direct separation of captopril diastereoisomers including their rotational isomers by RP-LC using a teicoplanin column.

A direct reversed-phase liquid chromatography (LC) method has been developed for the separation and analysis of captopril and its 2R,2S diastereoisomer using a teicoplanin stationary phase. The proline containing diastereoisomers, which are known to form conformers in aqueous solution, were also separated from their rotational isomers. The influence of temperature, different organic modifiers and buffer type, concentration and pH were optimised to obtain a working resolution between the two diastereoisomers and their respective rotational isomers. The diastereoisomeric purity of several commercial captopril batches was subsequently evaluated using a 0.05% triethylammonium acetate (TEAA) buffer (pH 3.8) run at 1.0 ml/min with mobile phase reservoir and column temperature controlled at 0 degrees C. Throughout the study online UV diode array and mass spectrometry detection was carried out simultaneously to confirm that peaks eluting from the teicoplanin column were in fact captopril and not its readily converted disulphide dimer. Additionally, as a result of the greater detection sensitivity of mass spectrometry, it also facilitated a more accurate optimisation study where trace amounts of the rotational isomers were found to be present in the baseline at temperatures higher than optimum.