Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK).

Non-particulate (continuous bed or monolithic) acrylate-based capillary columns for reversed-phase liquid chromatography and electrochromatography.

Three approaches are described to synthesize acrylic non-particulate beds (also called continuous beds or monoliths) in aqueous polymerization media for reversed-phase capillary liquid chromatography/electrochromatography. In the first, hexyl acrylate comonomer was dissolved together with water soluble polar comonomers using a non-ionic detergent. In the second, a new alkyl ammonium salt comonomer, (3-allylamino-2-hydroxypropyl)dodecyldimethylammonium chloride was used, which is water soluble and has detergent properties itself. The alkyl group of this comonomer provides hydrophobicity while the ionic groups generate electroosmosis in the non-particulate bed. In the third approach, the alkyl comonomer was used as a detergent to dissolve another hydrophobic comonomer in an aqueous polymerization medium. All three approaches were evaluated with respect to hydrophobicity, efficiency and electroosmotic properties of the beds. Hydrophobicity expressed as methylene group selectivity for the three types of the beds in 50% methanol mobile phase was 1.86, 1.16 and 1.78, electroosmotic mobility -5.14 x 10(-5), 6.89 x 10(-5) and 6.37 x 10(-5) cm2 V(-1) s(-1) and efficiency for the retained compound (methylparabene) 67,000, 93,000 and 110,000 plates m(-1) correspondingly. The columns were tested using pressure driven capillary chromatography and capillary electrochromatography. The influence of polymerization temperature on hydrodynamic permeability, separation impedance and inverse size exclusion porosimetry characteristics were used to evaluate the separation columns. The increase of the polymerization temperature resulted higher permeability of the bed, separation impedance and lower polymeric skeleton porosity. Further characterisation was provided by examining the separation efficiency observed for a series of benzoic acid esters and alkyl parabens.

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 microm ID) by in 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 x 10(-5) cm2 V(-1) s(-1) and the aqueous reversed-phase modes of operation (8.5 x 10(-5) cm2 V(-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.

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.

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.

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.

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.

Evaluation of a vancomycin chiral stationary phase in capillary electrochromatography using polar organic and reversed-phase modes.

A vancomycin chiral stationary phase (CSP) was fully evaluated in capillary electrochromatography (CEC) in reversed-phase and polar organic modes for a number of racemic pharmaceutical compounds. High efficiency and resolution values were obtained for a number of compound classes including thalidomide in both the polar organic mode (190000 plates meter(-1) and Rs = 13.8) and reversed-phase mode (125000 plates meter(-1) and Rs = 13.0). Experimental parameters, including organic modifier, organic solvent ratio, ionic strength, pH, temperature, and voltage, were examined in both the aqueous and nonaqueous modes to deduce their effect on the resultant EOF, retention times, resolution, and efficiency of chiral separations. All results were consistent with and found to be a combination of what is known from existing literature on CEC theory and experience obtained with macrocyclic antibiotic CSPs in LC. Column stability was excellent, and each column packed was found to offer repeatable separations even when switching from the aqueous to the nonaqueous mode.

Enantioselective supercritical fluid chromatography using ristocetin A chiral stationary phases.

Racemic mixtures of five acidic drugs have been successfully separated by supercritical fluid chromatography (SFC) using macrocyclic antibiotic chiral stationary phases (CSPs). A ristocetin A CSP has been prepared 'in-house' and effectively applied in packed capillary SFC to separate the enantiomers of dichlorprop (R(s) = 1.4), ketoprofen (R(s) = 0.9) and warfarin (R(s) = 0.9). The commercial ristocetin A CSP (Chirobiotic R) was subsequently studied in packed column SFC with similar results where the enantiomers of warfarin (R(s) = 2.2), coumachlor (R(s) = 2.5) and thalidomide (R(s) = 0.6) were separated. Interestingly, differences were observed between the two differently immobilised CSPs where the enantiomers of dichlorprop and ketoprofen, which were separated on the 'in-house' CSP, could not be separated on the commercial phase.

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.

Separation of the voriconazole stereoisomers by capillary electrophoresis and liquid chromatography.

A chiral capillary electrophoresis (CE) method has been developed for the direct separation of the four stereoisomers of a new broad spectrum antifungal agent, voriconazole. Cyclodextrin (CD) modified micellar electrokinetic chromatography employing, alpha-CD, beta-CD, gamma-CD, hydroxypropyl-beta-CD and hydroxyethyl-beta-CD was not sufficiently selective for the four neutral stereoisomers. Three anionic sulphobutyl-ether-beta-CD (SBE-beta-CD) electrolyte additives, each having a defined degree of substitution (DS) (6.5, 4.5 and 1.0) were subsequently examined. The complete CE separation of all four stereoisomers was obtained when using the medium substituted additive DS = 4.5. In liquid chromatography (LC), two approaches were examined for the direct chiral separation of the stereoisomers of voriconazole: (a) use of the neutral and anionic CD mobile phase additives and (b) a vancomycin chiral stationary phase. The CD additives were shown to be extremely selective for two stereoisomers of voriconazole (active drug and its enantiomer) but unable to discriminate between the opposite two stereoisomers. The converse was observed, however, when the vancomycin chiral stationary phase was employed.

Use of 1H-NMR spectroscopy to determine the enantioselective mechanism of neutral and anionic cyclodextrins in capillary electrophoresis.

One-dimensional (ID) and two-dimensional (2D) 1H nuclear magnetic resonance (NMR) techniques have been used to investigate the chiral recognition process in capillary electrophoresis (CE) for seven different cyclodextrins (CDs) with the calcium channel blocker amlodipine as a model compound. These include five neutral CDs (alpha-CD, beta-CD, gamma-CD, hydroxypropyl-beta-CD and hydroxyethyl-beta-CD) and two anionic CDs (sulphobutyl-ether-beta-CD and carboxymethyl-beta-CD) where mixtures of amlodipine with each of the seven CDs were examined by 1D NMR in deuterated phosphate buffer at pD 3.4. The resonance shift of signals with added CD, relative to the CD-free position (shift displacement, delta delta) and shift non-equivalence (delta delta *) of enantiomeric signals shifted relative to each other after addition of CD were examined for non-overlapped protons of amlodipine. The possible correlations of NMR shift non-equivalence data with chiral separation in CE for amlodipine have been critically assessed. Qualitative differences in the 1D NMR shifts and enhanced enantioselectivity in CE were observed for amlodipine with sulphobutyl-ether-beta-CD. Further experiments on the through-space interactions using 2D rotating frame nuclear Overhauser effect spectroscopy (ROESY) indicated that there was no association between internal glucopyranose hydrogen atoms and the aromatic hydrogens of amlodipine. This gives evidence for the aromatic ring not being included in this CD. Moreover, data from spin-lattice relaxation times (T1) measured for amlodipine in the free state and after addition of the anionic sulphobutyl-ether-beta-CD indicate that the aromatic moiety of amlodipine is not included into the sulphobutyl-ether-beta-CD cavity. There is evidence that it interacts with the sulphobutyl side chains, and may adopt a preferred orientation outside the sulphobutyl-ether-beta-CD toroid itself.

Chiral recognition in liquid chromatography utilising chargeable cyclodextrins for resolution of doxazosin enantiomers.

The chromatographic resolution of rac-doxazosin using reversed-phase high performance liquid chromatography (HPLC) with the chargeable chiral mobile phase additive, carboxymethyl-beta-cyclodextrin (CM-beta-CD), is described. The effects of different modifiers (acetonitrile, methanol and tetrahydrofuran), pH, temperature, and cyclodextrin concentration were investigated to a) assess the key chromatographic parameters for subsequent chemometric optimisation, and b) explore the enantioselective mechanism. Assuming a 1:1 complex between each doxazosin enantiomer and CM-beta-CD, studies of the relationship between the capacity factors (k') and functions of CM-beta-CD concentration indicate that the mechanisms for retention and chiral selectivity are comparable with those proposed earlier by Sybilska et al. Stability constants (KG) calculated for rac-doxazosin complexed with CM-beta-CD (647 +/- 55 and 594 +/- 45 M-1 for each enantiomer respectively) are significantly larger than those calculated for the barbiturates complexed with beta-CD (ca. 101-108 M-1). Investigations on pH indicate an ionic or ino-pair interaction between the anionic CM-beta-CD and the cationic doxazosin enantiomers. A central composite design was used to optimise the key chromatographic parameters: pH, methanol (v/v) and CM-beta-CD concentration. The Kaiser peak separation index, Pi, was used for the response function. The predicted response for this chiral separation has been compared with that observed experimentally and samples of the four-dimensional response surface have been assessed for their value in showing robustness.

Method development in liquid chromatography with a charged cyclodextrin additive for chiral resolution of rac-amlodipine utilising a central composite design.

A negatively charged derivative of beta-cyclodextrin, sulphobutyl ether-beta-cyclodextrin (SBE-beta-CD), was examined as a chiral mobile phase additive in reversed-phase high-performance liquid chromatography for the enantiomeric resolution of the calcium channel blocker rac-amlodipine. Theoretical and practical aspects are discussed for setting up a central composite design applicable to any analytical method. These include the correct location of factor points for maintaining orthogonality within the design and the augmentation of centrepoint experiments to allow a larger factor space by increasing the distance of axial star points. Optimised separation was achieved using a reverse-phase column with eluent comprising: acetonitrile (ACN)-potassium dihydrogen phosphate (pH 3.93) containing 2.66 mM SBE-beta-CD (26.5:73.5% v/v) at a flow rate of 1.0 ml/min. This yielded a Kaiser peak separation index, Pi = 0.96, at tR2 = 52 min with satisfactory reproducibility, relative standard deviation values: tR1, 0.39%; tR2, 0.47% (n = 5). These experimental results were in excellent agreement with those predicted by the SAS software package for a chromatographic response function model. Multiple regression analysis in four dimensions, with three response models based on Rs, Pi, and a function of Pi, produced response surfaces which revealed zones of optimum robustness and illustrated the interactions involved between the key chromatographic factors. Putative proposals for a mechanism involving the interaction of each of the positively charged enantiomers with the negatively charged cyclodextrin are also discussed. These examine the possibility of ion-pairing and inclusion phenomena to account for the excellent resolution observed.