Gradient stationary phase optimized selectivity liquid chromatography with conventional columns.

Stationary phase optimized selectivity liquid chromatography (SOSLC) is a promising technique to optimize the selectivity of a given separation. By combination of different stationary phases, SOSLC offers excellent possibilities for method development under both isocratic and gradient conditions. The so far available commercial SOSLC protocol utilizes dedicated column cartridges and corresponding cartridge holders to build up the combined column of different stationary phases. The present work is aimed at developing and extending the gradient SOSLC approach towards coupling conventional columns. Generic tubing was used to connect short commercially available LC columns. Fast and base-line separation of a mixture of 12 compounds containing phenones, benzoic acids and hydroxybenzoates under both isocratic and linear gradient conditions was selected to demonstrate the potential of SOSLC. The influence of the connecting tubing on the deviation of predictions is also discussed.

Determination of pharmaceutically related compounds by suppressed ion chromatography: IV. Interfacing ion chromatography with universal detectors.

This work forms the final part of a study investigating gradient elution ion-exchange chromatography of pharmaceutically relevant compounds, aiming at achieving complementary selectivity to reversed-phase HPLC. In this study the coupling of three universal detectors (electro-spray ionisation mass spectrometer (ESI-MS); corona charged aerosol detector (CAD); and evaporative light scattering detector (ELSD)) to suppressed IC using complex elution profiles with potassium hydroxide eluents is demonstrated. The non-volatile ions were removed from the eluent by the suppressor prior to detection, thus allowing a stable detector response, especially with the prototype electrolytic suppressor. The detector response for ten weakly anionic pharmaceuticals followed the expected models and the limits of detection obtained were not compromised by the use of a suppressor, yielding values below 50 ng/mL with MS, low to sub µg/mL levels with CAD and 2-20 µg/mL with ELSD (25 µL injections). When coupled to MS and CAD, the prototype electrolytic suppressor showed percentage relative standard deviations (%RSDs) in peak areas of 0.4-2.5% on average, compared to the chemical suppressor which yielded 1.5-3 fold higher %RSD values for the test analytes. The prototype electrolytic suppressor also generally exhibited wider linear response ranges than the chemical suppressor.

Determination of pharmaceutically related compounds by suppressed ion chromatography: III. Role of electrolytic suppressor design.

For the hyphenation of ion chromatography to nebulising detectors or mass spectrometry, suppression of the non-volatile ionic eluent to water is a required step to avoid elevated detector baselines. Presented here is a study of three new designs of electrolytic suppressors, incorporating high ion-exchange capacity screens and high ion-exchange capacity membranes in different thickness and compositions. These designs aim to minimise hydrophobic interactions of the suppressor with organic analytes and to provide higher compatibility with eluents containing acetonitrile. In comparison with a commercially available electrolytic suppressor and also a commercially available chemical suppressor, the new high-capacity suppressor showed superior performance, exhibiting minimal interactions with a test set of analytes under the examined conditions. This led to the attainment of high recoveries of the analytes after suppression (93-99% recovery) and significantly reduced band broadening during suppression. The new suppressor has been shown to perform well under both isocratic and gradient elution conditions.

Determination of pharmaceutically related compounds by suppressed ion chromatography: II. Interactions of analytes with the suppressor.

For the hyphenation of ion chromatography to nebulising detectors or mass spectrometry, suppression of the non-volatile ionic eluent to water is a required step. However, suppression of weakly acidic or weakly basic organic analytes can potentially lead to losses of analytes during suppression resulting from precipitation, hydrophobic adsorption onto the suppressor, or permeation of the analyte through the suppressor membranes. This study investigates the interactions between the suppressor and weak organic acid analytes, including pharmaceutically related compounds, for eluents containing organic solvent. Correlations were observed between analyte recovery rates after electrolytic suppression and the eluent composition, the suppression conditions, and the physico-chemical properties of the analytes. These results suggest that hydrophobic adsorption interactions occur in the electrolytic suppressor and that these interactions are ameliorated by the addition to the eluent of high levels of organic solvents, especially acetonitrile. Use of eluents containing 80% acetonitrile resulted in very low losses of analyte during suppression. Recovery experiments conducted in various compartments of the electrolytic suppressor showed that some analytes permeated through the suppressor membrane into the regenerant chambers, but this could be prevented by adding organic solvent to the regenerant solution. It was also noted that analyte losses increased with ageing of the electrolytic suppressors. Chemical suppression avoids some of the analyte losses observed with an electrolytic suppressor, but when used under the correct conditions, electrolytic suppressors gave close to equivalent performance to chemical suppressors.

Development of the first sphingomyelin biomimetic stationary phase for immobilized artificial membrane (IAM) chromatography.

A prototype sphingomyelin stationary phase for Immobilized Artificial Membrane (IAM) chromatography was synthesized by an ultra-short, solid-phase inspired methodology, in which an oxidative release monitoring strategy played a vital role. Evaluated in a proof-of-concept model for blood-brain barrier passage, partial least squares regression demonstrated its potential as an in vitro prediction tool.

Determination of pharmaceutically related compounds by suppressed ion chromatography: I. Effects of organic solvent on suppressor performance.

This overall study aims to investigate gradient elution ion-exchange chromatography of pharmaceutically relevant compounds using universal nebulisation detectors, such as evaporative light scattering detection (ELSD). Addition of organic solvents to the eluent is necessary to minimise hydrophobic adsorption on the polymeric stationary phase and improve solubility of analytes. It is also necessary to de-salt the eluent prior to detection, and in this work, ion chromatography suppressors were used for this step. Such suppressors have been designed for aqueous eluents, so the purpose of the present study was to investigate the effects of methanol and acetonitrile on suppressor performance. Chemical and electrolytic suppressors were evaluated for baseline drift, noise and efficiency of suppression using aqueous/organic eluents containing up to 40% (v/v) methanol or acetonitrile. Chemical suppression of aqueous/organic eluents showed minimal noise levels, uniform low baseline and low gradient drift. Electrolytic suppression gave good performance, but with higher baseline conductivity levels and baseline drift than chemical suppression. The elevated baseline was found not to be caused by incomplete suppression of the eluent, but was attributed to chemical reactions involving the organic solvents and facilitated by high electric currents and heat generation. It was demonstrated that suppressed ion-exchange separation using a complex KOH elution profile could be coupled with ELSD, with the suppressor effectively de-salting the eluent, producing a stable baseline. Finally, complementary separation selectivity was demonstrated using a set of pharmaceutically related organic acids separated by reversed-phase and ion-exchange methods.

Selectivity optimization in green chromatography by gradient stationary phase optimized selectivity liquid chromatography.

Stationary phase optimized selectivity liquid chromatography (SOSLC) is a promising technique to optimize the selectivity of a given separation by using a combination of different stationary phases. Previous work has shown that SOSLC offers excellent possibilities for method development, especially after the recent modification towards linear gradient SOSLC. The present work is aimed at developing and extending the SOSLC approach towards selectivity optimization and method development for green chromatography. Contrary to current LC practices, a green mobile phase (water/ethanol/formic acid) is hereby preselected and the composition of the stationary phase is optimized under a given gradient profile to obtain baseline resolution of all target solutes in the shortest possible analysis time. With the algorithm adapted to the high viscosity property of ethanol, the principle is illustrated with a fast, full baseline resolution for a randomly selected mixture composed of sulphonamides, xanthine alkaloids and steroids.

Prediction of the effects of methanol and competing ion concentration on retention in the ion chromatographic separation of anionic and cationic pharmaceutically related compounds.

The mixed-mode separation of a selection of anionic and cationic pharmaceutically related compounds is studied using ion-exchange columns and eluents consisting of ionic salts (potassium hydroxide or methanesulfonic acid) and an organic modifier (methanol). All separations were performed using commercially available ion-exchange columns and an ion chromatography instrument modified to allow introduction of methanol into the eluent without introducing compatibility problems with the eluent generation system. Isocratic retention prediction was undertaken over the two-dimensional space defined by the concentration of the competing ion and the percentage of organic modifier in the eluent. Various empirical models describing the observed relationships between analyte retention and both the competing ion concentration and the percentage of methanol were evaluated, with the resultant model being capable of describing the separation, including peak width, over the entire experimental space based on six initial experiments. Average errors in retention time and peak width were less than 6% and 27%, respectively, for runs taken from both inside and outside of the experimental space. Separations performed under methanol gradient conditions (while holding the competing ion concentration constant) were also modelled. The observed effect on retention of varying the methanol composition differed between analytes with several analytes exhibiting increased retention with increased percentage methanol in the eluent. An empirical model was derived based on integration of the observed t(R) vs. %methanol plot for each analyte. A combination of the isocratic and gradient models allowed for the prediction of retention time using multi-step methanol gradient profiles with average errors in predicted retention times being less than 4% over 30 different 2- and 3-step gradient profiles for anions and less than 6% over 14 different 2- and 3-step gradient profiles for cations. A modified peak compression model was used to estimate peak widths under these conditions. This provided adequate width prediction with the average error between observed and predicted peak widths being less than 15% for 40 1-, 2- and 3-step gradients for anions and less than 13% over 14 1-, 2- and 3-step gradients for cations.

Stationary-phase optimized selectivity liquid chromatography: development of a linear gradient prediction algorithm.

Stationary-phase optimized selectivity liquid chromatography (SOS-LC) is a tool in reversed-phase LC (RP-LC) to optimize the selectivity for a given separation by combining stationary phases in a multisegment column. The presently (commercially) available SOS-LC optimization procedure and algorithm are only applicable to isocratic analyses. Step gradient SOS-LC has been developed, but this is still not very elegant for the analysis of complex mixtures composed of components covering a broad hydrophobicity range. A linear gradient prediction algorithm has been developed allowing one to apply SOS-LC as a generic RP-LC optimization method. The algorithm allows operation in isocratic, stepwise, and linear gradient run modes. The features of SOS-LC in the linear gradient mode are demonstrated by means of a mixture of 13 steroids, whereby baseline separation is predicted and experimentally demonstrated.

High-efficiency hydrophilic interaction chromatography by coupling 25 cm x 4.6mm ID x 5 microm silica columns and operation at 80 degrees C.

Recently, hydrophilic interaction chromatography (HILIC) has emerged as a valuable orthogonal tool to reversed-phase liquid chromatography (RP-LC) as it allows for resolution of highly polar ionisable compounds. The relationships between separation efficiency, column length and speed of analysis for 4.6 mm ID x 5 microm silica particle columns in HILIC are demonstrated using kinetic plots. The kinetic plots constructed for conventional pressure systems operating at 350 bar and at 30 degrees C and 80 degrees C are confirmed using experimental data for different column lengths. Efficiencies of more than 130,000 theoretical plates could be achieved by connecting up to six columns of 25 cm. As expected, a significant gain in analysis speed without loss of efficiency could be obtained by operating at 80 degrees C compared to 30 degrees C. The advantages of using long columns in HILIC in combination with elevated column temperature for the pharmaceutical industry are illustrated using test mixtures comprised of commercially available ionisable compounds (including some containing functional groups with potential genotoxic typical structural alerts) as well as real polar ionisable pharmaceuticals.

Application of retention modelling to the simulation of separation of organic anions in suppressed ion chromatography.

The ion-exchange separation of organic anions of varying molecular mass has been demonstrated using ion chromatography with isocratic, gradient and multi-step eluent profiles on commercially available columns with UV detection. A retention model derived previously for inorganic ions and based solely on electrostatic interactions between the analytes and the stationary phase was applied. This model was found to accurately describe the observed elution of all the anions under isocratic, gradient and multi-step eluent conditions. Hydrophobic interactions, although likely to be present to varying degrees, did not limit the applicability of the ion-exchange retention model. Various instrumental configurations were investigated to overcome problems associated with the use of organic modifiers in the eluent which caused compatibility issues with the electrolytically derived, and subsequently suppressed, eluent. The preferred configuration allowed the organic modifier stream to bypass the eluent generator, followed by subsequent mixing before entering the injection valve and column. Accurate elution prediction was achieved even when using 5-step eluent profiles with errors in retention time generally being less than 1% relative standard deviation (RSD) and all being less than 5% RSD. Peak widths for linear gradient separations were also modelled and showed good agreement with experimentally determined values.

Serial coupling of reversed-phase and hydrophilic interaction liquid chromatography to broaden the elution window for the analysis of pharmaceutical compounds.

It is presently a common practice in drug discovery to analyse samples by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). To increase throughput, HILIC was connected in series to RPLC by means of a T-piece with make-up flow. The first column is a 2mm I.D. column having an optimal flow between 0.1 and 0.2mL/min. Via the T-piece, the flow for the second dimension column with an I.D. of 4.6mm is adjusted to 1.5-2.0mL/min with a high acetonitrile content (i.e. >/=80%) mobile phase. Therefore, even in gradient RPLC analysis starting with a mobile phase with high water content, the HILIC column is always operated at high acetonitrile concentration which is required to obtain retention on the HILIC column. The performance of the hyphenated RPLC/HILIC set-up is illustrated with the analysis of two model samples of pharmaceutical interest. Optimization of the conditions in the HILIC dimension is discussed.

Metabolic fingerprinting of Schistosoma mansoni infection in mice urine with capillary electrophoresis.

Schistosoma mansoni infection in mice has been fingerprinted using CE to study the capabilities of this technique as a diagnostic tool for this parasitic disease. Two modes of separation were used in generating the electrophoretic data, with each untreated urine sample the following methods were applied: (i) a fused-silica capillary, operating with an applied potential of 18 kV, in micellar EKC (MEKC) and (ii) a polyacrylamide-coated capillary, operating with an applied potential of -20 kV under zonal CZE conditions. By combining normal and reverse polarities in the data treatment we have extracted more information from the samples, which is a better approach for CE metabolomics. The traditional problems associated with variability in electrophoretic peak migration times for analytes were countered by using a dynamic programming algorithm for the electropherograms alignment. Principal component analyses of these aligned electropherograms and partial least square discriminant analysis (PLS-DA) data are shown to provide a valuable means of rapid and sample classification. This approach may become an important tool for the identification of biomarkers, diagnosis and disease surveillance.

Evaluation of multidimensional capillary electrophoretic methodologies for determination of amino bisphosphonate pharmaceuticals.

Alendronate and pamidronate are amino bisphosphonate analogues of pyrophosphate used for the treatment of a variety of bone diseases. Analysis of these compounds is problematic due to their polar ionic nature, lack of a suitable chromophore and chelation properties and current analytical approaches involve extensive sample preparation and derivatization procedures. The potential of multidimensional capillary electrophoretic methodological approaches, which eliminate sample preparation have been evaluated for the analysis of these compounds both in aqueous and urinary matrices. Capillary isotachophoresis (cITP) was employed as a pre-separation and on-line sample concentration step prior to analytical determination using either cITP or capillary zone electrophoresis (CZE) with conductivity detection. Single cITP, cITP-cITP and cITP-CZE approaches were partially validated with respect to repeatability, recovery and linearity of response for both compounds. The increases in sensitivity achievable through increasing injection volume from 30 to 300 microL may render such strategies appropriate for determination of these agents at biologically relevant concentrations with minimal sample work-up.

Enantiomeric resolution of 2-arylpropionic acid nonsteroidal anti-inflammatory drugs by capillary electrophoresis: methods and applications.

The 2-arylpropionic acids (2-APAs) are an important group of nonsteroidal anti-inflammatory drugs. These agents, the majority of which are available as racemates, exhibit stereoselectivity in both their action and disposition. Developments in stereoselective separation science methodology, mainly chromatographic, have facilitated an evaluation of the pharmacological properties of the individual enantiomers of these drugs and contributed to our understanding of both their mode(s) of action and disposition. While a number of electrophoretic techniques, including capillary electrophoresis, capillary electrochromatography and isotachophoresis, have been applied to the stereoselective resolution and stereospecific analysis of these agents using a variety of chiral selectors, e.g., cyclodextrins, oligosaccharides, macrocyclic antibiotics, and proteins, the number of published applications in pharmaceutical and biomedical analysis remains relatively limited. However, the utility of electrophoretic techniques for stereospecific analysis may be illustrated using the 2-APAs as typical examples of chiral acidic pharmaceuticals. Applications include: determination of enantiomeric composition following biosynthetic stereoselective hydrolysis; examination of both achiral and chiral impurity profiles in bulk drugs and formulated products; determination of enantiomeric impurities in both bulk drugs and formulated products; examination of configurational stability following stress testing of formulated products; determination of enantiomeric composition and metabolite profile in biological fluids following administration of the racemates and individual enantiomers. It may be anticipated that future exploitation of electrophoretic approaches to the stereospecific analysis of these agents will result in further contributions to our understanding of their stereoselective biological properties and therapeutic use.

Toxicological classification of urine samples using pattern recognition techniques and capillary electrophoresis.

In toxicology, hazardous substances detected in organisms may often lead to different pathological conditions depending on the type of exposure and level of dosage; hence, further analysis on this can suggest the best cure. Urine profiling may serve the purpose because samples typically contain hundreds of compounds representing an effective metabolic fingerprint. This paper proposes a pattern recognition procedure for determining the type of cadmium dosage, acute or chronic, administrated to laboratory rats, where urinary profiles are detected using capillary electrophoresis. The procedure is based on the composition of a sample data matrix consisting of areas of common peaks, with appropriate pre-processing aimed at reducing the lack of reproducibility and enhancing the potential contribution of low-level metabolites in discrimination. The matrix is then used for pattern recognition including principal components analysis, cluster analysis, discriminant analysis and support vector machines. Attention is particularly focussed on the last of these techniques, because of its novelty and some attractive features such as its suitability to work with datasets that are small and/or have low samples/variable ratios. The type of cadmium administration is detected as a relevant feature that contributes to the structure of the sample matrix, and samples are classified according to the class membership, with discriminant analysis and support vector machines performing complementarily on a training and on a test set.

Micellar electrokinetic capillary chromatography and data alignment analysis: a new tool in urine profiling.

The complex nature of biofluids demands efficient, sensitive and high-resolution analytical methodologies to examine how the 'metabolic fingerprint' changes during disease. This paper describes how sulphated beta-cyclodextrin-modified micellar electrokinetic capillary chromatography (SbetaCD-MECC) has been combined with data alignment analysis and may prove a useful new tool in urine profiling, allowing for separation of over 80 urinary analytes in under 25 min. The optimised and validated SbetaCD-MECC methodology combined with data alignment analysis provides rapid identification of 'mismatches' between urine profiles which are not easily detected with the naked eye as well as a 'similarity score' which indicates the total sum of differences between one profile and another. The combination of SbetaCD-MECC with data alignment software should prove a useful alternative tool in metabonomic studies for rapid comparison of urine profiles.