Just Published: Journal of Chromatography A

A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:

Journal of Chromatography A http://rss.sciencedirect.com/publication/science/5248

Selected papers from the latest issue:

Overpressured layer chromatography: From the pressurized ultramicro chamber to BioArena system

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Ernő Tyihák, Emil Mincsovics, Ágnes M. Móricz
The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms.

Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography–mass spectrometry with positive-ion electrospray ionization

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
D. Kloos, R.J.E. Derks, M. Wijtmans, H. Lingeman, O.A. Mayboroda, A.M. Deelder, W.M.A. Niessen, M. Giera
The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core–shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3–4 orders of magnitude. Detection limits range from 12 to 1000nM, depending on the analyte.

Electromembrane extraction of stimulating drugs from undiluted whole blood

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Ragnhild Elén Gjulem Jamt, Astrid Gjelstad, Lars Erik Eng Eibak, Elisabeth Leere Øiestad, Asbjørg Solberg Christophersen, Knut Einar Rasmussen, Stig Pedersen-Bjergaard
For the first time, electromembrane extraction (EME) of six basic drugs of abuse from undiluted whole blood and post mortem blood in a totally stagnant system is reported. Cathinone, methamphetamine, 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-methamphet-amine (MDMA), ketamine and 2,5-dimethoxy-4-iodoamphetamine (DOI) were extracted from the whole blood sample, through a supported liquid membrane (SLM) consisting of 1-ethyl-2-nitrobenzene (ENB) immobilized in the pores of a hollow fiber, and into an aqueous acceptor solution inside the lumen of the hollow fiber. The SLM acts as a barrier with efficient exclusion of all macromolecules and acidic substances in the sample. Due to the application of the electrical field, only the cationic compounds of interest are extracted efficiently across the membrane, thus providing extremely clean extracts for analysis with liquid chromatography–mass spectrometry, LC–MS. Recoveries in the range 10–30% were obtained from 80μl whole blood within 5min extraction time and an applied voltage of 15V across the SLM. The optimized technique was tested on real forensic whole blood samples taken from three forensic autopsy cases and on five forensic whole blood samples from living persons. The results were in agreement with the analysis using standard sample preparation methods (liquid–liquid extraction) performed on the same samples by Norwegian Institute of Public Health (NIPH), Division of Forensic Toxicology and Drug Abuse Research. Evaluation data were acceptable, with limit of detections (LODs) in the range 40–2610pg/mL, well below concentrations associated with drug abuse; linearites in the range between 10 and 250ng/mL with r ² values above 0.9939, and with repeatability (RSD) of 7–32%.

Separation of phenolic acids from natural plant extracts using molecularly imprinted anion-exchange polymer confined ionic liquids

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Wentao Bi, Minglei Tian, Kyung Ho Row
Polymer-confined ionic liquids were used for the separation of phenolic acids from natural plant extract by utilizing an anion-exchange mechanism. They were synthesized using molecular imprinting technique to reduce non-directional ion–ion interactions during anion-exchange and other interactions with interference substances that could decrease selectivity. A suitable sorbent for phenolic acid separation could be identified based on the adsorption behaviors of phenolic acids on different polymer-confined ionic liquids. Thus, the developed ionic liquid-based molecularly imprinted anion-exchange polymer (IMAP) achieved high recovery rates by solid-phase extraction of phenolic acids from Salicornia herbacea L. extract: 90.1% for protocatechuic acid, 95.5% for ferulic acid and 96.6% for caffeic acid. Moreover, the phenolic acids were separable from each other by repeated solid phase extraction cycles. The proposed method could be used to separate other phenolic acids or organic acids from complex samples.

Determination of accessible silanols groups on silica gel surfaces using microcalorimetric measurements

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Bogusław Buszewski, Szymon Bocian, Gerhard Rychlicki, Maria Matyska, Joseph Pesek
The calorimetric measurements of methanol and hexane heats of immersion were carried out on different silica gels. Based on the difference in immersion heats, a methodology for the determination of the number of silanols on the surface is presented. The calculated concentration of residual silanols on the silica gel surface agreed with data found in the literature. The proposed methodology, based on a calculation of possible hydrogen bond formation, was also tested on the series of bonded stationary phases with different coverage densities. A very good correlation between the calculated number of accessible residual silanols and the coverage density of bonded ligands was observed.

Enhanced separation performance using a new column technology: Parallel segmented outlet flow

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Michelle Camenzuli, Harald J. Ritchie, James R. Ladine, R. Andrew Shalliker
A new column technology – termed parallel segmented outlet flow was employed here to illustrate gains in separation performance that are achievable by the active management of flow as it exits from the outlet of the chromatography column. Parallel segmented outlet flow requires a column be fitted with an outlet fitting that separates flow from the central region of the column from that of wall region. Each region of flow is able to be processed independently, such that post column detection emulates end column localised detection. As a result of this flow segmentation and the subsequent more efficient means of detection, column efficiency was observed to increase by more than 20%, with gains in sensitivity by as much as 22%, and a decrease in peak volume by up to 85%.

Highly sensitive oligosaccharide analysis in capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Takayuki Kawai, Masato Watanabe, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka
To obtain high sensitivity in capillary electrophoresis of oligosaccharide without reducing the high resolution with an easy experimental procedure, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. As a fundamental study, effect of the conductivity of a sample solution in LVSEP was examined. It was revealed that LVSEP was successfully carried out even in using a sample solution with the ionic strength of 150μM and the conductivity ratio of 20, indicating a good applicability of LVSEP to the analysis of real samples containing salts. When glucose oligomer was analyzed as a model sample in LVSEP-capillary zone electrophoresis (CZE), all peaks were well resolved with decreasing only 5% of the peak-to-peak distance, which suggested 95% of the whole capillary could be used for the effective separation. In the analysis of maltoheptaose, a good calibration line with correlation coefficient of 0.9995 was obtained. The limit of detection was estimated as 2pM, which was 500-fold lower than that in the conventional CZE. N-linked glycans released from three glycoproteins, bovine ribonuclease B, bovine fetuin, and human α1-acid glycoprotein were also analyzed by LVSEP-CZE. By the sample purification with a gel filtration column, further sample dilution to reduce the sample conductivity for LVSEP was not needed. All glycan samples were well concentrated and separated with up to a 770-fold sensitivity increase. The run-to-run repeatabilities of the migration time, peak height, and peak area were good with relative standard deviations of 0.1–1.3%, 1.2–1.7%, and 2.8–4.9%, respectively.

Combination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Anne-Catherine Servais, Anne Rousseau, Georges Dive, Michel Frederich, Jacques Crommen, Marianne Fillet
In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged β-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-β-CD or HDAS-β-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained.

Comparison of the quantitative performance of constant pressure versus constant flow rate gradient elution separations using concentration-sensitive detectors

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
M. Verstraeten, K. Broeckhoven, F. Lynen, K. Choikhet, M. Dittmann, K. Witt, P. Sandra, G. Desmet
This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes.

A multi-fiber handling device for in vivo solid phase microextraction–liquid chromatography–mass spectrometry applications

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Erasmus Cudjoe, Janusz Pawliszyn
Solid phase microextraction, an in vivo and ex vivo sample preparation method, continues to capture growing interest among researchers for bioanalytical applications. When coupled with liquid chromatography mass spectrometry, the procedure often involves large numbers of fibers in, for example, both pharmacokinetic and pharmadynamic studies as well as other bioapplications. In this regard, appropriate and adequate precaution will be critical in preventing the fibers firstly from any possible external contamination and damage to maintain high analytical data integrity. In addition, improving the offline desorption of fibers specifically for in vivo SPME will not only help in improving data quality, but will also significantly decrease the overall analysis time. This article introduces a prototype multi-fiber handling device capable of simultaneous extraction/desorption of multiple solid phase microextraction (SPME) fibers on a 96-deep well plate format. This device thus provides an alternative approach to improving higher sample throughput for in vivo SPME liquid chromatography mass spectrometry applications. The portable design of the device ensures effective protection and prevention of fibers against damage and possible contamination and thus maintains analytical data reliability. To ensure its suitability for parallel extraction/desorption, the device was carefully evaluated using four benzodiazepines (diazepam, nordiazepam, oxazepam and lorazepam) as model drugs by monitoring inter- and intra-well variability. The effect of agitation speed on data precision and accuracy, effect of device weight on data precision, and comparison of the overall performance of the device with traditional manual desorption approach were also assessed. Results obtained from evaluation of the device with particular focus on the desorption process indicated that the weight of the device has no effect on the reliability and reproducibility of data acquired using the device. The average amount of diazepam obtained for 20 selected wells with and without device was 48.8pg and 49.4pg, respectively. Intra-, inter-well, and inter fiber variations recorded were all ≤13% indicating an excellent precision and reproducibility can be attained with the device.

Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: Ligand affinity screening and stereoselective ligand–macromolecule interaction

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
E. Calleri, G. Fracchiolla, R. Montanari, G. Pochetti, A. Lavecchia, F. Loiodice, A. Laghezza, L. Piemontese, G. Massolini, C. Temporini
In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC–MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC50 values reported in literature. The optimized PPARγ-OT capillary was validated by determining the K d values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.

Preparation and full characterization of a micro-immunoaffinity monolithic column and its in-line coupling with capillary zone electrophoresis with Ochratoxin A as model solute

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
J. Chamieh, C. Faye, V. Dugas, T. Moreau, O. Vandenabeele-Trambouze, C. Demesmay
A micro-immunoaffinity monolithic column (μIAC) was developed and in-line coupled with capillary zone electrophoresis in a fully automated way with Ochratoxin A as test solute. The in-line micro-immunoaffinity columns based on monolithic methacrylate polymers (EDMA-GMA) were prepared in situ at the inlet end of a PTFE coated fused silica capillary by UV initiated polymerization and subsequently grafted with antibodies. These μIACs were thoroughly characterized. The synthesis of the polymeric support was first demonstrated to be reproducible in terms of permeability, surface properties and efficiency. The antibodies immobilization was then studied by a new original hydrodynamic method (ADECA) allowing the in situ quantitative determination (at a miniaturized scale) of the total amount of immobilized antibodies. The combination of this measurement with the binding capacity of the μIAC allowed, for the first time, the in situ determination of immobilized antibody activity. A total of 260±15ng (1.6±0.1pmol) of IgG antibodies/cm in 75μm i.d. monolithic column (i.e. 18μgmg⁻¹) was obtained with (anti-Ochratoxin A/Ochratoxin A) as antibody/antigen model. 40% of the immobilized antibodies remain active corresponding to a binding capacity of 1.2±0.2pmol antigen/cm (i.e. 600pg/cm of our test solute OTA), a very high capacity when dealing with trace analysis and with regard to the detection limits (30pg and 0.5pg with UV and LIF detection, respectively). The recovery yields were quantitative with negligible non-specific adsorption and allow analysis of diluted samples (1ngmL⁻¹) for a percolated volume of 10μL. It was also demonstrated that despite the progressive denaturation of antibodies consecutive to the elution step, the binding capacity of the μIAC remained high enough to implement at least 15 consecutive analyses with the same column and in a fully automated way.

Is it really necessary to validate an analytical method or not? That is the question

15 March 2012, 11:37:29

Publication year: 2012
Source:Journal of Chromatography A, Volume 1232
Maria Rambla-Alegre, Josep Esteve-Romero, Samuel Carda-Broch
Method validation is an important requirement in the practice of chemical analysis. However, awareness of its importance, why it should be done and when, and exactly what needs to be done, seems to be poor amongst analytical chemists. Much advice related to method validation already exists in the literature, especially related to particular methods, but more often than not is underused. Some analysts see method validation as something that can only be done by collaborating with other laboratories and therefore do not go about it. In addition, analysts’ understanding of method validation is inhibited by the fact that many of the technical terms used in the processes for evaluating methods vary in different sectors of analytical measurement, both in terms of their meaning and the way they are determined. Validation applies to a defined protocol, for the determination of a specified analyte and range of concentrations in a particular type of test material, used for a specified purpose. In general, validation should check that the method performs adequately for the purpose throughout the range of analyte concentrations and test materials to which it is applied. It follows that these features, together with a statement of any fitness-for-purpose criteria, should be completely specified before any validation takes place.