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Analytical and Bioanalytical Chemistry (v.404, #6-7)

Steen Hansen, Stig Pedersen-Bjergaard, Knut Rasmussen: Introduction to pharmaceutical chemical analysis by Mira Petrovic (pp. 1617-1618).

Williams, S. Kim R.; Caldwell, Karin D. (Eds.): Field-flow fractionation in biopolymer analysis by Patrice Castignolles (pp. 1619-1620).

Mike S. Lee and Mingshe Zhu (Eds.): Mass spectrometry in drug metabolism and disposition: basic principles and applications by Martin Vogel (pp. 1621-1622).

ProSafeBeef and anthelmintic drug residues—a case study in collaborative application of multi-analyte mass spectrometry to enhance consumer safety by Kevin M. Cooper; D. Glenn Kennedy; Martin Danaher (pp. 1623-1630).

(K.Cooper@qub.ac.uk) is a post-doctoral research fellow with 18 years experience in analysis of veterinary drug residues in food and food-producing livestock by immunoassay and mass spectrometry at Queen’s University Belfast and the Agri-Food and Biosciences Institute of Northern Ireland. In addition to R&D collaborations with the N Ireland poultry sector, he has been particularly active in European framework programme research consortia addressing enforcement of the ban on use of the anabolic growth promoter zeranol (the Natural Zeranol project), the global nitrofuran antibiotics crisis (the FoodBRAND FP5 project) and anti-parasitic compounds in beef (the ProSafeBeef FP6 project). He is currently applying similar techniques to analysis of compounds implicated in human carcinogenesis at the Institute of Agri-Food and Land Use at Queen’s University Belfast. (glenn.kennedy@afbini.gov.uk) is Head of the Chemical Surveillance Branch in AFBI, Belfast, which is the UK National Reference Laboratory for most of the substances banned by European legislation (hormones, nitroimidazoles, ß-agonists, nitrofurans, etc). His laboratory is responsible for all testing for veterinary drug residues and marine biotoxins in Northern Ireland. He has authored more than 180 refereed scientific papers. He was a member of the Expert Group responsible for drafting Commission Decision 2002/657/EC, concerning analytical method validation. He has co-ordinated 2 EU-funded research projects and has been a partner in many others. He sits, as an Assessor, on the UK’s Advisory Committee on Animal Feedingstuffs (ACAF) and on the UK’s Veterinary Residues Committee (VRC). He has acted as a National Expert on several European Commission Food and Veterinary Office audits of residues testing programmes over the last twelve years. He has also acted as an advisor to government and/or industry in relation to the analysis of veterinary drug residues, especially the illegal drugs, in food of animal origin in a range of countries, including Thailand, China, Brazil, Chile, Bangladesh, Myanmar, Georgia, Malaysia, and Ecuador. (Martin.Danaher@teagasc.ie) is a senior research officer with Teagasc, the agriculture and food-development authority in Ireland, based at Teagasc Food Research Centre, Dublin. He is head of one of Ireland’s national reference laboratories for veterinary drug-residue analysis. In this role he is responsible for monitoring nitrofuran, antiparasitics, anticoccidials, carbamates, and pyrethroids in food of animal origin, and providing analytical testing services and expert advice. He is the lead scientist in chemical contaminants research with Teagasc. His research covers a range of contaminants including mycotoxins, plants, algal toxins, and antibiotics.

Novel method to investigate protein carbonylation by iTRAQ strategy by Angelo Palmese; Chiara De Rosa; Giovanni Chiappetta; Gennaro Marino; Angela Amoresano (pp. 1631-1635).

This paper reports a novel methodology for relative quantitative analysis of carbonylation sites in proteins by exploiting a new isobaric tag for relative and absolute quantitation (iTRAQ) derivative, iTRAQ hydrazide (iTRAQH), and the analytical power of linear ion trap instruments (QqLIT). Because of its operational simplicity, avoiding time-consuming enrichment procedures, this new strategy seems to be well suited for quantitative large-scale proteomic profiling of carbonylation. Figure Modification of alpha-aminoadipic semialdehyde by a novel iTRAQ reagent, namely iTRAQ-hydrazide; by using this reagent it is possible to selectively label carbonylated residues in order to perform qualitative and quantitative analysis of protein carbonylation.

Keywords: Protein carbonylation; iTRAQ; Quantitative proteomics

Palladium nanoparticle-decorated iron nanotubes hosted in a polycarbonate porous membrane: development, characterization, and performance as electrocatalysts of ascorbic acid by Elsayed M. Zahran; Mamas I. Prodromidis; Dibakar Bhattacharyya; Leonidas G. Bachas (pp. 1637-1642).

One-dimensional iron metallic nanotubes were prepared by electroless deposition within the pores of polycarbonate (PC) membranes. The longitudinal nucleation of the nanotubes along the pore walls was achieved by mounting the PC membrane between two halves of a U-shaped reaction tube. Palladium nanoparticles were post-deposited on the inner wall of the nanotubes. The composition, morphology, and structure of the Pd/Fe nanotubes were characterized by transmission electron microscopy, scanning electron microscopy, and inductively coupled plasma–atomic emission spectroscopy. A glassy carbon (GC) electrode modified with the free Pd/Fe bimetallic nanotubes (isolated after the dissolution of the host membranes) showed small improvement on the overpotential oxidation of ascorbic acid in comparison to the bare GC electrode. Alternatively, the Pd/Fe-polycarbonate membrane was covered with a sputtered gold thin layer of 10 nm from one side and mounted in a homemade electrochemical cell acting as the working electrode. The potential use of these functional membranes as catalytic surfaces for the electrochemical monitoring of ascorbic acid was investigated by cyclic voltammetry and amperometry. In the presence of a phosphate buffer solution, pH 7, Pd/Fe-polycarbonate membranes showed excellent electrocatalytic properties toward the oxidation of ascorbic acid even at potentials as low as 0 mV versus a Ag/AgCl reference electrode. In addition to the substantial lower overpotential, these electrodes offered selectivity over acetaminophen and uric acid, and a prolonged working stability without the need for maintenance. The electrodes were kept dry between different working days and retained their original activity for more than 1 week. Pd-polycarbonate and Fe-polycarbonate membranes were also developed for comparison purposes. Figure Palladium/iron bimetallic nanotubes is a promising material architecture for electrocatalysis

Keywords: Nanoparticles/nanotechnology; Chemical sensors; Electroanalytical methods

Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors by Karin Fromell; Pontus Forsberg; Mikael Karlsson; Karin Larsson; Fredrik Nikolajeff; Lars Baltzer (pp. 1643-1651).

A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.

Keywords: Protein binders; Nanocrystalline diamond; CRP; Biosensor; Surface characterization

An electrochemical sensor for rapid determination of ractopamine based on a molecularly imprinted electrosynthesized o-aminothiophenol film by Ling-Jie Kong; Ming-Fei Pan; Guo-Zhen Fang; Kun Qian; Shuo Wang (pp. 1653-1660).

A simple electrochemical sensor based on a molecularly imprinted polymer film as the recognition element was developed for ractopamine (RAC) detection. This is the first report of a RAC-imprinted film on a gold electrode surface, synthesized through an electrochemical method using o-aminothiophenol as the functional monomer. The imprinting mechanism and experimental parameters affecting the capability of the imprinted film are discussed here. The sensor was successfully applied with constant potential amperometry for RAC detection in an indirect process with potassium ferricyanide as an electrochemical probe. The sensor had a rapid equilibrium time (120 s), high binding affinity and selectivity towards RAC, and with good reproducibility and stability. Under the experimental conditions applied, a linear relationship between the relative amperometric response and RAC ranged from 2.0 × 10⁻⁷ to 1.4 × 10⁻⁶ mol L⁻¹, with a lower limit of detection (LOD) of 2.38 × 10⁻⁸ mol L⁻¹ (signal to noise ratio = 3). The sensor was tested with feed samples spiked with trace amounts of RAC, with good recoveries between 87.4 and 90.5 %. Fig Preparation procedures of the RAC-imprinted film electrode

Keywords: Electrochemical sensor; Molecularly imprinted electropolymerization film; Ractopamine; o-Aminothiophenol

Novel sensor based on carbon paste/Nafion^® modified with gold nanoparticles for the determination of glutathione by Nada F. Atta; Ahmed Galal; Shereen M. Azab (pp. 1661-1672).

Several problems for the direct electrochemical oxidation of reduced glutathione (GSH) challenge the usage of electroanalytical techniques for its determination. In this work, the electrochemical oxidation of GSH catalyzed by gold nanoparticles electrodeposited on Nafion modified carbon paste electrode in 0.04 mol L⁻¹ universal buffer solution (pH 7.4) is proved successful. The effect of various experimental parameters including pH, scan rate and stability on the voltammetric response of GSH was investigated. At the optimum conditions, the concentration of GSH was determined using differential pulse voltammetry (DPV) in two concentration ranges: 0.1 × 10⁻⁷ to 1.6 × 10⁻⁵ mol L⁻¹ and 2.0 × 10⁻⁵ to 2.0 × 10⁻⁴ mol L⁻¹ with correlation coefficients 0.9988, 0.9949 and the limit of detections (LOD) are 3.9 × 10⁻⁹ mol L⁻¹ and 8.2 × 10⁻⁸ mol L⁻¹, respectively, which confirmed the sensitivity of the electrode. The high sensitivity, wide linear range, good stability and reproducibility, and the minimal surface fouling make this modified electrode useful for the determination of spiked GSH in urine samples and in tablet with excellent recovery results obtained.

Keywords: Glutathione; Sensor; Carbon paste electrode; Gold nanoparticles; Nafion

A resonance light scattering sensor based on methylene blue–sodium dodecyl benzene sulfonate for ultrasensitive detection of guanine base associated mutations by Zhanguang Chen; Sihua Qian; Junhui Chen; Xi Chen; Liwen Zheng; Jinbin Liu (pp. 1673-1679).

A resonance light scattering (RLS) sensor for guanine base associated mutations has been developed on the basis of the high selectivity of methylene blue (MB) for guanine bases in the presence of sodium dodecyl benzene sulfonate (SDBS). MB, when bound to SDBS, underwent a dramatic enhancement of its RLS intensity. However, the addition of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) caused the strong RLS intensity of MB–SDBS to decrease, and the RLS intensity of MB–SDBS–ssDNA was much lower than that of MB–SDBS–dsDNA. Consequently, it can be concluded that the binding abilities of MB–SDBS with ssDNA and dsDNA were different. Besides, the experimental results showed that MB–SDBS could bind specifically to oligonucleotides rich in guanine bases. Short DNA targets with sequences related to β-thalassaemia, thrombophilia and psoriasis, all of which are guanine base relevant mutations, were synthesized. It was found that MB–SDBS could recognize the single-base mismatches in the mutational DNA, followed by different RLS signal changes between MB–SDBS–normal DNA systems and MB–SDBS–mutational DNA systems. The ultrasensitive sensor allows simple, rapid, sensitive and selective detection of guanine base associated mutations, indicating its potential application in the medical field. Figure An RLS sensor for the detection of guanine base associated mutations

Keywords: Resonance light scattering; Methylene blue; Sodium dodecyl benzene sulfonate; Guanine bases; Mutations

Direct detection of peptides and proteins on a microfluidic platform with MALDI mass spectrometry by Mian Yang; Tzu-Chiao Chao; Randall Nelson; Alexandra Ros (pp. 1681-1689).

The ability to detect and quantify proteins of individual cells in high throughput is of enormous biological and clinical relevance. Most methods currently in use either require the measurement of large cell populations or are limited to the investigation of few cells at a time. In this report, we present the combination of a polydimethylsiloxane-based microfluidic device to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) that allows the detection of as few as 300 molecules at the peptide level and ∼10⁶ to 10⁷ molecules at the protein level. Moreover, we performed an immunoassay with subsequent MALDI-TOF-MS to capture and detect insulin immobilized on a surface (∼0.05 mm²) in this device with a detection limit of 10⁶ insulin molecules. This microfluidic-based approach therefore begins to approach the sample handling and sensitivity requirements for MS-based single-cell analysis of proteins and peptides and holds the potential for easy parallelization of immunoassays and other highly sensitive protein analyses. Figure Combined immunoassay and MALDI/TOF-MS for insulin in a microfluidic platform

Keywords: Microfluidic; Mass spectrometry; Immunoassay; Single-cell sensitivity

Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group by Varvara Tsilia; Bart Devreese; Ilse de Baenst; Bart Mesuere; Andreja Rajkovic; Mieke Uyttendaele; Tom Van de Wiele; Marc Heyndrickx (pp. 1691-1702).

Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5 Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.

Keywords: B. cereus ; Foodborne pathogen; Enterotoxins; 1D electrophoresis; MALDI-TOF/MS; Biomarkers

Thermally annealed gold nanoparticles for surface-assisted laser desorption ionisation–mass spectrometry of low molecular weight analytes by Rosa Pilolli; Nicoletta Ditaranto; Cinzia Di Franco; Francesco Palmisano; Nicola Cioffi (pp. 1703-1711).

Metal nanomaterials have an emerging role in surface-assisted laser desorption ionisation–mass spectrometry (SALDI-MS) providing a useful tool to overcome some limitations intrinsically related to the use of conventional organic matrices in matrix-assisted LDI-MS. In this contribution, the possibility to use a stainless-steel-supported gold nanoparticle (AuNP) film as a versatile platform for SALDI-MS was assessed. A sacrificial anode electrosynthetic route was chosen in order to obtain morphologically controlled core–shell AuNPs; the colloidal AuNPs were, thereafter, drop cast onto a stainless-steel sample plate and the resulting AuNP film was thermally annealed in order to improve its effectiveness as LDI-MS promoter. Spectroscopic characterization of the nanostructured film by X-ray photoelectron spectroscopy was crucial for understanding how annealing induced changes in the surface chemistry and influenced the performance of AuNPs as desorption/ionisation promoter. In particular, it was demonstrated that the post-deposition treatments were essential to enhance the AuNP core/analyte interaction, thus resulting in SALDI-MS spectra of significantly improved quality. The AuNP films were applied to the detection of three different classes of low molecular weight (LMW) analytes, i.e. amino acids, peptides and LMW polymers, in order to demonstrate the versatility of this nanostructured material.

Keywords: XPS; SALDI; Mass spectrometry; Gold nanoparticle; PEG

Analyzing small samples with high efficiency: capillary batch injection–capillary electrophoresis–mass spectrometry by Marco Grundmann; Frank-Michael Matysik (pp. 1713-1721).

We present an experimental approach to conducting fast capillary electrophoresis–mass spectrometry (CE-MS) measurements of very small samples in the nanoliter range. This is achieved by injecting sample very efficiently into a CE-MS system. Injection efficiency represents the ratio of injected sample to the amount of sample needed for carrying out the injection process (v/v). In order to increase this injection efficiency from typical values of 10^–3 to 10⁻⁷, the concept of capillary batch injection is used to build an automated, small-footprint injection device for CE-MS. This device is capable of running true multi-sample measurement series, using minimal sample volumes and delivering an injection efficiency of up to 100 %. It is compatible with both aqueous and non-aqueous background electrolytes. As an additional benefit, CE-MS separations of a catecholamine model system in capillaries of 15 cm length under conditions of high electric field strength could be accomplished in 20 s with high separation efficiency. This report details design and specifications of the injection device and shows optimal parameter choices for injections with both high injection efficiency and high separation efficiency. Furthermore, a procedure is presented to coat the tip of a fused silica capillary with a silicone elastomer which acts as a seal between two capillaries. Figure An approach to transfer nL-samples into the separation capillary of a CE-MS system is presented. The automated and computer-controlled setup can transfer samples with up to 100 % efficiency from the point of sampling into the separation capillary, where highly efficient and fast CE-MS separations are conducted

Keywords: Injection efficiency; Fast capillary electrophoresis; Hyphenation; Mass spectrometry; Small sample; Fused silica capillaries; High electric field strengths; Catecholamines

The ExoMars Raman spectrometer and the identification of biogeological spectroscopic signatures using a flight-like prototype by Howell G. M. Edwards; Ian Hutchinson; Richard Ingley (pp. 1723-1731).

The molecular specificity of Raman spectroscopy provides a powerful tool for the analytical interrogation of mineralogical and many biological specimens. The Raman Laser Spectrometer (RLS) is a compact Raman spectrometer under development for deployment on the Martian surface as part of the forthcoming ESA ExoMars mission. This will be the first Raman instrument deployed in space. The scientific interpretation of the data emerging from such an instrument not only addresses the geological and mineral composition of the specimens but also enables an assessment to be made of organic biomaterials that may be preserved in the planetary geological record. The latter evidence centres on the residual and distinctive chemistry relating to the biological adaptation of the geological matrix that has occurred as a result of extremophilic organisms colonizing suitable geological niches for their survival in environmentally stressed habitats on Mars. These biogeological modifications have been studied terrestrially for Mars analogue sites and consist of both a geological component and residual key organic biomarkers, the recognition of which would be a prime factor in life detection surveys of a planetary surface and subsurface. In this paper, the protocols required for the Raman spectral discrimination of key biogeological features that may be detectable on the Martian planetary surface or subsurface are developed using the UK breadboard (UKBB) instrument. This instrument has been constructed to be functionally equivalent to the RLS flight instrument design in order to evaluate the feasible science return of the instrument which will finally be delivered to Mars. Initial Raman measurements using the UKBB are presented and compared with the performance of a commercial laboratory Raman microscope. The initial measurements reported here demonstrate this flight-like prototype achieves straightforward detection of biological signatures contained in geological matrices with Raman band signal to noise ratios high enough to determine sample composition by inspection and without the need for deconvolution or further processing. Figure Raman excitation of sample at 532nm using commercial optical head.

Keywords: Raman spectroscopy; Molecular analysis; Biomineralogy; Astrobiology; Mars; ExoMars mission; Extreme environments

Basis of a FTIR spectroscopy methodology for automated evaluation of Akt kinase inhibitor on leukemic cell lines used as model by Adrian Travo; Vanessa Desplat; Emmanuelle Barron; Emilie Poychicot-Coustau; Jean Guillon; Gérard Déléris; Isabelle Forfar (pp. 1733-1743).

The PI3K/Akt-signaling pathway, associated with cancer development and disease progression, is recognized to be an anti-tumor drug target that could present important therapeutic benefit. However, no targeted Akt medicines have been commercialized yet, reflecting that drug selection procedures requires significant improvement from early research to clinical trials. Thus, new methods permitting both the evaluation of cytotoxic and proliferation inhibition effect on cancer cells but also to provide a global fingerprint of the drug action mechanism of new Akt inhibitor candidates are of major interest. Because it can detect very subtle molecular changes and could provide a global fingerprint of drug effects on cells, Fourier-transform infrared (FTIR) spectroscopy appears to be a promising method to develop new time- and cost-saving tools for chemical library screening improvements. In this study, we combine FTIR spectroscopy, advanced chemometrics analysis and cross-validation by standard biological assays to establish a basis of a mid-throughput methodology for rapid and automated assessment of cell response to Akt inhibitors and quantitative evaluation of their anti-proliferative effects. Our results shows that our methodology is able (1) to detect cell response to an Akt inhibitor exposure even for very low doses, (2) to provide biochemical information of interest about its effects on the cell metabolism, lipidome, and proteome, (3) to predict accurately resulting cell proliferation inhibition rate. Thus, further based on a large spectral data base, our methodology could contribute to facilitate preliminary screening of chemical libraries and improving the selection procedure of drug candidates in laboratory routine. Figure Validation performance in prediction with PLS regression of the cell proliferation inhibition induced by A6730 on K562 cells. Blue average proliferation inhibition (±std) as assessed by MTS on five experiments; Red average proliferation inhibition predicted by PLS regression on three independent experiments; R ² correlation coefficient; RMSEV root mean square error of validation

Keywords: FTIR spectroscopy; Cancer; Inhibitor; Drug; Anti-proliferative activity

Isolating stem cells in the inter-follicular epidermis employing synchrotron radiation-based Fourier-transform infrared microspectroscopy and focal plane array imaging by Imran I. Patel; Wesley J. Harrison; Jemma G. Kerns; Jacob Filik; Katia Wehbe; Paul L. Carmichael; Andrew D. Scott; Mike P. Philpott; Mark D. Frogley; Gianfelice Cinque; Francis L. Martin (pp. 1745-1758).

Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ = 2.5–25 μm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell’s inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IR-derived markers. Paraffin-embedded samples of human scalp skin (n = 4) were obtained, and 10-μm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15 × 15 μm) and also imaged employing globar-source FTIR focal plane array (FPA) imaging (5.4 × 5.4 μm). Dependent on the location of derived spectra, wavenumber–absorbance/intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO ₂ ⁻ vibrational modes (1,225 and 1,080 cm⁻¹), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO ₂ ⁻ vibrational modes as potential putative SC markers. Figure “Delineating the putative stem cell lineage in interfollicular skin based on position-derived infrared spectral fingerprints”.

Keywords: Biospectroscopy; Focal plane array; Fourier-transform infrared; Skin; Stem cells; Symmetric phosphate

Near-infrared (NIR) hyperspectral imaging and multivariate image analysis to study growth characteristics and differences between species and strains of members of the genus Fusarium by Paul J. Williams; Paul Geladi; Trevor J. Britz; Marena Manley (pp. 1759-1769).

Near-infrared (NIR) hyperspectral imaging was used to study three strains of each of three Fusarium spp. (Fusarium subglutinans, Fusarium proliferatum and Fusarium verticillioides) inoculated on potato dextrose agar in Petri dishes after either 72 or 96 h of incubation. Multivariate image analysis was used for cleaning the images and for making principal component analysis (PCA) score plots and score images and local partial least squares discriminant analysis (PLS-DA) models. The score images, including all strains, showed how different the strains were from each other. Using classification gradients, it was possible to show the change in mycelium growth over time. Loading line plots for principal component (PC) 1 and PC2 explained variation between the different Fusarium spp. as scattering and chemical differences (protein production), respectively. PLS-DA prediction results (including only the most important strain of each species) showed that it was possible to discriminate between species with F. verticillioides the least correctly predicted (between 16 and 47 % pixels correctly predicted). For F. subglutinans, 78–100 % pixels were correctly predicted depending on the training and test sets used. Similarly, the percentage correctly predicted values of F. proliferatum were 60–80 %. Visualisation of the mycelium radial growth in the PCA score images was made possible due to the use of NIR hyperspectral imaging. This is not possible with bulk spectroscopy in the visible or NIR regions. Figure Principal component 1 score image showing differences between colonies. F. subglutinans (MRC 0115) are top left followed by F. proliferatum (MRC 2301) and F. verticillioides (MRC 0826).

Keywords: Near-infrared hyperspectral imaging; Fusarium ; PCA; PLS-DA; Classification gradients

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum) by V. A. Huck-Pezzei; J. D. Pallua; C. Pezzei; L. K. Bittner; S. A. Schönbichler; G. Abel; M. Popp; G. K. Bonn; C. W. Huck (pp. 1771-1778).

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

Keywords: Bioanalytical methods; Biological samples; Drug monitoring; Drug screening; IR spectroscopy; Raman spectroscopy

Occurrence of 2-methylthiazolidine-4-carboxylic acid, a condensation product of cysteine and acetaldehyde, in human blood as a consequence of ethanol consumption by Roland J. Reischl; Wolfgang Bicker; Thomas Keller; Günther Lamprecht; Wolfgang Lindner (pp. 1779-1787).

Acetaldehyde is a strongly electrophilic compound that is endogenously produced as a first intermediate in oxidative ethanol metabolism. Its high reactivity towards biogenic nucleophiles has toxicity as a consequence. Acetaldehyde readily undergoes a non-enzymatic condensation reaction and consecutive ring formation with cysteine to form 2-methylthiazolidine-4-carboxylic acid (MTCA). For analytical purposes, N-acetylation of MTCA was required for stabilization and to enable its quantification by reversed-phase chromatography combined with electrospray ionization–tandem mass spectrometry. Qualitative screening of post mortem blood samples with negative blood alcohol concentration (BAC) mostly showed low basal levels of MTCA. In BAC-positive post mortem samples, but not in corresponding urine specimens, strongly increased levels were present. To estimate the association between ethanol consumption and the occurrence of MTCA in human blood, the time curves of BAC and MTCA concentration were determined after a single oral dose of 0.5 g ethanol per kilogram of body weight. The blood elimination kinetics of MTCA was slower than that of ethanol. The peak concentration of MTCA (12.6 mg L^-1) was observed 4 h after ethanol intake (BAC 0.07‰) and MTCA was still detectable after 13 h. Although intermediary acetaldehyde scavenging by formation of MTCA is interesting from a toxicological point of view, lack of hydrolytic stability under physiological conditions may hamper the use of MTCA as a quantitative marker of acetaldehyde exposure, such as resulting from alcohol consumption.

Keywords: Ethanol consumption markers; Forensics/toxicology; High-performance liquid chromatography; Mass spectrometry

The toxicity outcome of silica nanoparticles (Ludox®) is influenced by testing techniques and treatment modalities by Caterina Fede; Francesco Selvestrel; Chiara Compagnin; Maddalena Mognato; Fabrizio Mancin; Elena Reddi; Lucia Celotti (pp. 1789-1802).

We analyzed the influence of the kind of cytotoxicity test and its application modality in defining the level of hazard of the in vitro exposures to nanostructures. We assessed the cytotoxicity induced by two different Ludox® silica nanoparticles (NPs), AS30 and SM30, on three human cell lines, CCD-34Lu, A549, and HT-1080. Dynamic light scattering measurements showed particle agglomeration when NPs are diluted in culture medium supplemented with fetal calf serum. We examined the impact of such particle aggregation on the cytotoxicity by exposing the cells to NPs under different treatment modalities: short incubation (2 h) in serum-free medium or long incubation (24–72 h) in serum-containing medium. Under this last modality, NP suspensions tended to form aggregates and were toxic at concentrations five- to tenfold higher than in serum-free medium. The results of cell survival varied considerably when the long-term clonogenic assay was performed to validate the data of the short-term MTS assay. Indeed, the half maximum effective concentrations (EC₅₀) in all the three cell lines were four- to fivefold lower when calculated from the data of clonogenic assay than of MTS. Moreover, the mechanisms of NP toxicity were cell-type-specific, showing that CCD-34Lu are prone to the induction of plasma membrane damages and HT-1080 are prone to DNA double-strand break and apoptosis induction. Taken together, our results demonstrate that the choice of testing strategy and treatment conditions plays an important role in assessing the in vitro toxicity of NPs. Figure

Keywords: Nanoparticles; Cell systems; Dynamic light scattering; MTS assay; Clonogenic assay

Analysis of cyclosporin A, tacrolimus, sirolimus, and everolimus in dried blood spot samples using liquid chromatography tandem mass spectrometry by J. C. G. den Burger; A. J. Wilhelm; A. Chahbouni; R. M. Vos; A. Sinjewel; E. L. Swart (pp. 1803-1811).

In the last few years, an increasing number of dried blood spot (DBS) sampling assays have been developed. With this increase, more insight is gained in the factors that possibly influence the performance of DBS assays. We have developed an assay for four commonly used immunosuppressants; some of them are possibly concomitantly prescribed: cyclosporin A (CsA), tacrolimus (TcR), sirolimus (SiR), and everolimus (EvE). Chromatographic separation from possible ion suppression was obtained within the total runtime of 4.2 min. Trifluoroacetic acid and ammonium acetate were used as mobile phase additives. The linearity ranged from 23.6 to 787, 1.14 to 30.3, 1.34 to 35.8, and 1.26 to 33.7 μg/L, for CsA, TcR, SiR, and EvE, respectively. Between- and within-run accuracy and precision were all within 15 % and extensive validation for DBS samples, such as hematocrit, blood spot volume, and spot punch location was performed. None of these factors were found to be of influence on the performance of the DBS assay.

Keywords: DBS; Cyclosporin A; Tacrolimus; Sirolimus; Everolimus; Dried blood spot; Immunosuppressants

Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry by Cornelius Hess; Andreas Thomas; Mario Thevis; Bernd Stratmann; Wulf Quester; Diethelm Tschoepe; Burkhard Madea; Frank Musshoff (pp. 1813-1822).

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 μU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.

Keywords: Human insulin; Diabetes mellitus; Liquid chromatography; Mass spectrometry; Oral antidiabetics; Synthetic insulins

Distribution measurements of 3,4-methylenedioxymethamphetamine and its metabolites in organs by matrix-assisted laser desorption/ionization imaging mass spectrometry using an automatic matrix spraying system with an air brush and a turntable by Kenji Kuwayama; Kenji Tsujikawa; Hajime Miyaguchi; Tatsuyuki Kanamori; Yuko T. Iwata; Hiroyuki Inoue (pp. 1823-1830).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI/IMS) is a useful tool for measuring drug distributions. To obtain reproducible analytical results with MALDI/IMS, it is essential to apply a homogeneous matrix coating onto sample surfaces. A simple and inexpensive automatic matrix spraying system (AMSS) with good reproducibility was developed in this study. In addition, drug distributions in organs were measured by MALDI/IMS using the AMSS for forensic toxicology applications. The AMSS was constructed from simple components, including an air brush, a turntable, and a microscope. Organ slices placed onto conductive sheets were attached to the turntable. The trigger of the air brush was held with a clamp to ensure that it sprayed continuously onto a defined area of the table. Periodic spraying of the matrix solution and evaporation of solvent were performed by rotating the turntable. The droplets and crystals on the sample surfaces were observed under a microscope attached to the turntable. The droplet size, rotation rate of the turntable, and the formulation of the matrix solution were optimized. The homogeneity of the matrix coating was evaluated using the coefficients of variation (CV) obtained by quantifying the color density of the sheet surface. The AMSS enabled more homogeneous matrix coating (intersheet CV = 5.4 %) than manual spraying (intersheet CV = 16.7 %) when 10 mL of 0.5 % aqueous trifluoroacetic acid/acetonitrile (1:3, v/v) containing 10 mg/mL α-cyano-4-hydroxycinnamic acid were sprayed as droplets less than 50 μm in diameter onto a turntable rotating at 30 rpm. The distributions of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites in the brain, liver, and kidney of a mouse that died from an MDMA overdose (58 mg/kg i.p.) were visualized by MALDI/IMS using the AMSS. The ion intensities of MDMA obtained from the same regions on three sequential kidney slices showed acceptable variations (CV = 2.9–8.8 % for five different regions), implying repeatable measurements with MALDI/IMS using the AMSS. It was revealed that MDMA was particularly concentrated around the brain stem and the major calix of the kidney. The AMSS would be suitable for preparing samples for measuring the distributions of drugs in organs at toxic dose levels in forensic toxicological applications.

Keywords: Matrix-assisted laser desorption/ionization; Drug distribution; Imaging mass spectrometry; Organ; 3,4-Methylenedioxymethamphetamine

A novel method for quantification of sulfolane (a metabolite of busulfan) in plasma by gas chromatography–tandem mass spectrometry by François Versace; Chakradhara Rao S. Uppugunduri; Maja Krajinovic; Yves Théorêt; Fabienne Gumy-Pause; Patrice Mangin; Christian Staub; Marc Ansari (pp. 1831-1838).

The role of busulfan (Bu) metabolites in the adverse events seen during hematopoietic stem cell transplantation and in drug interactions is not explored. Lack of availability of established analytical methods limits our understanding in this area. The present work describes a novel gas chromatography–tandem mass spectrometric assay for the analysis of sulfolane (Su) in plasma of patients receiving high-dose Bu. Su and Bu were extracted from a single 100 μL plasma sample by liquid–liquid extraction. Bu was separately derivatized with 2,3,5,6-tetrafluorothiophenolfluorinated agent. Mass spectrometric detection of the analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after electronic impact ionization. Bu and Su were analyzed with separate chromatographic programs, lasting 5 min each. The assay for Su was found to be linear in the concentration range of 20–400 ng/mL. The method has satisfactory sensitivity (lower limit of quantification, 20 ng/mL) and precision (relative standard deviation less than 15 %) for all the concentrations tested with a good trueness (100 ± 5 %). This method was applied to measure Su from pediatric patients with samples collected 4 h after dose 1 (n = 46), before dose 7 (n = 56), and after dose 9 (n = 54) infusions of Bu. Su (mean ± SD) was detectable in plasma of patients 4 h after dose 1, and higher levels were observed after dose 9 (249.9 ± 123.4 ng/mL). This method may be used in clinical studies investigating the role of Su on adverse events and drug interactions associated with Bu therapy. Figure Overall sample preparation procedure for quantification of sulfolane and busulfan in plasma from patients receiving higher doses of busulfan

Keywords: GC–MS/MS; Sulfolane; Busulfan; Metabolite; Pharmacokinetics; Therapeutic drug monitoring; Plasma

Rapid analysis of NSAIDs binding to β-cyclodextrin using the simultaneous measurement of absorption and circular dichroism with a novel multi-cell low-volume device by Ali Aboel Dahab; Dhia El-Hag (pp. 1839-1850).

One of the relatively recent and most widely used approaches to reduce side effects associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is the complexation of NSAIDs with Cyclodextrins (CyD). So far, CyD interaction with drugs is not well understood. There have been many reports along these lines; however, rarely do these studies exploit the full potential of optical techniques. The purpose of this work is to produce a versatile, compact, low-volume, routine apparatus for the simultaneous measurements of absorbance and circular dichroism (CD) which allows for the concurrent use of three different pathlengths for binding studies of NSAIDs/CyD as a function of pH. A new rotating multi-cell holder which holds four cells was designed and manufactured. The work was achieved using an effective novel method for binding titration employing four separate flow cells connected in series in a flow system involving a titration flask and a pump. The pK _a, binding constants, stoichiometry and structural co-conformations of NSAIDs/β-CyD complexes were elucidated and determined with accuracy. The system proved to be efficient and the analysis time was reduced to less than or equal to one fourth of total analysis time used in one-cell systems, with possible automation for high-throughput analysis. Figure A multi-cell low volume device for pH and binding studies using UV and CD measurements

Keywords: Absorbance and circular dichroism; Binding constants; Cyclodextrins; Multi-cell holder; Nonsteroidal anti-inflammatory drugs; Stoichiometry

A new strategy for quality control and qualitative analysis of Yinhuang preparations by HPLC-DAD-MS/MS by Hui Chen; Xu Chen; Qiang Han; Jing Wu; Dao-quan Tang; Qian Du; Xiao-xing Yin; Dong-zhi Yang (pp. 1851-1865).

A combinative method using fingerprint analysis (FA) and multi-ingredients quantification (MIQ) was developed and validated for the quality control of Yinhuang (YH) preparations including granule, capsule, and lozenge by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). Common peaks with or without standard references in FA were confirmed or identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The chromatographic separations were achieved on a Sepax GP-C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) with a gradient elution using a mixture of 0.1 % formic acid methanol solution and 0.1 % formic acid water solution. In quantitative analysis, nine bioactive constituents (chlorogenic acid, caffeic acid, luteoloside, baicalin, luteolin, wogonoside, baicalein, wogonin, and oroxylin A) were simultaneously determined. The detection wavelength was set at 275 nm, 320 nm, and 350 nm according to the absorption properties of the nine quantified compounds. The linearity, recovery, intraday and interday precision, accuracy, limit of detection (LOD) and quantification (LOQ), repeatability and stability were all tested and good results were obtained. In the FA, 320 nm was selected. The correlation coefficients of similarity were determined on the basis of the relative retention time (RRT) and relative peak area (RPA) of 20 common peaks in chromatographic fingerprints. Results indicated that both the RRT and RPA of 20 common peaks shared a close similarity. From the 20 common peaks, 18 compounds, including the nine quantified compounds, were identified or tentatively characterized by comparing their retention times, UV spectra, and MS spectra with those of standard compounds or literature data. The study not only presents a powerful and reliable analytical tool for the quality control of YH preparations, but also provides the chemical evidence for revealing the material basis of their therapeutic effects. Figure The HPLC-DAD and LC-ESI-MS/MS chromatogram of Yinhuang granule

Keywords: Yinhuang preparations; Multi-ingredients quantification; Fingerprint analysis; Qualitative analysis; HPLC-DAD; ESI-MS/MS

Capture and identification of proteins that bind to a GGA-rich sequence from the ERBB2 gene promoter region by Tian Zhang; Huiping Zhang; Yuexi Wang; Linda B. McGown (pp. 1867-1876).

The ERBB2 gene (HER2/neu) is overexpressed in many human breast cancers. It is an important therapeutic target and its product protein is a key biomarker for breast cancer. A 28-bp GGA repeat sequence (Pu28-mer) in the nuclease hypersensitive site of the ERBB2 promoter region may play an important role in the regulation of ERBB2 transcription, possibly involving the formation of a G-quadruplex. In order to investigate this possibility, an affinity MALDI-MS approach was used for in vitro protein capture from nuclear extracts from cultured MCF-7 and BT-474 cancer cells at Pu28-mer and control oligonucleotide-modified surfaces. Captured proteins from MCF-7 cells were analyzed by LC-MS/MS. Based on these results, Western blot was then used to interrogate captured proteins from both MCF-7 and the Her-2/neu-positive BT-474 cells. Results support the formation of a G-quadruplex by Pu28-mer, indicated by circular dichroism spectroscopy, that selectively captures transcription factors including Ku70, Ku80, PURA, nucleolin, and hnRNP K. Chromatin immunoprecipitation confirmed binding of Ku70, Ku80, PURA, and nucleolin to ERBB2 promoter in the live BT-474 cells. These findings may lead to a better understanding of the role of non-duplex DNA structures in gene regulation and provide a more complete picture of the regulation of ErbB2 expression in breast cancer. The results also provide a blueprint for development of “genome-inspired” aptamers based on the Pu28-mer sequence for in vitro and in vivo detection of proteins related to regulation of ERBB2 gene expression and breast cancer.

Keywords: Bioanalytical methods; Nucleic acids (DNA | RNA); Genomics; Proteomics; Cell systems; Single-cell analysis

Characterization of perchlorate in a new frozen human urine standard reference material by Lee L. Yu; Jeffery M. Jarrett; W. Clay Davis; Eric L. Kilpatrick; Rabia Oflaz; Gregory C. Turk; Dennis D. Leber; Liza Valentin; Maria Morel-Espinosa; Benjamin C. Blount (pp. 1877-1886).

Perchlorate, an inorganic anion, has recently been recognized as an environmental contaminant by the US Environmental Protection Agency. Urine is the preferred matrix for assessment of human exposure to perchlorate. Although the measurement technique for perchlorate in urine was developed in 2005, the calibration and quality assurance aspects of the metrology infrastructure for perchlorate are still lacking in that there is no certified reference material (CRM) traceable to the International System of Units. To meet the quality assurance needs in biomonitoring measurements of perchlorate and the related anions that affect thyroid health, the National Institute of Standards and Technology (NIST), in collaboration with the Centers for Disease Control and Prevention (CDC), developed Standard Reference Material (SRM) 3668 Mercury, Perchlorate, and Iodide in Frozen Human Urine. SRM 3668 consists of perchlorate, nitrate, thiocyanate, iodine, and mercury in urine at two levels that represent the 50th and 95th percentiles, respectively, of the concentrations (with some adjustments) in the US population. It is the first CRM being certified for perchlorate. Measurements leading to the certification of perchlorate were made collaboratively at NIST and CDC using three methods based on liquid or ion chromatography tandem mass spectrometry. Potential sources of bias were analyzed, and results were compared for the three methods. Perchlorate in SRM 3668 Level I urine was certified to be 2.70 ± 0.21 μg L⁻¹, and for SRM 3668 Level II urine, the certified value is 13.47 ± 0.96 μg L⁻¹. Figure Producing a urine SRM for certification of perchlorate-an emerging environmental contaminant and a potential health hazard

Keywords: Perchlorate; Urine; Standard reference material; Certified reference material

Intercomparison of inductively coupled plasma mass spectrometry, quantitative neutron capture radiography, and prompt gamma activation analysis for the determination of boron in biological samples by C. L. Schütz; C. Brochhausen; G. Hampel; D. Iffland; B. Kuczewski; G. Otto; T. Schmitz; C. Stieghorst; J. V. Kratz (pp. 1887-1895).

Boron determination in blood and tissue samples is a crucial task especially for treatment planning, preclinical research, and clinical application of boron neutron capture therapy (BNCT). Comparison of clinical findings remains difficult due to a variety of analytical methods, protocols, and standard reference materials in use. This paper addresses the comparability of inductively coupled plasma mass spectrometry, quantitative neutron capture radiography, and prompt gamma activation analysis for the determination of boron in biological samples. It was possible to demonstrate that three different methods relying on three different principles of sample preparation and boron detection can be validated against each other and yield consistent results for both blood and tissue samples. The samples were obtained during a clinical study for the application of BNCT for liver malignancies and therefore represent a realistic situation for boron analysis. Figure

Keywords: Boron neutron capture therapy; Boron analysis; Quantitative neutron capture radiography; Inductively coupled plasma mass spectrometry; Prompt gamma activation analysis

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities by Yi H. Nai; Oliver Zemb; Maria-Luisa Gutierrez-Zamora; Mike Manefield; Shane M. Powell; Michael C. Breadmore (pp. 1897-1906).

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24 h to 60 min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities. Fig Schematic representation of direct rRNA microbial community characterization workflow using CE-LIF

Keywords: Capillary electrophoresis; Laser-induced fluorescence; RNA; Fluorescence-labeled hybridization probe; Polydimethylacrylamide; 16S ribosomal RNA; Fingerprint; Single-stranded conformation polymorphism; Microbial community characterization

Mercapturic acids derived from toluene in rat urine samples: identification and measurement by gas chromatography–tandem mass spectrometry by Frédéric Cosnier; Céline Brochard; Manuella Burgart; Benoît Cossec (pp. 1907-1917).

Toluene is one of the most widely used CMR chemicals in industry. Worker exposure to this compound is regulated in France, but new, more sensitive methods are required to effectively monitor this exposure. A gas chromatography–tandem mass spectrometry (GC-MS/MS) method was developed and fully validated for the simultaneous determination of urinary toluene mercapturic acids derived from side chain and ring oxidation, i.e., benzylmercapturic acid and the three isomers o-, m- and p-toluylmercapturic acids, respectively. The method involves a simple and efficient two-step preparation procedure consisting of liquid–liquid extraction of the urinary acids followed by a microwave-assisted esterification of the isolated compounds using 2-propanol. The method meets all the required validation criteria: high selectivity, intra-day and inter-day precision ranges between 1.0 % and 12.4 %, with close to 100 % recovery. Linearity has been shown over the reduced concentration range 0.03–0.5 mg/L whereas a multiplicative model (ln–ln transformation) had to be used to describe the full range of concentrations 0.03–20 mg/L. The limits of detection for the four analytes, ranging from 2.8 to 5.5 μg/L, made the method suitable for their identification and quantification in urine from rats inhaling toluene in the 2 to 200 ppm concentration range. All urine samples from exposed rats contained measurable amounts of all metabolites. This is the first time that o- and m-toluylmercapturic acids have been shown to occur. Our results confirm the hypothesis that toluene mercapturic acids derived from ring oxidation exist in three forms. Figure Simplified metabolisation scheme of toluene in rat.

Keywords: Mercapturic acid; Toluene; GC-MS/MS; Microwave esterification

Chemoselective reduction-based fluorescence probe for detection of hydrogen sulfide in living cells by Bifeng Chen; Cong Lv; Xinjing Tang (pp. 1919-1923).

A selective and sensitive fluorescence probe for hydrogen sulfide (H₂S) detection was synthesized and evaluated in PBS buffer and fetal bovine serum. The effect of pH on the probe was also studied. In addition, visualization of H₂S in Hela cells was achieved using confocal laser scanning fluorescence microscopy.

Keywords: Hydrogen sulfide; Fluorescence probes; Azides; Resorufamine

Simultaneous determination of dl-lactic acid and dl-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS by Haruhito Tsutsui; Toshiki Mochizuki; Toshio Maeda; Ichiro Noge; Yutaka Kitagawa; Jun Zhe Min; Kenichiro Todoroki; Koichi Inoue; Toshimasa Toyo’oka (pp. 1925-1934).

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC–MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2′-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of dl-LA and dl-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of dl-LA and dl-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5–12 fg). The proposed procedure was successfully applied for the determination of the d- and l-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The d-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC–ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume. Figure Concentration of HA and LA enantiomers in saliva of DM patients and healthy volunteers (n = 10). DM diabetes mellitus patients, C healthy volunteers

Keywords: Lactic acid; 3-Hydroxybutyric acid; Enantiomeric separation; Diabetes mellitus; Saliva; UPLC–ESI-MS/MS

Novel plastic biochips for colorimetric detection of biomolecules by Jing Wen; Xiaoli Shi; Yining He; Jianjun Zhou; Yunchao Li (pp. 1935-1944).

We report a novel plastic biochip for the sensitive colorimetric detection of analytes of interest. This type of biochip is designed to perform bioassays in a sandwich format, i.e., employing the immobilized probe molecules to capture target and then utilizing gold nanoparticle (AuNP)-labeled reporters to screen the biorecognition events. To fabricate and implement such plastic biochips, not only have we demonstrated the probe immobilization, sensor unit formation, signal transduction and visualization on the plastic substrate, but we have also introduced new methods for imaging and analysis of them. As two proof-of-concept detection applications, plastic immunochips and DNA biochips have been fabricated and their responses to human IgG and DNA have been examined respectively. To further assess the detection sensitivity of the colorimetric-based biochip, we have compared it with an enzyme-catalyzed-based biochip and with a conventional fluorescent-based biochip. We believe the colorimetric-based plastic biochip presented herein is able to fully combine the advantage of colorimetric detection and plastic substrate, thus making it an ideal platform for point-of-care analysis and diagnostics. Figure Schematic illustration of the construction and the application of a plastic biochip for colorimetric detection of biomolecules

Keywords: Plastic biochips; Bioassay; Colorimetric detection; Silver enhancement

UPLC-Q-TOF-MS analysis of non-volatile migrants from new active packaging materials by M. Aznar; A. Rodriguez-Lafuente; P. Alfaro; C. Nerin (pp. 1945-1957).

Ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) is a useful tool in the analysis of non-volatile compounds, and the use of a quadrupole-time-of-flight (Q-TOF) mass analyzer allows a high sensitivity and accuracy when acquiring full fragment mode, providing a high assurance of correct identification of unknown compounds. In this work, UPLC-Q-TOF-MS technology has been applied to the analysis of non-volatile migrants from new active packaging materials. The materials tested were based on polypropylene (PP), ethylene–vinyl alcohol copolymer (EVOH), and poly(ethylene terephthalate) (PET). The active packaging materials studied were one PP film containing a natural antioxidant, and two PP/EVOH films, two PET/EVOH films and one coextruded PP/EVOH/PP film containing natural antimicrobials. The chemical structure of several compounds was unequivocally identified. The analysis revealed the migration of some of the active substances used in the manufacture of active packaging, such as caffeine (0.07 ± 0.01 μg/g), carvacrol (0.31 ± 0.03 μg/g) and citral (0.20 ± 0.01 μg/g). Unintentionally added substances were also found, such as citral reaction compounds, or citral impurities present in the raw materials.

Keywords: TOF; Q-TOF; Packaging; Migration; NIAS; Active packaging

Application of the molecularly imprinted solid-phase extraction to the organophosphate residues determination in strawberries by Isabela Mendes Baldim; Marília Cristina de Oliveira Souza; João Carlos Jacinto da Cunha Souza; Eduardo Costa Figueiredo; Isarita Martins (pp. 1959-1966).

This study describe an analytical method employing gas chromatography (GC) using flame photometric detection that has been developed for the simultaneous determination of organophosphate pesticides (diazinon, disulfoton, parathion, chlorpyrifos and malathion) in strawberry samples. For this purpose, molecularly imprinted solid-phase extraction was applied as a sample preparation technique. The method was linear in the ranges from 0.10 to 1.00 μg g⁻¹, for diazinon, disulfoton, parathion and chlorpyrifos, and 0.10 to 2.00 μg g⁻¹ for malathion with r > 0.99. The detection limits (LD) ranged from 0.02 to 0.05 μg g⁻¹. Recovery studies yielded average recoveries in the range of 65.25 to 87.70 %. These results showed the potential of this technique for organophosphate residue monitoring in strawberry samples.

Keywords: Molecularly imprinted polymer; Solid-phase extraction; Organophosphate residues; Strawberry; GC-FPD

Biological response of free-living mouse Mus spretus from Doñana National Park under environmental stress based on assessment of metal-binding biomolecules by SEC-ICP-MS by M. A. García-Sevillano; M. González-Fernández; R. Jara-Biedma; T. García-Barrera; J. López-Barea; C. Pueyo; J. L. Gómez-Ariza (pp. 1967-1981).

A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination. Figure Metallomics assessment of biological response in natural ecosystems

Keywords: Speciation; SEC-ICP-MS; Mus spretus ; Environmental metallomics; Biological response; Doñana National Park

Enantioselective separation and simultaneous determination of fenarimol and nuarimol in fruits, vegetables, and soil by liquid chromatography–tandem mass spectrometry by Hu Zhang; Xinquan Wang; Shulin Zhuang; Mingrong Qian; Kezhi Jiang; Xiangyun Wang; Hao Xu; Peipei Qi; Qiang Wang (pp. 1983-1991).

A method for simultaneous enantioselective determination of fenarimol and nuarimol in apple, grape, cucumber, tomato, and soil was developed using liquid chromatography–tandem mass spectrometry. The enantioseparation results of the two fungicides through three different cellulose-based chiral columns are discussed. The influence of column temperature on the resolution of the enantiomers of the two fungicides was examined. Complete enantioseparation of the two fungicides’ enantiomers was obtained on a cellulose tris(4-methylbenzoate) column (Lux Cellulose-3) at 25 °C using methanol and 0.1 % formic acid solution (80:20, v/v) as mobile phase. The linearity, matrix effect, recovery, and precision were evaluated. Good linearity was obtained over the concentration range of 1–500 μg L⁻¹ for each enantiomer in the standard solution and sample matrix calibration solution. There was no significant matrix effect in apple, grape, cucumber, or tomato samples, but signal suppression was typically observed with the soil extracts. The mean recoveries, repeatability, and reproducibility were 76.5–103 %, 2.1–9.0 %, and 4.2–11.8 %, respectively. The limit of quantification for enantiomers of the two fungicides in fruits, vegetables and soil was 5 μg kg⁻¹. Moreover, the absolute configuration of the enantiomers of fenarimol and nuarimol was determined from a combination of experimentally determined and predicted electronic circular dichroism spectra. Figure Predicted ECD spectra of nuarimol enantiomers (a) and fenarimol enantiomers (b). Experimentally measured ECD spectra of nuarimol enantiomers (c) and fenarimol enantiomers (d) in acetonitrile (20 mg L⁻¹)

Keywords: Chiral analysis; High-performance liquid chromatography–tandem mass spectrometry; Absolute configuration; Fenarimol; Nuarimol

Parts per trillion level determination of endocrine-disrupting chlorinated compounds in river water and wastewater effluent by stir-bar-sorptive extraction followed by gas chromatography–triple quadrupole mass spectrometry by N. Sridhara Chary; Sonia Herrera; Maria Jose Gómez; Amadeo R. Fernández-Alba (pp. 1993-2006).

A new analytical method using stir-bar-sorptive extraction (SBSE) followed by liquid desorption (LD) and gas chromatography with triple-quadrupole mass spectrometric detection (GC–QqQ–MS–MS) has been used for quantitative determination of 25 chlorinated endocrine-disrupting compounds (EDCs) in river water and wastewater. The experimental conditions affecting the SBSE–LD performance were studied and are discussed in detail. Results from systematic assay revealed that a 100-mL water sample, stir bars coated with 47 μL PDMS, an extraction time of 14 h (at 900 rpm), 5 % MeOH as modifier and 10 % NaCl resulted in the best analytical recovery of all the target compounds studied. Use of 1:1 ACN–MeOH as back-extraction solvent and two successive sonication steps, each for 5 min, resulted in the best performance for monitoring EDCs in water matrices. The method detection limits for most of the target compounds were very good— ≤ 2 ng L⁻¹ and ≤10 ng L⁻¹ for river water and wastewater effluents respectively. Experimental recovery for all the compounds was >70 %, with the exception of simazine for which recovery from the matrix was 65 %. Signal enhancement observed for a few of the compounds in wastewater effluents was managed by use of matrix-matched standards and different injection liners. The method was successfully used for analysis of river water samples from Henares River (Spain) and wastewater effluent samples from wastewater-treatment plants (WWTP). Eleven of the 25 compounds studied were detected in both river water and wastewater effluents. Terbutylazine and methoxychlor were detected in almost all the river water and effluent samples; amounts varied between 37–58.5 ng L⁻¹ and 15.2–46.8 ng L⁻¹, respectively. This method was shown enable reliable, effective, and sensitive monitoring of chlorinated EDCs at nanogram levels in surface water and wastewater effluent. Figure Theoretical and Experimental recoveries as function of Log K_OW for the target Chlorinated EDCs

Keywords: Chlorinated EDCs; GC–QqQ–MS; Stir-bar-sorptive extraction (SBSE); Liquid desorption; River water; Wastewater effluents

A method based on microextraction by packed sorbent-programmed temperature vaporizer–fast gas chromatography–mass spectrometry for the determination of aromatic amines in environmental water samples by Miguel del Nogal Sánchez; Cristina Pérez Sappó; José Luis Pérez Pavón; Bernardo Moreno Cordero (pp. 2007-2015).

We report a sensitive method for the determination of 15 aromatic amines in environmental water samples. They have been included in the list of priority pollutants in surface water by the European Union. The method is based on analyte enrichment using microextraction by packed sorbent (MEPS) and later analysis using programmed temperature vaporizer–gas chromatography–mass spectrometry (PTV-GC-MS). All MEPS steps were carried out manually. The detection limits were of the order of nanograms per liter for most of the compounds. The results were compared with those obtained without MEPS using the method exclusively based on direct injection of the sample into the PTV-GC-MS. External calibration in ultrapure water was used in the determination of the compounds studied in five types of water samples (sea, river, tap, influent, and effluent waste water) since no significant matrix effect was found. The results obtained can be considered highly satisfactory and they revealed the presence of aniline in the sea and the influent and effluent waste water samples.

Keywords: Aromatic amines; Microextraction by packed sorbent; Gas chromatography; Mass spectrometry; Water samples

Simultaneous enantioselective determination of triazole fungicide difenoconazole and its main chiral metabolite in vegetables and soil by normal-phase high-performance liquid chromatography by Jing Li; Fengshou Dong; Youpu Cheng; Xingang Liu; Jun Xu; Yuanbo Li; Xiu Chen; Zhiqiang Kong; Yongquan Zheng (pp. 2017-2031).

Herein is reported, for the first time, a simple and highly sensitive chiral high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of difenoconazole stereoisomers and their hydroxylated metabolite difenoconazole alcohol (CGA-205375) enantiomers in vegetables and soil matrix. The separation of difenoconazole and CGA-205375 including their simultaneous enantioseparation was studied using four different polysaccharide-type chiral stationary phases (CSPs) in combination with n-hexane–polar organic alcohols mobile phase. Chiralcel OJ consisting of 25 % of cellulose tris(4-methylbenzoate) coated on wide-pore polysaccharide silica gel exhibited higher resolving ability compared to cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) as well as to its similar amylose derivative (Chiralpak AD) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of difenoconazole and its metabolite CGA-205375 could be achieved under optimized separation conditions. Based on the established HPLC method, enantioselective analysis method for this fungicide and its main chiral metabolite in vegetables and soil matrix were developed and validated. Parameters including the matrix effect, linearity, precision, accuracy, and stability were evaluated. Under the optimal conditions, the mean recoveries from cucumber, tomato, and soil matrix ranged from 81.65 to 94.52 %, with relative standard deviations in the range of 1.05–8.32 % for all stereoisomers. Coefficients of determination R ² ≥ 0.998 were achieved for each enantiomer in the cucumber, tomato and soil matrix calibration curves within the range of 0.5–50 μg mL^-1. The limits of quantification for all enantiomers in three matrices were all below 0.1 μg mL^-1. The methodology was successfully applied for simultaneous enantioselective analysis of difenoconazole stereoisomers and their metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of difenoconazole in food and environmental matrix. Figure Typical chromatograms of difenoconazole and its chiral metabolite CGA-205375 in real soil sample detected by chiral HPLC system and GC-MS

Keywords: Difenoconazole; Chiral metabolite; Enantioselective separation; Chiral high-performance liquid chromatography; Stereoisomer

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells by James Z. Xing; Stephan Gabos; Biao Huang; Tianhong Pan; Min Huang; Jie Chen (pp. 2033-2041).

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell’s kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays. Figure Quantitative analysis of low copy number mutant cells with cell growth kinetic curves. a Low copy number HPRT gene mutant cells are growning on electronic chip and the cell growth kinetic curve is monitored by a real-time cell electronic sensor (RT-CES) system. b When set up a threshold is set at certain level of cell index (CI), the samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells

Keywords: RT-CES mutagenicity test; Cell growth kinetic curve; High-throughput assay; Low copy number mutant cell; Baseline and threshold analyses

Anidulafungin—challenges in development and validation of an LC-MS/MS bioanalytical method validated for regulated clinical studies by Tanja Alebic-Kolbah; Michael S. Modesitt (pp. 2043-2055).

Anidulafungin is a semi-synthetic echinocandin with antifungal activity, usually administered as an intravenous infusion. In order to determine the pharmacokinetics (PK) of anidulafungin in pediatric patients, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) bioanalytical method (M1) was developed and validated for quantification of anidulafungin in plasma. During analysis of incurred samples (samples collected from patients enrolled in a clinical study) an isobaric chromatographic interference was observed. The source of interference was identified as an anidulafungin open-ring form (D1) and its impact on the quantification of anidulafungin was investigated. It was found that accurately quantifying anidulafungin in incurred samples required chromatographic separation of the open-ring form from anidulafungin. The method was redeveloped to achieve the appropriate baseline separation and to avoid experimental conditions that favored opening the anidulafungin ring. The extraction of anidulafungin from plasma by protein precipitation remained unchanged, but the changes in chromatography warranted validation of a new method, M2, 2 years after M1 was validated. Incurred samples from three studies that were previously analyzed by M1 and were within confirmed long-term frozen stability were then reanalyzed by M2. Although the incurred sample reproducibility tests on those samples passed for each of the two methods, comparison of concentrations from the same samples obtained by M1 and M2 revealed that an overestimation of anidulafungin following the M1 method exceeded acceptance criteria. The new HPLC-MS/MS method (M2) is applicable for quantification of anidulafungin within a nominal range 50–20,000 ng/mL and requires a 50 μL human plasma aliquot. A linear, 1/concentration squared weighted, least-squares regression algorithm was used to generate the calibration curve and its parameters were used to quantitate the incurred samples. The inter-assay accuracy in heparin human plasma validation ranged from −4.33 to 0.0386 % and precision was ≤7.32 %. The method M2 was validated for use in regulated bioanalysis and is presently used to quantitate anidulafungin in plasma samples from clinical studies.

Keywords: Anidulafungin; HPLC-MS/MS; Anidulafungin open-ring form; Pediatric blood collection; Regulated bioanalysis; Validation

Enhancing the lateral-flow immunoassay for detection of proteins using an aqueous two-phase micellar system by Foad Mashayekhi; Alexander M. Le; Parsa M. Nafisi; Benjamin M. Wu; Daniel T. Kamei (pp. 2057-2066).

The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05 μg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays. Figure A schematic representation of concentrating a target protein into the top phase of an aqueous two-phase micellar system prior to its detection via the lateral-flow immunoassay

Keywords: Lateral-flow immunoassay; Aqueous two-phase micellar systems; Point-of-care; Protein concentration and detection; Diagnostics

Screening test of silver nanoparticles in biological samples by graphite furnace-atomic absorption spectrometry by F. Gagné; P. Turcotte; C. Gagnon (pp. 2067-2072).

A simple, rapid and inexpensive screening test is presented to determine the presence of silver (Ag) nanoparticles in biological samples. The method is based on graphite furnace-atomic absorption spectrometry (Zeeman background correction) where an increase in the atomization temperature is observed with an increase in the particle size of Ag. The method is able to determine the presence of Ag ions from the presence of nano-Ag of 20, 60 and 80 nm, but the methodology was less apt to resolve nanoparticles between 20 and 60 nm. The proposed methodology was capable of determining the presence of dissolved Ag⁺ from 20 nm in prepared mixtures, and in the liver of rainbow trout exposed to either dissolved or 20-nm nano-Ag.

Keywords: Silver; Nanoparticles; Graphite furnace-atomic absorption spectrometry; Size analysis

Multiplexed protein detection using an affinity aptamer amplification assay by Kris P. F. Janssen; Karel Knez; Dragana Spasic; Jan Schrooten; Jeroen Lammertyn (pp. 2073-2081).

Affinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer–target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time. Figure Multiplexed affinity probe capillary electrophoresis (APCE) is enhanced through the use of PCR affinity aptamer amplification, improving both the limits of detection as well as the resolution of a typical APCE assay.

Keywords: Bioanalytical methods; Bioassays; Biochips/high-throughput screening; Biosensors; Capillary electrophoresis/electrophoresis; Nucleic acids (DNA/RNA)

Protein kinase inhibitor analysis with the core–shell separation technique by Marek Dziadosz; Rüdiger Lessig; Heidemarie Bartels (pp. 2083-2086).

Here, we describe the application of the core–shell separation technology in the field of protein kinase inhibitor analysis with the HPLC–diode-array detector (DAD) technique. A Kinetex 2.6 μm C8 100 Å, 150 × 4.6 mm column maintained at 30 ± 1 °C was used for the separation, and further, a connection of two core–shell HPLC columns with a total column dimension of 250 × 4.6 mm was also applied. The analytes were eluted with a mobile phase consisting of 0.05 M H₃PO₄/KH₂PO₄ (pH = 2.3)-acetonitrile (7:3, v/v), with a flow rate of 0.7 mL/min. A liquid–liquid extraction with 1-chlorobutane was used for the sample preparation. The validation of the canertinib analytical method resulted in recoveries in the range of 74–79 % and in relative standard deviation and accuracy lower than 15 %, both for between- and within-batch calculations. A very good linearity in the validated range (5–10,000 ng/mL) and a limit of quantification (LOQ) of 5 ng/mL were achieved. Peak width, height and peak area of six protein kinase inhibitors analysed with a core–shell column and with a conventional fully porous particle column were compared. The results showed that, for most inhibitors analysed with the core–shell analytical column, the peaks were about two to three times narrower and two to three times higher, with areas that remained almost unchanged. The presented study revealed that the application of this separation technology is a very cost-effective way to bring the LOQ of the protein kinase inhibitor analysis with an HPLC closer to the possibilities of the LC–MS/MS technique.

Keywords: Canertinib; CI-1033; Dasatinib; Core–shell; Protein kinase

Pulsed supersonic beams with nucleobases by Adnan Sarfraz; Klaus Rademann; Wolfgang Christen (pp. 2087-2090).

The dissolution of the primary nucleobases in supercritical fluids has been investigated using pulsed molecular beam mass spectrometry. Due to the low critical temperatures of ethylene and carbon dioxide, their adiabatic jet expansion permits transferring thermally sensitive solutes into the gas phase. This feature is particularly attractive for pharmaceutical and biomedical applications. In this study, adenine, guanine, cytosine, thymine, and uracil have been dissolved in supercritical ethylene with a few percent of ethanol as cosolvent. At source temperatures of 313 K, these solutions have been expanded from supercritical pressures into high vacuum using a customized pulsed nozzle. A mass spectrometer was used to monitor the relative amounts of solute, solvent, and cosolvent in the supersonic beam. The results suggest a paramount influence of the cosolvent.

Keywords: Biomolecules; Nucleobases; Solubility; Supercritical fluids; Supersonic beams

Determination of imatinib and its active metabolite N-desmethyl imatinib in human plasma by liquid chromatography/tandem mass spectrometry by Mei Zhang; Grant A. Moore; Liam J. Fernyhough; Murray L. Barclay; Evan J Begg (pp. 2091-2096).

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3 μm C18 column using gradient elution of 0.05 % formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r > 0.999) over the concentration range of 10 to 2,000 ng/mL which encompasses clinical concentrations. Bias was ≤±8.3 %, intra- and inter-day coefficients of variation (imprecision) were ≤8.0 % and the limit of quantification was 10 ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib. Figure Representative LC-MS/MS chromatogram of imatinib, N–desmethyl imatinib and the internal standard D8-imatinib in human plasma

Keywords: Imatinib; N-desmethyl imatinib; Plasma; LC-MS/MS

Quantification of p-cresol sulphate in human plasma by selected reaction monitoring by Aurora Cuoghi; Marialuisa Caiazzo; Elisa Bellei; Emanuela Monari; Stefania Bergamini; Giuseppe Palladino; Tomris Ozben; Aldo Tomasi (pp. 2097-2104).

Chronic renal failure patients accumulate in the blood molecules that are normally excreted into the urine. p-Cresol Sulphate (pCS), the most representative retained toxin, shows a high level of toxicity. Therefore, its quantification could represent a prediction factor to determine the risk of endothelial dysfunction and cardiovascular complication and response to the haemodialysis treatment. The aim of this study was to evaluate the suitability of the multiple reaction monitoring (MRM) technique in order to improve the sensibility, the selectivity and the timing of pCS detection in a small amount of plasma. Deproteinized plasma of uremic patients was concentrated and dissolved in liquid chromatography (LC) mobile phase solution. pCS was quantified by LC coupled to tandem mass spectrometry (LC-MS/MS) on a triple-quadrupole mass spectrometer. Selective and sensitive detection of pCS was achieved by selecting the specific parent ion and monitoring two specific fragment ions. The MRM assay was carried out using the following transitions: m/z 187 → 80.00 and m/z 187 → 107.00. A good linearity was observed for each calibration curve. The intra-day and inter-day results showed a good precision and repeatability. The percentage recoveries indicate an optimal selectivity of the analytical method. The MRM assay to quantify pCS in a small amount of human plasma is rapid, highly sensitive, selective and with a good repeatability. Figure pCS workflow of the SRM method. The m/z of the precursor ion (selected in Q1) is fragmented in Q2 (not showed) and selected fragment ions are monitored from the detector (after Q3) and quantified by the detector

Keywords: Biological samples; Process analysis; Mass spectrometry/ICP-MS; HPLC; Separations/instrumentation

A calculation mode for the correct GC/ECNI-MS-SIM determination of polychlorinated terphenyls in the presence of high excesses of polychlorinated biphenyls by Natalie Rosenfelder; Walter Vetter (pp. 2105-2111).

Polychlorinated terphenyls (PCTs) are a class of persistent organic pollutants difficult to analyze by gas chromatography with mass spectrometry operated in the selected ion monitoring mode (GC/MS-SIM) in environmental samples due to the retention time and mass range overlap with polychlorinated biphenyls (PCBs). To overcome these drawbacks, we developed and evaluated a mathematical calculation algorithm which allows to detail the interference of PCT congeners in GC/electron capture negative ion (ECNI)-MS-SIM chromatograms by PCBs. The calculation takes advantage of the abundance and ratio of two suitable isotope peaks of the molecular ion of PCTs. With the help of this method, we detected at least 63 tetra- to nonachlorinated terphenyls in the blubber of a harbour porpoise (Phocoena phocoena) from the North Sea. The interference of these peaks by PCBs ranged from >100 to 0 %. The novel calculation method used in combination with GC/ECNI-MS-SIM is suitable to analyze PCTs in environmental and food samples. However, it can also be applied to GC/EI-MS measurements.

Keywords: Gas Chromatography-Mass Spectrometry; Organic compounds/trace organic compounds; Polychlorinated compounds; Mathematical correction

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Analytical and Bioanalytical Chemistry (v.404, #6-7)