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Analytical and Bioanalytical Chemistry (v.403, #3)

Quality assurance challenge 10 by Manfred Reichenbächer; Jürgen W. Einax (pp. 633-634).

Solution to Plato’s elements challenge by Juris Meija (pp. 635-635).

2012 Winter Conference on Plasma Spectrochemistry, Tucson, Arizona, January 8–14, 2012 by Jan O. Orlandini v. Niessen (pp. 637-638).

Luigi Mondello (Ed.): Comprehensive chromatography in combination with mass spectrometry by James Harynuk (pp. 639-640).

16th Conference on Solid State Analysis by Gernot Friedbacher (pp. 641-642).

is Associate Professor of Analytical Chemistry at the Vienna University of Technology. His research activities focus on the investigation of surfaces and surface processes with scanning probe microscopy and electron probe x-ray microanalysis. He has published over 120 research articles, reviews, and book chapters, and he is editor of the 2nd edition of Surface and Thin Film Analysis published in 2011.

Characterization of non-stoichiometric co-sputtered Ba_0.6Sr_0.4(Ti1 − xFe x)1 + xO3 − δ thin films for tunable passive microwave applications by F. Stemme; H. Geßwein; M. D. Drahus; B. Holländer; C. Azucena; J. R. Binder; R.-A. Eichel; J. Haußelt; M. Bruns (pp. 643-650).

The fabrication of novel iron-doped barium strontium titanate thin films by means of radio frequency (RF) magnetron co-sputtering is shown. Investigations of the elemental composition and the dopant distribution in the thin films obtained by X-ray photoelectron spectroscopy, Rutherford backscattering spectrometry, and time-of-flight secondary ion mass spectroscopy reveal a homogeneous dopant concentration throughout the thin film. The incorporation of the iron dopant and the temperature-dependent evolution of the crystal structure and morphology are analyzed by electron paramagnetic resonance spectroscopy, X-ray diffraction, Raman spectroscopy, atomic force microscopy, and scanning electron microscopy. In summary, these results emphasize the RF magnetron co-sputter process as a versatile way to fabricate doped thin films. Figure Cross section of the RF magnetron co-sputter setup and the X-ray phototelectron spectroscopy iron spectrum of a co-sputtered iron doped Barium strontium titanate thin film

Keywords: XPS; XRD; EPR; RF magnetron co-sputtering; BST thin film

Nanoscale analysis of surface oxides on ZnMgAl hot-dip-coated steel sheets by M. Arndt; J. Duchoslav; H. Itani; G. Hesser; C. K. Riener; G. Angeli; K. Preis; D. Stifter; K. Hingerl (pp. 651-661).

In this work, the first few nanometres of the surface of ZnMgAl hot-dip-galvanised steel sheets were analysed by scanning Auger electron spectroscopy, angle-resolved X-ray photoelectron spectroscopy and atomic force microscopy. Although the ZnMgAl coating itself is exhibiting a complex micro-structure composed of several different phases, it is shown that the topmost surface is covered by a smooth, homogeneous oxide layer consisting of a mixture of magnesium oxide and aluminium oxide, exhibiting a higher amount of magnesium than aluminium and a total film thickness of 4.5 to 5 nm. Especially by the combined analytical approach of surface-sensitive methods, it is directly demonstrated for the first time that within surface imprints—created by industrial skin rolling of the steel sheet which ensures a smooth surface appearance as well as reduced yield-point phenomenon—the original, smooth oxide layer is partly removed and that a layer of native oxides, exactly corresponding to the chemical structure of the underlying metal phases, is formed. Figure The picture shows an overlay of the topography in a skin pass imprint of a hot-dip-galvanized ZnMgAl-coated steel sheet taken by an AFM scan and the chemical composition on the same area taken by AES mapping (Zn red, Mg green and Al blue). The smooth blue green area in the middle is a still remaining part of the originally present MgAl oxide layer whereas the surrounding area shows the metals Zn, Mg and Al

Keywords: Hot-dip galvanizing; ZnMgAl-coating; Surface characterisation; Auger electron spectroscopy; X-ray photoelectron spectroscopy; Atomic force microscopy

X-ray photoelectron and scanning Auger electron spectroscopy study of electrodeposited ZnCr coatings on steel by H. Itani; J. Duchoslav; M. Arndt; T. Steck; J. Gerdenitsch; J. Faderl; K. Preis; W. Winkler; D. Stifter (pp. 663-673).

Zn–Cr alloyed coatings electrochemically deposited are of high interest for leading steel manufacturing companies because of their novel properties and high corrosion resistance compared with conventional Zn coatings on steel. For tuning and optimizing the properties of the electrodeposited Zn–Cr coatings, a broad range of the deposition conditions must be studied. For this reason, two different types of material were investigated in this study, one with a low electrolyte temperature and one with an elevated electrolyte pH, compared with the standard values. Because different corrosion performance and delamination behaviour of the layers were observed for the two types, advanced surface analysis was conducted to understand the origin of this behaviour and to discover differences in the formation of the coatings. The topmost surface, the shallow subsurface region, and the whole bulk down to the coating–steel interface surface were analysed in detail by X-ray photoelectron spectroscopy (XPS) and high-resolution scanning Auger electron spectroscopy to determine the elemental and the chemical composition. For better understanding of the resulting layer structure, multiple reference samples and materials were measured and their Auger and XPS spectra were fitted to the experimental data. The results showed that one coating type is composed of metallic Zn and Cr, with oxide residing only on the surface and interface, whereas the other type contains significant amounts of Zn and Cr oxides throughout the whole coating thickness.

Keywords: AES; X-ray spectroscopy (XPS| XRF|EDX); Steel; Spectroscopy/instrumentation

Characterisation of diamond coatings with different morphologies by Raman spectroscopy using various laser wavelengths by Moritz Rudigier; Roland Haubner (pp. 675-681).

Since the beginning of low-pressure diamond synthesis, Raman spectroscopy has been widely used to identify and characterise the quality of diamonds. The diamond crystal is characterised by a Raman peak at about 1,332 cm^-1. Other peaks are associated with miscellaneous carbon structures, e.g. graphite and amorphous phases. In recent years, both well-faceted crystalline diamonds and nanocrystalline and ultrananocrystalline diamonds have been investigated. For these fine-grained materials, the diamond peak at 1,332 cm^-1 disappears and the intensities of peaks at other wavelengths increase. To study the influence of the Raman laser wavelength, three lasers were used (472.681 nm, blue; 532.1 nm, green; 632.81 nm, red). For well-faceted diamonds, the Raman spectra with blue and green laser light were similar. A shift of the peak maxima and different intensities were observed. With use of the red laser, a strong luminescence peak and low peak intensities for the various carbon-related peaks occurred. When the diamond morphology changes from well-faceted to fine-grained ballas diamond, the spectra are similar for all three lasers.

Keywords: Diamond; Raman; Morphology; Nanodiamond; Ballas

Visualisation and characterisation of ageing induced changes of polymeric surfaces by spectroscopic imaging methods by Gabriele C. Eder; Lidija Spoljaric-Lukacic; Boril S. Chernev (pp. 683-695).

A polymeric resin material was chosen as the model system to visualise the ageing-induced chemical surface changes with molecular spectroscopic imaging techniques and correlate these results to physical properties such as colour changes. The influence of light radiation, temperature and humidity on the polymeric surfaces was analysed by means of attenuated total reflection infrared imaging, Raman imaging spectroscopy and scanning electron microscopy. Samples were analysed before, during and after the weathering/ageing tests. From these combined data, the mechanisms for the damaging of the resin surface under the various environmental conditions (as applied in the accelerated ageing tests) were deduced. Photo-oxidative decay of the resin leading to a degradation of the uppermost surface layers as well as hydrolysis of the aged surface was identified. The combination of the spectral and spatial data as obtained from spectroscopic imaging with the morphological and elemental information of scanning electron microscopic mapping experiments turned out to be highly advantageous for the elucidation of ageing processes. A correlation between the molecular spectroscopic data and the results from the macroscopic colour difference measurements was found.

Keywords: ATR FT-IR imaging; Raman imaging; Spectroscopic imaging; Accelerated ageing; Polymeric surfaces

Electron spray ionization mass spectrometry and 2D ³¹P NMR for monitoring ¹⁸O/¹⁶O isotope exchange and turnover rates of metabolic oligophosphates by Emirhan Nemutlu; Nenad Juranic; Song Zhang; Lawrence E. Ward; Tumpa Dutta; K. Sreekumaran Nair; Andre Terzic; Slobodan Macura; Petras P. Dzeja (pp. 697-706).

A new method was here developed for the determination of ¹⁸O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, β-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of ¹⁸O/¹⁶O exchange was validated with gas chromatography–mass spectrometry and 2D ³¹P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D ³¹P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, ¹⁸O-labeling kinetics and turnover rates of α-, β-, and γ-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system. Figure Monitoring of ¹⁸O enrichment in ATP at α-, β- and γ-phosphoryl moieties using ESI-MS, GC-MS, 1D and 2D ³¹P NMR.

Keywords: ¹⁸O isotope labeling; ESI-MS; ³¹P NMR; ATP; Energy metabolism; Phosphotransfer networks

Acoustic deposition with NIMS as a high-throughput enzyme activity assay by Matthew Greving; Xiaoliang Cheng; Wolfgang Reindl; Benjamin Bowen; Kai Deng; Katherine Louie; Michael Nyman; Joseph Cohen; Anup Singh; Blake Simmons; Paul Adams; Gary Siuzdak; Trent Northen (pp. 707-711).

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid–liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.

Keywords: Nanostructure initiator mass spectrometry; NIMS; Nimzyme; Enzyme assay; Glycoside hydrolase

A biomimetic sensor surface to detect anti-β₂-glycoprotein-I antibodies as a marker for antiphospholipid syndrome by Urs Hilbig; Oliver Bleher; Alexander Le Blanc; Günter Gauglitz (pp. 713-717).

A biomimetic sensor has been developed, that allows for quantification of autoantibodies related to the antiphospholipid syndrome (APS). Autoantibodies directed against the β₂-glycoprotein-I (β₂GP-I) are known as the best markers for diagnosis of APS, however, detection of such antibodies is still a challenge. The epitopes of β₂GP-I are exposed upon binding to negatively charged membranes. The surface of the sensor chips was therefore modified with such type of membranes, on which β₂GP-I molecules were subsequently immobilized as recognition elements. Using the label-free method, reflectometric interference spectroscopy, it was possible to quantify anti-β₂GP-I antibodies and to calibrate the sensor chip in buffer. A mild regeneration procedure allows for many consecutive measurements without stripping off the membrane in between.

Keywords: Biosensor; Reflectometric interference spectroscopy (RIfS); Optical sensors; Antiphospholipid syndrome (APS); β₂-glycoprotein-I; Lipid membranes

Unsupervised unmixing of Raman microspectroscopic images for morphochemical analysis of non-dried brain tumor specimens by Norbert Bergner; Christoph Krafft; Kathrin D. Geiger; Matthias Kirsch; Gabriele Schackert; Jürgen Popp (pp. 719-725).

Raman microspectroscopic imaging provides molecular contrast in a label-free manner with subcellular spatial resolution. These properties might complement clinical tools for diagnosis of tissue and cells in the future. Eight Raman spectroscopic images were collected with 785 nm excitation from five non-dried brain specimens immersed in aqueous buffer. The specimens were assigned to molecular and granular layers of cerebellum, cerebrum with and without scattered tumor cells of astrocytoma WHO grade III, ependymoma WHO grade II, astrocytoma WHO grade III, and glioblastoma multiforme WHO grade IV with subnecrotic and necrotic regions. In contrast with dried tissue section, these samples were not affected by drying effects such as crystallization of lipids or denaturation of proteins and nucleic acids. The combined data sets were processed by use of the hyperspectral unmixing algorithms N-FINDR and VCA. Both unsupervised approaches calculated seven endmembers that reveal the abundance plots and spectral signatures of cholesterol, cholesterol ester, nucleic acids, carotene, proteins, lipids, and buffer. The endmembers were correlated with Raman spectra of reference materials. The focus of the single mode laser near 1 μm and the step size of 2 μm were sufficiently small to resolve morphological details, for example cholesterol ester islets and cell nuclei. The results are compared for both unmixing algorithms and with previously reported supervised spectral decomposition techniques. Figure Morphological details in tissue sections are resolved by Raman imaging and might contribute together with chemical information to improved diagnosis.

Keywords: Raman imaging; Brain tumors; Non-dried specimens; Hyperspectral unmixing

Label-free differentiation of human pituitary adenomas by FT-IR spectroscopic imaging by Gerald Steiner; Luisa Mackenroth; Kathrin D. Geiger; Allison Stelling; Thomas Pinzer; Ortrud Uckermann; Valdas Sablinskas; Gabriele Schackert; Edmund Koch; Matthias Kirsch (pp. 727-735).

Fourier transform infrared (FT-IR) spectroscopic imaging has been used to characterize different types of pituitary gland tumors and normal pituitary tissue. Freshly resected tumor tissue from surgery was prepared as thin cryosections and examined by FT-IR spectroscopic imaging. Tissue types were discriminated via k-means cluster analysis and a supervised classification algorithm based on linear discriminant analysis. Spectral classification allowed us to discriminate between tumor and non-tumor cells, as well as between tumor cells that produce human growth hormone (hGH+) and tumor cells that do not produce that hormone (hGH−).The spectral classification was compared and contrasted with a histological PAS and orange G stained image. It was further shown that hGH+ pituitary tumor cells show stronger amide bands than tumor cells that do not produce hGH. This study demonstrates that FT-IR spectroscopic imaging can not only potentially serve as a fast and objective approach for discriminating pituitary gland tumors from normal tissue, but that it can also detect hGH-producing tumor cells.

Keywords: Pituitary gland tumors; Infrared spectroscopic imaging; Supervised classification

Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP by Sebastian Wierer; Sébastien Peter; Kirstin Elgass; Hans-Georg Mack; Stefan Bieker; Alfred J. Meixner; Ulrike Zentgraf; Frank Schleifenbaum (pp. 737-744).

For the quantitative analysis of molecular processes in living (plant) cells, such as the perception and processing of environmental and endogenous signals, new combinatorial approaches in optical and spectroscopic technologies are required and partly already became established in many fields of the life sciences. One hallmark of the in vivo analysis of cell biological processes is the use of visible fluorescent proteins to create fluorescent fusion proteins. Recent progress has been made in generating a redox-sensitive mutant of green fluorescent proteins (roGFP), which exhibits alterations in its spectral properties in response to changes in the redox state of the surrounding medium. An established method to probe the local redox potential using roGFP is based on a ratiometric protocol. This readout modality requires two excitation wavelengths, which makes the technique less suited for in vivo studies of e.g. dynamic samples. We clarify the origin of the redox sensitivity of roGFP by ab initio calculations, which reveal a changed protonation equilibrium of the chromophore in dependence on the redox potential. Based on this finding, we test and compare different spectroscopic readout modalities with single wavelength excitation to determine the local redox potential and apply these techniques to live cell analytics.

Keywords: roGFP; FLIM; Redox potential; Spectro-microscopy; Theory; Computational chemistry

A study of Docetaxel-induced effects in MCF-7 cells by means of Raman microspectroscopy by Katharina Hartmann; Melanie Becker-Putsche; Thomas Bocklitz; Katharina Pachmann; Axel Niendorf; Petra Rösch; Jürgen Popp (pp. 745-753).

Chemotherapies feature a low success rate of about 25%, and therefore, the choice of the most effective cytostatic drug for the individual patient and monitoring the efficiency of an ongoing chemotherapy are important steps towards personalized therapy. Thereby, an objective method able to differentiate between treated and untreated cancer cells would be essential. In this study, we provide molecular insights into Docetaxel-induced effects in MCF-7 cells, as a model system for adenocarcinoma, by means of Raman microspectroscopy combined with powerful chemometric methods. The analysis of the Raman data is divided into two steps. In the first part, the morphology of cell organelles, e.g. the cell nucleus has been visualized by analysing the Raman spectra with k-means cluster analysis and artificial neural networks and compared to the histopathologic gold standard method hematoxylin and eosin staining. This comparison showed that Raman microscopy is capable of displaying the cell morphology; however, this is in contrast to hematoxylin and eosin staining label free and can therefore be applied potentially in vivo. Because Docetaxel is a drug acting within the cell nucleus, Raman spectra originating from the cell nucleus region were further investigated in a next step. Thereby we were able to differentiate treated from untreated MCF-7 cells and to quantify the cell–drug response by utilizing linear discriminant analysis models. Figure Raman microspectroscopy in combination with powerful chemometric methods (e.g. artificial neural networks) indicates morphological (nucleus fragmentation) and spectral changes in Docetaxel treated breast cancer cells (MCF-7) in comparison to untreated cell samples

Keywords: Raman microspectroscopy; Docetaxel; Breast cancer; MCF-7

Rapid detection and quantification of 35 benzodiazepines in urine by GC-TOF-MS by Kathrin Arnhard; Rupert Schmid; Uwe Kobold; Roland Thiele (pp. 755-768).

A rapid and sensitive method for the screening and quantification of 35 benzodiazepines in human urine by gas chromatography/time-of-flight mass spectrometry was developed and validated. Target analytes were isolated from 1 ml urine by solid-phase extraction using Oasis MCX extraction columns (extraction recovery between 35 and 99 %). With a supported liquid–liquid extraction method, a new modification of conventional liquid–liquid-extraction, a less time intensive alternative for benzodiazepine extraction is presented. The sample pretreatment entails the derivatization of the benzodiazepines with N,O-bis(trimethylsilyl)trifluoroacetamide plus 1 % trimethylchlorosilane. Separation of all benzodiazepines was done within 9.5 min, and detection was based on full mass spectra for each analyte. A deconvolution algorithm was used for unresolved chromatographic peaks to identify coeluted substances. The subsequent quantification was done using significant masses. The limit of quantification is 10 ng/ml for most of the compounds. Linearity is in the range between 10 and 350 ng/ml. Reproducibility was observed with coefficients of variation below 2 % at concentrations of 50 and 200 ng/ml. The accuracy is between 88 and 108 % depending on the respective analyte and the concentration. Figure Chromatogram of an urinary calibrator (200 ng/ml) after extraction with Oasis MCX cartridges and derivatization containing all 35 benzodiazepines plus the internal standard (ISTD) oxazepam-d ₅ (100 ng/ml). TMS trimethylsilane

Keywords: Benzodiazepines; Gas chromatography/time-of-flight mass spectrometry; Urine; Screening; Drug of abuse testing; Signal deconvolution

Investigations on the influence of different grinding procedures on measured ethyl glucuronide concentrations in hair determined with an optimized and validated LC-MS/MS method by M. E. Albermann; F. Musshoff; L. Aengenheister; B. Madea (pp. 769-776).

Ethyl glucuronide (EtG) analysis in hair is a suitable method for the retrospective determination of previous alcohol consumption. According to the German guidelines, EtG abstinence is improbable at c _EtG > 7 pg/mg in the proximal 3 cm of scalp hair. The chromatography of the routinely used liquid chromatography-tandem mass spectrometry procedure was optimized by replacing the stationary phase. To simplify sample preparation, two different mills were tested, and an optimized grinding process was developed. The new method was successfully validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry. Despite a simple extraction procedure without any cleaning steps, a very high sensitivity (limit of detection, 1.7 pg/mg; limit of quantitation, 2.3 pg/mg) could be achieved. Competitive analysis showed significantly higher EtG concentrations in pulverized versus cut hair samples. The strong impact of sample preparation on the determined EtG concentrations suggests the introduction of a standardized sample preparation method to produce comparable results.

Keywords: Hair analysis; Ethyl glucuronide; LC-MS/MS; Hypercarb column; Cut vs. pulverizing

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine by Igor Botello; Francesc Borrull; Marta Calull; Carme Aguilar; Govert W. Somsen; Gerhardus J. de Jong (pp. 777-784).

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping. Figure Schematic diagram of In-line SPE-CE device

Keywords: Drugs of abuse; Capillary electrophoresis; In-line solid-phase extraction; Preconcentration; Urine; Mass spectrometry

Development of novel molecularly imprinted solid-phase microextraction fibers and their application for the determination of antibiotic drugs in biological samples by SPME-LC/MSⁿ by Malgorzata Szultka; Jacek Szeliga; Marek Jackowski; Boguslaw Buszewski (pp. 785-796).

Novel molecularly imprinted polymer (MIP)-coated fibers for solid-phase microextraction (SPME) fibers were prepared by using linezolid as the template molecule. The characteristics and application of these fibers were investigated. The polypyrrole, polythiophene, and poly(3-methylthiophene) coatings were prepared in the electrochemical polymerization way. The molecularly imprinted SPME coatings display a high selectivity toward linezolid. Molecularly imprinted coatings showed a stable and reproducible response without any influence of interferents commonly existing in biological samples. High-performance liquid chromatography with spectroscopic UV and mass spectrometry (MS) detectors were used for the determination of selected antibiotic drugs (linezolid, daptomycin, amoxicillin). The isolation and preconcentration of selected antibiotic drugs from new types of biological samples (acellular and protein-free simulated body fluid) and human plasma samples were performed. The SPME MIP-coated fibers are suitable for the selective extraction of antibiotic drugs in biological samples.

Keywords: Solid-phase microextraction; Antibiotic drugs; High-performance liquid chromatography; Mass spectrometry

Multivariate statistics for the differentiation of erythropoietin preparations based on intact glycoforms determined by CE-MS by Angelina Taichrib; Markus Pioch; Christian Neusüß (pp. 797-805).

Owing to the increasing number of erythropoietin biosimilars being approved, the comparison of different erythropoietin preparations in the pharmaceutical area is gaining in importance. Erythropoietin has a distinct natural heterogeneity arising from its glycosylation, which shows strong composition variations. This heterogeneity increases the complexity of the analysis of erythropoietin considerably, but may also be used to distinguish different preparations. Here, a method is presented for the differentiation of various erythropoietin preparations by capillary electrophoresis–mass spectrometry and the subsequent application of multivariate statistics. Relative peak areas of selected intact erythropoietin isoforms were used as variables in principal component analysis and hierarchical agglomerative clustering. Both of these strategies were suited for the clear differentiation of all erythropoietin preparations, including marketed products and preproduction preparations, which differ in the manufacturer, the production cell line, and the batch number. By this means, even closely related preparations were distinguished on the basis of the combined information on the antennarity, the sialoform, and the acetylation of the observed isoforms.

Keywords: Capillary electrophoresis–mass spectrometry; Biosimilars; Erythropoietin; Principal component analysis; Cluster analysis

Low–medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay by Hamdi Joda; Valerio Beni; Deirdre Curnane; Ioanis Katakis; Noora Alakulppi; Jukka Partanen; Kristina Lind; Linda Strömbom; Ciara K. O’Sullivan (pp. 807-819).

Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained. Figure Example of the results obtained during the colorimetric HLA typing of a real sample

Keywords: Coeliac disease; HLA typing; Sequence specific oligonucleotide probes; SPR; ELONA

Simultaneous determination of two major snow crab aeroallergens in processing plants by use of isotopic dilution tandem mass spectrometry by Anas M. Abdel Rahman; Sébastien Gagné; Robert J. Helleur (pp. 821-831).

Snow crab is a major fishery in the North Atlantic region. During crab processing the proteins are aerosolized and some are responsible for development of occupational asthma. Tropomyosin and arginine kinase have recently been reported as major snow crab allergens. A liquid chromatographic tandem mass spectrometric method has been developed for simultaneous analysis of these two proteins in air samples collected from processing plants. These proteins were initially isolated then characterized by use of mass spectrometry to determine their primary structure and signature peptides. The signature peptides were chemically synthesized in light and heavy forms and used as standards for developing the multiple-reaction monitoring transitions to monitor allergen levels. A validation study was performed; precision and accuracy were 1.8–8% and 91–104%, respectively. Replicate air samples were collected on air filters from two crab-processing plants in Newfoundland and Labrador (NL) and four located in Quebec. In NL, measured levels of both tropomyosin and arginine kinase were between 1 and 20 ng m⁻³. In Quebec plants, however, levels were found to be much higher at 2–2400 ng m⁻³. Significant differences were also observed among the plants and individual processing workstations. For the first time arginine kinase has been detected in its aerosolized form in processing plants. In general, levels of the allergens were highest in the butchering and cooking areas; plant design can, however, have a significant effect on levels of the allergens. Online Abstract Figure

Keywords: Aeroallergens; Bioanalytical method; Tandem mass spectrometry; Proteins; Peptides; High throughput screening; Occupational health; Asthma

Quantitative analysis of azaspiracids in Azadinium spinosum cultures by Thierry Jauffrais; Christine Herrenknecht; Véronique Séchet; Manoella Sibat; Urban Tillmann; Bernd Krock; Jane Kilcoyne; Christopher O. Miles; Pearse McCarron; Zouher Amzil; Philipp Hess (pp. 833-846).

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 10⁹ μm³. Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29–32.

Keywords: Extraction procedure; Extraction artefact; Matrix effects; LC-MS/MS; Azaspiracid methyl ester; Dinoflagellate; Liquid chromatography–mass spectrometry

A combination of metabolomics and metallomics studies of urine and serum from hypercholesterolaemic rats after berberine injection by Feng Liu; Pei Pei Gan; Huanan Wu; Wei Shan Woo; Eng Shi Ong; Sam Fong Yau Li (pp. 847-856).

Berberine, long used as a remedy in China and India for intestinal infections, has been discovered in recent years in western countries and is now being used to treat ailments ranging from urinary tract infections to diabetes and obesity. In order to study the effect of berberine more deeply, a combined metabolomic and metallomic approach was developed in this study using the hypercholesterolaemic rat model, which involved the use of proton nuclear magnetic resonance for the analysis of rat urine to achieve metabolic fingerprinting and inductively coupled plasma mass spectrometry for the analysis of rat blood serum to achieve metallomic fingerprinting. The results obtained indicated that major metabolic processes like Krebs cycle, cholesterol metabolism and osmoregulation in hypercholesterolaemic rats are perturbed upon berberine injection. In addition, the changes of some elements, such as V, Mn, Na and K, revealed in the metallomic study may contribute to the search of new biomarkers for hypercholesterolaemic disease. We concluded that both the metabolomic and metallomic profiles of berberine-treated hypercholesterolaemic rats were different from those of the control group and that the selected metabolites and elements could probably be applied as potential biomarkers for the understanding of the effect of berberine on biochemical process in the animal model. Such a multi-analytical approach will potentially provide an information-rich platform for the elucidation of effects of xenobiotics and drug efficacy studies. Figure The process of the metabolomics and metallomic fingerprinting analysis.

Keywords: Metabolomics; Metallomics; Berberine; ¹H NMR; ICP-MS

Preparation of a sewage sludge laboratory quality control material for butyltin compounds and their determination by isotope-dilution mass spectrometry by Tea Zuliani; Radmila Milačič; Janez Ščančar (pp. 857-865).

The characterisation of a laboratory quality control material (QCM) for dibutyltin (DBT) and tributyltin (TBT) in sewage sludge is described. The reference values were determined by the use of two different types of isotope-dilution mass spectrometry: gas chromatography–mass spectrometry and gas chromatography–inductively coupled plasma mass spectrometry. To avoid possible analytical errors such as non-quantitative extraction and species degradation during sample preparation, different extraction methods were tested (microwave- and ultrasound-assisted extraction and mechanical stirring). The reference values were based on the unweighted means of results from the homogenisation and characterisation studies. The reference values obtained were 1,553 ± 87 and 534 ± 38 ng Sn g^-1 for DBT and TBT, respectively. In the uncertainty budget estimation, the sample inhomogeneity and between-method imprecision were taken into account. The concentrations of DBT and TBT in QCM are similar to those in the harbour sediment certified reference material PACS-2. Likewise, the levels of DBT and TBT are in the range of these compounds normally present in sewage sludge worldwide. In the future, the QCM will be used for an intercomparison study on DBT and TBT in sewage sludge, and as a day-to-day QCM during studies concerning the application of sewage sludge as an additive to artificial soil or as a raw material in civil engineering construction. Figure From landfill to quality control material; butyltin compounds in sewage sludge

Keywords: Butyltin compounds; Sewage sludge; Isotope dilution; Gas chromatography–mass spectrometry; Gas chromatography–inductively coupled plasma mass spectrometry

On-line solid-phase extraction coupled to ultra-performance liquid chromatography with tandem mass spectrometry detection for the determination of benzotriazole UV stabilizers in coastal marine and wastewater samples by Sarah Montesdeoca-Esponda; Zoraida Sosa-Ferrera; José Juan Santana-Rodríguez (pp. 867-876).

Benzotriazoles are a group of UV absorbing compounds considered emerging contaminants that are used in different personal care products, and therefore, it is of high interest to develop sensitive and fast methods for investigating their presence in the environment. In this work, we present the development and application of a novel method based on on-line solid-phase extraction coupled to ultra-performance liquid chromatography with tandem mass spectrometry detection (SPE-UPLC-MS/MS) for the determination of seven benzotriazole UV stabilizers (BUVSs) in coastal marine and wastewater samples. This process is compared with a conventional off-line SPE procedure followed by UPLC-MS/MS. The parameters affecting the performance of the sample preparation and determination processes were evaluated. The results indicate that the on-line procedure provides for better sensitivity and reproducibility and is faster and easier than the off-line procedure. The detection limits and quantification limits achieved were in the range of 0.6–4.1 ng∙L⁻¹ and 2.1–14 ng∙L⁻¹ and relative standard deviation between 6.2 and 10 %. The developed method was applied to coastal marine and wastewater samples from Gran Canaria Island (Spain). All of the BUVSs studied were detected in the samples from wastewater treatment plants and two were found in the seawater samples (UV P in the range of 2.8–4.4 ng∙L⁻¹ and UV 360 between 3.6 and 5.2 ng∙L⁻¹).

Keywords: Benzotriazole UV stabilizers; On-line SPE; UPLC-MS/MS; Water analysis

Rapid determination of anilines in water samples by dispersive liquid–liquid microextraction based on solidification of floating organic drop prior to gas chromatography–mass spectrometry by Chun-Peng Diao; Chao-Hai Wei (pp. 877-884).

A rapid, sensitive and environmentally friendly method for the analysis of 14 anilines in water samples by dispersive liquid–liquid microextraction based on solidification of floating organic drop (DLLME-SFO) prior to gas chromatography–mass spectrometry (GC-MS) was developed and optimized. In the proposed method, cyclohexane was used as the extraction solvent as its toxicity was much lower than that of the solvent usually used in dispersive liquid–liquid microextraction (DLLME). In the optimized conditions, the method exhibited good analytical performance. Based on a signal-to-noise ratio of 3, limits of detection for anilines were in the range of 0.07 to 0.29 μg L⁻¹, and the linear range was 0.5–200 μg L⁻¹ with regression coefficients (r ²) higher than 0.9977. It was efficient for qualitative and quantitative analysis of anilines in water samples. The relative standard deviations varied from 2.9 to 8.6 % depending on different compounds indicating good precision. Tap water and river water were selected for evaluating the application to real water samples. The relative recoveries of anilines for the two real samples spiked with 10 μg L⁻¹ anilines were in the scope of 78.2–114.6 % and 77.3–115.6 %, respectively. Figure Flow diagram of DLLME-SFO

Keywords: Dispersive liquid–liquid microextraction; Floating organic droplet; Anilines; Gas chromatography–mass spectrometry; Water sample

Development and validation of a method for determination of residues of 15 pyrethroids and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography–tandem mass spectrometry by Stephen W. C. Chung; C. H. Lam (pp. 885-896).

This paper reports a novel approach for the detection, confirmation, and quantification of 15 selected pyrethroid pesticides, including pyrethins, and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS). The proposed method makes use of a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure that combines isolation of the pesticides and sample cleanup in a single step. Analysis of pyrethroids and dithiocarbamate metabolites was performed by UPLC–MS–MS operated with electrospray and atmospheric pressure chemical ionization, respectively. Two specific precursor–product ion transitions were acquired per target compound in multiple reaction monitoring (MRM) mode. Such acquisition achieved the minimum number of identification points according to European Commission (EC) document no. SANCO/10684/2009, thus fulfilling the EC point system requirement for identification of contaminants in samples. The method was validated with a variety of food samples. Calibration curves were linear and covered from 1 to 800 μg kg⁻¹ in the sample for all target compounds. Average recoveries, measured at mass fractions of 10 and 100 μg kg⁻¹ for pyrethroids and 5 and 50 μg kg⁻¹ for dithiocarbamate metabolites, were in the range of 70–120% for all target compounds with relative standard deviations below 20%. Method limits of quantification (MLOQ) were 10 μg kg⁻¹ and 5 μg kg⁻¹ for pyrethroids and dithiocarbamate metabolites, respectively. The method has been successfully applied to the analysis of 600 food samples in the course of the first Hong Kong total diet study with pyrethroids and metabolites of dithiocarbamates being the pesticides determined. Figure UPLC–MS/MS chromatogram for twenty target analytes spiked at the MLOQ level

Keywords: Pyrethroids; Pyrethins; Dithiocarbamate metabolites; Pesticide residue; Food; QuEChERS; UPLC-MS-MS

Erratum to: Identification of volatile degradation products from Baltic amber by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry by Gianluca Pastorelli; Jens Glastrup (pp. 897-897).

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Analytical and Bioanalytical Chemistry (v.403, #3)