Analytical and Bioanalytical Chemistry (v.397, #6)

Imaging techniques with synchrotron radiation by Cyril Petibois (2031-2032).
is Assistant Professor at the University of Bordeaux 2, France, where he is a biochemistry teacher and a scientist involved in the development of imaging methodology for biosample analysis (CNRS UMR 5248 research unit; “Vibrational spectroscopy group”, headed by Dr. Bernard Desbat). C. Petibois’ research interest is in the development of multimodal imaging for full characterization of small samples, such as individual cells, combining elemental, morphological, molecular, and chemical techniques. His research unit develops all aspects of the multimodal imaging methodology, i.e. cell cultures dedicated to imaging, post-processing image manipulation for multimodality, specific chemometrics, image functionalization, etc. The laboratory is also engaged in the development of high-performance imaging using synchrotron radiation, notably using FTIR and X-ray beamlines in major European and Asian synchrotron radiation facilities.

HERSCHEL/SCORE, imaging the solar corona in visible and EUV light: CCD camera characterization by M. Pancrazzi; M. Focardi; F. Landini; M. Romoli; S. Fineschi; A. Gherardi; E. Pace; G. Massone; E. Antonucci; D. Moses; J. Newmark; D. Wang; G. Rossi (2033-2038).
The HERSCHEL (helium resonant scattering in the corona and heliosphere) experiment is a rocket mission that was successfully launched last September from White Sands Missile Range, New Mexico, USA. HERSCHEL was conceived to investigate the solar corona in the extreme UV (EUV) and in the visible broadband polarized brightness and provided, for the first time, a global map of helium in the solar environment. The HERSCHEL payload consisted of a telescope, HERSCHEL EUV Imaging Telescope (HEIT), and two coronagraphs, HECOR (helium coronagraph) and SCORE (sounding coronagraph experiment). The SCORE instrument was designed and developed mainly by Italian research institutes and it is an imaging coronagraph to observe the solar corona from 1.4 to 4 solar radii. SCORE has two detectors for the EUV lines at 121.6 nm (HI) and 30.4 nm (HeII) and the visible broadband polarized brightness. The SCORE UV detector is an intensified CCD with a microchannel plate coupled to a CCD through a fiber-optic bundle. The SCORE visible light detector is a frame-transfer CCD coupled to a polarimeter based on a liquid crystal variable retarder plate. The SCORE coronagraph is described together with the performances of the cameras for imaging the solar corona. Figure During an eclipse the Sun shows a bright region surrounding its disk: the solar corona. The corona is composed by a continuous flux of charged particles that leave the Sun surface and spread all over the space. This flux is known as the solar wind and our planet is wrapped by this breeze of particles. The temperature of the solar corona is surprisingly high, and the most part of emitted radiation falls into ultraviolet band. Studying the dynamic processes driving the solar wind is not only relevant to understand how our star works, but also for the safety of our own lives
Keywords: UV/vis detectors; Imaging; Coronagraph; Solar corona; CCD cameras

Application of micro-FTIR imaging in the Earth sciences by G. Della Ventura; F. Bellatreccia; A. Marcelli; M. Cestelli Guidi; M. Piccinini; A. Cavallo; M. Piochi (2039-2049).
In this paper we describe recent applications of micro-infrared imaging in the Earth sciences. We address, in particular, the use of Fourier-transform infrared (FTIR) spectroscopy in characterizing the zoning and speciation of H and C in a variety of geological materials, including microporous minerals, nominally anhydrous volcanic minerals (NAMs), and crystal inclusions. These investigations show that use of the modern techniques of FTIR imaging enables detection of the zoning of volatile species across the studied samples, and possible configuration changes of structurally-bound carbon molecular species (e.g., CO2 vs CO3) during crystal growth. Such features, which are not accessible with other micro-analytical techniques, may provide information about the physicochemical properties which act as constraints in the genesis of the samples, and important information about the evolution of the geological system. Tests performed with focal-plane-array detectors (FPA) show that resolution close to the diffraction limit can be achieved if the amounts of the target molecules in the sample are substantially different. We also point out the possibility of using FTIR imaging for investigations under non-ambient conditions. Figure FTIR-FPA image of fluid inclusions within a haüyine matrix
Keywords: FTIR spectroscopy; Imaging; Hydrogen; Carbon; Geological materials

Combining elemental, chemical, molecular, and morphological imaging information from individual cells with a lateral resolution well below 1 × 1 μm2 is the current technological challenge for investigating the smallest dimensions of living systems. In the race for such analytical performance, several techniques have been successfully developed; some use probes to determine given cellular contents whereas others use possible interactions between cellular matter with light or elements for characterization of contents. Morphological techniques providing information about cell dimensions have, when combined with other techniques, also opened the way to quantitative studies. New analytical opportunities are now being considered in cell biology, combining top-performance imaging techniques, applied to the same biosystem, with microscopy (nm–μm range) techniques providing elemental (micro-X-ray fluorescence, particle-induced X-ray emission, secondary-ion mass spectrometry), chemical (Raman, coherent anti-stokes Raman, Fourier-transform infrared, and near-field), molecular (UV–visible confocal and multiphoton), and morphological (AFM, ellipsometry, X-ray phase contrast, digital holography) information. Dedicated cell-culture methods have been proposed for multimodal imaging in vitro and/or ex vivo. This review shows that in addition to UV–fluorescent techniques, the imaging modalities able to provide interesting information about a cell, with high spatial and time resolution, have grown sufficiently to envisage quantitative analysis of chemical species inside subcellular compartments.
Keywords: Bioimaging; Microscopy; Metrology; Elements; Molecules; Cells

Progress of diffraction enhanced imaging at the Beijing Synchrotron Radiation Facility by Kai Zhang; Peiping Zhu; Qingxi Yuan; Wanxia Huang; Xiaosong Liu; Youli Hong; Gun Gao; Xin Ge; Zhili Wang; Ziyu Wu (2067-2078).
A simple framework that allows a new general diffraction enhanced imaging (DEI) equation to be derived is presented. This latter equation may explain all open problems associated with the equation introduced by Chapman and those not explained by the first DEI equation, such as the noise background due to the small-angle scattering reflected by the analyzer. Combing the DEI equation with computed tomography (CT) theory, we propose a new DEI–CT formula that explains qualitatively the contour contrast caused by extinction of the refraction. Two formulae with a new method to extract the refraction angle are also introduced. Within this new theoretical framework the three components of the gradient of the refractive index can be reconstructed.
Keywords: Phase-contrast image; Computed technology; Diffraction-enhanced imaging; Synchrotron radiation

Analytical characterization of cell–asbestos fiber interactions in lung pathogenesis by Seydou Yao; Giancarlo DellaVentura; Cyril Petibois (2079-2089).
Asbestos is a fiber causing lung diseases such as asbestosis and mesothelioma. Although the process involving these diseases remains to be elucidated for developing drugs and treatments, direct consequences of fiber exposure in humans have been clearly demonstrated. These diseases are first characterized by histological heterogeneity and combine chronic inflammation with fibrosis and cellular alterations. As a consequence, asbestosis is usually diagnosed at advanced stages of the disease and treatments are usually inefficient to cure the patients. Here, we review the links established between asbestos fiber chemistry and morphology with the occurrence of associated lung diseases. Cytological and histological aspects of diseases are described with respect to current analytical capabilities, notably for microscopy techniques.
Keywords: Clinical/biomedical analysis; Bioanalytical methods; Asbestos fibers; Lung disease; Bioinorganic chemistry

Quantitative coherence analysis with an X-ray Talbot–Lau interferometer by Zhili Wang; Peiping Zhu; Wanxia Huang; Qingxi Yuan; Xiaosong Liu; Kai Zhang; Youli Hong; Huitao Zhang; Xin Ge; Kun Gao; Ziyu Wu (2091-2094).
Differential phase-contrast (DPC) X-ray imaging has been performed in the Talbot–Lau configuration, in which the X-ray source was a combination of an absorption grating and a laboratory X-ray generator. We report here quantitative analysis of partial coherence effects on the X-ray Talbot–Lau interferometer. Based on the visibility of the self-image, the well-known geometry condition is reproduced. It is shown that effects of partial coherence are determined by the opening ratio of the source grating, and that the effects are independent of the Talbot order and the type of the phase grating, a condition quite different from those in a Talbot interferometer. A possible explanation is discussed from the point of view of the effective spatial coherence length. Taking into account the available X-ray flux and experimental fluctuations, we present the optimum opening ratio. Furthermore, we mention that our results can also be successfully used to discuss the properties of a multiline X-ray source.
Keywords: Partial coherence; Talbot–Lau interferometer; Self-image; Fractional Talbot distance; Visibility

Infrared and X-ray simultaneous spectroscopy: a novel conceptual beamline design for time resolved experiments by Augusto Marcelli; Wei Xu; Dariush Hampai; Luca Malfatti; Plinio Innocenzi; Ulrich Schade; Ziyu Wu (2095-2108).
Many physical/chemical processes such as metal–insulator transitions or self-assembly phenomena involve correlated changes of electronic and atomic structure in a wide time range from microseconds to minutes. To investigate these dynamic processes we not only need a highly brilliant photon source in order to achieve high spatial and time resolution but new experimental methods have to be implemented. Here we present a new optical layout for performing simultaneous or concurrent infrared and X-ray measurements. This approach may indeed return unique information for example the interplay between structural changes and chemical processes occurring in the investigated sample. A beamline combining two X-ray and IR beams may really take advantage of the unique synchrotron radiation properties: the high brilliance and the broad spectrum. In this contribution we will describe the conceptual layout and the expected performance of a complex system designed to collect IR and X-ray radiation from the same bending magnet on a third-generation synchrotron radiation ring. If realized, this beamline will enable time-resolved spectroscopy experiments offering new scientific opportunities at the frontiers of science.
Keywords: Synchrotron radiation instrumentation; X-ray absorption spectroscopy; EXAFS, XANES, etc.; Infrared spectrometers, auxiliary equipment, and techniques; Time resolved spectroscopy

Synchrotron microangiography studies of angiogenesis in mice with microemulsions and gold nanoparticles by Chia-Chi Chien; C. H. Wang; C. L. Wang; E. R. Li; K. H. Lee; Y. Hwu; Chien-Yi Lin; Shing-Jyh Chang; C. S. Yang; Cyril Petibois; G. Margaritondo (2109-2116).
We present an effective solution for the problem of contrast enhancement in phase-contrast microangiography, with the specific objective of visualising small (<8 µm) vessels in tumor-related microangiogenesis. Different hydrophilic and hydrophobic contrast agents were explored in this context. We found that an emulsified version of the hydrophobic contrast agents Lipiodol® provides the best contrast and minimal distortion of the circulation and vessel structure. Such emulsions are reasonably biocompatible and, with sizes of 0 ± 0.8 µm, sufficient to diffuse to the smallest vessel and still provide reasonable contrast. We also explored the use of Au nanoparticle colloids that could be used not only to enhance contrast but also for interesting applications in nanomedicine. Both the Lipiodol microemulsions and Au nanoparticle colloids can be conjugated with medicines or cell specific labeling agents and their small size can allow the study of the diffusion of contrast agents through the vessel leakage. This enables direct imaging of drug delivery which is important for cancer treatment. Figure
Keywords: Microradiology; Microangiography; Angiogenesis; Micro-emulsion; Gold nanoparticles; Contrast agent

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV by Jie Chen; Yunhao Yang; Xiaobo Zhang; Joy C. Andrews; Piero Pianetta; Yong Guan; Gang Liu; Ying Xiong; Ziyu Wu; Yangchao Tian (2117-2121).
Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples. Figure Absorption contrast tomographic results of five single cells with diameter of 2.5–3.5 μm progressed through different stages of the cell cycle. The first column is the projection X-ray image of each yeast cell. The second to fifth columns are reconstructed slices through the corresponding reconstruction data showing different regions of the cells. Each slice is about 30 nm thick. The sixth column contains the 3D renderings of the cells.
Keywords: X-ray microscopy; Tomography; Imaging; Schizosaccharomyces pombe ; Yeast subcellular structure

Synchrotron radiation FTIR imaging in minutes: a first step towards real-time cell imaging by C. Petibois; M. Cestelli-Guidi; M. Piccinini; M. Moenner; A. Marcelli (2123-2129).
FTIR microscopy with a focal plane array (FPA) of detectors enables routine chemical imaging on individual cells in only a few minutes. The brilliance of synchrotron radiation (SR) IR sources may enhance the signal obtained from such small biosamples containing small amounts of organic matter. We investigated individual cells obtained from a cell culture specifically developed for transmission FTIR imaging using either a Globar or an SR source coupled to the same instrumentation. SR-IR source focussing was optimized to control the energy distribution on the FPA of detectors. Here we show that accessing the IR absorption distribution from all the organic contents of cells at 1 × 1 μm pixel resolution was possible only with high circulating current (≥1.2 A) illuminating a limited number of the FPA’s detectors to increase the signal-to-noise ratio of IR images. Finally, a high-current SR ring is mandatory for collecting FTIR images of biosamples with a high contrast in minutes.
Keywords: Spectroscopy/instrumentation; IR spectroscopy/Raman spectroscopy; Cell systems/single-cell analysis; Synchrotron radiation; FTIR; Focal plane array; Cell imaging; Chemical analysis

X-ray diffraction microtomography (XRD-CT), a novel tool for non-invasive mapping of phase development in cement materials by G. Artioli; T. Cerulli; G. Cruciani; M. C. Dalconi; G. Ferrari; M. Parisatto; A. Rack; R. Tucoulou (2131-2136).
A recently developed synchrotron-based imaging technique, X-ray diffraction microtomography (XRD-CT), has been applied here for the first time to a complex system, the hydrating Portland cement paste, in order to monitor the evolution of microstructure and phase formation with a 3D non-invasive imaging approach. The ettringite-XRD-peak-based image reconstructions, combined with transmission microtomography (X-μCT) images, allowed to assess the ubiquitous distribution of this phase, which appears early in the hydration process and showed its preferential concentration in the relatively less compact regions of the paste. The comparison of greyscale histograms for cement pastes after 9 and 58 h from hydration showed an increase of ettringite content with age, in agreement with the quantitative Rietveld analysis of the sum patterns. By renormalizing the greyscale histograms to the relative weight fraction, as obtained from Rietveld refinements, a new technique which allows estimation of phase contents with spatial resolution has been developed. The results achievable by combining XRD-CT, X-μCT and Rietveld appear very promising to provide experimental snapshots of the cement hydration process to be compared with results obtained from computer simulations. Figure Experimental set-up of X-ray diffraction microtomography
Keywords: Powder diffraction; Synchrotron; Microtomography; Imaging; Portland cements; Rietveld

Analysis of polychromaticity effects in X-ray Talbot interferometer by Zhili Wang; Peiping Zhu; Wanxia Huang; Qingxi Yuan; Xiaosong Liu; Kai Zhang; Youli Hong; Huitao Zhang; Xin Ge; Kun Gao; Ziyu Wu (2137-2141).
The influence of polychromaticity of the X-ray source on the performance of an X-ray Talbot interferometer applied for phase-contrast imaging is analyzed through numerical simulations based on the Fresnel diffraction theory. The presented simulation results show that the visibility of the self-image is fairly insensitive to the source polychromaticity and explain why the interferometer could be well combined with polychromatic X-ray sources in recent experiments. Furthermore, the self-image with a high visibility can be obtained under polychromatic illumination even at a high-order fractional Talbot distance. This fact implies that the acquired image quality for phase measurements can be improved, since the primary signal for phase measurement is proportional to the inter-grating distance. Finally, we mention that the results are also valid for Talbot–Lau interferometer and scanning double-grating configuration.
Keywords: Polychromaticity; X-ray Talbot interferometer; Fractional Talbot distance; Self-image; Visibility

3D visualization of the microstructure of Quedius beesoni Cameron using micro-CT by Kai Zhang; De-e Li; Peiping Zhu; Qingxi Yuan; Wanxia Huang; Xiaosong Liu; Youli Hong; Gun Gao; Xin Ge; Hongzhang Zhou; Ziyu Wu (2143-2148).
The investigation of the internal morphology of insects is usually performed using classical microtomy yielding optical micrographs of stained thin sections. The achievement of high-quality cross sections for microtomy is time-consuming and the risk of damaging sections is unavoidable. Moreover, the approach is impractical, in particular when quick acquisition of 3D structural information is required. Recently, X-ray computed microtomography (micro-CT) with a high spatial resolution was considered as a potential tool for the morphological classification of insects. We used micro-CT to investigate Quedius beesoni Cameron at the cellular length scale. This method provides a new powerful and nondestructive approach to obtain 3D structural information on the biological organization of insects. The preliminary images presented in this contribution clearly reveal the endoskeleton and the muscles of the head and the thorax with a full 3D structure. We also reconstructed the 3D structure of the brain of Quedius beesoni Cameron, and this is the first reconstruction in Staphylinidae, which will be a great advancement for morphological and phylogenic research. We claim that both the spatial resolution and the contrast characteristic of micro-CT imaging may fulfill the requirements necessary for zoological insect morphology and phylogeny, in particular, when a classification of a rare and unique insect specimen is required.
Keywords: Tomography; Staphylinidae; Morphology; X-ray image

IMA 09—6th International Conference on Instrumental Methods of Analysis: Modern Trends and Applications by Maria Ochsenkühn-Petropoulou; Antony C. Calokerinos (2149-2150).
is Professor at the School of Chemical Engineering of the National Technical University of Athens (NTUA) and head of the Instrumental Methods of Analysis—Environment Group within the Laboratory of Inorganic and Analytical Chemistry. Since 1976 she has been Professor of Instrumental Methods of Analysis at the NTUA, teaching instrumental methods of analysis, environmental control and advanced inorganic chemistry to graduate and postgraduate students of the NTUA. Her research interests include speciation analysis, investigation of airborne particulate composition and of particulates emitted from vehicle catalysts, trace element analysis by voltammetric, spectrometric, and hyphenated chromatographic techniques, utilization of industrial byproducts in the laboratory and on a pilot-plant scale, and production and characterization of superconducting powders and coatings. She has published more than 200 scientific papers in international journals and conference proceedings, and has been the coordinator of more than 40 European and Greek research projects. She is the founder of the IMA International Conference series. is Professor of Analytical Chemistry in the Department of Chemistry of the National and Kapodistrian University of Athens. His current research interests include molecular emission analytical techniques, analytical chemiluminescence, flow techniques, separation techniques and natural products. He has authored more than 150 scientific papers and reviews in international journals. He is a member of the National Council of Education and the Council of Higher Academic Education.

The combined effect of the ion-pairing reagent concentration, C ipr, and organic modifier content, φ, on the retention under φ-gradient conditions at different constant C ipr was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D φ-gradient retention data obtained under the same φ-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C ipr values in the eluent. The second approach is based on a retention model describing the combined effect of C ipr and φ on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C ipr values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C ipr values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by φ-variation.
Keywords: Ion-pair reversed-phase chromatography; Isocratic-gradient retention prediction; Combined organic modifier and ion-pair concentration effect; Underivatized amino acids separation optimization

A validated method based on liquid chromatography/positive ion electrospray–mass spectrometry (LC-ESI/MS) is described for the quantification of perindopril and its active metabolite, perindoprilat, in human plasma. The assay was based on 500-μL plasma samples, following solid-phase extraction using Oasis HLB cartridges. All analytes and the internal standard (trandolapril) were separated by hydrophilic interaction liquid chromatography using a SeQuant Zic-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 μm, 200 Å) with isocratic elution. The mobile phase consisted of 10% 5.0 mM ammonium acetate water solution in a binary mixture of acetonitrile/methanol (60:40, v/v) and pumped at a flow rate of 0.10 mL min−1. Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 5.0–500.0 ng mL−1 for perindopril and perindoprilat. Intermediate precision were found less than 3.5% over the tested concentration ranges. A run time of less than 6.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method is the first reported application of HILIC in the analysis of angiotensin-converting enzyme inhibitors and can be used to quantify perindopril and perindoprilat in human plasma covering a variety of pharmacokinetic or bioequivalence studies.
Keywords: Hydrophilic interaction liquid chromatography (HILIC); Liquid chromatography/mass spectrometry; Perindopril; Perindoprilat; Trandolapril; Human plasma

Biomimetic chromatographic analysis of selenium species: Application for the estimation of their pharmacokinetic properties by Fotios Tsopelas; Anna Tsantili-Kakoulidou; Maria Ochsenkühn-Petropoulou (2171-2180).
The retention behavior of selenites, selenates, seleno-dl-methionine, selenocystine, selenocystamine, selenourea, dimethyl selenide, and dimethyl diselenide was investigated by means of biomimetic liquid chromatography. For this purpose, two immobilized artificial membrane (IAM) columns, namely, IAM.PC.DD2 and IAM.PC.MG, and two immobilized plasma protein columns, human serum albumin (HSA) and α1-acid glycoprotein (AGP) columns, were employed using different mobile phase conditions in respect to pH and buffer composition. In general, satisfactory interrelations between retention factors obtained with the two IAM stationary phases and HSA/AGP columns were obtained. Large differences were observed between biomimetic retention factors and octanol–water logD values, since the latter fail to describe electrostatic interactions. In contrast, despite the column diversity, the net retention outcome on all four biomimetic columns was quite similar, especially in the presence of phosphate-buffered saline, which by its effective shielding alleviates the differences between the stationary phases. Of the two IAM columns, IAM.PC.DD2 showed better performance when compared with HSA and AGP columns as well as to octanol–water partitioning. Biomimetic chromatographic indices were further used to estimate the percentage of human oral absorption and plasma protein binding of the eight selenium species investigated, according to equations previously reported in the literature. The estimated values of human oral absorption imply moderate absorption only for dimethyl diselenide, which also may exhibit considerable plasma protein binding. Moderate affinity for plasma proteins should also be expected for dimethyl selenide and selenocystamine. Figure Biomimetic chromatography in estimating pharmacokinetic properties of Se species
Keywords: Selenium speciation; Immobilized artificial membrane chromatography; Human serum albumin/α1-acid glycoprotein columns; Biomimetic chromatography; Oral absorption; Plasma protein binding

A multi-residue method for pesticide residue analysis in rice grains using matrix solid-phase dispersion extraction and high-performance liquid chromatography–diode array detection by Emmanouil D. Tsochatzis; Urania Menkissoglu-Spiroudi; Dimitrios G. Karpouzas; Roxani Tzimou-Tsitouridou (2181-2190).
Pesticides are widely used in rice cultivation, often resulting in detection of their residues in rice grains. So far, no analytical method has been available for the simultaneous determination of most rice pesticides in rice grains. This paper reports the development and validation of such a method for the determination of eight rice pesticides (penoxsulam tricyclazole, propanil, azoxystrobin, molinate, profoxydim, cyhalofop-butyl, deltamethrin) and 3,4-dichloroaniline, the main metabolite of propanil. Pesticide extraction and clean-up was performed by an optimized matrix solid-phase dispersion (MSPD) protocol on neutral alumina (5 g) using acetonitrile as the elution solvent. Samples were analyzed in a high-performance liquid chromatography–diode array detection (HPLC-DAD) system. Pesticide separation was achieved with a mobile phase of acetonitrile/water in a linear elution gradient from 30:70% (v/v) to 100:0% (v/v) in 14 min at a flow rate of 0.8 mL min−1. Method validation was performed by means of linearity, intra-day accuracy, inter-day precision and sensitivity. Linear regression coefficients (R 2) were always above 0.9948. Limits of detection (LOD) and quantification (LOQ) varied from 0.002 to 0.200 mg kg−1 and 0.006 to 0.600 mg kg−1, respectively. Recoveries were investigated at three fortification levels and were found to be acceptable (74–127%) with relative standard deviations (RSD) below 12%. Application of the method for the analysis of five commercial rice grain samples showed that the pesticide levels were below the LOD. Overall, the method developed is suitable for the determination of residues of most rice pesticides in rice grains at levels below the established MRLs.
Keywords: Rice pesticides; Rice grains; HPLC; Matrix solid-phase dispersion (MSPD)

A 13-min LC–MS method was developed for the determination of daptomycin, a new potent antibiotic, in peritoneal fluid, blood plasma, and urine of patients receiving renal replacement therapy. Chromatography was performed on a C18 column and detection was performed by a single-quadrupole mass spectrometer coupled to LC via an electrospray interface (ESI). The column effluent was also monitored at 370 nm using a photodiode-array detector. The developed method provided a linear dynamic range for concentrations from 0.5 μg mL−1 to 100 μg mL−1. Method precision and accuracy were found to be satisfactory for clinical application, thus the method was successfully used for the analysis of daptomycin in pharmacokinetic studies. The drug was preventively administered against Gram-positive infections to 19 clinical patients undergoing peritoneal dialysis. Peritoneal fluid, blood plasma, and urine samples were collected at 13 time points over a period of 48 h. Clinical samples were analysed following simple sample-preparation procedures and daptomycin was unambiguously detected and quantified.
Keywords: LC–MS; Daptomycin; Pharmacokinetics; Plasma; Peritoneal fluid; Urine; Peritoneal dialysis

A new hydrophilic interaction liquid chromatographic (HILIC) method for the simultaneous determination of isoascorbic (IAA) and ascorbic acid (AA) was developed. The separation of IAA and AA was studied in various HILIC stationary phases and the influence of the composition of the mobile phase, the ionic strength and the column temperature to the chromatographic characteristics is presented. The final method used an aminopropyl column under isocratic elution with acetonitrile–100 mM ammonium acetate solution (90:10, v/v) at a flow rate of 0.4 mL/min and a detection wavelength of 240 nm. This method was validated and the calibration curves were found to be linear in the range of 1.0–65 μg/mL for both IAA and AA. The method limit of detection for IAA determination in fish tissue was 2.3 μg/g. Inter-day precision (as %RSDR) was ranged between 0.56% and 8.3% at three concentration levels, whereas the respected recoveries ranged between 82% and 98%. This method was applied to the determination of IAA (as additive E315) in frozen redfish samples. The hyphenation of the HILIC separation with a tandem mass spectrometer was also studied and the problems encountered with negative electrospray ionization under HILIC separation conditions are discussed. Figure A new HILIC-UV method has been developed for the determination of isoascorbic acid in frozen redfish tissues
Keywords: HILIC; MS/MS; Erythorbic acid; Ascorbic acid; Food; E315; Isoascorbic acid

Capillary electrophoresis based on the principles of frontal analysis (CE-FA) was used to characterize the binding of flavonoids to human serum albumin (HSA) at near-physiological conditions: 67 mM phosphate buffer (pH 7.4), temperature 36.5 °C. The studied flavonoids (flavone, rutin, quercitrin) displayed moderate affinities toward the human serum albumin with binding constants in the range 103−104 M−1. The binding of the flavonoids to the protein noticeably depended on their lipophilicity and decreased in the case of glycosylation. The corresponding thermodynamic parameters characterized the acting forces between the HSA and flavonoids as mainly hydrophobic forces and electrostatic interactions. Based on the results of the displacement experiments, the binding of the flavonoids took place at site I of the HSA molecule. The results demonstrated by CE-FA were similar to those obtained by fluorescence spectroscopy. The developed method proved to be a reliable alternative to conventional methods, providing a lot of useful parameters for characterization of ligand–protein interactions. Figure Study of flavonoid-HSA interactions by capillary electrophoresis frontal analysis
Keywords: Capillary electrophoresis; Frontal analysis; Binding constant; Flavonoids; Human serum albumin

Solid-phase extraction for purification of alkannin/shikonin samples and isolation of monomeric and dimeric fractions by E. Noula; V. F. Samanidou; A. N. Assimopoulou; V. P. Papageorgiou; I. N. Papadoyannis (2221-2232).
Isohexenylnaphthazarins (IHN), commonly known as alkannins and shikonins (A/S), are potent pharmaceutical substances with a wide spectrum of wound healing, antimicrobial, anti-inflammatory, and antitumor activity. Purification of A/S is crucial for their use in pharmaceuticals and for biological experimentation. Dimeric and oligomeric A/S derivatives co-exist with the active monomeric ones in most of the samples produced either by (semi)-synthesis or biotechnologically or isolated from natural products. Oligomeric A/S derivatives have not been studied for biological activity hitherto and a method to isolate them is essential.In the present study, solid-phase extraction (SPE) was applied for purification of commercial samples and isolation of monomeric and oligomeric A/S fractions, testing several stationary phases. Sephadex LH-20 cartridges achieved efficient purification for commercial samples containing both monomeric and dimeric A/S derivatives and also separation and isolation of both pure monomeric and dimeric A/S fractions for biological experiments.A high-performance liquid chromatography-diode array detection method was applied for detection, identification and quantification of monomeric and oligomeric shikonin fractions.
Keywords: Alkannin; Shikonin; Solid-phase extraction; Naphthoquinones; Alkanna tinctoria ; Dimeric and trimeric quinones

Determination of selected pesticides in environmental water by employing liquid-phase microextraction and liquid chromatography–tandem mass spectrometry by Tatjana Trtić-Petrović; Jelena Ðorđević; Nikolina Dujaković; Ksenija Kumrić; Tatjana Vasiljević; Mila Laušević (2233-2243).
An optimised extraction and cleanup method for the analysis of pesticide in natural water samples is presented. Sixteen pesticides of different polarity and from the different chemical classes (organophosphates, triazines, benzimidazoles, carbamates, carbamides, neonicotinoides, methylureas, phenylureas and benzohydrazides), most frequently used in Serbia, were selected for the analysis. Liquid-phase microextraction in a single hollow fibre (HF-LPME) has been applied for sample preparation. The concentrations of pesticides were determined using HPLC-MS/MS method with electrospray ionisation. The extraction behaviour and selection of the experimental conditions was predicted based on log D and pK a values of targeted pesticides, which were calculated applying the computer software ACD/Labs PhysChem Suite v12. The influence of the donor pH and concentration of pesticides, organic phase composition as well as the extraction time on the extraction efficiency was investigated. Optimum extraction conditions were evaluated with respect to the investigated parameters of the extraction. The extraction method was validated for 10 out of 16 studied pesticides. Linear range of the pesticides was 0.1–5 µg L−1 with the correlation coefficient from 0.991 to 0.9998, and the relative standard deviation for three standard measurements was between 0.2 and 11.8%. The limits of detections ranged from 0.026 to 0.237 µg L−1 and the limits of quantifications from 0.094 to 0.793 µg L−1. The optimised two-phase HF-LPME method was successfully applied for determination of moderately polar as well low-polar pesticides in the environmental water samples.
Keywords: HPLC-MS/MS; Liquid-phase microextraction; Pesticides; Sample preparation

Development of a fast and selective method for the sensitive determination of anatoxin-a in lake waters using liquid chromatography–tandem mass spectrometry and phenylalanine-d 5 as internal standard by Ioannis K. Dimitrakopoulos; Triantafyllos S. Kaloudis; Anastasia E. Hiskia; Nikolaos S. Thomaidis; Michael A. Koupparis (2245-2252).
Anatoxin-a is a potent alkaloid neurotoxin produced by a number of cyanobacterial species and released in freshwaters during cyanobacterial blooms. Its high toxicity is responsible for several incidents of lethal intoxications of birds and mammals around the world; therefore anatoxin-a has to be regarded as a health risk and its concentration in lakes and water reservoirs should be monitored. Phenylalanine is a natural amino acid, also present in freshwaters, isobaric to anatoxin-a, with a very similar fragmentation pattern and LC retention. Since misidentification of phenylalanine as anatoxin-a has been reported in forensic investigations, special care must be taken in order to selectively determine traces of anatoxin-a in the presence of naturally occurring phenylalanine. A fast LC tandem MS method was developed by using a 1.8 μm 50 × 2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d 5 was employed as internal standard to compensate for electrospray ion suppression and sample preconcentration losses. Both compounds were preconcentrated 1,000-fold on a porous graphitic carbon solid-phase extraction (SPE) cartridge after adjustment of sample pH to 10.5. The method was validated by using lake water spiked at four different levels from 0.01 to 1 μg L−1. Anatoxin-a recovery ranged from 73 to 97%, intra-day precision (RSD%) ranged from 4.2 to 5.9, while inter-day precision (RSD%) ranged from 4.2 to 9.1%. Limits of detection and quantification were 0.65 and 1.96 ng L−1 respectively. The method was successfully applied for the detection of anatoxin-a in Greek lakes at concentrations ranging from less than 0.6 to 9.1 ng L−1. A novel LC-MS/MS method has been developed for the determination of anatoxin-a in lake waters during cyanobacterial blooms, using phenylalanine-d5 as internal standard
Keywords: Anatoxin-a; Cyanotoxins; Water analysis; LC-ESI-MS/MS; Isotope-labelled internal standard; Porous graphitic carbon SPE

This work deals with the design, synthesis, and characterization of a new solvatochromic dye. The intense solvatochromic behavior of this new synthesized non symmetric viologen was investigated using UV–Vis spectrophotometry. A further purpose was the study of the interactions between the solvent and solute molecules responsible for the solvatochromism. Several protic and aprotic solvents were used, and the resulting absorption maxima wavenumbers obtained via UV–Vis spectrophotometry, were correlated with the solvent polarity parameters, E T (30) (Dimroth–Reichardt solvent polarity parameter) and the Gutmann’s donor number (DN) using the biparametric model introduced by Krygowski and Fawcett. The analysis of the relative contribution of each parameter has clearly shown that the dominating interaction responsible for the solvatochromic behavior observed is the proton donation by the solute molecules to the solvent molecules, the latter acting as a Lewis bases. This is an interaction which can be described by DN. Additionally, the good correlation with the Kamlet–Taft parameter β is in good agreement. Figure The impact of solvent polarity and solvent basicity on the electronic absorption of a new synthesized viologen
Keywords: Solvatochromism; UV–Vis spectrophotometry; LSER’s; Viologens; Solvent–solute interactions

Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as ∼25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity.
Keywords: Protein sensing; Multiplexing; Encoded nanoparticle; Solution array; Fluorescent-conjugated polymer; Cancer marker detection

Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay by Min Jung Kim; Su Chul Lee; Seong Ho Kang; Jaebum Choo; Joon Myong Song (2271-2277).
A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m2). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.
Keywords: Oligonucleotide chip; Lesion-specific enzyme-induced break assay; Cyclobutane pyrimidine dimer; T4 endonuclease V

DNA phosphorylation is a vital process in the repair, replication, and recombination of nucleic acids. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive, simple, and economical method for DNA phosphorylation detection and T4 polynucleotide kinase (T4 PNK) activity assay based on marking DNA phosphorylation/biotinylation events by the attachment of fluorescent nanoparticles. Enzyme activity of T4 PNK is measured down to a limit of 5 × 10−6 U/ml, which is 400 times lower than previous reports. We also studied DNA phosphorylation specificity with different DNA substrates. Furthermore, T4 PNK inhibition by the inhibitor ADP and activation by the activator spermine are shown, demonstrating the potential for high-throughput screening for inhibitors and activators. Figure Sensitive detection of DNA phosphorylation is achieved by counting single nanoparticles, providing direct measurement of T4 PNK down to 5 x 10-6 U/mL, or 400 times lower than previous reports.
Keywords: DNA; Phosphorylation; Kinase activity; Single particle

Commercial phosgene dosimeter badges are lacking precise and sensitive analysis when used only by visual comparison to a color reference. To meet the discussed occupational standard set to 54 ppm min, objective quantification by reflectance measurement is proposed. At 573 nm, the pink dye (“Koenig´s salt”) formed at the membrane surface by reaction of phosgene, and aromatic amine shows a strict linear relationship in reflectance between doses of 10 and 300 ppm min. The detection limit is calculated to 29 ppm min.
Keywords: Phosgene; Dosimetry; Reflectance

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants by Iryna N. Berdichevets; Hristina R. Shimshilashvili; Iryna M. Gerasymenko; Yana R. Sindarovska; Yuriy V. Sheludko; Irina V. Goldenkova-Pavlova (2289-2293).
Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.
Keywords: PCR; Genetically modified plants; Reporter proteins; Thermostable lichenase; Fatty acid desaturases

The aim of this study was to develop an efficient, sensitive and reliable analytical method for the determination of traces of steroid hormones (including oestrogen, androgens and progestagens) and corticosteroids in soil. A method of sample preparation involving pressurized liquid extraction (PLE) and solid-phase extraction (SPE) was developed for the determination of six steroids and five corticosteroids in soils, followed by analysis by liquid chromatography-tandem mass spectrometry. The conditions employed for PLE involved acetone/methanol (50:50) as the extracting solvent, a temperature of 80 °C, two cycles and a static time of 5 min. The extraction was followed by a SPE clean-up based on a polymeric phase. With use of protocol, a residual matrix effect was, however, highlighted. The limit of detection in soil was 0.08–0.89 ng/g for steroids and 0.09–2.84 ng/g for corticosteroids.
Keywords: Soil; Emerging pollutants; Traces; Analytical method; Pressurized liquid extraction; Liquid chromatography-tandem mass spectrometry

In clinical and forensic toxicology, different extraction procedures as well as analytical methods are used to monitor different drug classes of interest in biosamples. Multi-analyte procedures are preferable because they make the analytical strategy much simpler and cheaper and allow monitoring of analytes of different drug classes in one single body sample. For development of such a multi-analyte liquid chromatography-tandem mass spectrometry approach, a rapid and simple method for the extraction of 136 analytes from the following drug classes has been established: antidepressants, neuroleptics, benzodiazepines, beta-blockers, oral antidiabetics, and analytes relevant in the context of brain death diagnosis. Recovery, matrix effects, and process efficiency were tested at two concentrations using six different lots of blank plasma. The recovery results obtained using absolute peak areas were compared with those calculated using area ratios analyte/internal standard. The recoveries ranged from 8% to 84% for antidepressants, from 10% to 79% for neuroleptics, from 60% to 81% for benzodiazepines, from 1% to 71% for beta-blockers, from 10% to 73% for antidiabetics, and from 60% to 86% for analytes relevant in the context of brain death diagnosis. With the exception of 52 analytes at low concentration and 37 at high concentration, all compounds showed recoveries with acceptable variability with less than 15% and 20% coefficients of variation. Recovery results obtained by comparing peak area ratios were nearly the same, but 35 analytes at low concentration and 17 at high concentration lay above the acceptance criteria. Matrix effects with more than 25% were observed for 18 analytes. The results were acceptable for 119 analytes at high concentrations.
Keywords: Liquid–liquid extraction; Recovery; Matrix effects; Internal standard; Multi-analyte LC-MS/MS method; Plasma

Discrimination of cancerous and non-cancerous cell lines by headspace-analysis with PTR-MS by C. Brunner; W. Szymczak; V. Höllriegl; S. Mörtl; H. Oelmez; A. Bergner; R. M. Huber; C. Hoeschen; U. Oeh (2315-2324).
Proton transfer reaction mass spectrometry (PTR-MS) has been used to analyze the volatile organic compounds (VOCs) emitted by in-vitro cultured human cells. For this purpose, two pairs of cancerous and non-cancerous human cell lines were selected:1. lung epithelium cells A-549 and retinal pigment epithelium cells hTERT-RPE1, cultured in different growth media; and 2. squamous lung carcinoma cells EPLC and immortalized human bronchial epithelial cells BEAS2B, cultured in identical growth medium. The VOCs in the headspace of the cell cultures were sampled: 1. online by drawing off the gas directly from the culture flask; and 2. by accumulation of the VOCs in PTFE bags connected to the flask for at least 12 h. The pure media were analyzed in the same way as the corresponding cells in order to provide a reference. Direct comparison of headspace VOCs from flasks with cells plus medium and from flasks with pure medium enabled the characterization of cell-line-specific production or consumption of VOCs. Among all identified VOCs in this respect, the most outstanding compound was m/z = 45 (acetaldehyde) revealing significant consumption by the cancerous cell lines but not by the non-cancerous cells. By applying multivariate statistical analysis using 42 selected marker VOCs, it was possible to clearly separate the cancerous and non-cancerous cell lines from each other. Figure The multivariate statistical analysis allowed to clearly separate the different cell lines from each other using 42 selected marker VOCs.
Keywords: Proton transfer reaction mass spectrometry; Volatile organic compounds; In vitro; Human cells

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification by Josep L. Acero Sanchez; Olivier Y. F. Henry; Teresa Mairal; Nadja Laddach; Anders Nygren; Siegfried Hauch; Jasmin Fetisch; Ciara K. O’Sullivan (2325-2334).
An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L−1, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.
Keywords: ELONA; MLPA; Rare tumour cell; mRNA expression; Breast cancer

Effect of hydrolysis on identifying prenatal cannabis exposure by Teresa R. Gray; Allan J. Barnes; Marilyn A. Huestis (2335-2347).
Identification of prenatal cannabis exposure is important due to potential cognitive and behavioral consequences. A two-dimensional gas chromatography–mass spectrometry method for cannabinol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 8β,11-dihydroxy-THC, and 11-nor-9-carboxy-THC (THCCOOH) quantification in human meconium was developed and validated. Alkaline, enzymatic, and enzyme–alkaline tandem hydrolysis conditions were optimized with THC- and THCCOOH-glucuronide reference standards. Limits of quantification ranged from 10 to 15 ng/g, and calibration curves were linear to 500 ng/g. Bias and intra-day and inter-day imprecision were <12.3%. Hydrolysis efficiencies were analyte-dependent; THC-glucuronide was effectively cleaved by enzyme, but not base. Conversely, THCCOOH-glucuronide was most sensitive to alkaline hydrolysis. Enzyme–alkaline tandem hydrolysis maximized efficiency for both glucuronides. Identification of cannabinoid-positive meconium specimens nearly doubled following alkaline and enzyme–alkaline hydrolysis. Although no 11-OH-THC glucuronide standard is available, enzymatic hydrolysis improved 11-OH-THC detection in authentic specimens. Maximal identification of cannabis-exposed neonates and the widest range of cannabis biomarkers are achieved with enzyme–alkaline tandem hydrolysis.
Keywords: Cannabinoids; Meconium; Hydrolysis; Glucuronide conjugates; β-glucuronidase

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys549 (KAm(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.
Keywords: Amadori product; Electrospray ionization (ESI); Glucose; Non-enzymatic glycosylation; Tandem mass spectrometry (MS/MS)

Separation and characterization of aggregated species of amyloid-beta peptides by Henning Wiberg; Patrik Ek; Frida Ekholm Pettersson; Lars Lannfelt; Åsa Emmer; Johan Roeraade (2357-2366).
We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Aβ) peptides from their aggregates. From solutions of Aβ1–40 or Aβ1–42 monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6–6.5. The formation of Aβ aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Aβ1–40 and Aβ1–42, respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Aβ1–42 peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Aβ-selective antibody mAb1C3, where a monomeric Aβ1–16 peptide was added to the protofibril preparation. Aβ1–16 is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
Keywords: Alzheimer’s disease; Protofibrils; Amyloid-beta (Aβ); Isoelectric focusing (IEF); Mass spectrometry (MS); Bioanalytical methods; Capillary electrophoresis / Electrophoresis

Evolving neural network optimization of cholesteryl ester separation by reversed-phase HPLC by Michael A. Jansen; Jacqueline Kiwata; Jennifer Arceo; Kym F. Faull; Grady Hanrahan; Edith Porter (2367-2374).
Cholesteryl esters have antimicrobial activity and likely contribute to the innate immunity system. Improved separation techniques are needed to characterize these compounds. In this study, optimization of the reversed-phase high-performance liquid chromatography separation of six analyte standards (four cholesteryl esters plus cholesterol and tri-palmitin) was accomplished by modeling with an artificial neural network–genetic algorithm (ANN-GA) approach. A fractional factorial design was employed to examine the significance of four experimental factors: organic component in the mobile phase (ethanol and methanol), column temperature, and flow rate. Three separation parameters were then merged into geometric means using Derringer’s desirability function and used as input sources for model training and testing. The use of genetic operators proved valuable for the determination of an effective neural network structure. Implementation of the optimized method resulted in complete separation of all six analytes, including the resolution of two previously co-eluting peaks. Model validation was performed with experimental responses in good agreement with model-predicted responses. Improved separation was also realized in a complex biological fluid, human milk. Thus, the first known use of ANN-GA modeling for improving the chromatographic separation of cholesteryl esters in biological fluids is presented and will likely prove valuable for future investigators involved in studying complex biological samples. Figure ANN-derived response surface plot for two interacting factors and overall response
Keywords: Lipids; Cholesteryl linoleate; Innate immunity; Biological fluids; Artificial neural networks; Genetic algorithms

We have developed a capillary electrophoresis (CE) method with universal fluorescent multiplex PCR to simultaneously detect the SMN1 and SMN2 genes in exons 7 and 8. Spinal muscular atrophy (SMA) is a very frequent inherited disease caused by the absence of the SMN1 gene in approximately 94% of patients. Those patients have deletion of the SMN1 gene or gene conversion between SMN1 and SMN2. However, most methods only focus on the analysis of whole gene deletion, and ignore gene conversion. Simultaneous quantification of SMN1 and SMN2 in exons 7 and 8 is a good strategy for estimating SMN1 deletion or SMN1 to SMN2 gene conversion. This study established a CE separation allowing differentiation of all copy ratios of SMN1 to SMN2 in exons 7 and 8. Among 212 detected individuals, there were 23 SMA patients, 45 carriers, and 144 normal subjects. Three individuals had different ratios of SMN1 to SMN2 in two exons, including an SMA patient having two SMN2 copies in exon 7 but one SMN1 copy in exon 8. This method could provide more information about SMN1 deletion or SMN1 to SMN2 gene conversion for SMA genotyping and diagnosis.
Keywords: Gene conversion; Spinal muscular atrophy; SMN1/SMN2 ; Universal fluorescent multiplex PCR; Capillary electrophoresis

Alignment of retention time obtained from multicapillary column gas chromatography used for VOC analysis with ion mobility spectrometry by Thorsten Perl; Bertram Bödeker; Melanie Jünger; Jürgen Nolte; Wolfgang Vautz (2385-2394).
Multicapillary column (MCC) ion mobility spectrometers (IMS) are increasingly in demand for medical diagnosis, biological applications and process control. In a MCC-IMS, volatile compounds are differentiated by specific retention time and ion mobility when rapid preseparation techniques are applied, e.g. for the analysis of complex and humid samples. Therefore, high accuracy in the determination of both parameters is required for reliable identification of the signals. The retention time in the MCC is the subject of the present investigation because, for such columns, small deviations in temperature and flow velocity may cause significant changes in retention time. Therefore, a universal correction procedure would be a helpful tool to increase the accuracy of the data obtained from a gas-chromatographic preseparation. Although the effect of the carrier gas flow velocity and temperature on retention time is not linear, it could be demonstrated that a linear alignment can compensate for the changes in retention time due to common minor deviations of both the carrier gas flow velocity and the column temperature around the MCC-IMS standard operation conditions. Therefore, an effective linear alignment procedure for the correction of those deviations has been developed from the analyses of defined gas mixtures under various experimental conditions. This procedure was then applied to data sets generated from real breath analyses obtained in clinical studies using different instruments at different measuring sites for validation. The variation in the retention time of known signals, especially for compounds with higher retention times, was significantly improved. The alignment of the retention time—an indispensable procedure to achieve a more precise identification of analytes—using the proposed method reduces the random error caused by small accidental deviations in column temperature and flow velocity significantly.
Keywords: Gas sensors; Chemical sensors; Gas chromatography; Bioanalytical methods

Halogenated phenols are widely used as biocides and are considered to be possibly carcinogenic to humans. In this report, a previously developed photoelectrochemical DNA sensor was employed to investigate DNA damage induced by tetra-halogenated quinones, the in vivo metabolites of halogenated phenols. The sensor surface was composed of a double-stranded DNA film assembled on a SnO2 semiconductor electrode. A DNA intercalator, Ru(bpy)2(dppz)2+, was allowed to bind to the DNA film and produce photocurrent upon light irradiation. After the DNA film was exposed to 300 μM tetrafluoro-1,4-benzoquinone (TFBQ), the photocurrent dropped by 20%. In a mixture of 300 μM TFBQ and 2 mM H2O2, the signal dropped by 40%. The signal reduction indicates less binding of Ru(bpy)2(dppz)2+ due to structural damage of ds-DNA in the film. Similar results were obtained with tetra-1,4-chlorobenzoquinone (TCBQ), although the signal was not reduced as much as TFBQ. Fluorescence measurement showed that TFBQ/H2O2 generated more hydroxyl radicals than TCBQ/H2O2. Gel electrophoresis proved that the two benzoquinones produced DNA strand breaks together with H2O2, but not by themselves. Using the photoelectrochemical sensor, it was also found that TCBQ covalently bound with DNA did not produce additional oxidative damage in the presence of H2O2. The combined photoelectrochemistry, gel electrophoresis, and fluorescence data revealed distinctive differences between TFBQ and TCBQ in terms of DNA adduct formation and hydroxyl radical generation. Figure
Keywords: DNA damage; Photoelectrochemistry sensor; Benzoquinone

Boronic acid lectin affinity chromatography (BLAC). 3. Temperature dependence of glycoprotein isolation and enrichment by Marcell Olajos; Ákos Szekrényes; Peter Hajos; Doug T. Gjerde; András Guttman (2401-2407).
In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5–65 °C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 °C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 °C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.
Keywords: Affinity chromatography; Glycoprotein enrichment; Temperature control

Reagent peak-free liquid chromatography–fluorescence analysis of carboxylic acids using a fluorous scavenging–derivatization method by Kenichiro Todoroki; Hiroki Hashimoto; Tomohiko Mikawa; Miki Itoyama; Tadashi Hayama; Eijiro Kojima; Hideyuki Yoshida; Hitoshi Nohta; Masatoshi Yamaguchi (2409-2416).
We developed a fluorous scavenging–derivatization method for reagent peak-free liquid chromatography (LC)–fluorescence analysis of carboxylic acids. In this method, carboxylic acids were fluorescently derivatized with 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and could be selectively removed by microfluorous solid-phase extraction before LC analysis. With use of this method, eight fluorescent derivatives of linear aliphatic carboxylic acids (C1–C8) can be separated within 30 min by reversed-phase LC with gradient elution. In the chromatogram obtained, the fluorous-tagged unreacted reagent peak is greatly decreased after microfluorous solid-phase extraction and does not interfere with the quantification of each acid. With use of microfluorous solid-phase extraction with 80% (v/v) aqueous methanol elution, over 99.9% of the unreacted fluorescent reagent was removed. The detection limits (signal-to-noise ratio of 3) for the carboxylic acids examined are 2.3–8.0 fmol per 10-μL injection. We also applied this method successfully to the analysis of highly polar carboxylic acids such as α-keto acids and tricarboxylic acid cycle metabolites.
Keywords: Liquid chromatography; Fluorous tag; Microfluorous solid-phase extraction; Fluorescence derivatization; Carboxylic acids

Oxidation of glycated phosphatidylethanolamines: evidence of oxidation in glycated polar head identified by LC-MS/MS by Cláudia Simões; Vanda Simões; Ana Reis; Pedro Domingues; M. Rosário M. Domingues (2417-2427).
Phosphatidylethanolamine glycation occurs in diabetic patients and was found to be related with oxidative stress and with diabetic complications. Glycated phosphatidylethanolamines seem to increase oxidation of other molecules; however, the reason why is not understood. In this work, we have studied the oxidation of glycated phosphatidylethanolamines (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylethanolamine (PLPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (dPPE)) using a Fenton system. Liquid chromatography–electrospray ionization (ESI)–mass spectrometry and ESI–tandem mass spectrometry in both positive and negative modes were used for detecting and identifying the oxidation products. We were able to identify several oxidation products with oxidation in unsaturated sn-2 acyl chain of PLPE, as long- and short-chain products with main oxidation sites on C-7, C-8, C-9, and C-12 carbons. Other products were identified in both glycated PLPE and glycated dPPE, revealing that oxidation also occurs in the glycated polar head. This fact has not been reported before. These products may be generated from oxidation of glycated phosphatidylethanolamines (PE) as Schiff base, leading to short-chain product without the amine moiety, due to cleavage of glycated polar head and long-chain product with two keto groups linked to the glycated polar head or from glycated PE as Amadori product, short-chain products with –NHCHO and –NHCHOHCHO terminal in polar head. Oxidation of glycated phosphatidylethanolamines occurred more quickly than the oxidation of non-glycated phosphatidylethanolamines probably because of the existence of more oxidation sites derived from glycation of polar head group. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients. Figure Glycated phosphatidylethanolamines (PEs) occur in diabetic patients and was found to be related with oxidative stress and with diabetic complications. LC-MS/MS allowed the identification, for the first time, that oxidation in glycated PEs also occurs in the glycated polar head, leading to the formation of short chain oxidation products due to cleavage in glycated moiety. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients
Keywords: Phospholipid; Phosphatidylethanolamines; Glycation; Oxidation; Mass spectrometry; LC-MS/MS

Vibrational and electronic characterisation of Staphylococcus aureus wall teichoic acids and relevant components in thin films by Florian Latteyer; Heiko Peisert; Nadine Göhring; Andreas Peschel; Thomas Chassé (2429-2437).
This work reports an investigation of S. aureus wall teichoic acid (WTA) and compares this biopolymer with its major occurring components, d-alanine and glycerol phosphate. Detailed insight into molecular structures and electronic properties is obtained by vibrational and photoemission spectroscopy. Calculations are performed to support the analysis of our experimental vibrational spectra. It is shown that there are contributions of positive and negative charges in WTAs, but the number of negative charges is expected to be higher. The presence of both positive and negative charges on WTA may offer a route for modification of surfaces with the objective of avoiding the formation of biofilms.
Keywords: FTIR; Photoemission; Raman; Staphylococcus aureus ; Wall teichoic acid

In this study, we investigated the feasibility of using a novel volatile organic compound (VOC)-based metabolic profiling approach with a newly devised chemometrics methodology which combined rapid multivariate analysis on total ion currents with in-depth peak deconvolution on selected regions to characterise the spoilage progress of pork. We also tested if such approach possessed enough discriminatory information to differentiate natural spoiled pork from pork contaminated with Salmonella typhimurium, a food poisoning pathogen commonly recovered from pork products. Spoilage was monitored in this study over a 72-h period at 0-, 24-, 48- and 72-h time points after the artificial contamination with the salmonellae. At each time point, the VOCs from six individual pork chops were collected for spoiled vs. contaminated meat. Analysis of the VOCs was performed by gas chromatography/mass spectrometry (GC/MS). The data generated by GC/MS analysis were initially subjected to multivariate analysis using principal component analysis (PCA) and multi-block PCA. The loading plots were then used to identify regions in the chromatograms which appeared important to the separation shown in the PCA/multi-block PCA scores plot. Peak deconvolution was then performed only on those regions using a modified hierarchical multivariate curve resolution procedure for curve resolution to generate a concentration profiles matrix C and the corresponding pure spectra matrix S. Following this, the pure mass spectra (S) of the peaks in those region were exported to NIST 02 mass library for chemical identification. A clear separation between the two types of samples was observed from the PCA models, and after deconvolution and univariate analysis using N-way ANOVA, a total of 16 significant metabolites were identified which showed difference between natural spoiled pork and those contaminated with S. typhimurium.
Keywords: VOC analysis; Pork; Salmonella typhimurium ; Peak deconvolution; Metabolic profiling

Trueness, precision, and detectability for sampling and analysis of organic species in airborne particulate matter by John M. Turlington; David A. Olson; Leonard Stockburger; Stephen R. McDow (2451-2463).
Recovery, precision, limits of detection and quantitation, blank levels, calibration linearity, and agreement with certified reference materials were determined for two classes of organic components of airborne particulate matter, polycyclic aromatic hydrocarbons and hopanes, using typical sampling and gas chromatography/mass spectrometry analysis methods. These determinations were based on initial method proficiency tests and on-going internal quality control procedures. Recoveries generally ranged from 75% to 85% for all target analytes and collocated sample precision estimates were generally better than 20% for polycyclic aromatic hydrocarbons and better than 25% for hopanes. Results indicated substantial differences in data quality between the polycyclic aromatic hydrocarbons and hopanes. Polycyclic aromatic hydrocarbons demonstrated better collocated precision, lower method detection limits, lower blank levels, and better agreement with certified reference materials than the hopanes. The most serious area of concern was the disagreement between measured and expected values in the standard reference material for hopanes. With this exception, good data quality was demonstrated for all target analytes on all other data quality indicators.
Keywords: Molecular marker; Source apportionment; Hopanes; Polycyclic aromatic hydrocarbons (PAHs); Particulate matter; Method proficiency

Rapid analysis of Gram-positive bacteria in water via membrane filtration coupled with nanoprobe-based MALDI-MS by Shuping Li; Zhongxian Guo; Hui-Fen Wu; Ying Liu; Zhaoguang Yang; Chee Hoe Woo (2465-2476).
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is challenging when it is directly applied to identify bacteria in water. This study demonstrates a rapid, sensitive, and selective technique for detection of Gram-positive bacteria in water. It involves a combination of membrane filtration (MF) and vancomycin-conjugated magnetite nanoparticles (VNPs) to selectively separate and concentrate Gram-positive bacteria in tap water and reservoir water, followed by rapid analysis of the isolates using whole-cell MALDI-MS. VNPs specifically recognize cells of Gram-positive bacteria, which serves as a basis for affinity capture of target Gram-positive bacteria. A two-step procedure of surface modification of bare magnetite nanoparticles was applied to synthesize VNPs. MF prior to VNP-based magnetic separation can effectively increase the enrichment factor and detection sensitivity and reduce time-consuming culture steps and the matrix effect for analysis of bacteria in MALDI-MS. The enrichment factor for the MF-VNP technique is about 6 × 104. A variety of bacteria, including Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, and Enterococcus faecium, were successfully analyzed from aqueous solutions and their mixtures with Gram-negative bacteria. The optimal conditions of the VNP/MALDI-MS technique, including selection of the MALDI matrix, the choice of cell-washing solution, and the VNP concentration, were also investigated. The capture efficiencies of Gram-positive bacteria with VNPs were 26.7–33.3%. The mass variations of characteristic peaks of the captured bacteria were within ±5 Da, which indicated good reproducibility of the proposed technique. The technique was applied to detect Gram-positive bacteria in tap water and reservoir water with an analysis time of around 2 h. The detection limit for Bacillus cereus, Enterococcus faecium, and Staphylococcus aureus was 5 × 102 cfu/ml for 2.0-l water samples. Figure Scheme of the MF-VNP/MALDI-MS technique to detect Gram-bacteria in water
Keywords: Bacteria; Matrix-assisted laser desorption/ionization mass spectrometry; Magnetic affinity separation; Functionalized nanoparticles; Water analysis; Membrane filtration

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry by Fulvia Caretti; Alessandra Gentili; Annalisa Ambrosi; Lucia Mainero Rocca; Maurizio Delfini; Maria Enrica Di Cocco; Giuseppe D’Ascenzo (2477-2490).
A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and 1H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.
Keywords: Glucocorticoids; Bovine milk; Electrospray; Triamcinolone; Matrix effect

Microscale imaging of the preservation state of 5,000-year-old archaeological bones by synchrotron infrared microspectroscopy by Ina Reiche; Matthieu Lebon; Céline Chadefaux; Katharina Müller; Anne-Solenn Le Hô; Michael Gensch; Ulrich Schade (2491-2499).
Archaeological bone materials record characteristic markers of life in prehistoric times (dating, climate, environment, diet, human migration) in their isotopic and chemical composition in addition to palaeontological, archaeozoological, anthropological and palaeogenetic information. Thus, the discovery and conservation of archaeological bone materials is of great importance to get access to this information. However, archaeological materials are altered by different postmortem processes and it appears necessary to estimate if the archaeological information is still reliable or if it has been modified during burial. As archaeological bone materials present a high structural hierarchy at the micro- and nanoscale, changes induced by diagenetic phenomena have to be observed at these scales. One method for revealing post mortem changes of the bone structure and composition at the microscale is synchrotron radiation micro-FTIR imaging (SR micro-FTIR). Thus, thin sections of about 5,000-year-old archaeological bones have been analysed in transmission mode at the IRIS beamline (BESSY II, HZB Berlin) to determine markers of the state of bone preservation at the microscale. The archaeological bone material comes from station 19 of the Neolithic site of the Chalain Lake. By using SR micro-FTIR it was possible to image characteristic bone structures, e.g. osteons (the constitutive histological unit of cortical bone), using the absorption band ratios corresponding to different chemical bone constituents (collagen content and quality, phosphate crystallinity, carbonate content). These data allow us to precisely evaluate the state of preservation of a 5,000-year-old bone at the histological level. Figure Chemical mapping of a thin section of the archaeological bone AB_CH19nb1 from the Neolithic station 19 at Chalain Lake
Keywords: Infrared spectroscopy; Mapping; Transmission mode; Archaeological bones; Diagenesis; Thin section

Neutron diffraction experiments have been performed on laboratory-prepared samples of copper alloys to determine their microscopic phase characteristics. The aim of this experiment is to set up a database that can be used in future neutron diffraction measurements on metal samples mainly of archaeological interest.
Keywords: Neutron diffraction; Copper alloys; Crystal structure; Phase analysis

Retention and selectivity properties of carbamate pesticides on novel polar-embedded stationary phases by Jesse O. Omamogho; Elaine M. Stack; Apichai Santalad; Supalax Srijaranai; Jeremy D. Glennon; Helen Yamen; Klaus Albert (2513-2524).
This study describes the use of stationary phases with polar functionality suitable for the chemical analysis of carbamates pesticides and comparing with conventional alkyl C8 and C18 phases. The emphasis of this study was to compare the selectivity and retention of the pesticides on different stationary phases, bonded onto 1.7 µm partially porous silica particles under isocratic separation condition. Four stationary phases including: phenylaminopropyl (PAP) phase, bidentate propylurea-C18 (BPUC18), C8 and C18, were successfully bonded on the partially porous silica spheres as evidenced by 29Si and 13C solid-state NMR analysis. The phenylaminopropyl phase exhibited smaller retentivity and enhanced selectivity compared to the alkyl C8 phase; the analysis time to run separation of the six carbamate pesticides (i.e., methomyl, propoxur, carbofuran, carbaryl, isoprocarb, and promecarb) on the PAP phase was threefold faster than alkyl C8 phase. In a similar manner, the BPUC18 phase shows similar selectivity to that of the PAP phase, but with longer retentivity; although the BPUC18 phase is characterized with a lesser degree of retentivity for the carbamate pesticides than the conventional alkyl C18 phase. We propose that π–π and weak polar interactions between the carbamate pesticides and the PAP phase dominates the separation mechanism and providing a superior selectivity; faster separation time was also achieved as a result of smaller retentivity. Whereas the C8 and C18 bonded phases exhibits only hydrophobic interactions with the pesticides, leading to larger retentivity. The BPUC18 phase is shown to interact via polar–polar interactions in addition to hydrophobic interactions with the pesticides, providing similar selectivity with the PAP phase but with larger retentivity. The Polar embedded phase studied (PAP) bonded onto core-shell silica particles and the chromatographic separation of six carbamate pesticides using the PAP phase.
Keywords: Polar-embedded phase; Seeded growth mesoporous shell; Core-shell silica; Carbamate pesticides

Application of the QuEChERS method for the analysis of pyrethrins and pyrethroids in fish tissues by Dorothea F. K. Rawn; Judy Judge; Veronica Roscoe (2525-2531).
The application of the quick easy cheap effective rugged and safe (QuEChERS) method has generally been used in the preparation of produce samples (e.g., lettuce and oranges) for pesticide analysis. In the present study, the QuEChERS method was successfully applied to the determination of the natural pyrethrins (cinerin I and II, jasmolin I and II, and pyrethrin I and II), as well as two pyrethroid insecticides, cypermethrin and deltamethrin, in fin and non-fin fish products. Analysis of these compounds was performed using gas chromatography–mass spectrometry. Although cypermethrin is not and has not been registered for pest control use in aquaculture operations in Canada, cypermethrin was detected in domestically produced salmon. Cypermethrin was detected in seven of the 18 Canadian farmed salmon samples (39%), although it was not detected in any wild domestic salmon (n = 3). Cypermethrin concentrations ranged from 0.3 to 6.5 ng/g in the positive samples. It was not, however, observed in any imported fish product or any other domestically produced fish product. Figure 20 ng/g standard prepared in salmon matrix
Keywords: QuEChERS; Pyrethroid; Pyrethrins; Fish; GC-MS; Matrix

Relevance of Sb(III), Sb(V), and Sb-containing nano-particles in urban atmospheric particulate matter by Silvia Canepari; Elisabetta Marconi; Maria Luisa Astolfi; Cinzia Perrino (2533-2542).
A quick and reliable analytical method for the separation and quantification of extractable Sb(III) and Sb(V) in atmospheric particulate matter (PM) by ion chromatography(IC)-inductively coupled plasma-mass spectrometry (ICP-MS) has been optimized, validated on pairs of real, equivalent PM10 samples and applied to a field monitoring campaign in a urban site. Both Sb(III) and Sb(V) forms were detected in real samples with Sb(III)/Sb(V) ratios up to 1.5. These two Sb species accounts only for a portion, of variable magnitude, of the total extractable Sb (10–70%); anyway, no other soluble Sb species were detected in the samples. The analysis of size-segregated samples collected by a 13-stage impactor showed that the recovery of [Sb(III) + Sb(V)] versus total extractable Sb is almost quantitative in the coarse fraction while it is below than 10% in the fine fraction. In the extracted solution from particles below 1 μm we could highlight the presence of Sb-containing suspended solid nano-particles, which probably constitute the missing fraction. The contribution of nano-particles can be estimated as the difference between ICP-MS and IC-ICP-MS data, as small size solid bodies are able to pass through the nebulizer and reach the plasma torch, while they are retained by the chromatographic column. The aggregation state of these nano-particles seems to be easily altered when they are suspended in a water solution; a similar behavior could be hypothesized when in contact with biological fluids. It has been confirmed that brake pad abrasion is the prevalent source of Sb(III) in PM and that Sb(V) may be formed by oxidation during the braking processes. Differing from other environmental matrices, there is no evidence of any spontaneous oxidative conversion within the two species. Figure Size distribution of [Sb(III) + Sb(V)] species determined by IC-ICP-MS compared to the total extractable Sb determined by ICP-MS. Differences have been attributed to Sb-containing nanoparticles
Keywords: Airborne particulate matter; Antimony; Nano-particles aggregates; Size-segregated samples; Ion chromatographic separation

A simple dispersive liquid–liquid microextraction based on solidification of floating organic droplet coupled with high-performance liquid chromatography–diode array detection was developed for the determination of five organophosphorus pesticides (OPs) in water samples. In this method, the extraction solvent used is of low density, low toxicity, and proper melting point near room temperature. The extractant droplet could be collected easily by solidifying it in the lower temperature. Some important experimental parameters that affect the extraction efficiencies were optimized. Under the optimum conditions, the calibration curve was linear in the concentration range from 1 to 200 ng mL−1 for the five OPs (triazophos, parathion, diazinon, phoxim, and parathion-methyl), with the correlation coefficients (r) varying from 0.9991 to 0.9998. High enrichment factors were achieved ranging from 215 to 557. The limits of detection were in the range between 0.1 and 0.3 ng mL−1. The recoveries of the target analytes from water samples at spiking levels of 5.0 and 50.0 ng mL−1 were 82.2–98.8% and 83.6–104.0%, respectively. The relative standard deviations fell in the range of 4.4% to 6.3%. The method was suitable for the determination of the OPs in real water samples.
Keywords: Dispersive liquid–liquid microextraction based on solidification of floating organic droplet; Organophosphorus pesticides; High-performance liquid chromatography; Water samples

Immunoassays have been regarded as a possible alternative or supplement for measuring polycyclic aromatic hydrocarbons (PAHs) in the environment. Since there are too many potential cross-reactants for PAH immunoassays, it is difficult to determine all the cross-reactivities (CRs) by experimental tests. The relationship between CR and the physical-chemical properties of PAHs and related compounds was investigated using the CR data from a commercial enzyme-linked immunosorbent assay (ELISA) kit test. Two quantitative structure-activity relationship (QSAR) techniques, regression analysis and comparative molecular field analysis (CoMFA), were applied for predicting the CR of PAHs in this ELISA kit. Parabolic regression indicates that the CRs are significantly correlated with the logarithm of the partition coefficient for the octanol-water system (log K ow) (r 2 = 0.643, n = 23, P < 0.0001), suggesting that hydrophobic interactions play an important role in the antigen-antibody binding and the cross-reactions in this ELISA test. The CoMFA model obtained shows that the CRs of the PAHs are correlated with the 3D structure of the molecules (r cv 2 = 0.663, r 2 = 0.873, F 4,32 = 55.086). The contributions of the steric and electrostatic fields to CR were 40.4 and 59.6%, respectively. Both of the QSAR models satisfactorily predict the CR in this PAH immunoassay kit, and help in understanding the mechanisms of antigen-antibody interaction. Figure Contour maps of CoMFA steric and electrostatic fields of phenanthrene
Keywords: Polycyclic aromatic hydrocarbons; Immunoassay; Cross-reactivity; Quantitative structure-activity relationship; Comparative molecular field analysis; Hapten

In-sample acetylation-non-porous membrane-assisted liquid–liquid extraction for the determination of parabens and triclosan in water samples by Eugenia Villaverde-de-Sáa; Iria González-Mariño; José Benito Quintana; Rosario Rodil; Isaac Rodríguez; Rafael Cela (2559-2568).
A procedure for the determination of seven parabens (esters of 4-hydroxybenzoic acid), including the distinction between branched and linear isomers of propyl- and butyl-parabens and triclosan in water samples, was developed and evaluated. The procedure includes in-sample acetylation-non-porous membrane-assisted liquid–liquid extraction and large volume injection–gas chromatography–ion trap–tandem mass spectrometry. Different derivatisation strategies were considered, i.e. post-extraction silylation with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and in situ acylation with acetic anhydride (Ac2O) and isobutylchloroformate. Moreover, acceptor solvent and the basic catalyser of the acylation reaction were investigated. Thus, in situ derivatisation with Ac2O and potassium hydrogenphosphate (as basic catalyser) was selected. Potassium hydrogenphosphate overcomes some drawbacks of other basic catalysers, e.g. toxicity and bubble formation, while leads to higher responses. Subsequently, other experimental variables affecting derivatisation–extraction yield such as pre-stirring time, salt addition and volume of Ac2O were optimised by an experimental design approach. Under optimised conditions, the proposed method achieved detection limits from 0.1 to 1.4 ng L−1 for a sample volume of 18 mL and extraction efficiencies, estimated by comparison with liquid–liquid extraction, between 46% (for methyl- and ethyl-parabens) and 110% (for benzylparaben). The reported sample preparation approach is free of matrix effects for parabens but affected for triclosan with a reduction of ≈ 40% when wastewater samples are analysed; therefore, both internal and external calibration can be used as quantification techniques for parabens, but internal standard calibration is mandatory for triclosan. The application of the method to real samples revealed the presence of these compounds in raw wastewater at concentrations up to 26 ng mL−1, the prevalence of the linear isomer of propylparaben (n-PrP), and the coexistence of the two isomers of butylparaben (i-BuP and n-BuP) at similar levels.
Keywords: Parabens; Triclosan; Polyethylene membranes; Membrane-assisted liquid–liquid extraction (MALLE); Membrane-assisted solvent extraction (MASE); Derivatisation; Gas chromatography–mass spectrometry (GC-MS); Water

Fish certified reference material (CRM), NMIJ CRM 7404-a, for the analysis of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) was developed by the National Metrology Institute of Japan, part of the National Institute of Advanced Industrial Science and Technology. Fish samples (Japanese seabass) used for the preparation of the CRM were collected from Tokyo Bay, and the edible part was freeze-dried, pulverized, sieved, homogenized, and sterilized by γ-irradiation. This sample is in the form of a powder comprising approximately 10 g stored in a brown glass bottle. The certification was carried out using multiple analytical methods such as pressurized liquid extraction, Soxhlet extraction, saponification, and homogenization to ensure the reliability of analytical results; the certified values of target PCBs (PCB 28, PCB 70, PCB 105, PCB 153, and PCB 170) and OCPs (trans-nonachlor, dieldrin, p,p′-DDE, p,p′-DDT, and p,p′-DDD) were 1.05–14.0 μg kg−1 and 1.57–18.0 μg kg−1 for PCBs and OCPs, respectively. This is the first fish powder CRM in which PCBs and OCPs were determined by isotope dilution mass spectrometry.
Keywords: Quality assurance/quality control; Certified reference material; Certification; Polychlorinated biphenyls (PCBs); Organochlorine pesticides (OCPs); Fish

In the present work, a strategy for the qualitative and quantitative analysis of 24 volatile compounds listed as suspected allergens in cosmetics by the European Union is reported. The list includes benzyl alcohol, limonene, linalool, methyl 2-octynoate, β-citronellol, geraniol, citral (two isomers), 7-hydroxycitronellal, anisyl alcohol, cinnamal, cinnamyl alcohol, eugenol, isoeugenol (two isomers), coumarin, α-isomethyl ionone, lilial®, α-amylcinnamal, lyral®, α-amylcinnamyl alcohol, farnesol (three isomers), α-hexyl cinnamal, benzyl cinnamate, benzyl benzoate, and benzyl salicylate. The applicability of a headspace (HS) autosampler in combination with a gas chromatograph (GC) equipped with a programmable temperature vaporizer (PTV) and a quadrupole mass spectrometry (qMS) detector is explored. By using a headspace sampler, sample preparation is reduced to introducing the sample into the vial. This reduces the analysis time and the experimental errors associated with this step of the analytical process. Two different injection techniques were used: solvent-vent injection and hot-split injection. The first offers a way to improve sensitivity at the same time maintaining the simple headspace instrumentation and it is recommended for compounds at trace levels. The use of a liner packed with Tenax-TA® allowed the compounds of interest to be retained during the venting process. The signals obtained when hot-split injection was used allowed quantification of all the compounds according to the thresholds of the European Cosmetics Directive. Monodimensional gas chromatography coupled to a conventional quadrupole mass spectrometry detector was used and the 24 analytes were separated appropriately along a run time of about 12 min. Use of the standard addition procedure as a quantification technique overcame the matrix effect. It should be emphasized that the method showed good precision and accuracy. Furthermore, it is rapid, simple, and—in view of the results—highly suitable for the determination of suspected allergens in different cosmetic products.
Keywords: Allergens; Headspace analysis; Programmed temperature vaporization; Cosmetic products

A sensitive and reliable method using capillary zone electrophoresis with UV-diode array detection has been developed and validated for trace determination of residues of sulfonylurea herbicides in environmental water samples and grapes from different origins. The analytes included are triasulfuron, rimsulfuron, flazasulfuron, metsulfuron-methyl, and chlorsulfuron. Optimum separation has been achieved on a 48.5-cm × 50-μm (effective length 40 cm) bubble cell capillary using 90 mM ammonium acetate buffer, pH 4.8, by applying a voltage of 20 kV at 25 °C and using p-aminobenzoic acid as the internal standard. In order to increase sensitivity, large volume sample stacking with polarity switching has been applied as on-line preconcentration methodology. For water samples, a solid-phase extraction (SPE) procedure based on the use of Oasis HLB cartridges was applied for off-line preconcentration and cleanup. For grape samples, the SPE procedure was achieved with C18 sorbent, after extraction of the compounds with MeOH:H2O (1:1) by sonication. The limits of detection for the studied compounds were between 0.04 and 0.12 μg/L for water samples and 0.97 and 8.30 μg/kg in the case of grape samples, lower in all cases than the maximum residue limits permitted by the EU for this kind of food. The developed methodology has demonstrated its suitability for the monitoring of these residues in environmental water and grape samples with high sensitivity, precision, and satisfactory recoveries.
Keywords: Capillary zone electrophoresis; Sample stacking; Water samples; Grapes; Sulfonylurea herbicides

Comparison of several chemometric techniques for the classification of orujo distillate alcoholic samples from Galicia (northwest Spain) according to their certified brand of origin by Roberto Iglesias Rodríguez; Manuel Fernández Delgado; Julia Barciela García; Rosa María Peña Crecente; Sagrario García Martín; Carlos Herrero Latorre (2603-2614).
This paper has a double objective. The first goal was to develop an authentication system to differentiate between traditional orujo alcoholic distillates with and without a certified brand of origin (CBO). Owing to their low price and quality, samples without a CBO can be used as substrates for falsification of genuine CBO ones. The second objective was to perform a comparison of the abilities of the different chemometric procedures employed for this classification. The classification was performed on the basis of the chemical information contained in the metal composition of the orujo distillates. Eight metals determined by electrothermal atomic absorption spectrometry and inductively coupled plasma optical emission spectrometry were considered (Ca, Cd, Cr, Cu, K, Mg, Na and Ni). After the appropriate pretreatment, the data were processed using different chemometric techniques. In the first stage, principal component analysis and cluster analysis were employed to reveal the latent structure contained in the data. Once it had been demonstrated that a relation exists between the metal composition and the raw materials, and not between the metal composition and the distillation systems employed for the orujo production, the second step consisted in the comparative application of different supervised pattern recognition procedures (such as linear discriminant analysis, K-nearest neighbours, soft independent modelling of class analogy, UNEQ and different artificial neural network approaches, including multilayer feed-forward, support vector machines, learning vector quantization and probabilistic neural networks). The results showed the different capabilities of the diverse classification techniques to discriminate between Galician orujo samples. The best results were those provided by probabilistic neural networks, in which the correct recognition abilities for CBO classes and without CBO classes were 98.6 ± 3.1 and 98.0 ± 4.5%; the prediction results were 87.7 ± 3.3 and 86.2 ± 5.0%, respectively. The usefulness of chemical metal analysis in combination with chemometric techniques to develop a classification procedure to authenticate Galician CBO orujo samples is demonstrated.
Keywords: Alcoholic distillates; Orujo ; Metals; Classification; Chemometrics

Determination of fluoroquinolone residues in poultry muscle in Portugal by A. Pena; L. J. G. Silva; A. Pereira; L. Meisel; C. M. Lino (2615-2621).
A total of 98 poultry samples, including chicken and turkey muscle, were analysed, using a sensitive and reliable analytical method based on liquid chromatography (LC) with spectrofluorimetric detection, for simultaneous determination of four fluoroquinolone (FQ) antibiotics, namely enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), and sarafloxacin (SARA). The method involved extraction with 0.15 mol L−1 HCl and clean-up by solid-phase extraction using Oasis HLB cartridges. Chromatographic separation was carried out on a C18 TSK gel column, in isocratic mode, with 0.025 mol L−1 H3PO4 solution, adjusted to pH 3.0 with tetrabutylammonium hydroxide-methanol (78:22) as mobile phase. Good linearity over the investigated concentration range was observed, with mean values of correlation coefficients higher than 0.9989 for all the analytes studied. The limits of quantification (LOQ), expressed as the lowest fortification level with acceptable precision were 15 μg kg−1 for ENRO, CIPRO, and NOR, and 30 μg kg−1 for SARA; these values are in compliance with requirements for monitoring of maximum residues levels (MRLs). Overall recoveries from spiked samples ranged from 80% to 92% with relative standard deviations (RSD) lower than 6.1%. Of the chicken and turkey samples analysed, 44.2% and 37.8%, respectively, were contaminated. The levels found in the analysed poultry samples, collected from markets of Oporto and Coimbra, located in the north and central zones of Portugal, respectively, were lower than 114.2 and 87.6 μg kg−1 in chicken and turkey muscle samples, respectively. One positive chicken sample was contaminated with ENRO at levels higher than the MRL.
Keywords: Fluoroquinolones; Chicken and turkey muscle tissue; Liquid chromatography; Spectrofluorimetric detection