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Analytical and Bioanalytical Chemistry (v.401, #4)

Oluwatosin O. Dada wins ABC best paper award by Andrea Pfeifer (pp. 1089-1090).

Molality–molarity challenge by Mahdi Hashemi; Thomas G. Chasteen (pp. 1091-1092).

Solution to Hofmeister effect challenge by Mahdi Hashemi; Thomas G. Chasteen (pp. 1093-1093).

Solution to radioactive Jeopardy challenge by Juris Meija (pp. 1095-1096).

Highlights of the 59th Conference on Mass Spectrometry and Allied Topics of the ASMS in Denver, Colorado by Stefanie Mädler (pp. 1097-1099).

On the nanostructure of micrometer-sized cellulose beads by Andreas F. Thünemann; Peter Klobes; Christoph Wieland; Stefano Bruzzano (pp. 1101-1108).

The analysis of the porosity of materials is an important and challenging field in analytical chemistry. The gas adsorption and mercury intrusion methods are the most established techniques for quantification of specific surface areas, but unfortunately, dry materials are mandatory for their applicability. All porous materials that contain water and other solvents in their functional state must be dried before analysis. In this process, care has to be taken since the removal of solvent bears the risk of an incalculable alteration of the pore structure, especially for soft materials. In the present paper, we report on the use of small-angle X-ray scattering (SAXS) as an alternative analysis method for the investigation of the micro and mesopores within cellulose beads in their native, i.e., water-swollen state; in this context, they represent a typical soft material. We show that even gentle removal of the bound water reduces the specific surface area dramatically from 161 to 109 m² g⁻¹ in cellulose bead sample type MT50 and from 417 to 220 m² g⁻¹ in MT100. Simulation of the SAXS curves with a bimodal pore size distribution model reveals that the smallest pores with radii up to 10 nm are greatly affected by drying, whereas pores with sizes in the range of 10 to 70 nm are barely affected. The SAXS results were compared with Brunauer–Emmett–Teller results from nitrogen sorption measurements and with mercury intrusion experiments. Figure Volume-weighted pore size distribution of wet and dry cellulose beads derived from small-angle X-ray scattering experiments using a bimodal pore structure model

Keywords: Small-angle X-ray scattering; Cellulose; Mesopores; Micropores; Porosimetry

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods by Jeroen Kool; Niels Jonker; Hubertus Irth; Wilfried M. A. Niessen (pp. 1109-1125).

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS. Figure Approach for analysis of protein complexes with MS

Keywords: Mass spectrometry; Protein–protein interactions; Chemical proteomics; affinity; Native mass spectrometry and interaction proteomics

Standardization of activated sludge for biodegradation tests by Gabriela A. Vázquez-Rodríguez; Rosa Icela Beltrán-Hernández; Claudia Coronel-Olivares; Jean-Luc Rols (pp. 1127-1137).

Activated sludges are an inoculum source commonly used in biodegradation studies, as wastewater treatment facilities constitute an entry point to the environment for many chemicals. In this paper, the main issues relating to the use of activated sludge in biodegradability tests are presented. Special attention is also devoted to discussing the factors affecting both the activity of the microbial communities and the test results. After a short survey of the state of the art of microbiology of activated sludge, the paper focuses on the methods used to reduce the variations in the diversity, quality and quantity of these communities. Finally, use of surrogates as reference materials in biodegradability tests is discussed. Figure Graphical abstract

Keywords: Bioassays; Bioanalytical methods; Sludge; Reference materials

Technological applications of chlorophyll a fluorescence for the assessment of environmental pollutants by K. Buonasera; M. Lambreva; G. Rea; E. Touloupakis; M. T. Giardi (pp. 1139-1151).

Chlorophyll a fluorescence has been extensively studied over the last few years. As demonstrated, this phenomenon is closely related to the state of photosystem II, which plays a leading role in the photosynthetic process, and therefore it has become a powerful tool to investigate this complex and any damage occurring in it as a result of physical or chemical stresses. This means that by using photosynthetic organisms as biological probes, one can consider chlorophyll a fluorescence as one of the techniques of choice to reveal the presence of some hazardous toxicants widely spread in the environment. Herbicides, pesticides, and heavy metals, whose concentration in water and food products is generally subject to extremely severe restrictions, are a concrete example of compounds detectable by chlorophyll a fluorescence. These dangerous substances react with the photosystem II, modifying the fluorescence emitted and giving responses which vary in a concentration-dependent manner. The possibility of performing easy, fast, and direct measurements of the fluorescence, even under light conditions, has opened new frontiers for the analysis in situ of pollutants. The aim of this review is to give an overview of the different techniques based on chlorophyll a fluorescence spectrometry, focusing in particular on those which represented the starting point for applications addressed to the assessment of toxic compounds in environmental samples.

Keywords: Photosynthetic proteins; Environmental monitoring; Herbicides; Heavy metals; Pesticides; Biosensors

Simultaneous measurement of endogenous cortisol, cortisone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in nails by use of UPLC–MS–MS by Mehdi Ben Khelil; Marion Tegethoff; Gunther Meinlschmidt; Carole Jamey; Bertrand Ludes; Jean-Sébastien Raul (pp. 1153-1162).

Steroid hormone concentrations are mostly determined by using different body fluids as matrices and applying immunoassay techniques. However, usability of these approaches may be restricted for several reasons, including ethical barriers to invasive sampling. Therefore, we developed an ultra-performance LC–MS–MS method for high-throughput determination of concentrations of cortisol, cortisone, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEAS) in small quantities of human nails. The method was validated for linearity, limits of detection and quantification, recovery, intra and interassay precision, accuracy, and matrix effect. Samples from 10 adult women were analyzed to provide proof-of-principle for the method’s applicability. Calibration curves were linear (r ² > 0.999) in the ranges 10–5000 pg mg⁻¹ for cortisol, cortisone, and DHEAS, and 50–5000 pg mg⁻¹ for DHEA. Limits of quantification were 10 pg mg⁻¹ for cortisol, cortisone, and DHEAS, and 50 pg mg⁻¹ for DHEA. The sensitivity and specificity of the method were good, and there was no interference with the analytes. Mean recovery of cortisol, cortisone, DHEA, and DHEAS was 90.5%, 94.1%, 84.9%, and 95.9%, respectively, with good precision (coefficient of variation <14% for all analytes) and accuracy (relative error (%) −8.3% to 12.2% for all analytes). The median (pg mg⁻¹, range) hormone concentrations were 69.5 (36–158), 65 (32–133), 212 (50–1077), and 246 (115–547) for cortisol, cortisone, DHEA, and DHEAS, respectively. This method enables measurement of cortisol, cortisone, DHEA, and DHEAS in small quantities of human nails, leading to the development of applications in endocrinology and beyond.

Keywords: Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS); Hypothalamic–pituitary–adrenal (HPA) axis; Keratin tissue; Steroid

LC-MS/MS analysis of phosphatidylethanol in dried blood spots versus conventional blood specimens by Andrea Faller; Barbara Richter; Matthias Kluge; Patrick Koenig; Helmut K. Seitz; Annette Thierauf; Heike Gnann; Michaela Winkler; Rainer Mattern; Gisela Skopp (pp. 1163-1166).

Phosphatidylethanol (PEth), which is formed extrahepatically by the action of phospholipase D on phosphatidylcholine in the presence of ethanol, has been suggested as a promising marker of alcohol misuse. Analysis of dried blood spots (DBS) is particularly advantageous for the determination of delicate analytes such as PEth. Therefore, measurement of PEth species (18:1/18:1, 16:0/18:1) in DBS versus whole blood was performed to ascertain whether respective results are directly comparable. Samples were obtained from subjects (n = 40) undergoing alcohol detoxification treatment. Analysis involved liquid–liquid extraction from both, DBS and whole blood (100 μL, respectively), with phosphatidylpropanol as the internal standard. Extracts were subjected to LC gradient separation using multiple reaction monitoring of deprotonated molecules. Results from measurements of corresponding DBS and whole blood specimens were compared by estimating the respective mean values and by a Bland and Altman analysis. Concentrations of PEth 18:1/18:1 ranged from 46.1 to 3,360 ng/mL in whole blood (mean, 461.7 ng/mL) and from 35.8 to 3,360 ng/mL in DBS (mean, 457.6 ng/mL); for PEth 16:0/18:1, concentrations were from 900 to 213,000 ng/mL (mean, 23,375 ng/mL) and 922–213,000 ng/mL (mean, 23,470 ng/mL) in blood and DBS, respectively. Estimated mean differences were −4.3 ng/mL for PEth 18:1/18:1 and 95.8 ng/mL for PEth 16:0/18:1. The Bland–Altman plot of both PEth species showed that the variation around the mean difference was similar all through the range of measured values and that all differences except one were within the limits of agreement. It could be shown that the determination of PEth species in DBS is as reliable as in whole blood samples. This assay may facilitate monitoring of alcohol misuse.

Keywords: Dried blood spots; Whole blood; Phosphatidylethanol; LC-MS/MS

A gas sensor based on Prussian blue film for the detection of chlorobenzene vapor by Tiexiang Fu (pp. 1167-1172).

A new resistance-type sensor based on Prussian blue film has been fabricated for the detection of chlorobenzene vapor. The effect of Prussian blue preparation conditions on the response of sensor was studied. The sensor exhibited good response and selectivity to chlorobenzene vapor. The sensor prepared with Fe₂(SO₄)₃ at 298 K has response 8.5 at operating voltage of 10 V. The selectivity of the sensor to chlorobenzene against all other tested gases is exceeding almost by 5.6 times. The sensor showed linear response to chlorobenzene vapor in the concentration range of 24–169 ppm at room temperature and at a 10 V operating voltage. The response and recovery time of the sensor was about 18 and 12 s, respectively. Sensor stability test indicated the sensor had a good stability. Furthermore, seven real samples of chlorobenzene vapor was measured using the sensor. The relative error was in the range of about ±1.3%. Figure Response to different gases of the sensor based on Prussian blue prepared with Fe₂(SO₄)₃ at 298 K

Keywords: Chlorobenzene; Prussian blue; Gas sensor; Thin film

Multiplex ELISA in a single microfluidic channel by Naoki Yanagisawa; James O. Mecham; Robert C. Corcoran; Debashis Dutta (pp. 1173-1181).

In this article, we demonstrate a novel approach to implementing multiplex enzyme-linked immunosorbent assay (ELISA) in a single microfluidic channel by exploiting the slow diffusion of the soluble enzyme reaction product across the different assay segments. The functionality of the reported device is realized by creating an array of ELISA regions within a straight conduit that are selectively patterned with chosen antibodies/antigens via a flow-based method. The different analytes are then captured in their respective assay segments by incubating a 5-μL aliquot of sample in the analysis channel for an hour under flow conditions. Once the ELISA surfaces have been prepared and the enzyme substrate introduced into the analysis channel, it is observed that the concentration of the soluble enzyme reaction product (resorufin) at the center of each assay region grows linearly with time. Further, the rate of resorufin generation at these locations is found to be proportional to the concentration of the analyte being assayed in that segment provided that the ELISA reaction time in the system (τ R) is kept much shorter than that required by the resorufin molecules to diffuse across an assay segment (τ D). Under the operating condition τ R << τ D, the reported device has been shown to have a 35% lower limit of detection for the target analyte concentration compared with that on a commercial microtiter plate using only a twentieth of the sample volume.

Keywords: Multiplex; ELISA; Viral antibodies; Microfluidic; Diffusion

Quantification of the six major α-dicarbonyl contaminants in peritoneal dialysis fluids by UHPLC/DAD/MSMS by Stefan Mittelmaier; Michael Fünfrocken; Dominik Fenn; Robert Berlich; Monika Pischetsrieder (pp. 1183-1193).

During heat sterilization of peritoneal dialysis solutions, glucose is partially transformed into glucose degradation products (GDPs), which significantly reduce the biocompatibility of these medicinal products. Targeted α-dicarbonyl screening identified glyoxal, methylglyoxal, 3-deoxyglucosone, 3,4-dideooxyglucosone-3-ene, glucosone, and 3-deoxygalactosone as the major six GDPs with α-dicarbonyl structure. In the present study, an ultra-high-performance liquid chromatography method was developed which allows the separation of all relevant α-dicarbonyl GDPs within a run time of 15 min after derivatization with o-phenylenediamine. Hyphenated diode array detection/tandem mass spectrometry detection provides very robust quantification and, at the same time, unequivocal peak confirmation. Systematic evaluation of the derivatization process resulted in an optimal derivatization period that provided maximal derivatization yield, minimal de novo formation (uncertainty range ±5%), and maximal sample throughput. The limit of detection of the method ranged from 0.13 to 0.19 μM and the limit of quantification from 0.40 to 0.57 μM. Relative standard deviations were below 5%, and recovery rates ranged between 91% and 154%, dependent on the type and concentration of the analyte (in 87 out of 90 samples, recovery rates were 100 ± 15%). The method was then applied for the analysis of commercial peritoneal dialysis fluids (nine different product types, samples from three lots of each). Figure A novel UHPLC/DAD/MSMS method allows the quantification of the six major α-dicarbonyl contaminants in peritoneal dialysis fluids

Keywords: Glucose degradation products; Peritoneal dialysis fluids; UHPLC; α-Dicarbonyl compounds; Tandem mass spectrometry; 3,4-DGE

Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface by Anna Laura Capriotti; Giulio Caracciolo; Chiara Cavaliere; Carlo Crescenzi; Daniela Pozzi; Aldo Laganà (pp. 1195-1202).

The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography–high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids.

Keywords: Cationic liposomes; Mass spectrometry; Nanoparticles; Plasma; Protein corona

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides by Gunnar Schwarz; Sebastian Beck; Michael G. Weller; Michael W. Linscheid (pp. 1203-1209).

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.

Keywords: Quantitative proteomics; Mass spectrometry; Metal labeling; Stable isotope labeling; Lanthanide DOTA

Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane by Md. Golam Azam; Takayuki Shibata; Tsutomu Kabashima; Masaaki Kai (pp. 1211-1217).

In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27–31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14–20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays. Figure A Schematic principle for the chemiluminescence (CL) detection of biotin-conjugated anti-rabbit IgG antibody (b-Ab) on a solid-phase membrane by using a dextran (Dex)-biotin (Bio)-ALP probe, B CL-imaging detection of Dex-Bio-ALP probes and ALP adsorbed on a nylon membrane, and C CL-imaging detection of b-Ab on a nylon membrane using Dex-Bio₁₂₀-ALP₂₈ probe. Amounts (femtomole) of b-Ab per spot: a ₁ = 0.32, a ₂ = 0.63, a ₃ = 1.25, a ₄ = 2.5, a ₅ = 5, and a ₆ = 10

Keywords: Chemiluminescence detection; Dextran probe; Alkaline phosphatase; Protein; Solid-phase membrane

Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins by Yu-Min Juang; Chao-Jung Chen; Chien-Chen Lai (pp. 1219-1229).

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples. Figure On-tape-based phosphoprotein preparation and digestion under microwave heating.

Keywords: Carbon tape; Microwave-assisted protein preparation and digestion; Protein identification; Phosphopeptide; MALDI; Enrichment

Combined Raman and IR spectroscopic study on the radical-based modifications of methionine by A. Torreggiani; S. Barata-Vallejo; C. Chatgilialoglu (pp. 1231-1239).

Among damages reported to occur on proteins, radical-based changes of methionine (Met) residues are one of the most important convalent post-translational modifications. The combined application of Raman and infrared (IR) spectroscopies for the characterisation of the radical-induced modifications of Met is described here. Gamma-irradiation was used to simulate the endogenous formation of reactive species such as hydrogen atoms (^•H), hydroxyl radicals (^•OH) and hydrogen peroxide (H₂O₂). These spectroscopic techniques coupled to mass experiments are suitable tools in detecting almost all the main radical-induced degradation products of Met that depend on the nature of the reactive species. In particular, Raman spectroscopy is useful in revealing the radical-induced modifications in the sulphur-containing moiety, whereas the IR spectra allow decarboxylation and deamination processes to be detected, as well as the formation of other degradation products. Thus, some band patterns useful for building a library of spectra–structure correlation for radical-based degradation of Met were identified. In particular, the bands due to the formation of methionine sulfoxide, the main oxidation product of Met, have been identified. All together, these results combine to produce a set of spectroscopic markers of the main processes occurring as a consequence of radical stress exposure, which can be used in a spectroscopic protocol for providing a first assessment of Met modifications in more complex systems such as peptides and proteins, and monitoring their impact on protein structure. Figure The combined use of Raman and IR spectroscopy allows to monitor the formation of the main degradation products of amino acids like methionine after radical stress exposure. In particular, Raman spectra are useful for revealing the occurrence of modifications in sulphur-containing moiety, whereas IR spectroscopy is able to detect decarboxylation and deamination processes, as well as the formation of new products

Keywords: Methionine; Radical stress; Raman spectroscopy; IR spectroscopy; Gamma-radiolysis

Sensoric potential of gold–silver core–shell nanoparticles by Andrea Steinbrück; Ondrej Stranik; Andrea Csaki; Wolfgang Fritzsche (pp. 1241-1249).

The sensitivities of five different core–shell nanostructures were investigated towards changes in the refractive index of the surrounding medium. The shift of the localized surface plasmon resonance (LSPR) maximum served as a measure of the (respective) sensitivity. Thus, gold–silver core–shell nanoparticles (NPs) were prepared with different shell thicknesses in a two-step chemical process without the use of any (possibly disturbing) surfactants. The measurements were supported by ultramicroscopic images in order to size the resulting core–shell structures. When compared to sensitivities of nanostructures reported in the literature with those of the (roughly spherical) gold–silver core–shell NPs, the latter showed comparable (or even higher) sensitivities than gold nanorods. The experimental finding is supported by theoretical calculation of optical properties of such core–shell NP. Extinction spectra of ideal spherical and deformed core–shell NPs with various core/shell sizes were calculated, and the presence of an optimal silver shell thickness with increased sensitivity was confirmed. This effect is explained by the existence of two overlapping plasmon bands in the NP, which change their relative intensity upon change of refractive index. Results of this research show a possibility of improving LSPR sensor by adding an extra metallic layer of certain thickness. Figure Figure: Left TEM image of gold-silver core-shell nanoparticle, Right Two images of E-field distribution in the core-shell nanoparticle at different excitation wavelengths.

Keywords: Core–shell nanoparticles; Gold–silver nanoparticles; Localized surface plasmon resonance; Biosensor; Sensing

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere by Xiajuan Zou; Dan Liu; Lijun Zhong; Bin Yang; Yaxin Lou; Baihe Hu; Yuxin Yin (pp. 1251-1261).

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan–glycidyl methacrylate (GMA)–iminodiacetic acid (IDA)–Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe³⁺-IMAC and TiO₂, these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe³⁺ nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS. Figure A method for highly specific enrichment of phosphopeptides with novel multifunctional chitosan-GMA-IDA-Fe (III) nanospheres for direct analysis by matrix assisted laser desorption-ionization mass spectrometry (MALDI MS).

Keywords: Chitosan-GMA-IDA-Fe (III); Phosphopeptide enrichment; Nanosphere; MALDI-MS; Ferrum ion; Immobilized metal ion affinity chromatography

Determination of the concentration of nitrogenous bio-organic compounds using an isotope ratio mass spectrometer operating in continuous flow mode by Illa Tea; Katarzyna Kosieradzka; Ingrid Antheaume; Emmanuel Gentil; Richard J. Robins (pp. 1263-1271).

The quality of the determination of compound-specific isotopic content at natural abundance by gas chromatography–isotope ratio measurement–mass spectrometry (GC-irm-MS) relies on the stability of the voltage generated by the ion detector Faraday cages. The application of GC-irm-MS to the determination of δ¹³C (‰) and δ¹⁵N (‰) is now routine. However, for numerous applications, it is necessary to determine both the isotope content (δ¹⁵N) and the quantity (in micromoles) of analyte present. We now show that it is possible for nitrogen-containing compounds to measure how much analyte is present with an irm mass spectrometer linked to a GC by exploiting the integrated N₂ total ion current intensity (Vs) generated by measuring the ¹⁵N/¹⁴N isotope ratio. The method is validated over a range of concentration (2–70 mmol/L) and δ¹⁵N (−70 to +50‰) values for six molecules of diverse chemical nature and functionality (nortropine, norpseudotropine, nortropinone, cysteine, taurine, glutathione). It is shown that once the ion current is calibrated, the quantitative values are of a comparable quality to those obtained from GC with flame ionization detection (GC-FID). In addition, it is demonstrated that over a definable range, the δ¹⁵N (‰) value is independent of the quantity of analyte introduced, confirming the validity of this method.

Keywords: Quantification; Concentration determination; Isotope ratio mass spectrometry; Nitrogen isotope ratio; Bio-organic compounds

Direct quantification of cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry by David M. Schwope; Karl B. Scheidweiler; Marilyn A. Huestis (pp. 1273-1283).

The first method for quantifying cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid-phase extraction followed protein precipitation with acetonitrile. High-performance liquid chromatography separation was achieved in 16 min via gradient elution. Electrospray ionization was utilized for cannabinoid detection; both positive (Δ⁹-tetrahydrocannabinol [THC] and cannabinol [CBN]) and negative (11-hydroxy-THC [11-OH-THC], 11-nor-9-carboxy-THC [THCCOOH], cannabidiol [CBD], THC-glucuronide, and THCCOOH-glucuronide) polarity were employed with multiple reaction monitoring. Calibration by linear regression analysis utilized deuterium-labeled internal standards and a 1/x ² weighting factor, yielding R ² values >0.997 for all analytes. Linearity ranged from 0.5 to 50 μg/L (THC-glucuronide), 1.0–100 μg/L (THC, 11-OH-THC, THCCOOH, CBD, and CBN), and 5.0–250 μg/L (THCCOOH-glucuronide). Imprecision was <10.5% CV, recovery was >50.5%, and bias within ±13.1% of target for all analytes at three concentrations across the linear range. No carryover and endogenous or exogenous interferences were observed. This new analytical method should be useful for quantifying cannabinoids in whole blood and further investigating cannabinoid glucuronides as markers of recent cannabis intake. Figure LC–MS/MS MRM ion chromatograms of extracted blank whole blood and cannabinoid analytes at limits of quantification

Keywords: THC; Cannabinoid glucuronides; LC–MS/MS; Whole blood

Immunochemical analysis of 3-phenoxybenzoic acid, a biomarker of forestry worker exposure to pyrethroid insecticides by Ki Chang Ahn; Shirley J. Gee; Hee-Joo Kim; Pavel A. Aronov; Helen Vega; Robert I. Krieger; Bruce D. Hammock (pp. 1285-1293).

Pyrethroid insecticides widely used in forestry, agricultural, industrial, and residential applications have potential for human exposure. Short sample preparation time and sensitive, economical high-throughput assays are needed for biomonitoring studies that analyze a large number of samples. An enzyme-linked immunosorbent assay (ELISA) was used for determining 3-phenoxybenzoic acid (3-PBA), a general urinary biomarker of exposure to some pyrethroid insecticides. A mixed-mode solid-phase extraction reduced interferences from acid hydrolyzed urine and gave 110 ± 6% recoveries from spiked samples. The method limit of quantification was 2 μg/L. Urine samples were collected from forestry workers that harvest pine cone seeds where pyrethroid insecticides were applied at ten different orchards. At least four samples for each worker were collected in a 1-week period. The 3-PBA in workers classified as high, low, or no exposure based on job analysis over all sampling days was 6.40 ± 9.60 (n = 200), 5.27 ± 5.39 (n = 52), and 3.56 ± 2.64 ng/mL (n = 34), respectively. Pair-wise comparison of the differences in least squares means of 3-PBA concentrations among groups only showed a significant difference between high and no exposure. Although this difference was not significant when 3-PBA excretion was normalized by creatinine excretion, the general trend was still apparent. No significant differences were observed among days or orchards. This ELISA method using a 96-well plate was performed as a high-throughput tool for analyzing around 300 urine samples measured in triplicate to provide data for workers exposure assessment.

Keywords: Pyrethroid insecticide; Biomonitoring; Immunoassay; Forestry workers

Collection and identification of human remains volatiles by non-contact, dynamic airflow sampling and SPME-GC/MS using various sorbent materials by Lauryn E. DeGreeff; Kenneth G. Furton (pp. 1295-1307).

Human remains detection canines are used in locating deceased humans in diverse scenarios and environments based on odor produced during the decay process of the human body. It has been established that human remains detection canines are capable of locating human remains specifically, as opposed to living humans or animal remains, thus suggesting a difference in odor between the different sources. This work explores the collection and determination of such odors using a dynamic headspace concentration device. The airflow rate and three sorbent materials—Dukal cotton gauze, Johnson & Johnson cotton-blend gauze, and polyester material—used for odor collection were evaluated using standard compounds. It was determined that higher airflow rates and openly woven material, e.g., Dukal cotton gauze, yielded significantly less total volatile compounds due to compound breakthrough through the sorbent material. Collection from polymer- and cellulose-based materials demonstrated that the molecular backbone of the material is a factor in compound collection as well. Volatiles, including cyclic and straight-chain hydrocarbons, organic acids, sulfides, aldehydes, ketones, and alcohols, were collected from a population of 27 deceased bodies from two collection locations. The common compounds between the subjects were compared and the odor profiles were determined. These odor profiles were compared with those of animal remains and living human subjects collected in the same manner. Principal component analysis showed that the odor profiles of the three sample types were distinct.

Keywords: Forensic/toxicology; Human remains; Odor; Detection canines; Odor sampling

C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP by Min S. Wang; Joshua C. Black; Michelle K. Knowles; Scott M. Reed (pp. 1309-1318).

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease. Figure Evidence for mCRP selectivity of aptamer by gel electrophoresis, fluorescence anisotropy, and TIRF microscopy

Keywords: C-reactive protein; Isoform; Aptamer; Cardiovascular disease; TIRF microscopy; Fluorescence anisotropy

Analysis of urinary estrogens, their oxidized metabolites, and other endogenous steroids by benchtop orbitrap LCMS versus traditional quadrupole GCMS by Adrian A. Franke; Laurie J. Custer; Yukiko Morimoto; Frank J. Nordt; Gertraud Maskarinec (pp. 1319-1330).

Estrogens and other endogenous steroids are known risk markers for cancer. Gas chromatography (GC) with mass spectrometry (MS) has traditionally predominated the analysis of estrogens and other endogenous steroids, but liquid chromatography (LC) MS is increasingly favored. Direct comparisons of the two technologies have hitherto not been performed. Steroids were analyzed from 232 urine samples of 78 premenopausal women in a blinded fashion by benchtop orbitrap LCMS and single quadrupole GCMS. Sixteen steroidal estrogens including oxidized metabolites could be analyzed by LCMS. LCMS–GCMS Spearman rank correlations of the major estrogens E₁, E₂, E₃, 16α-OHE₁, and 2-OHE₁ were very high (r = 0.72–0.91), and absolute concentrations also agreed (<5% difference for E₁, E₂, E₃, 16α-OHE₁). LCMS allowed reinterrogation of the acquired data due to orbitrap technology, which permitted post-analysis quantitation of progesterone, cortisol, and cortisone (LCMS–GCMS Spearman rank correlations = 0.80–0.84; absolute difference, <7%; n = 137). GCMS allows the measurement of a wide range of steroids including non-polar analytes that escape the presented LCMS assay. In contrast, orbitrap-based LCMS can detect more estrogens, is faster, less costly, allows post-data acquisition reinterrogation of certain analytes that had not been targeted a priori, and requires much less urine. Figure Orbitrap based LCMS trace of steroids after hydrolysis and dansylation

Keywords: Estrogen metabolites; Corticoids; Progesterone; Orbitrap LCMS; Urine; Breast cancer biomarkers

Secretome of the preimplantation human embryo by bottom-up label-free proteomics by Sylvia S. Cortezzi; Jerusa S. Garcia; Christina R. Ferreira; Daniela P. A. F. Braga; Rita C. S. Figueira; Assumpto Iaconelli Jr; Gustavo H. M. F. Souza; Edson Borges Jr; Marcos N. Eberlin (pp. 1331-1339).

A bottom-up label-free mass spectrometric proteomic strategy was used to analyse the protein profiles of the human embryonic secretome. Culture media samples used for embryonic culture of patients undergoing intracytoplasmic sperm injection cycles were selected as a test case for this exploratory proof-of-principle study. The media were stored after embryo transfer and then pooled into positive (n = 8) and negative (n = 8) implantation groups. The absolute quantitative bottom-up technique employed a multidimensional protein identification technology based on separation by nano-ultra-high pressure chromatography and identification via tandem nano-electrospray ionization mass spectrometry with data-independent scanning in a hydrid QqTOF mass spectrometer. By applying quantitative bottom-up proteomics, unique proteins were found exclusively in both the positive- and negative-implantation groups, which suggest that competent embryos express and secrete unique biomarker proteins into the surrounding culture medium. The selective monitoring of these possible secretome biomarkers could make viable procedures using single-embryo transfer. Figure A bottom-up label-free proteomic mass spectrometric analysis of the human embryo secretome is described. This approach seems to allow quantification and identification of unique proteins related to positive- and negative-implantation groups, which can be further validated as biomarkers for selection and transfer of a single embryo.

Keywords: Bioanalytical methods; Mass spectrometry; Proteomics; Label-free quantitation; In vitro fertilization; Embryo secretome

Ultra high performance liquid chromatographic-tandem mass spectrometric multi-analyte procedure for target screening and quantification in human blood plasma: validation and application for 31 neuroleptics, 28 benzodiazepines, and Z-drugs by Daniela Remane; Markus R. Meyer; Dirk K. Wissenbach; Hans H. Maurer (pp. 1341-1352).

For fast and reliable screening, identification, and quantification of as many analytes as possible, multi-analyte approaches are very useful in clinical and forensic toxicology. Using ultra high performance liquid chromatography-tandem mass spectrometry, such an approach has been developed for blood plasma analysis after simple liquid–liquid extraction. In the present paper, validation and application is described for 31 neuroleptics, 28 benzodiazepines, and Z-drugs (zaleplone, zolpidem, and zopiclone). The validation parameters included recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes, selectivity, crosstalk, accuracy and precision, stabilities, and limits of quantification and detection. The results showed that the approach was selective, sensitive, accurate, and precise for 24 neuroleptics and 21 benzodiazepines and Z-drugs. The remaining analytes were unstable and/or too low dosed. Cost- and time-saving one-point calibration was applicable only for half of the analytes. The applicability was successfully shown for most of the drugs by analyzing authentic plasma samples and external quality control samples.

Keywords: UHPLC-MS/MS; Benzodiazepines; Neuroleptics; Plasma; Validation

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine by Byoung Wook Jeon; Hye Hyun Yoo; Eun Sook Jeong; Ho Jun Kim; Changbae Jin; Dong Hyun Kim; Jaeick Lee (pp. 1353-1363).

A sensitive and rapid method based on liquid chromatography–triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid–liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers. Figure Two-step procedure for the screening and confirmation of anabolic steroids in doping control analyses: simultaneous multiresidue screening method based on LC-MS/MS with selected reaction monitoring and generic confirmatory method based on LC-MS/MS with product ion scan

Keywords: Anabolic steroids; Screening; Confirmation; Doping analysis; Liquid chromatography–tandem mass spectrometry

Identification and characterization of impurities of tetracosactide by capillary electrophoresis and liquid chromatography coupled to time-of-flight mass spectrometry by Angelina Taichrib; Gerhard K. E. Scriba; Christian Neusüß (pp. 1365-1375).

Tetracosactide is a synthetic peptide analogue of the human adrenocorticotropic hormone that stimulates the production of cortisol in the adrenal cortex. The medical use of the compound is primarily the diagnosis of the adrenal cortex function. In order to characterize impurities of the drug, tetracosactide samples were analysed by both liquid chromatography and capillary electrophoresis coupled to a quadrupole time-of-flight mass spectrometer. The identification of the impurities was carried out based on accurate mass determination and fragment ion spectra. The presence of several peptides of lower and higher masses than tetracosactide could be shown, including N- and C-terminally truncated peptides as well as peptides which still contained protecting groups or additional amino acids. Furthermore, a semi-quantitative estimation of the relative amounts of the impurities in different samples as well as a commercial preparation revealed that the number and the type of the impurities varied between the samples. Comparing the selectivity of liquid chromatography and capillary electrophoresis regarding the separation of tetracosactide impurities, it can be stated that capillary electrophoresis showed a higher suitability for the separation of tetracosactide fragments (smaller peptides) while the larger peptides, i.e. those wearing protecting groups, were separated more efficiently by liquid chromatography.

Keywords: Tetracosactide; Impurity identification; LC/MS; CE/MS

Poly(3-alkylthiophenes): new sorption materials for solid phase microextraction of drugs isolated from human plasma by Pawel Olszowy; Malgorzata Szultka; Boguslaw Buszewski (pp. 1377-1384).

A novel sorbent in solid phase microextraction (SPME) method based on poly(3-alkylthiophenes) was used in the isolation of linezolid from human plasma samples following liquid chromatography determination. The effect of extraction time on the sorption capacity of the SPME process was studied and pointed at 10 min both for adsorption and desorption. Poly(3-methylthiophene) and poly(3-nonylthiophene) were applied for the extraction of linezolid from water solutions. In plasma samples, four coatings including polythiophene and poly(3-penthylthiophene) were investigated. With these measurements, correlation coefficients were calculated in the range from 0.9820 to 0.9995, and the relative standard deviations were below 15%. That allowed claiming that the synthesized and described materials can be successfully applied in the analysis of linezolid also from other matrices such as urine or blood.

Keywords: Solid phase microextraction; Poly(3-alkylthiophenes); Linezolid; High-performance liquid chromatography

Simultaneous determination of histamine and prostaglandin D₂ using an LC-ESI-MS/MS method with positive/negative ion-switching ionization modes: application to the study of anti-allergic flavonoids on the degranulation of KU812 cells by Junko Koyama; Shiori Taga; Kae Shimizu; Maki Shimizu; Izumi Morita; Atsuko Takeuchi (pp. 1385-1392).

A new method for simultaneous determination of histamine and prostaglandin D₂ (PGD₂) by liquid chromatography–electrospray ionization tandem mass spectrometry operated in positive and negative ionization switching modes was developed and validated without a previous derivatization step. This method was used to measure histamine and PGD₂ release following degranulation of KU812 human basophilic cells, using pyrazol and d₄-PGD₂ as internal standards. Analyses were performed on a liquid chromatography system employing a Cosmosil 5C₁₈ PAQ column and an isocratic elution with mixed solution of methanol–water (7:3, v/v) with 0.0015% trifluoroacetic acid at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer operating in selected reaction monitoring mode simultaneously monitored using the following transitions: positive m/z 112/95 for histamine and negative m/z 351/271 for PGD₂. The retention times of histamine and pyrazol were 4.2 and 5.0 min, respectively. PGD₂ and d₄-PGD₂ had retention times of 8.5 min. The limits of detection were 0.3 and 0.5 ng/mL for histamine and PGD₂, respectively. The relative standard deviations of the retention time and peak area for histamine were between 1.6% and 7.7%, and were 1.2% and 7.8% for PGD₂. This method was used to evaluate the anti-allergic effects of 26 flavonoids and sodium cromoglicate which are first-line anti-allergic drugs. Of these compounds, baicalein and morin were the most potent inhibitors.

Keywords: LC-ESI-MS/MS; Histamine; Prostaglandin D₂ ; KU812 cell

Multi-walled carbon nanotubes as solid-phase extraction adsorbents for the speciation of cobalamins in seafoods by liquid chromatography by Pilar Viñas; Ignacio López-García; María Bravo Bravo; Manuel Hernández-Córdoba (pp. 1393-1399).

Multi-walled carbon nanotubes (MWCNTs) were evaluated as potential adsorbents for miniaturized solid-phase extraction coupled to liquid chromatography. The adsorption capacity of this sorbent was applied to assess the speciation of four cobalamins representing the various forms of vitamin B₁₂. The preconcentration on the MWCNTs was based on the retention of analytes by introducing the sample online into the mini-column system. Dimethyl sulfoxide was used to elute the retained vitamins for liquid chromatographic analysis. The experimental conditions of the continuous flow device, which affect the enrichment procedure, such as the type and amount of nanotubes, the volume, pH and flow rate of the sample solution, and the eluent and its volume, were optimized. For detection purposes, a diode array device was used and good resolution was obtained with a mobile-phase acetonitrile–phosphate buffer and gradient elution. Specificity was demonstrated by the retention characteristics and UV spectra and by comparing the peak purity index with commercial standards. Linearity, precision, recovery, and sensitivity were satisfactory. Detection limits ranged from 0.35 to 30 ng mL⁻¹. The method was successfully applied to the determination of cobalamins in seafoods, which were extracted from the sample with a buffer solution using an ultrasonic probe. The reliability of the procedure was checked by analyzing a certified reference material.

Keywords: Multi-walled carbon nanotubes; Solid-phase extraction; Liquid chromatography; Speciation; Cobalamins; Seafoods

Development of a combined SEM and ICP-MS approach for the qualitative and quantitative analyses of metal microparticles and sub-microparticles in food products by D. Beltrami; D. Calestani; M. Maffini; M. Suman; B. Melegari; A. Zappettini; L. Zanotti; U. Casellato; M. Careri; A. Mangia (pp. 1401-1409).

An integrated approach based on the use of inductively coupled plasma mass spectrometry (ICP-MS) and scanning electron microscopy (SEM) for the qualitative and quantitative analyses of metal particles in foods was devised and validated. Different raw materials and food products, like wheat, durum wheat, wheat flour, semolina, cookies, and pasta were considered. Attention was paid to the development of sample treatment protocols for each type of sample to avoid potential artifacts such as aggregation or agglomeration. The analytical protocols developed followed by ICP-MS and SEM investigations allowed us the quantitative determination and the morphological and dimensional characterization of metal nano- and microparticles isolated from the raw materials and finished food products considered. The ICP-MS method was validated in terms of linearity (0.8–80 μg/g and 0.09–9 μg/g for Fe and Ti, respectively), quantification limits (0.73 μg/g for Fe and 0.09 μg/g for Ti), repeatability (relative standard deviation (RSD) % equal to 10% for Fe and 20% in a wheat matrix as an example), and extraction recoveries (93 ± 2–101 ± 2%). Validation of the scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDS) measurements was performed working in a dimensional range from 1 to 100 μm with an estimated error in the size determination equal to 0.5 μm. ICP-MS data as well as SEM measurements showed a decrease in the concentration of metal particles from wheat to flour and from durum wheat to semolina samples, thus indicating an external contamination of grains by metal particles. These findings were confirmed by environmental SEM analysis, which allowed investigation of particles of lower dimensions. Generally, the largest number of particles was found in the case of iron and titanium, whereas particles of copper and zinc were only occasionally found without any possibility of quantifying their number.

Keywords: Metal particles; Food; Inductively coupled plasma mass spectrometry; Scanning electron microscopy; Environmental scanning electron microscopy; Sample treatment

Development of a simple gel permeation clean-up procedure coupled to a rapid disequilibrium enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I dye in spices and sauces by Michalina Oplatowska; Paul J. Stevenson; Claudia Schulz; Lutz Hartig; Christopher T. Elliott (pp. 1411-1422).

Sudan dyes have been found to be added to chilli and chilli products for illegal colour enhancement purposes. Due to the possible carcinogenic effect, they are not authorized to be used in food in the European Union or the USA. However, over the last few years, many products imported from Asian and African countries have been reported via the Rapid Alert System for Food and Feed in the European Union to be contaminated with these dyes. In order to provide fast screening method for the detection of Sudan I (SI), which is the most widely abused member of Sudan dyes family, a unique (20 min without sample preparation) direct disequilibrium enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on polyclonal antibodies highly specific to SI. A novel, simple gel permeation chromatography clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The assay was validated according to the Commission Decision 2002/657/EC criteria. The detection capability was determined to be 15 ng g⁻¹ in sauces and 50 ng g⁻¹ in spices. The recoveries found ranged from 81% to 116% and inter- and intra-assay coefficients of variation from 6% to 20%. The assay was used to screen a range of products (85 samples) collected from different retail sources within and outside the European Union. Three samples were found to contain high amounts (1,649, 722 and 1,461 ng g⁻¹) of SI by ELISA. These results were confirmed by liquid chromatography-tandem mass spectrometry method. The innovative procedure allows for the fast, sensitive and high throughput screening of different foodstuffs for the presence of the illegal colorant SI. Figure Figure (A) Typical standard curve obtained with a rapid disequilibrium Sudan I ELISA, (B) structure of Sudan I and (C) gel permeation chromatography procedure for the clean-up of spices samples

Keywords: Sudan I; Sudan dyes; ELISA; Food; Gel permeation chromatography

Synthesis of molecularly imprinted polymers via ring-opening metathesis polymerization for solid-phase extraction of bisphenol A by Xiaohong Wang; Lirong Chen; Xiaojie Xu; Yuanzong Li (pp. 1423-1432).

The use of molecularly imprinted polymers (MIPs) prepared by ring-opening metathesis polymerization (ROMP) for bisphenol A (BPA) was reported in this article. The resulting MIPs have high imprinting and adsorption capacities, and can be used for separation and determination of BPA in environmental water samples. The successful application of ROMP in the molecular imprinting field is described here. For the first time, two cross-linkers (dicyclopentadiene and 2,5-norbornadiene) and two Grubbs catalysts (first and second generation) were investigated to compare their effects on the binding performance of MIPs. The ROMP technique is able to create the imprinted polymers within 1 h under mild conditions. Furthermore, it can provide MIPs with obvious imprinting effects towards the template, very fast template rebinding kinetics, high binding capacity and appreciable selectivity over structurally related compounds. The adsorption process for MIPs in this study can be completed within 45 min, which is much faster than that of bulk MIPs synthesized by traditional free-radical polymerization. The resulting imprinting polymer was evaluated for its use as a sorbent support in an off-line solid-phase extraction approach to recover BPA from diluted aqueous samples. The optimized extraction protocol resulted in a reliable MISPE method suitable for selective extraction and preconcentration of BPA from tap water, human urine and liquid milk samples. This article demonstrates the practical feasibility of the MIPs prepared via ROMP as solid-phase extraction materials. Figure Synthesis of biaphenol A-imprinted polymer via Ringopening metathesis polymerization (ROMP)

Keywords: Molecular imprinted polymer; Ring-opening metathesis polymerization; Solid-phase extraction

Striking presence of Egyptian blue identified in a painting by Giovanni Battista Benvenuto from 1524 by Jørn Bredal-Jørgensen; Jana Sanyova; Vibeke Rask; Maria Louise Sargent; Rikke Hoberg Therkildsen (pp. 1433-1439).

Egyptian blue has been identified in a painting from 1524 by the Italian artist Ortolano Ferrarese (Giovanni Battista Benvenuto). Egyptian blue is the oldest known synthetic pigment, invented by the Egyptians in the fourth dynasty (2613–2494 bc) of the Old Kingdom and extensively used throughout Antiquity. From about 1000 a.d., it disappeared from the historical record and was only reinvented in the late nineteenth and early twentieth century. The discovery of Egyptian blue in Ortolano Ferrarese’s painting from 1524 shows that Egyptian blue was in fact available in the period from which it is normally considered not to exist. The identification of Egyptian blue is based on optical microscopy supported by energy-dispersive spectroscopy and visual light photon-induced spectroscopy, and finally confirmed by Raman microspectroscopy. Figure St. Margaret by Giovanni Batista Benvenuto. National Gallery of Denmark.

Keywords: Egyptian blue; Giovanni Battista Benvenuto; Optical analysis; Raman micro spectroscopy; Scanning electron microscopy; Photo-induced luminescence

Ancient Roman wall paintings mapped nondestructively by portable NMR by Agnes Haber; Bernhard Blümich; Daria Souvorova; Eleonora Del Federico (pp. 1441-1452).

The stratigraphies of decorated walls in ancient Herculaneum, Italy, were analyzed by single-sided ¹H NMR. A large version of the NMR-MOUSE® with a maximum penetration depth of 25 mm was used to map proton density profiles at different positions of the Mosaic of Neptune and Amphitrite showing considerable differences between different tesserae and the mortar bed at different times of the year. In the House of the Black Room, different mortar layers were observed on painted walls as well as different proton content in different areas due to different moisture levels and different conservation treatments. The proton density profiles of the differently treated areas indicated that one method leads to higher moisture content than the other. Untreated wall paintings from different times were profiled in a recently excavated room at the Villa of the Papyri showing two different types of mortar layer structures which identify two different techniques of preparing the walls for painting. Reflectance Fourier mid-infrared spectroscopy and in situ X-ray fluorescence measurements complemented the NMR measurements and provided additional insight into the identification of organic coatings as well as the nature of the pigments used, respectively. The information acquired nondestructively by NMR is valued for elaborating conservation strategies and for identifying different schools of craftsmen who prepared the mortar supports of the wall paintings.

Keywords: Nuclear magnetic resonance; NMR-MOUSE; X-ray fluorescence; Reflectance mid-FTIR; Nondestructive analysis; Wall paintings; Mosaic; Cultural heritage

Erratum to: Development of a combined SEM and ICP-MS approach for the qualitative and quantitative analyses of metal microparticles and sub-microparticles in food products by D. Beltrami; D. Calestani; M. Maffini; M. Suman; B. Melegari; A. Zappettini; L. Zanotti; U. Casellato; M. Careri; A. Mangia (pp. 1453-1453).

Erratum to: Atomic Layer Deposition (ALD) as a coating tool for reinforcing fibers by A. K. Roy; W. Baumann; I. König; G. Baumann; S. Schulze; M. Hietschold; T. Mäder; D. J. Nestler; B. Wielage; W. A. Goedel (pp. 1455-1455).

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Analytical and Bioanalytical Chemistry (v.401, #4)