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Analytical and Bioanalytical Chemistry (v.405, #2-3)

Analytical science in Italy by Aldo Roda (pp. 441-441).

is Professor of Analytical Chemistry at Bologna University. His main research interests center on the area of analytical and bioanalytical chemistry applied to clinical chemistry, pharmacotoxicology, medicinal chemistry, environmental and food analysis. He published more than 400 original articles on international journal and he is the owner of many patents on new bile acids drugs, new chemiluminescent and luciferases labels, and miniaturized devices.

Application of quality by design to the development of analytical separation methods by Serena Orlandini; Sergio Pinzauti; Sandra Furlanetto (pp. 443-450).

Recent pharmaceutical regulatory documents have stressed the critical importance of applying quality by design (QbD) principles for in-depth process understanding to ensure that product quality is built in by design. This article outlines the application of QbD concepts to the development of analytical separation methods, for example chromatography and capillary electrophoresis. QbD tools, for example risk assessment and design of experiments, enable enhanced quality to be integrated into the analytical method, enabling earlier understanding and identification of variables affecting method performance. A QbD guide is described, from identification of quality target product profile to definition of control strategy, emphasizing the main differences from the traditional quality by testing (QbT) approach. The different ways several authors have treated single QbD steps of method development are reviewed and compared. In a final section on outlook, attention is focused on general issues which have arisen from the surveyed literature, and on the need to change the researcher’s mindset from the QbT to QbD approach as an important analytical trend for the near future. Figure Quality by design guide for analytical method development

Keywords: Capillary electrophoresis; Chromatography; Design of experiments; Design space; Pharmaceuticals; Quality by design

Carbon nanostructured materials for applications in nano-medicine, cultural heritage, and electrochemical biosensors by F. Valentini; M. Carbone; G. Palleschi (pp. 451-465).

This review covers applications of pristine and functionalized single-wall carbon nanotubes (SWCNTs) in nano-medicine, cultural heritage, and biosensors. The physicochemical properties of these engineered nanoparticles are similar to those of ultrafine components of airborne pollution (UF) and might have similar adverse effects. UF may impair cardiovascular autonomic control (inducing a high-risk condition for adverse cardiovascular effects), cause mammalian embryo toxicity, and increase geno-cytotoxic risk. SWCNTs coated with a biopolymer, for example polyethylenimine (PEI), become extremely biocompatible, hence are useful for in-vivo and in-vitro drug delivery and gene transfection. It is also possible to successfully immobilize a human enteric virus on PEI/SWCNT composites, suggesting application as a carrier in non-permissive media. The effectiveness of carbon nanostructured materials in the cleaning, restoration, and consolidation of deteriorated historical surfaces has been widely shown by the use of carbon nanomicelles to remove black dendritic crust from stone surfaces. The nanomicelles, here, have the twofold role of delivery and controlled release of the cleaning agents. The high biocompatibility of functionalized SWCNTs with enzymes and proteins is a fundamental feature used in the assembly of electrochemical biosensors. In particular, a third-generation protoporphyrin IX-based biosensor has been assembled for amperometric detection of nitrite, an environmental pollutant involved in the biodeterioration and black encrustation of historical surfaces.

Keywords: SWCNTs; Biopolymers; Nano-medicine; Biosensors; Electrochemistry; Cultural heritage

Lateral-flow immunoassays for mycotoxins and phycotoxins: a review by Laura Anfossi; Claudio Baggiani; Cristina Giovannoli; Gilda D’Arco; Gianfranco Giraudi (pp. 467-480).

Natural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters. Figure 1 Competitive lateral flow immunoassay for myco- or phycotoxin: the Test zone is formed by adsorbing a conjugate of the target compound (toxin); Control zone is formed by anti-species antibodies (white), reporters are specific (anti-toxin antibodies, black) and non-specific (grey) antibodies labelled with gold nanoparticles (GNP). Focalization of GNP-labelled antibodies determines a visible/detectable colour appearance on both the Test and Control lines, which can be related to analyte amount in a liquid sample.

Keywords: Rapid methods; Food analysis; Immunogold colloidal immunoassay

Capillary electrophoresis: a useful tool for the management of plant genetic resources by Angela R. Piergiovanni (pp. 481-491).

Capillary electrophoresis (CE) is a powerful analytical tool that is widely applied to the analysis of biological samples. Proteins, peptides, nonprotein amino acids, phenolic compounds, and ions can be analysed using this electrophoretic methodology. This review summarises some applications of CE to the evaluation and characterisation of plant genetic resources of both Triticum and legume species, as carried out at the Istituto di Genetica Vegetale, National Research Council (IGV-CNR) in Bari (Italy). Different protein fractions as well as nonprotein amino acids were investigated by capillary zone electrophoresis (CZE), the most user-friendly mode of CE application. The described case studies show that CZE can be applied to some institutional activities of gene banks such as the evaluation of genetic diversity within stored collections, the acquisition of new samples, the differentiation of species belonging to the same genus, the identification of misclassified accessions, and the measurement of compounds relevant to nutrition or health.

Keywords: Capillary zone electrophoresis; Characterisation; Genetic resources; Legume species; Triticum species

Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry by Tommaso R. I. Cataldi; Giuliana Bianco; Juliano Fonseca; Philippe Schmitt-Kopplin (pp. 493-507).

Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC–ESI-qTOF MS has also proved to be suitable for identification of 3O-C₁₀HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized. Figure LC separation and FTICR MS identification of 3-oxo-C12-HSL in bacterial isolates of P. aeruginosa (strain ATCC 9027)

Keywords: N-Acyl-homoserine lactones; Gram-negative bacteria; Quorum sensing; FTICR MS; UPLC-ESI-qTOF MS; Mass spectrometry

Polythiophenes and polythiophene-based composites in amperometric sensing by C. Zanardi; F. Terzi; R. Seeber (pp. 509-531).

This overview of polythiophene-based materials provides a critical examination of meaningful examples of applications of similar electrode materials in electroanalysis. The advantages arising from the use of polythiophene derivatives in such an applicative context is discussed by considering the organic conductive material as such, and as one of the components of hybrid materials. The rationale at the basis of the combination of two or even more individual components into a hybrid material is discussed with reference to the active electrode processes and the consequent possible improvements of the electroanalytical performance. In this respect, study cases are presented considering different analytes chosen among those that are most frequently reported within the classes of organics and inorganics. The use of a polythiophene matrix to stably fix biological elements at the electrode surface for the development of catalytic biosensors and genosensors is also discussed. Finally, a few possible lines along which the next research in the field could be fruitfully pursued are outlined. Furthermore, the work still to be done to exploit the possibilities offered by novel products of organic synthesis, even along paths already traced in other fields of electrochemistry, is discussed. Prototypical voltammetric responses obtained in a solution of two analytes on: bare electrode (left); polythiophine-based coated electrode (right).

Keywords: Electrochemical sensors; Electroanalytical methods; Polythiophene; Nanoparticles/nanotechnology; Composite materials; Biosensors

Thin-layer chromatography enantioseparations on chiral stationary phases: a review by Massimo Del Bubba; Leonardo Checchini; Luciano Lepri (pp. 533-554).

The current state of chiral separations by thin-layer chromatography using chiral stationary phases is reviewed. Both stationary phases essentially constituted by the chiral selector and those obtained by the impregnation of achiral plates with appropriate chiral selectors are described. Particular attention is paid to commercial and non-commercial cellulose and cellulose-derivative plates, as well as commercially available Chiralplate™, which are currently the most widely used. Some of the most important results obtained to date are reported and discussed; the examples provided illustrate the very wide range of structurally different solutes that can be readily resolved into their enantiomers by planar chromatographic methods. Special attention is paid to the discussion of the retention and resolution factors that influence chiral discrimination. The quantitative analysis of enantiomers is also discussed, especially from the point of view of determination of enantiomeric purity.

Keywords: Chiral analysis; Thin-layer chromatography; Chiral stationary phase; Chiral coated stationary phase; Enantiomers

Trace analysis of pollutants by use of honeybees, immunoassays, and chemiluminescence detection by S. Girotti; S. Ghini; E. Maiolini; L. Bolelli; E. N. Ferri (pp. 555-571).

Specific and sensitive analysis to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and food is urgently required because of the increasing attention devoted to the environment and health protection. Our research group has been involved in monitoring the presence and distribution of agrochemicals by monitoring beehives distributed throughout the area studied. Honeybees have been used both as biosensors, because the pesticides affect their viability, and as “contaminant collectors” for all environmental pollutants. We focused our research on the development of analytical procedures able to reveal and quantify pesticides in different samples but with a special attention to the complex honeybee matrix. Specific extraction and purification procedures have been developed and some are still under optimization. The analytes of interest were determined by gas or liquid chromatographic methods and by compound-specific or group-specific immunoassays in the ELISA format, the analytical performance of which was improved by introducing luminescence detection. The range of chemiluminescent immunoassays developed was extended to include the determination of completely different pollutants, for example explosives, volatile organic compounds (including benzene, toluene, ethylbenzene, xylenes), and components of plastics, for example bisphenol A. An easier and portable format, a lateral flow immunoassay (LFIA) was added to the ELISA format to increase application flexibility in these assays. Aspects of the novelty, the specific characteristics, the analytical performance, and possible future development of the different chromatographic and immunological methods are described and discussed. Fig Map of a territory monitored by using honeybees, showing the areas covered by each beehive station (circles) and thedifferent agricultural products included in it (different colours).

Keywords: Honeybees; Pesticides; Chromatographic analysis; Immunoassays; Chemiluminescence; Explosives

Surface plasmon resonance imaging for nucleic acid detection by Roberta D’Agata; Giuseppe Spoto (pp. 573-584).

Surface plasmon resonance imaging (SPRI) is a powerful tool for simple, fast and cheap nucleic acid detection. Great efforts have been made during the last decade with the aim of developing even more sensitive and specific SPRI-based methods to be used for the direct detection of DNA and RNA. Here, after a description of the fundamentals of SPRI, the state of the art of recent platform and assay developments is presented, with special attention given to advances in SPRI signal enhancement procedures.

Keywords: Surface plasmon resonance imaging; DNA detection; Gold nanoparticles; PCR-free; Biosensors

Different approaches to the study of chelating agents for iron and aluminium overload pathologies by Guido Crisponi; Annalisa Dean; Valerio Di Marco; Joanna I. Lachowicz; Valeria M. Nurchi; Maurizio Remelli; Andrea Tapparo (pp. 585-601).

Our objective is to illustrate the activity of the groups operating in Italy involved in identification and study of new chelating agents, mainly intended for treatment of human pathology correlated with metal overload. The objective of “chelation therapy” is removal of toxic metal ions from the human body or attenuation of their toxicity by transforming them into less toxic compounds or by dislocating them from the site at which they exert a toxic action. Because most of this research activity is related to chelating agents for iron and aluminium, diseases related to these two metal ions are briefly treated. Iron overload is the most common metal toxicity disease worldwide. The toxicity of aluminium in dialysis patients was a serious problem for haemodialysis units in the seventies and eighties of the last century. In particular, this review focuses on research performed by the group at Cagliari and Ferrara, and by that at Padova. The former is studying, above all, bisphosphonate and kojic acid derivatives, and the latter is investigating 3,4-hydroxypyridinecarboxylic acids with differently substituted pyridinic rings. Figure Aim of this paper is to illustrate the research on different classes of ligands, which are intended as possible chelating agents for the treatment of human pathologies correlated to iron and aluminium overload.

Keywords: Chelating agent; Metal toxicity; Iron; Aluminium; Complex-formation equilibria

Layered-double-hydroxide-modified electrodes: electroanalytical applications by Domenica Tonelli; Erika Scavetta; Marco Giorgetti (pp. 603-614).

Two-dimensional inorganic solids, such as layered double hydroxides (LDHs), also defined as anionic clays, have open structures and unique anion-exchange properties which make them very appropriate materials for the immobilization of anions and biomolecules that often bear an overall negative charge. This review aims to describe the important aspects and new developments of electrochemical sensors and biosensors based on LDHs, evidencing the research from our own laboratory and other groups. It is intended to provide an overview of the various types of chemically modified electrodes that have been developed with these 2D layered materials, along with the significant advances made over the last several years. In particular, we report the main methods used for the deposition of LDH films on different substrates, the conductive properties of these materials, the possibility to use them in the development of membranes for potentiometric anion analysis, the early analytical applications of chemically modified electrodes based on the ability of LDHs to preconcentrate redox-active anions and finally the most recent applications exploiting their electrocatalytic properties. Another promising application field of LDHs, when they are employed as host structures for enzymes, is biosensing, which is described considering glucose as an example. Figure

Keywords: Layered double hydroxides; Amperometric sensors; Amperometric glucose biosensors; Potentiometric sensors; Electrocatalysis

Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices by Laura Maria Zanoli; Marco Licciardello; Roberta D’Agata; Claudia Lantano; Alessandro Calabretta; Roberto Corradini; Rosangela Marchelli; Giuseppe Spoto (pp. 615-624).

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar–attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms. Figure The fluorescence generated by peptide nucleic acid molecular beacons allows the detection of 100-200 attomoles of PCR-amplified DNA sequences from solutions encapsulated in nanoliter-sized droplets.

Keywords: DNA detection; Droplet microfluidics; Peptide nucleic acids; Molecular beacon; PCR

Analysis of paint cross-sections: a combined multivariate approach for the interpretation of μATR-FTIR hyperspectral data arrays by Giorgia Sciutto; Paolo Oliveri; Silvia Prati; Marta Quaranta; Silvia Lanteri; Rocco Mazzeo (pp. 625-633).

The present research is aimed at introducing a suitable approach for the exploitation of the hyperspectral data obtained by μATR-FTIR analyses of paint cross-sections. The application of principal component analysis for chemical mapping is well-established, even if a very limited number of applications to μFTIR data have been reported so far in the field of analytical chemistry for cultural heritage. Moreover, in many cases, chemometric tools are under-utilized and the outcomes under-interpreted. As a consequence, results and conclusions may be considerably compromised. In an attempt to overcome such drawbacks, the present work is proposing a comprehensive and efficient procedure based on an interactive brushing approach, which combines the structural information of the score scatter plots and the spatial information of the principal component (PC) score maps. In particular, the study demonstrates not only how the multivariate approach may provide more information than the univariate one, but also how the integration of different chemometric tools may allow a more comprehensive interpretation of the results with respect to the studies up to now reported in the literature. The examination of the average spectral profile of each score cluster, jointly with the loading analysis, is functional to characterize each area investigated on the basis of its spectral features. A multivariate comparison with spectra of standard compounds, projected in the PC score space, helps in supporting the chemical identification. The approach was validated on two real case studies.

Keywords: Paint cross-section; μATR-FTIR spectroscopy; Multivariate chemical mapping; Principal component analysis (PCA); Score map; Brushing

Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins by Anna Laura Capriotti; Giulio Caracciolo; Giuseppe Caruso; Chiara Cavaliere; Daniela Pozzi; Roberto Samperi; Aldo Laganà (pp. 635-645).

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA–cationic lipid complexes (lipoplexes), and lipid–polycation–DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

Keywords: Label-free quantification; Proteomics; Mass spectrometry; Protein corona; Liposome; Lipoplexes; DOTAP; Gene delivery

Speciation analysis of iodine and bromine at picogram-per-gram levels in polar ice by Andrea Spolaor; Paul Vallelonga; Jacopo Gabrieli; Natalie Kehrwald; Clara Turetta; Giulio Cozzi; Luisa Poto; John M. C. Plane; C. Boutron; Carlo Barbante (pp. 647-654).

Iodine and bromine species participate in key atmospheric reactions including the formation of cloud condensation nuclei and ozone depletion. We present a novel method coupling a high-performance liquid chromatography with ion chromatography and inductively coupled plasma mass spectrometry, which allows the determination of iodine (I) and bromine (Br) species (IO ₃ ⁻ , I⁻, Br⁻, BrO ₃ ⁻ ) at the picogram-per-gram levels presents in Antarctic ice. Chromatographic separation was achieved using an IONPAC® AS16 Analytical Column with NaOH as eluent. Detection limits for I and Br species were 5 to 9 pg g⁻¹ with an uncertainty of less than 2.5% for all considered species. Inorganic iodine and bromine species have been determined in Antarctic ice core samples, with concentrations close to the detection limits for iodine species, and approximately 150 pg g⁻¹ for Br⁻. Although iodate (IO ₃ ⁻ ) is the most abundant iodine species in the atmosphere, only the much rarer iodide (I⁻) species was present in Antarctic Holocene ice. Bromine was found to be present in Antarctic ice as Br⁻.

Keywords: Iodine; Bromine; Speciation; Ice cores; Antarctica

Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads by G. Volpe; U. Sozzo; S. Piermarini; E. Delibato; G. Palleschi; D. Moscone (pp. 655-663).

This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small “reservoirs” containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each “reservoir” is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H₂O₂ produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H₂O₂, is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H₂O₂ into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution. Figure Schematic representation of the single-step immunoassay: in the upper part, the content of the ‘reservoirs’ (containing MB-rabbit IgG, TPi, HQ and various concentration levels of anti-rabbit IgG-HRP) are transferred to an 8 well/sensor strip coupled with an special magnetic support which can draw the IMBs to the electrode surface; the lower part shows the two electrochemical approaches proposed to monitor the immunological reaction

Keywords: Single-step amperometric immunosensor; 8-well/sensor strip; Magnetic beads; Enzyme channelling principle; SPE-based immunoassay

Ionomic profiling of Nicotiana langsdorffii wild-type and mutant genotypes exposed to abiotic stresses by Francisco Ardini; Francesco Soggia; Maria Luisa Abelmoschi; Emanuele Magi; Marco Grotti (pp. 665-677).

To provide a new insight into the response of plants to abiotic stresses, the ionomic profiles of Nicotiana langsdorffii specimens have been determined before and after exposure to toxic metals (chromium) or drought conditions. The plants were genetically transformed with the rat glucocorticoid receptor (GR) or the gene for Agrobacterium rhizogenes rolC, because these modifications are known to produce an imbalance in phytohormone equilibria and a significant change in the defence response of the plant. Elemental profiles were obtained by developing and applying analytical procedures based on inductively coupled plasma atomic emission and mass spectrometry (ICP–AES/MS). In particular, the removal of isobaric interferences affecting the determination of Cr and V by ICP–MS was accomplished by use of a dynamic reaction cell, after optimization of the relevant conditions. The combined use of ICP atomic emission and mass spectrometry enabled the determination of 29 major and trace elements (Ba, Bi, Ca, Cd, Co, Cr, Cu, Eu, Fe, Ga, K, Li, Mg, Mn, Mo, Na, P, Pb, Pt, Rb, S, Sb, Sn, Sr, Te, V, W, Y, and Zn) in different parts of the plants (roots, stems, and leaves), with high accuracy and precision. Multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the target organism to chemical treatment or water stress. Genetic modification mainly affected the distribution of Bi, Cr, Mo, Na, and S, indicating that these elements were involved in biochemical processes controlled by the GR or rolC genes. Chemical stress strongly affected accumulation of several elements (Ba, Ca, Fe, Ga, K, Li, Mn, Mo, Na, P, Pb, Rb, S, Sn, Te, V, and Zn) in different ways; for Ca, Fe, K, Mn, Na, and P the effect was quite similar to that observed in other studies after treatment with other transition elements, for example Cu and Cd. The effect of water deficit was less evident, mainly consisting in a decrease of Ba, Cr, Na, and Sr in roots. Figure Roots, stems and leaves of different Nicotiana langsdorffii genotypes exposed to abiotic stresses were analysed by ICP-AES and ICP-MS, obtaining information on the distribution of 29 major and trace elements in the samples

Keywords: Inductively coupled plasma; Atomic emission spectrometry; Inorganic mass spectrometry; Dynamic reaction cell; Plants; Ionomics; Abiotic stress

Determination of petitgrain oils landmark parameters by using gas chromatography–combustion–isotope ratio mass spectrometry and enantioselective multidimensional gas chromatography by Luisa Schipilliti; Ivana Bonaccorsi; Danilo Sciarrone; Laura Dugo; Luigi Mondello; Giovanni Dugo (pp. 679-690).

Gas chromatography–combustion–isotope mass spectrometry was employed for the assessment of the Carbon isotope ratios of volatiles in Italian mandarin and lemon petitgrain oils. In addition, the composition of the whole oil and the enantiomeric distribution of selected chiral compounds were determined for all the samples by using gas chromatography and by multidimensional and conventional enantioselective gas chromatography. The composition of the oils was compared with previous studies. The enantiomeric distribution of lemon petitgrain oils is here reported for the first time. On the composition of mandarin petitgrain oil, the information available in literature, to date, is relative only to one sample from Egypt. Carbon isotope ratio of several terpene hydrocarbons and of their oxygenated derivatives contained in petitgrains was compared with the δ ¹³C_VPDB values of the same compounds present in the corresponding genuine Italian Citrus peel oil. The results prove that the isotopic values obtained for lemon and mandarin petitgrain oils are very close to those relative to the corresponding peel oils determined in previous studies.

Keywords: Lemon petitgrain oil; Mandarin petitgrain oil; Composition; Stable carbon isotope ratio analysis; Chiral analysis

Analysis of egg-based model wall paintings by use of an innovative combined dot-ELISA and UPLC-based approach by Mariangela Potenza; Giuseppina Sabatino; Francesca Giambi; Luca Rosi; Anna Maria Papini; Luigi Dei (pp. 691-701).

The chemical analysis of egg-based wall paintings—the mezzo fresco technique—is an interesting topic in the characterisation of organic binders. A revised procedure for a dot-enzyme-linked immunosorbent assay (dot-ELISA) able to detect protein components of egg-based wall paintings is reported. In the new dot-ELISA procedure we succeeded in maximizing the staining colour by adjusting the temperature during the staining reaction. Quantification of the colour intensity by visible reflectance spectroscopy resulted in a straight line plot of protein concentration against reflectance in the wavelength range 380–780 nm. The modified dot-ELISA procedure is proposed as a semi-quantitative analytical method for characterisation of protein binders in egg-based paintings. To evaluate its performance, the method was first applied to standard samples (ovalbumin, whole egg, egg white), then to model specimens, and finally to real samples (Giotto’s wall paintings). Moreover, amino acid analysis performed by innovative ultra-performance liquid chromatography was applied both to standards and to model samples and the results were compared with those from the dot-ELISA tests. In particular, after protein hydrolysis (24 h, 114 °C, 6 mol L⁻¹ HCl) of the samples, amino acid derivatization by use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate enabled reproducible analysis of amino acids. This UPLC amino acid analysis was rapid and reproducible and was applied for the first time to egg-based paintings. Because the painting technique involved the use of egg-based tempera on fresh lime-based mortar, the study enabled investigation of the effect of the alkaline environment on egg-protein detection by both methods. Figure Model wall paintings specimens and typical dot-ELISA stains for egg proteins.

Keywords: UPLC-based amino acid analysis; dot-ELISA; Egg-based wall paintings; Cultural heritage conservation

Influence of secondary preparative parameters and aging effects on PLGA particle size distribution: a sedimentation field flow fractionation investigation by Catia Contado; Eleonora Vighi; Alessandro Dalpiaz; Eliana Leo (pp. 703-711).

Poly(lactic-co-glycolic acid) particles in the 200–400-nm size range were formulated through nanoprecipitation and solvent evaporation methods. Different concentrations of the polymer and stabilizer (Pluronic® F 68) were tested in order to identify the best conditions for making poly(lactic-co-glycolic acid) particles of suitable size, stable in time, and to be used as carriers for brain-targeting drugs. The particles with the best characteristics for delivery system design were those formulated by nanoprecipitation with an organic/water phase ratio of 2:30, a polymer concentration of 25 mg/mL, and a surfactant concentration of 0.83 mg/mL; their surface charge was reasonably negative (approximately -27 mV) and the average size of the almost monodisperse population was roughly 250 nm. Particle characterization was obtained through ζ-potential measurements, scanning electron microscope observations, and particle size distribution determinations; the latter achieved by both photon-correlation spectroscopy and sedimentation field flow fractionation. Sedimentation field flow fractionation, which is considered more reliable than photon-correlation spectroscopy in describing the possible particle size distribution modifications, was used to investigate the effects of 3 months of storage at 4 °C had on the lyophilized particles. Figure Particle size ditribution from the SdFFF and the PCS techniques

Keywords: Poly(lactic-co-glycolic acid); Nanoparticles; Size analysis; Sedimentation field flow fractionation; Photon-correlation spectroscopy

Non-destructive depth profile reconstruction of bio-engineered surfaces by parallel-angle-resolved X-ray photoelectron spectroscopy by Rosa Pilolli; Nicoletta Ditaranto; Nicola Cioffi; Luigia Sabbatini (pp. 713-724).

In the present, contribution angle-resolved X-ray photoelectron spectroscopy (AR-XPS) was proposed as a useful tool to address the challenge of probing the near-surface region of bio-active sensor surfaces. A model bio-functionalised surface was characterised by parallel AR-XPS and commercially available Thermo Avantage-ARProcess software was used to generate non-destructive concentration depth profiles of protein-functionalised silicon oxide substrates. At each step of the functionalisation procedure, the surface composition, the overlayer thickness, the in-depth organisation and the in-plane homogeneity were evaluated. The critical discussion of the generated profiles highlighted the relevance of the information provided by PAR-XPS technique. Figure Schematic representation of the functionalization procedure along with the generated profiles

Keywords: Parallel-angle-resolved XPS; Concentration depth profile; Overlayer thickness; Protein covalent immobilisation

Pressurized-liquid extraction for determination of illicit drugs in hair by LC–MS–MS by Manuel Sergi; Sabino Napoletano; Camilla Montesano; Roberto Iofrida; Roberta Curini; Dario Compagnone (pp. 725-735).

An LC–MS–MS-based procedure for determination in hair of 14 different drugs of abuse belonging to the classes cocaine, amphetamine-like compounds, opiates, and hallucinogens has been developed. A pressurized-liquid extraction procedure was used and proved useful for quantitative recovery of all the analytes tested. This procedure, in conjunction with a simple decontamination step, performed to avoid false-positive samples, enabled the detection of all the analytes with LOQ ranging from 1.8 to 16 pg mg⁻¹ and accuracy varying from 85 to 111 %. The procedure was validated in accordance with the SOFT/AAFS guidelines and seems to be suitable for routine determination of the drugs tested in hair.

Keywords: Pressurized-liquid extraction; LC–MS–MS; Hair; Illicit drugs

Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene by Marco Giannetto; Elisabetta Maiolini; Elida Nora Ferri; Stefano Girotti; Giovanni Mori; Maria Careri (pp. 737-743).

A new amperometric immunosensor for 2,4,6-trinitrotoluene based on the working principle of competitive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled monolayers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our approach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determination of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidencing a good recovery rate (113 %). Figure Setup of sensor and immunoassay for TNT

Keywords: Biosensors; Immunoassay; Nanoparticles; TNT; Explosives; Dendrimer; SAM

Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst by Immacolata Serra; Daniela Ubiali; Davide A. Cecchini; Enrica Calleri; Alessandra M. Albertini; Marco Terreni; Caterina Temporini (pp. 745-753).

The mutant penicillin G acylase (PGA) 3K-PGA contains three additional Lys residues on the surface opposite the active site. This protein was designed to selectively drive its immobilization on aldehyde supports. We describe here a modified bottom-up proteomic method to assess the orientation of the immobilized wild-type and mutant proteins to verify our hypothesis of a driven immobilization induced by the mutations introduced. Tryptic digestion of the immobilized enzymes followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of released peptides was performed. This protocol generated peptides from the most accessible surface areas of the immobilized protein, thus not directly bound to the solid support, providing direct evidence of the areas involved in the linkage to the solid matrix. The results obtained suggest that 72 % of the wild-type PGA is immobilized on aldehyde agarose mainly through the Lys residues on the same side of the active site, whereas 3K-PGA reacted with the same support preferentially through the additional Lys residues introduced by mutation on the opposite side. This demonstrates that the active site of the 3K-PGA faces mostly (63 %) toward the reaction medium, resulting in significantly improved accessibility to the substrates. This finding is supported by the catalytic properties of the immobilized biocatalysts. The two immobilized preparations were tested in the synthesis of mandelyl-7-aminocephalosporanic acid (mandelyl-7-ACA) by N-acylation of the β-lactam nucleus (7-aminocephalosporanic acid) with mandelic acid methyl ester: upon immobilization, the synthetic properties of wild-type PGA strongly decreased, whereas those of 3K-PGA were unaffected. Furthermore, the activity of 3K-PGA was not influenced by the physicochemical nature of the support used for immobilization (glyoxyl agarose or aldehyde Sepabeads) unlike that of wild-type PGA, whose active site is close to the matrix. The results obtained from the analytical characterization correlate well with those obtained by investigation of the synthetic properties of the immobilized enzymes both in the synthesis of mandelyl-7-ACA and in the preparative synthesis of cefazolin. This work highlights the effect exerted by site-directed mutagenesis on the orientation of PGA upon immobilization on solid matrices and suggests how protein engineering tools can be exploited in a synergistic fashion to rationally develop efficient biocatalysts. Figure Site directed mutagenesis & enzyme orientation

Keywords: Liquid chromatography–tandem mass spectrometry; Penicillin G acylase; Immobilized protein orientation; Site-directed mutagenesis

Quantitative structure/eluent–retention relationships in reversed-phase high-performance liquid chromatography based on the solvatochromic method by Angelo Antonio D’Archivio; Maria Anna Maggi; Fabrizio Ruggieri (pp. 755-766).

Some predictive approaches aimed at modelling the combined effect of solute molecular structure and mobile phase composition on retention in reversed-phase high-performance chromatography (RP-HPLC) have been developed in the literature. These models are established for a given binary eluent (normally acetonitrile–water or methanol–water) by non-linear (curvilinear or artificial neural network) regression assuming as the mobile phase descriptor the volume fraction φ of the organic modifier. In the present investigation, we propose a model applicable simultaneously to acetonitrile–water and methanol–water eluents. To this end, the Kamlet-Taft solvatochromic descriptors of the eluent and the solvatochromic descriptors of the analytes are considered as the input variables of a multi-layer artificial neural network (ANN) providing the solute retention as the response. This approach is applied to a set of 31 molecules analyzed with five different columns in the φ range 20–70 % at 10 % steps for both acetonitrile- and methanol-containing mobile phases. For each column, an ANN-based model is built using retention data of 25 molecules selected by the Kennard-Stones algorithm while retention data of the unselected six solutes are considered in the final evaluation of predictive performance of the trained network. To test cross-eluent prediction, the network optimized for a given column was successively trained with data collected in eight out of 12 eluents and applied to deduce retention in the four remaining mobile phases. The results reveal that RP-HPLC behavior of external solutes is quite accurately modelled in the whole explored composition range of acetonitrile– and methanol–water mobile phases. Moreover, the model exhibits a promising capability of deducing retention of external solutes even in unknown eluents. Figure Quantitative structure/eluent–retention relationship established by artificial neural network regression

Keywords: Retention prediction; Artificial neural network; Reversed-phase high-performance liquid chromatography; Mobile phase; Solvatochromic descriptors

Continuous flow analysis method for determination of soluble iron and aluminium in ice cores by A. Spolaor; P. Vallelonga; J. Gabrieli; M. Roman; C. Barbante (pp. 767-774).

Iron and aluminium are the two most abundant metals on the Earth's crust, but they display quite different biogeochemical properties. While iron is essential to many biological processes, aluminium has not been found to have any biological function at all. In environmental studies, iron has been studied in detail for its limiting role in the bioproductivity of high nutrient, low carbon oceanic zones, while aluminium is routinely used as a reference of crustal contributions to atmospheric deposition archives including peat bogs, lacustrine and marine sediments and ice sheets and glaciers. We report here the development of a flow injection analysis technique, which has been optimised for the simultaneous determination of soluble iron and aluminium in polar ice cores. Iron was determined by its catalytic role in the reduction of N,N-dimethyl-p-phenylenediamene (DPD) to a semiquinonic form (DPDQ) and subsequent absorption spectroscopy at 514 nm. Aluminium was determined by spectroscopic analysis of an aluminium–lumogallion complex that exhibits fluorescence at 560 nm. These techniques have been applied to a section of Greenland ice dated to 1729–1733 ad and indicate that volcanism is a source of highly soluble aluminium and iron. Figure The micro volume flow cell used in the continuous flow detections of iron illuminated by a 525-nm LED light source.

Keywords: Continuous flow analysis; Solubility; Dust; Greenland; Ice core; Volcano

Innate immunity probed by lipopolysaccharides affinity strategy and proteomics by Chiara Giangrande; Lucia Colarusso; Rosa Lanzetta; Antonio Molinaro; Piero Pucci; Angela Amoresano (pp. 775-784).

Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.

Keywords: Innate immunity; Lipopolysaccharide; Proteomics; Affinity

Determination of the two major endocannabinoids in human plasma by μ-SPE followed by HPLC-MS/MS by Manuel Sergi; Natalia Battista; Camilla Montesano; Roberta Curini; Mauro Maccarrone; Dario Compagnone (pp. 785-793).

Endocannabinoids (ECs) are endogenous compounds that interact with type-1 and type-2 cannabinoid receptors (CB₁ and CB₂), as well as non-cannabinoid receptors. The multitude of roles attributed to ECs makes them an emerging target of pharmacotherapy for a number of disparate diseases. Here a high-throughput bioanalytical method based on micro SPE (μ-SPE) followed by LC-MS/MS analysis for the simultaneous determination of the two major endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) in human plasma is presented. The chromatographic conditions obtained with the fused-core column allowed a good separation in 10 min also of the AG isomers. A very simple and reliable extraction has been optimised by means of C18-modified tips: it requires only 100 μL of plasma and allows the use of minimal volumes of organic solvent. The present method allows a rapid and effective clean-up, which also minimises the isomerisation of 2-AG. The whole procedure has been validated following the FDA guidelines for bioanalytical methods validation: the satisfactory recovery values, the negligible matrix effect and the good values of accuracy and reproducibility make it a simple and high-throughput analytical tool for clinical and biochemical studies on endocannabinoid signaling in humans. Figure Determination of the two major endocannabinoids in human plasma by μ-SPE followed by HPLC-MS/MS

Keywords: Endocannabinoids; Plasma; LC-MS/MS; Arachidonoylethanolamine (anandamide, AEA); 2-Arachidonoylglycerol (2-AG)

Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase by Manuela Bartolini; Marina Naldi; Dan V. Nicolau; Falco C. M. J. M. van Delft; Jeroen van Zijl; Jaap Snijder; Eric F. C. van den Heuvel; Emile P. Naburgh; Natalia Calonghi; Vincenza Andrisano (pp. 795-804).

Human acetylcholinesterase (AChE) is a widely studied target enzyme in drug discovery for Alzheimer’s disease (AD). In this paper we report evaluation of the optimum structure and chemistry of the supporting material for a new AChE-based fluorescence sensing surface. To achieve this objective, multilayered silicon wafers with spatially controlled geometry and chemical diversity were fabricated. Specifically, silicon wafers with silicon oxide patterns (SiO₂/Si wafers), platinum-coated silicon wafers with SiO₂ patterns (SiO₂/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO₂ and SiO₂ (SiO₂/TiO₂/Pt/Ti/Si wafers) were labelled with the fluorescent conjugation agent HiLyte Fluor 555. Selection of a suitable material and the optimum pattern thickness required to maximize the fluorescence signal and maintain chemical stability was performed by confocal laser-scanning microscopy (CLSM). Results showed that the highest signal-to-background ratio was always obtained on wafers with 100 nm thick SiO₂ features. Hence, these wafers were selected for covalent binding of human AChE. Batch-wise kinetic studies revealed that enzyme activity was retained after immobilization. Combined use of atomic-force microscopy and CLSM revealed that AChE was homogeneously and selectively distributed on the SiO₂ microstructures at a suitable distance from the reflective surface. In the optimum design, efficient fluorescence emission was obtained from the AChE-based biosensing surface after labelling with propidium, a selective fluorescent probe of the peripheral binding site of AChE. Figure Micropatterned silicon wafers containing covalently bound human acetylcholinesterase. The binding and displacement of the fluorescent probe propidium (red areas) from the enzyme's peripheral binding site is visualized by scanning laser microscopy

Keywords: Acetylcholinesterase; Micropatterned silicon wafer; Biosensing surface; Atomic-force microscopy; Confocal scanning microscopy; Fluorescence labelling

An innovative, easily fabricated, silver nanoparticle-based titanium implant coating: development and analytical characterization by E. De Giglio; D. Cafagna; S. Cometa; A. Allegretta; A. Pedico; L. C. Giannossa; L. Sabbatini; M. Mattioli-Belmonte; R. Iatta (pp. 805-816).

Microbial colonization and biofilm formation on implanted devices represent an important complication in orthopaedic and dental surgery and may result in implant failure. Controlled release of antibacterial agents directly at the implant site may represent an effective approach to treat these chronic complications. Resistance to conventional antibiotics by pathogenic bacteria has emerged in recent years as a major problem of public health. In order to overcome this problem, non-conventional antimicrobial agents have been under investigation. In this study, polyacrylate-based hydrogel thin coatings have been electrosynthesised on titanium substrates starting from poly(ethylene glycol diacrylate)–co–acrylic acid. Silver nanoparticles (AgNPs) with a narrow size distribution have been synthesized using a “green” procedure and immobilized on Ti implant surfaces exploiting hydrogel coatings’ swelling capabilities. The coatings have been characterized by XPS and SEM/EDX, while their silver release performances have been monitored by ICP–MS. The antibacterial activity of these AgNP-modified hydrogel coatings was tested evaluating in vitro inhibition growth of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, among the most common pathogens in orthopaedic infections. Moreover, a preliminary investigation of the biocompatibility of silver-loaded coatings versus MG63 human osteoblast-like cells has been performed. An important point of strength of this paper, in fact, is the concern about the effect of silver species on the surrounding cell system in implanted medical devices. Silver ion release has been properly tuned in order to assure antibacterial activity while preserving osteoblasts’ response at the implant interface. Figure Silver nanoparticles-loaded PEGDA-AA hydrogel coatings for inhibition of titanium implants associated infections

Keywords: Silver nanoparticles; Hydrogels; Electrosynthesis; Titanium implants; Antibacterial activity; MG63 human osteoblast-like cells

Study of 1,4-naphthoquinone as a new useful derivatization reagent for LC analysis of aliphatic thiols in dietary supplements and pharmaceuticals by Rita Gatti; Paolo Andreatta; Silvia Boschetti (pp. 817-825).

The use of 1,4-naphthoquinone as an advantageous pre-column reagent for liquid chromatography analysis of aliphatic thiol compounds is proposed. The compound reacts selectively in mild conditions (5 min at room temperature; pH 7.5) with thiol function. The resulting adducts were separated under isocratic conditions by using a reversed-phase column (C-12n) with a mobile phase corresponding to methanol/triethylammonium phosphate buffer (pH 3; 0.05 mol L⁻¹) 65:35, v/v, at a flow rate of 0.4 mL min⁻¹ in presence of quercetin as internal standard. Detection was set at a wavelength of 420 nm. The effect of the derivatization reaction conditions on the N-acetylcysteine (NAC) reaction yield was investigated by a series of experiments. The yield of NAC derivative was found to be quantitative at a reagent thiol molar ratio of about 3 by comparison with an authentic specimen of synthesized NAC adduct, which was characterized by ¹ H NMR, IR, and UV. Similar linear responses were observed by standard and placebo solutions (determination coefficient, 0.9998). The within- and between-day standard deviations (RSD) were ≤0.47 %. Recovery studies showed good results (100.03 %) with RSD 0.76 %. The limit of detection was about 20 pmol. The utility of the validated method for the determination of NAC in a new dietary supplement and commercial formulations is demonstrated.

Keywords: LC; 1,4-Naphthoquinone; Pre-column derivatization; Dietary supplements; Thiol drugs

Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors by Angela De Simone; Francesca Mancini; Feliciana Real Fernàndez; Paolo Rovero; Carlo Bertucci; Vincenza Andrisano (pp. 827-835).

The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors. Figure SPR competition studies for BACE-1 inhibitors

Keywords: BACE-1; Surface plasmon resonance; Peptidic and nonpeptidic inhibitors; Circular dichroism; Fluorescence spectroscopy

Development of new chromatographic tools based on A_2A adenosine receptor subtype for ligand characterization and screening by FAC-MS by Caterina Temporini; Gabriella Massolini; Gabriella Marucci; Catia Lambertucci; Michela Buccioni; Rosaria Volpini; Enrica Calleri (pp. 837-845).

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A_2A adenosine receptor (A_2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A_2AAR vector (A_2A(+)) and from the same cell line transfected with the corresponding empty vector (A_2A(−)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A_2A(+)-IAM and A_2A(−)-IAM stationary phases. Frontal chromatography experiments on both A_2A(+)-IAM and A_2A(−)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A_2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K _d values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm × 100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A_2A(+)-OT and A_2A(−)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates. Figure Immobilized A_2A receptor for Kd determination and ligand screening

Keywords: FAC-MS; A_2A receptor; Binding assay; Biochromatography; Drug discovery

Simultaneous diastereo- and enantioseparation of farnesoid X receptor (FXR) agonists with a quinine carbamate-based chiral stationary phase by Roccaldo Sardella; Maura Marinozzi; Federica Ianni; Antonella Lisanti; Benedetto Natalini (pp. 847-862).

In the frame of a project aimed at finding non-steroidal farnesoid X receptor (FXR) agonists, we identified 4-(2,4-dimethoxyphenyl)-3,6-dimethyl-1-(2-tolyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-one (1) as a hit endowed with FXR activity. Most of the compounds synthesised during the hit-to-lead optimisation work were characterised by the presence of two chiral centres and were therefore obtained as mixtures of anti(±)- and syn(±)-diastereoisomers. A restricted sub-set of species harboured with a carboxylic acid group on the distal phenyl ring of the biphenyl (a(±)5 (A1) and s(±)5 (S1)) or the phenoxyphenyl (a(±)6 (A2) and s(±)6 (S2)) moiety at C-4 position of the pyrazole[3,4-e][1,4]thiazepin-7-one core, resulted in suitable diastereo- and enantioresolution with a quinine (QN) carbamate-derived chiral stationary phase (CSP). Differently from the compounds usually analysed with QN-based CSPs, the couples A1/S1 and A2/S2 were atypical selectands, in which the two chiral carbon atoms reside at a remote position with respect to the carboxylic function, the main “point of attack” to the CSP. We produced evidence that the scarcely employed normal-phase (NP) eluent systems represent the elective choice for achieving the simultaneous diastereo- and enantioseparation of this class of compounds over the usually preferred reversed-phase (RP) and polar-organic (PO) modes of elution. Indeed, after the optimisation of the eluent composition, NP conditions allowed to obtain profitable enantioselectivity profiles, along with excellent diastereoselectivity levels (α(A1) = 1.07, R _S(A1) = 1.15; α(S1) = 1.09, R _S(S1) = 1.47; α(A2) = 1.08, R _S(A2) = 1.31; and α(S2) = 1.06, R _S(S2) = 1.18). The optimised NP methods are suitable for simultaneously providing information on the diastereo- and enantiopurity of the investigated compounds. Figure Simultaneous diastereoand enantioseparation of two non-steroidal FXR agonists with a quinine carbamate-based chiral stationary phase, in the normal-phase mode of elution.

Keywords: Farnesoid X receptor agonists; Quinine carbamate-based chiral stationary phase; Enantioseparation; Diastereoseparation; Ion-exchange liquid chromatography; Normal-phase conditions

Fast screening of 88 pharmaceutical drugs and metabolites in whole blood by ultrahigh-performance liquid chromatography–tandem mass spectrometry by Marco Vincenti; Daniele Cavanna; Enrico Gerace; Valentina Pirro; Michele Petrarulo; Daniele Di Corcia; Alberto Salomone (pp. 863-879).

Forensic investigations involving acute or lethal intoxication, drug-facilitated sexual assault, driving or workplace impairment frequently require the analysis of fresh or postmortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after single-dose consumption. A sensitive and selective ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) screening method was developed for fast screening of 88 psychoactive drugs and metabolites in blood samples, including the ones most frequently involved in acute intoxications and forensic investigations in Italy. The new method allows short sample processing and analysis time (the whole procedure can be accomplished in less than 30 min) together with the simultaneous monitoring of a large number of pharmaceutical substances. These features represent crucial factors in the approach of acute intoxications, when the patient requires urgent and appropriate therapy. Blood sample treatment was limited to protein precipitation. Two UHPLC–MS/MS runs in positive and negative electrospray ionization modes were performed. The data were acquired at unit mass resolution in the selected reaction monitoring mode. According to international guidelines, linearity range, precision, trueness, detection and quantification limits, recovery, selectivity, specificity, carryover, and matrix effect phenomena were determined. Despite the limited sample purification and the inherent decreased chance of eliminating any potential interference, the present multiresidue screening method proved extremely effective and sensitive, allowing the detection of all tested drugs, even those belonging to structurally different classes of substances. Moreover, the developed method is easily susceptible to further expansion to encompass more drugs, either new or those becoming important for criminal investigation. This protocol was also applied to the analysis of authentic blood samples collected from victims of various crimes in routine casework, whose relevance in forensic investigations is presented in five cases.

Keywords: UHPLC–MS/MS; Whole blood; Multiresidue screening; Pharmaceutical drugs

Sequestering ability of some chelating agents towards methylmercury(II) by Gabriella Falcone; Claudia Foti; Antonio Gianguzza; Ottavia Giuffrè; Anna Napoli; Alberto Pettignano; Daniela Piazzese (pp. 881-893).

A study on the interactions between CH₃Hg⁺ and some S, N and O donor ligands (2-mercaptopropanoic acid (thiolactic acid (H₂ TLA)), 3-mercaptopropanoic acid (H₂ MPA), 2-mercaptosuccinic acid (thiomalic acid (H₃ TMA)), d,l-penicillamine (H₂ PSH), l-cysteine (H₂ CYS), glutathione (H₃ GSH), N,N′-bis(3-aminopropyl)-1-4-diaminobutane (spermine (SPER)), 1,2,3,4,5,6-benzenehexacarboxylic acid (mellitic acid (H₆ MLT)) and ethylenediaminetetraacetic acid (H₄ EDTA)) is reported. The speciation models in aqueous solution and the possible structures of the complexes formed are discussed on the basis of potentiometric, calorimetric, UV spectrophotometric and electrospray mass spectrometric results. For the CH₃Hg⁺–S donor ligand systems, the formation of ML1–z and MLH2–z complex species is observed, together with a diprotonated MLH ₂ ^3–z species for CYS ²⁻, PSH ²⁻ and GSH ³⁻ and the mixed hydrolytic one ML(OH)^−z for TLA ²⁻ and MPA ²⁻. The dependence of the stability on ionic strength and on temperature is also analysed. In the other CH₃Hg⁺-L systems (L = MLT ⁶⁻, SPER and EDTA ⁴⁻), ML1–z, MLH2–z and MLH₂ 3–z complex species are formed, together with the MLH₃ 4–z species for SPER, the mixed hydrolytic ML(OH)^–z one for SPER and EDTA, and the M₂L^2–z for EDTA only. On the basis of the speciation models proposed, the sequestering ability of the ligands towards methylmercury(II) cation is evaluated. All S donor ligands show a good sequestering power (at 10⁻¹¹ mol L⁻¹ level, in the pH range 4 to 8) following the trend MPA ²⁻ < PSH ²⁻ < GSH ³⁻ < TLA ²⁻ < CYS ²⁻ < TMA ³⁻, while significantly lower is the sequestering ability of MLT, SPER and EDTA (at 10⁻³–10⁻⁵ mol L⁻¹ level, in the pH range 4 to 8). Figure Sum of fractions of CH₃Hg⁺-L z^– species (L = S, O and N donor ligands vs. pL

Keywords: Methylmercury(II) cation; Sequestering ability of S, N and O donor ligands; UV–spectrophotometry; Electrospray ionisation mass spectrometry (ESI-MS); Potentiometry; Calorimetry

Development of innovative embedding procedures for the analyses of paint cross sections in ATR FITR microscopy by S. Prati; G. Sciutto; E. Catelli; A. Ashashina; R. Mazzeo (pp. 895-905).

We report the development of innovative embedding procedures for the analysis of paint cross sections by attenuated total reflection (ATR) Fourier transform IR microscopy. This technique was chosen because it is widely employed for the characterization and spatial location of organic and inorganic components in artistic samples. Moreover, the performance of the technique may be critically affected by sample preparation in terms of surface morphology and the presence of contamination. First, we evaluated the use of KBr as a barrier to contamination by the embedding synthetic medium. In this way, the sample cross section can be polished by means of a sample holder, which allows a controlled pressure to be applied to the sample, thus improving the reproducibility and quality of the surface cross section. In addition, argon ion milling was used for the polishing of samples embedded in KBr, and provided very promising results in terms of surface planarity and reduction of superficial contamination by KBr. Finally, the use of NaCl as an alternative to KBr was proposed thanks to its advantages in terms of hygroscopicity, cost, and toxicity. In addition, cross sections embedded in NaCl were characterized by greater hardness, a feature that allowed us to obtain improved contact with the ATR crystal.

Keywords: Preparation of cross sections; Fourier transform IR microscopy in attenuated total reflection mode; Paint samples; Spectral quality

Simultaneous determination of sixteen underivatized biogenic amines in human urine by HPLC-MS/MS by Fabio Gosetti; Eleonora Mazzucco; Maria Carla Gennaro; Emilio Marengo (pp. 907-916).

The broad group of biogenic amines includes polyamines and catecholamines, whose presence in human tissues and biological fluids can give important diagnostic information and act as marker of many pathologies. In particular, polyamines are involved in cancer cell growth while catecholamines act as neurotransmitters and hormones. Their simultaneous determination in biological tissues and fluids is therefore an important task. A high-performance liquid chromatography tandem mass spectrometry method is presented here for the simultaneous determination in urine of 16 biogenic amines: adrenaline (epinephrine), agmatine, cadaverine, dopamine, histamine, 3-methoxytyramine, noradrenaline (norepinephrine), norephedrine, octopamine, 2-phenylethylamine, putrescine, serotonin, spermidine, spermine, tryptamine, and tyramine. The method does not require any derivatization step. To guarantee the maximum of sensitivity, the mass spectrometer works in selected reaction monitoring mode, monitoring for each analyte the two most intensive transitions. Method validation includes the evaluation of limits of detection (that range from 0.3 to 6.6 μg L⁻¹), limits of quantitation (that range from 1.0 to 21.9 μg L⁻¹), linearity range (three orders of magnitude), recovery, intra- and inter-day precision on both concentration, and retention time. Recovery (R) is shown not to depend on the analyte concentration: the average R percent ranges from 72.9 to 100.0 %. Particular attention is devoted to the matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection. Figure HPLC-MS/MS determination of 16 biogenic amines

Keywords: Biogenic amines; Neurotransmitters; Catecholamines; Human urine; HPLC; Mass spectrometry

Development of a method for simultaneous analysis of PCDDs, PCDFs, PCBs, PBDEs, PCNs and PAHs in Antarctic air by R. Piazza; A. Gambaro; E. Argiriadis; M. Vecchiato; S. Zambon; P. Cescon; C. Barbante (pp. 917-932).

The development of a unique analytical method for the determination of five classes of persistent organic pollutants (POPs) in atmospheric gas and the particle phase through gas chromatography coupled to high-resolution mass spectrometry is presented. Every step of the pre-analytical and analytical optimization process is described. Great effort was put into simplifying the traditional techniques, with reference to EPA and literature methods. Automated instruments were used for sample extraction and cleanup in order to enhance repeatability and reduce contamination risks. Unlike most common approaches, no separation of the analytes was performed before the GC analysis in order to avoid sample fractionation and to save time and materials. This allowed low instrumental and method detection limits (pg to sub-pg) to be achieved. Accuracy and precision were tested by fortifying the matrix and analysing standard reference materials (NIST SRM 1649b Urban Dust and 2585 Organic Contaminants in House Dust). The method was applied to five samples from Terra Nova Bay, Antarctica. Concentrations of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) are presented. Lighter compounds dominate the distribution and are mainly present in the gaseous phase. The observed pattern may be attributable to long-range transport. Results are in general agreement with literature data, where available.

Keywords: POPs; Aerosol; Dioxins; Antarctica

Single and multiplexed immunoassays for the chemiluminescent imaging detection of animal glues in historical paint cross-sections by G. Sciutto; L. S. Dolci; M. Guardigli; M. Zangheri; S. Prati; R. Mazzeo; A. Roda (pp. 933-940).

The characterization of the organic components in a complex, multilayered paint structure is fundamental for studying painting techniques and for authentication and restoration purposes. Proteinaceous materials, such as animal glue, are of particular importance since they are widely used as binders, adhesives and for gilding. Even though proteins are usually detected by chromatographic and proteomic techniques, immunological methods represent an alternative powerful approach to protein analysis thanks to the high specificity of antigen–antibody reactions. Our previous studies demonstrated that ovalbumin and casein could be localized in paint cross-sections with high sensitivity and good spatial resolution (i.e. within the single painting layers) by using chemiluminescent (CL) immunochemical microscope imaging. In the present research work, we describe for the first time the immunolocalization of collagen (the main protein of animal glue) in paint cross-sections by CL imaging microscopy. Two different analytical protocols have been developed, allowing either the detection of collagen or the simultaneous detection of collagen and ovalbumin in the same paint sample. The assays were used to detect collagen and ovalbumin in cross-sections from model samples and historical paintings (a wall painting dated to 1773–1774 and a painted wood panel of the Renaissance period) in order to achieve information on paint techniques and past restoration interventions. Figure Left Reflected light image of a cross-section of a sample taken from a Renaissance painted wood panel. Right Localization of the proteins collagen (from animal glue) and ovalbumin in a painting cross-section assessed by multiplexed chemiluminescence immunochemical imaging (the chemiluminescent signals corresponding to collagen and ovalbumin are displayed in shades of blue and red, respectively)

Keywords: Animal glue; Chemiluminescence; Imaging; Immunoassay; Ovalbumin; Painting cross-section

Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2′deoxyuridine-labeled DNA in parvovirus B19-infected cells by Francesca Bonvicini; Mara Mirasoli; Elisabetta Manaresi; Giovanna Angela Gentilomi; Aldo Roda; Giorgio Gallinella (pp. 941-949).

Incorporation of exogenous analogues is a widely used method to evaluate DNA synthesis in cultured cells exposed to exogenous factors such as infectious agents. Herein, two new quantitative methodologies exploiting ultrasensitive chemiluminescence (CL) detection of 5-bromo-2′-deoxyuridine (BrdU) have been developed: a CL microscope imaging assay to evaluate BrdU labelling at single-cell level and a CL dot-blot assay to measure the amounts of DNA produced in the course of an in vitro infection of proliferating cells. The assays have been optimized on UT7/EpoS1 cells cultured in presence of different concentrations of BrdU (from 3 to 100 μM) and used to monitor parvovirus B19 (B19) life cycle in infected cells. The CL microscope imaging assay provided a detailed localization of BrdU-labelled nuclei allowing to count positive cells and measure their related CL intensity signals. The CL dot-blot assay, coupled with a B19 capture procedure performed with a specific peptide nucleic acid probe, has been designed to discriminate and selectively quantify cellular and viral BrdU-labelled genomes. Quantitative evaluation of BrdU-labelled B19 DNA has been achieved by means of a CL calibration curve. The high detectability, down to 2 × 10⁶ B19 genome copies, and the linear range extending up to 5 × 10⁸ copies make the method suitable to evaluate the amounts of B19 DNA produced throughout a replicative viral cycle. Figure A chemiluminescence (CL) quantitative dot-blot assay was developed to study the parvovirus B19 life cycle following in vitro infection of different susceptible cells. The procedure, involving the labelling of newly synthesized DNA with 5-bromo-2′deoxyuridine (BrdU), allows for (a) detecting the sum of viral and cellular DNAs and (b) selective detecting only viral DNA, exploiting the ability of a specific peptide nucleic acid probe to capture B19 DNA

Keywords: 5-Bromo-2′-deoxyuridine; Chemiluminescence; Parvovirus B19; Chemiluminescence microscope imaging assay; Chemiluminescence dot-blot assay

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis by Annalinda Contino; Giuseppe Maccarrone; Maurizio Remelli (pp. 951-959).

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm⁻³ (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L = l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄. Figure Schematic view of the separation process

Keywords: Chiral analysis; Ligand exchange capillary electrophoresis; Thermodynamic parameters; Copper(II); Amino acids

Occupational exposure to complex mixtures of volatile organic compounds in ambient air: desorption from activated charcoal using accelerated solvent extraction can replace carbon disulfide? by Giovanni Fabrizi; Marzia Fioretti; Lucia Mainero Rocca (pp. 961-976).

A desorption study of 57 volatile organic compounds (VOCs) has been conducted by use of accelerated solvent extraction (ASE) and gas chromatography–mass spectrometry. Different solvents were tested to extract activated charcoal tubes with the objective of replacing carbon disulfide, used in official methods, because of its highly toxic health and environmental effects. Extraction conditions, for example temperature and number of cycles, were investigated and optimized. The definitive extraction procedure selected was use of acetone at 150 °C and two consecutive extraction cycles at a pressure of 1,500 psi. Considering a sample volume of 0.005 Nm³, corresponding to a sampling time of 8 h at a flow rate of 0.01 L min⁻¹, the method was validated over the concentration range 65–26,300 μg Nm⁻³. The lowest limit of quantification was 6 μg Nm⁻³, and recovery for the 93 % of analytes ranged from 65 to 102 %. For most of the compounds, relative standard deviations were less than 15 % for inter and intra-day precision. Uncertainty of measurement was also determined: the relative expanded uncertainty was always below 29.6 %, except for dichlorodifluoromethane. This work shows that use of friendlier solvent, for example acetone, coupled with use of ASE, can replace use of CS₂ for chemical removal of VOCs from activated charcoal. ASE has several advantages over traditional solvent-extraction methods, including shorter extraction time, minimum sample manipulation, high reproducibility, and less extraction discrimination. No loss of sensitivity occurs and there is also a salutary effect on bench workers’ health and on the smell of laboratory air. Figure Acetone molecule, vials, cells and sorbent tubes used for VOCs extraction with Accelerated Solvent Extractor.

Keywords: VOCs; ASE; GC–MS; Occupational exposure; Method validation; Air sample

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation by Debora Lattuada; Barbara Roda; Chiara Pignatari; Ruben Magni; Federico Colombo; Alessandra Cattaneo; Andrea Zattoni; Irene Cetin; Pierluigi Reschiglian; Giorgio Bolis (pp. 977-984).

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

Keywords: Tag-less cell sorting; Gravitational field-flow fractionation (GrFFF); HUVEC (Human umbilical vein endothelial cells)

Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification by M. L. Ermini; S. Mariani; S. Scarano; D. Campa; R. Barale; M. Minunni (pp. 985-993).

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Keywords: Surface plasmon resonance imaging; Whole genome amplification; DNA-based sensing; Pharmacogenomics; Single nucleotide polymorphism; ABCB1

Ensembles of nanoelectrodes modified with gold nanoparticles: characterization and application to DNA-hybridization detection by Morena Silvestrini; Paolo Ugo (pp. 995-1005).

A new method to increase the active area (A _act) of nanoelectrode ensembles (NEEs) is described. To this aim, gold nanoparticles (AuNPs) are immobilized onto the surface of NEEs using cysteamine as a cross-linker able to bind the AuNPs to the heads of the nanoelectrodes to obtain the so-called AuNPs-NEEs. The analysis of the cyclic voltammograms recorded in pure supporting electrolyte showed that the presence of the nanoparticles reflects in an, approximately, ten-times increase in the electrochemically active area of the ensemble. The measurement of the amount of electroactive polyoxometalates, which can be adsorbed on the gold surface of NEEs vs. AuNPs-NEEs, confirmed a significant increase of active area for the latter. These evidences indicate that there is a good electronic connection between the AuNPs and the underlying nanoelectrodes. The possibility to exploit AuNPs-NEEs for biosensing application was tested for the case of DNA-hybridization detection. After immobilization on the gold surface of AuNPs-NEEs of a thiolated single-stranded DNA, the hybridization with complementary sequences labeled with glucose oxidase (GOx) was performed. The detection of the hybridization was achieved by adding to the electrolyte solution the GOx substrate (i.e., glucose) and a suitable redox mediator, namely the (ferrocenylmethyl) trimethylammonium (FA⁺) cation; when the hybridization occurs, an electrocatalytic increase of the oxidation current of FA⁺ is recorded. Comparison of electrocatalytic current recorded at DNA modified NEEs and AuNPs-NEEs indicate, for the latter, a significant increase in sensitivity in the detection of the DNA-hybridization event.

Keywords: Nanoelectrode; Ensembles; Gold nanoparticles; DNA hybridization; Voltammetry

UHPLC-DAD method for the determination of neonicotinoid insecticides in single bees and its relevance in honeybee colony loss investigations by Andrea Tapparo; Chiara Giorio; Lidia Soldà; Sara Bogialli; Daniele Marton; Matteo Marzaro; Vincenzo Girolami (pp. 1007-1014).

In the understanding of colony loss phenomena, a worldwide crisis of honeybee colonies which has serious consequences for both apiculture and bee-pollination-dependent farm production, analytical chemistry can play an important role. For instance, rapid and accurate analytical procedures are currently required to better assess the effects of neonicotinoid insecticides on honeybee health. Since their introduction in agriculture, neonicotinoid insecticides have been blamed for being highly toxic to honeybees, possibly at the nanogram per bee level or lower. As a consequence, most of the analytical methods recently optimized have focused on the analysis of ultratraces of neonicotinoids using liquid chromatography–mass spectrometry techniques to study the effects of sublethal doses. However, recent evidences on two novel routes—seedling guttations and seed coating particulate, both associated with corn crops—that may expose honeybees to huge amounts of neonicotinoids in the field, with instantly lethal effects, suggest that selected procedures need optimizing. In the present work, a simplified ultra-high-performance liquid chromatography–diode-array detection method for the determination of neonicotinoids in single bees has been optimized and validated. The method ensures good selectivity, good accuracy, and adequate detection limits, which make it suitable for the purpose, while maintaining its ability to evaluate exposure variability of individual bees. It has been successfully applied to the analysis of bees in free flight over an experimental sowing field, with the bees therefore being exposed to seed coating particulate released by the pneumatic drilling machine.

Keywords: Honeybees; Neonicotinoid insecticides; Liquid chromatography; Quick, easy, cheap, effective, rugged, and safe extraction; Mass spectrometry

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages by Carmen Palermo; Marilena Muscarella; Donatella Nardiello; Marco Iammarino; Diego Centonze (pp. 1015-1023).

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r > 0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23–65 μg/kg and quantification limits in spiked blank real samples in the range 65–198 μg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03–0.32 % (n = 4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami. Figure IEC‐CD multiresidual method for accurate determinations of biogenic amines in foodstuffs

Keywords: Ion chromatography; Conductivity detection; Biogenic amines; Linear gradient elution; Cation suppression; Food

Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay by Francesca Bettazzi; Ezat Hamid-Asl; Carla Lucia Esposito; Cristina Quintavalle; Nello Formisano; Serena Laschi; Silvia Catuogno; Margherita Iaboni; Giovanna Marrazza; Marco Mascini; Laura Cerchia; Vittorio De Franciscis; Gerolama Condorelli; Ilaria Palchetti (pp. 1025-1034).

MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin–alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L⁻¹ and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.

Keywords: MicroRNA; Magnetic beads; Electrochemical assay; miRNA-222

Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs by Luca Cevenini; Elisa Michelini; Marcello D’Elia; Massimo Guardigli; Aldo Roda (pp. 1035-1045).

Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ⁹-tetrahydrocannabinol as major constituent. Figure Evaluation of (anti)-androgenic activity of seized drugs with the dual-color bioluminescent bioreporter

Keywords: Bioassays; Bioluminescence; Androgen receptor; Forensic/toxicology; Doping; Bioanalytical methods

Mass spectrometric techniques for characterizing low-molecular-weight resins used as paint varnishes by I. Bonaduce; M. P. Colombini; I. Degano; F. Di Girolamo; J. La Nasa; F. Modugno; S. Orsini (pp. 1047-1065).

The molecular structure of three low-molecular-weight resins used as paint varnishes has been characterized by use of an approach based on three different mass spectrometric techniques. We investigated the ketone resin MS2A, the aldehyde resin Laropal A81, and the hydrocarbon resin Regalrez 1094, now commonly used in restoration. To date, the molecular structures of these resins have not been completely elucidated. To improve current knowledge of the chemical composition of these materials, information obtained by gas chromatography–mass spectrometry (GC/MS), pyrolysis–gas chromatography–mass spectrometry (Py/GC/MS), and electrospray ionization mass spectrometry (ESI–Q–ToF) was combined. Analysis, in solution, of the whole polymeric fraction of the resins by flow-injection ESI–Q–ToF, and of the non-polymeric fraction by GC–MS, enabled us to identify previously unreported features of the polymer structures. In addition, the Py–GC/MS profiles that we obtained will help to enhance the databases currently available in the literature. The proposed approach can be extended to other low-molecular-weight resins used as varnishes in conservation.

Keywords: Low molecular weight resins; MS2A; Laropal A81; Regalrez 1094; ESI-Q-ToF; GC/MS; Py-GC/MS

An innovative bovine odorant binding protein-based filtering cartridge for the removal of triazine herbicides from water by Federica Bianchi; Giuseppina Basini; Stefano Grolli; Virna Conti; Francesco Bianchi; Francesca Grasselli; Maria Careri; Roberto Ramoni (pp. 1067-1075).

Odorant binding protein (OBP) is a multi-functional scavenger for small hydrophobic molecules dissolved in the mucus lining the nasal epithelia of mammals, characterized by broad ligand binding specificity towards a large number of structurally unrelated natural and synthetic molecules of different chemical classes. Here, we demonstrate for the first time the application of OBP as the active element of an innovative filtering matrix for the removal of environmental pollutants such as triazine herbicides from water samples. The filtering device, obtained by coupling histidine-tagged bovine OBP to a nickel nitrilotriacetic acid (Ni-NTA) agarose resin, was characterized in terms of retention capacity for the herbicides atrazine, simazine, and propazine. Analysis of these herbicides at trace levels with solid-phase microextraction followed by gas chromatography–mass spectrometry using the selected ion monitoring mode proved the capabilities of the proposed device for the decontamination of surface and groundwater samples in the 0.2–2,300 μg/L concentration range, obtaining a reduction in the triazine content greater than 97 %, thus suggesting its possible use for the potabilization of water. Figure Tridimensional structure of bOBP with AMA inside the cavity

Keywords: Atrazine; Triazines; Odorant binding protein; Filtering cartridge; Endocrine disruptors; Solid-phase microextraction

Comparison of two different multidimensional liquid–gas chromatography interfaces for determination of mineral oil saturated hydrocarbons in foodstuffs by Giorgia Purcaro; Mariosimone Zoccali; Peter Quinto Tranchida; Laura Barp; Sabrina Moret; Lanfranco Conte; Paola Dugo; Luigi Mondello (pp. 1077-1084).

This investigation focused on direct comparison of two popular multidimensional liquid–gas chromatography (LC–GC) systems, the Y-interface (retention gap approach) and the syringe-based interface (programmed temperature vaporizer approach). Such transfer devices are structurally very different, and could potentially have a substantial effect on the outcome of a specific application. In this work the application was a topic of much current interest, determination of mineral oil saturated hydrocarbon (MOSH) contamination of a series of food products (rice, pasta, icing sugar, olive oil); the final results were then compared. The two LC–GC methods developed were validated for linearity over the calibration range, analyte discrimination, precision, accuracy, and limits of detection and quantification. No significant differences were found between the two approaches. Figure Direct comparison between the Y/LC-GC and the PTV/LC-GC results, on two rice samples

Keywords: Multidimensional liquid–gas chromatography; LC–GC; Food analysis; Paraffins; Retention gap technique; Programmed-temperature vaporizer interface

Conditions for calibration of an isothermal titration calorimeter using chemical reactions by Carmelo Sgarlata; Valeria Zito; Giuseppe Arena (pp. 1085-1094).

The reaction of protonation of 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) is a suitable one for the calibration of isothermal titration calorimeter (ITC), providing that experimental conditions are appropriately chosen. The conditions and methods for handling experimental data from a nanowatt-ITC are discussed. Also, the binding of Ba²⁺ to 18-Crown-6 is successfully used to check the accuracy and precision of the chemical calibration performed with TRIS. This latter reaction has the additional advantage that the data can also be used for a check on the determination of the value of a binding constant. The anomaly of the first injection in ITC is analyzed and, by combining calorimetric and spectroscopic measurements, it is shown that it mainly results from a backlash effect of the syringe plunger rather than from a diffusion effect.

Keywords: Calorimetry; ITC; Chemical calibration; Solution equilibria; Backlash vs. diffusion

GC/MS analysis of water-soluble organics in atmospheric aerosol: optimization of a solvent extraction procedure for simultaneous analysis of carboxylic acids and sugars by Maria Chiara Pietrogrande; Dimitri Bacco; Sara Chiereghin (pp. 1095-1104).

The main goal of the present study was to develop a procedure for the simultaneous GC/MS analysis of a wide range of water-soluble organic compounds (WSOCs, mainly dicarboxylic acids and sugars) in atmospheric aerosol as chemical markers of atmospheric processes. The response surface methodology, including central composite design, was applied to systematically investigate and optimize the operating conditions of a solvent extraction method as the most widely used procedure for determining WSOCs in particulate matter (PM) samples. A solvent mixture of methanol/dichloromethane (90:10) and a volume of 10 ml were the optimum conditions that permit the simultaneous analysis of 30 target WSOCs, including dicarboxylic acids and sugars. Recoveries and limits of detection were determined for a standard mixture containing target analytes, and they varied from 79 to 103 % and 0.3 to 1 ng m⁻³, respectively. The optimized procedure was used for the analysis of field PM filters and demonstrated its feasibility in detecting several important environmental WSOCs.

Keywords: Multiresidue GC/MS method; Experimental design and response surface methodology; Dicarboxylic acids and sugars; Atmospheric aerosol; Solvent extraction optimization

Study of the photocatalytic transformation of synephrine: a biogenic amine relevant in anti-doping analysis by Claudio Medana; Paola Calza; Valeria Giancotti; Federica Dal Bello; Manuela Aragno; Claudio Baiocchi (pp. 1105-1113).

The occurrence of some cases of positive results in anti-doping analysis of octopamine requires clarification as to whether its methylated derivative synephrine could be a metabolic precursor of octopamine itself. Synephrine is a natural phenylethylamine derivative present in some food supplements containing Citrus aurantium, permitted in sport regulations. A simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in biological samples obtained from three human volunteers and four rats treated with synephrine; the parent compound and its new potential metabolic products were investigated in human urine and rat plasma samples. The transformation of synephrine and octopamine and the formation of intermediate products were evaluated, adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC-HRMSⁿ technique. The main intermediates identified in these experimental conditions were compared with the major synephrine metabolites found in in vivo studies on rats and humans. Some more oxidized species, already formed in the photocatalytic process, were also found in urine and plasma samples of treated animals. These new findings could be of interest in further metabolism studies. The main photocatalytic pathway involving synephrine appears to be N-demethylation to give octopamine. On the contrary, we demonstrate the inconsistency of this reaction in both rat and human in vivo determinations, resulting in forensic importance.

Keywords: Synephrine; Octopamine; Metabolism; Doping control; Photocatalysis; HRMS

Effect of drying conditions during sample pre-treatment on the determination of polycyclic aromatic hydrocarbons in soils by Riccardo Narizzano; Fulvia Risso; Alfredo Magherini; Emanuele Magi; Michele Giampieri; Carla Devia; Gloria Venturelli; Eugenio Carlini (pp. 1115-1121).

In the context of the entire analytical process, pre-treatment of soil samples is often inadequately considered although the reliability of the results is definitely compromised if the sample is not properly prepared. In this paper, the effect of drying conditions in soil sample pre-treatment on the determination of polycyclic aromatic hydrocarbons (PAHs) has been studied. A systematic approach has been adopted by varying soil type, drying temperatures and solvent polarity to highlight the effect on the analyte recovery; the relationship between PAH molecular structure and their evaporation process from soils is discussed. Experimental data demonstrate that, concerning temperature-assisted drying procedures, PAHs are divided in two distinct groups: PAHs lighter than pyrene, which are seriously affected by drying temperature; and heavier PAHs that can be considered as non-volatile compounds. For studies involving the analysis of lighter PAHs in environmental samples, working on as-received samples is necessary.

Keywords: PAHs; Air-drying; Sample pre-treatment; Soil analysis; Semi-volatile organic compounds

Optimisation of analytical procedures for the quantification of ionic and carbonaceous fractions in the atmospheric aerosol and applications to ambient samples by Andrea Piazzalunga; Vera Bernardoni; Paola Fermo; Roberta Vecchi (pp. 1123-1132).

In the last decade, our research group set up and optimised analytical techniques for the characterisation of the major components of atmospheric aerosol (i.e. secondary inorganic ions and carbonaceous material) and source markers (e.g. levoglucosan, carbonates). In this study, we present a complete overview on the most problematic aspects that can be encountered during the quantification of the two main components of aerosol, i.e. the ionic and carbonaceous fractions. More in detail, different liquid chromatographic approaches were set up for main ions and anhydrosugars determination. Quality assurance procedures (i.e. tests on data reliability) were applied during the set-up phase and they are presented in this work. As regards the carbonaceous component characterisation, two evolved gas analysis techniques were set up and applied: the thermogravimetric technique coupled to the Fourier transformed infrared spectroscopy (TGA/FTIR) and the thermal–optical transmittance method (TOT). A suitable protocol for organic and elemental carbon separation was set up for the TGA/FTIR system and a comparison with the results obtained by the TOT method was carried out. Studies on the impact of filter load, field blanks, and sample composition on OC/EC quantification by the TOT method were performed. Moreover, approaches for carbonate carbon quantification on different kinds of filters were developed. It was demonstrated that this approach allows to reach the ionic balance in samples impacted by carbonate compounds. The optimised methods have been applied for the analysis of thousands of PM filters allowing the obtainment of reliable results.

Keywords: Aerosol characterisation; Liquid chromatographic techniques; Thermal-optical transmittance method; Thermogravimetric analysis; Carbonate quantification

Selective sensing of Hg²⁺ by a proton-ionizable calix[4]arene fluoroionophore by Giuseppe Arena; Francesco Attanasio; Dongmei Zhang; Yanfei Yang; Richard A. Bartsch; Carmelo Sgarlata (pp. 1133-1137).

A fluorogenic derivative of a calix[4]arene with two proton-ionizable N-(phenyl)sulfonyl carboxamide-containing side arms in the 1,3-positions on the lower rim is employed for the selective sensing of Hg²⁺ at low concentration levels in water/MeCN (1:1, v/v) solutions containing Pb²⁺ and Cd²⁺. All three metal ions quench the fluorescence of the ligand in pure MeCN. However, in water/MeCN mixed solvent, the recognition of such cations occurs differently as only Hg²⁺ complexation quenches the fluorescence of the calixarene. Experiments carried out in the presence of an acid and a bulky non-complexing cation shows that the quenching of the calixarene fluorescence upon Hg²⁺ addition is likely due to proton displacement from the proton-ionizable side arms of the ligand. The system may be employed as a simple tool for the selective and efficient mercury sensing in mixed water/organic solvent.

Keywords: Fluorescent sensor; Selective detection; Mercury; Heavy metals; Calixarenes

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes by Mara Mirasoli; Francesca Bonvicini; Luisa Stella Dolci; Martina Zangheri; Giorgio Gallinella; Aldo Roda (pp. 1139-1143).

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol L⁻¹ for all B19 genotype oligonucleotides and 650 pmol L⁻¹ for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products. Figure A portable multiplex biosensor was developed for detection and genotyping of parvovirus B19 DNA, exploiting lensless CL imaging. The reaction chip is composed of a polydimethylsiloxane microfluidic layer coupled with a glass slide on which oligonucleotide probes specific for three different B19 genotypes are covalently immobilized in a 3 × 3 array. The reaction chip was used in hybridization reactions with biotin-labeled targets and then hybrids were then detected by means of an avidin-HRP conjugate, upon addition of a CL substrate for HRP

Keywords: Biosensor; Chemiluminescence lensless imaging; Point-of-care testing; Gene probe hybridization; Multiplex assay; Parvovirus B19 genotyping

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Analytical and Bioanalytical Chemistry (v.405, #2-3)