ALBERT

Description: Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [ 1 ], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of • OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of • OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics. Figure Barrel structure of ECFP: residues that were found to be oxidized by .OH radicals are highlighted

Description: A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.

Description: The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics. Figure Proposed interactions occurring between arginine–agarose matrix and RNA molecules. Given the multiplicity of arginine side-chain interactions and depending upon RNA folding state, arginine will preferably bind to phosphate groups of RNA backbone or RNA bases.

Description: Functionalized magnetic nanoparticles have attracted much attention in sample preparation because of their excellent performance compared with traditional sample-preparation sorbents. In this review, we describe the application of magnetic nanoparticles functionalized with silica, octadecylsilane, carbon-based material, surfactants, and polymers as adsorbents for separation and preconcentration of analytes from a variety of matrices. Magnetic solid-phase extraction (MSPE) techniques, mainly reported in the last five years, are presented and discussed.

Description: Racemic mixtures of the promising anti-malarial bisindole alkoids, flinderole A–C, desmethyl flinderole C, borreverine and isoborreverine, are baseline-separated for the first time by HPLC using vancomycin-based stationary phases and partially separated by capillary electrophoresis (CE) using cyclodextrin selectors. The HPLC results compare the performance of Chirobiotic V and V2 in the polar organic and reversed phase modes and their complementary selectivity is discussed. The performance of the cyclodextrin selectors in CE, while less effective, are discussed in terms of their selectivity in normal and reversed polarity modes.

Description: A multidimensional, on-line coupled liquid chromatographic/gas chromatographic system was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs). A two-dimensional liquid chromatographic system (2D-liquid chromatography (LC)), with three columns having different selectivities, was connected on-line to a two-dimensional gas chromatographic system (2D-gas chromatography (GC)). Samples were cleaned up by combining normal elution and column back-flush of the LC columns to selectively remove matrix constituents and isolate well-defined, PAH enriched fractions. Using this system, the sequential removal of polar, mono/diaromatic, olefinic and alkane compounds from crude extracts was achieved. The LC/GC coupling was performed using a fused silica transfer line into a programmable temperature vaporizer (PTV) GC injector. Using the PTV in the solvent vent mode, excess solvent was removed and the enriched PAH sample extract was injected into the GC. The 2D-GC setup consisted of two capillary columns with different stationary phase selectivities. Heart-cutting of selected PAH compounds in the first GC column (first dimension) and transfer of these to the second GC column (second dimension) increased the baseline resolutions of closely eluting PAHs. The on-line system was validated using the standard reference materials SRM 1649a (urban dust) and SRM 1975 (diesel particulate extract). The PAH concentrations measured were comparable to the certified values and the fully automated LC/GC system performed the clean-up, separation and detection of PAHs in 16 extracts in less than 24 h. The multidimensional, on-line 2D-LC/2D-GC system eliminated manual handling of the sample extracts and minimised the risk of sample loss and contamination, while increasing accuracy and precision. Figure Scheme of the 2D-LC/2D-GC system

Description: Recently, an atomic force microscopy (AFM)-based approach for quantifying the number of biological molecules conjugated to a nanoparticle surface at low number densities was reported. The number of target molecules conjugated to the analyte nanoparticle can be determined with single nanoparticle fidelity using antibody-mediated self-assembly to decorate the analyte nanoparticles with probe nanoparticles (i.e., quantitative immunostaining). This work refines the statistical models used to quantitatively interpret the observations when AFM is used to image the resulting structures. The refinements add terms to the previous statistical models to account for the physical sizes of the analyte nanoparticles, conjugated molecules, antibodies, and probe nanoparticles. Thus, a more physically realistic statistical computation can be implemented for a given sample of known qualitative composition, using the software scripts provided. Example AFM data sets, using horseradish peroxidase conjugated to gold nanoparticles, are presented to illustrate how to implement this method successfully.

Description: Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2′-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes. Figure Myoblasts were marked with DiI ( red ) and EdU ( purple ), and injected into lesioned skeletal muscle. At day 9 following transplantation, only DiI positive cells were observed and had participated in myofibre formation as (indicated by arrowheads) visualized by red fluorescence signals inside laminin ( green ) positive multinucleated myofibres. EdU was toxic to the engrafted cells, suggesting that this reagent is non-applicaple for tracing of stem cells.

Description: Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled “not for human consumption” and considered “legal highs”. Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3’-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5–1 to 100 μg/L, with limits of detection of 0.25–1 μg/L. Imprecision ( n  = 20) was 〈15.9 % and accuracy ( n  = 20) 85.2–118.1 %. Extraction efficiency was 78.9–116.7 % (CV 1.4–16.7 %, n  = 5), process efficiency 57.7–104.9 %, and matrix effects from −29.5 % to 1.5 % (CV 1.9–13.1 %, n  = 10). Most synthetic cathinones were stable at 4 °C for 72 h ( n  = 27) and after 3 freeze-thaw cycles ( n  = 26), but many ( n  = 19) were not stable at room temperature for 24 h (losses up to −67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.

Description: Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV–Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis. Figure Reaction‐detection scheme for the fluorescent derivative of proline with o‐pthalaldehyde reagent

Description: The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

Description: Direct analysis in real time mass spectrometry (DART-MS) has become an established technique for rapid mass spectral analysis of a large variety of samples. DART-MS is capable of analyzing the sample at atmospheric pressure, essentially in the open laboratory environment. DART-MS can be applied to compounds that have been deposited or adsorbed on to surfaces or that are being desorbed therefrom into the atmosphere. This makes DART-MS suitable and well-known for analysis of ingredients of plant materials, pesticide monitoring on vegetables, forensic and safety applications such as screening for traces of explosives, warfare agents, or illicit drugs on luggage, clothes, or bank notes, etc. DART can also be used for analysis of either solid or liquid bulk materials, as may be required in quality control, or to quickly investigate the identity of a compound from chemical synthesis. Even living organisms can be subjected to DART-MS. Driven by different needs in analytical practice, the combination of the DART ionization source and interface can be configured in multiple geometries and with various accessories to adapt the setup as required. Analysis by DART-MS relies on some sort of gas-phase ionization mechanism. In DART, initial generation of the ionizing species is by use of a corona discharge in a pure helium atmosphere which delivers excited helium atoms that, upon their release into the atmosphere, will initiate a cascade of gas-phase reactions. In the end, this results in reagent ions created from atmospheric water or (solvent) vapor in the vicinity of the surface subject to analysis where they effect a chemical ionization process. DART ionization processes may generate positive or negative ions, predominantly even-electron species, but odd-electron species do also occur. The prevailing process of analyte ion formation from a given sample is highly dependent on analyte properties.

Description: In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column—a so-called dress-up chiral column—featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53 % in 20-times repeated analysis, and were all less than or equal to 18.7 % in four different preparations, respectively.

Description: A method for evaluating the interactions between metal ions and nonionic surfactants in aqueous solutions containing high-concentration HCl, using gas pressure-driven low-pressure high-performance liquid chromatography (LP-HPLC) as a highly acid-resistant HPLC system, was developed. To construct the LP-HPLC for this purpose, poly(styrene- co -divinylbenzene)-based low-flow-resistance monolithic columns tolerant to highly acidic conditions were prepared using low-conversion thermal polymerization. Thermal polymerization at 65 °C for 1.5 h (monomer conversions, 33 % for styrene and 59 % for divinylbenzene) allowed preparation of a column with both high separation efficiency (around 60,000 plates m −1 for alkylbenzenes) and a quite low back pressure of 0.14 MPa at a linear flow rate of 1 mm s −1 (2.8 × 10 −13  m 2 in permeability). The base column prepared under the above conditions was coated with a nonionic surfactant, polyoxyethylene nonylphenyl ether (PONPE, average oxyethylene unit numbers ( n ) = 3, 7.5, 15, and 20), and used for evaluation of the interactions between PONPEs and metal ions in 6 M HCl. The interactions between PONPEs and Au(III), Ga(III), Fe(III), Zn(II), and Cu(II) were successfully evaluated using both breakthrough and chromatographic methods. Furthermore, a study of the effect of the polyoxyethylene (POE) chain length revealed that the use of PONPE with the longer POE moiety enhanced the magnitude of the interaction together with the increase in the amount of oxyethylene (OE) units coated on the monolith. Moreover, the interactions of metal ions with a single OE unit were almost constant in the range of n  = 7.5–20, whereas the suppression of the interaction between Au(III) with the shortest PONPE chain ( n  = 3) was also observed. Figure Acid-resistive gas pressure-driven low-pressure high-performance liquid chromatography was developed and applied to the evaluation of interactions between metal ions and nonionic surfactants in high-concentration HCl, in particular for the effect of polyoxyethylene length on the interaction.

Description: In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm 2 microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4–20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42–3.80 and 2.74–6.97 %, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1 %. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.

Description: The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites—17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one—using a fungus ( Cunninghamella elegans ) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete’s urine, different analytical approaches were followed, such as liquid chromatography–tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography–tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity ( R 2  〉 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation 〈20 %). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Description: Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations ( r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D ® ) and a more cost-efficient and transportable planar imaging detector (MAGPIX ® ), hence demonstrating adequate transferability.

Description: Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 96/22/EC. However, this kind of substances is sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semisynthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e., estradiol-17β or progesterone) still remains a challenge for European authorities. These target compounds are indeed always present in the animal matrix, and the establishment of reference thresholds appears very difficult because of the extreme variability existing among animals. In 2011, a metabolomics study was performed on serum samples obtained from cows treated with estradiol-17β (or its ester estradiol benzoate) and from control animals using a high-performance liquid chromatography (HPLC)-LTQ-Orbitrap system. After appropriate data processing and multivariate statistical analysis (orthogonal partial least squares discriminant analysis), it was possible to highlight one potential biomarker candidate of estradiol treatments in bovine animals. Now, this biomarker has been structurally elucidated as a dipeptide, and its usefulness has been tested through a targeted HPLC-MS/MS method. Its presence in the previous samples has been confirmed and also in additional samples from estradiol-treated animals.

Description: A bootstrapped fuzzy rule-building expert system (FuRES) and a bootstrapped t -statistical weight feature selection method were individually used to select informative features from gas chromatography/mass spectrometry (GC/MS) chemical profiles of basil plants cultivated by organic and conventional farming practices. Feature subsets were selected from two-way GC/MS data objects, total ion chromatograms, and total mass spectra, separately. Four economic classifiers based on the bootstrapped FuRES approach, i.e., fuzzy optimal associative memory (e-FOAM), e-FuRES, partial least-squares–discriminant analysis (e-PLS-DA), and soft independent modeling by class analogy (e-SIMCA), and four economic classifiers based on the bootstrapped t-weight approach, i.e., e-PLS-DA-t, e-FOAM-t, e-FuRES-t, and e-SIMCA-t, were constructed thereafter to be compared with full-size classifiers obtained from the entire GC/MS data objects (i.e., FOAM, FuRES, PLS-DA, and SIMCA). By using three features selected from two-way data objects, the average classification rates with e-FOAM, e-FuRES, e-PLS-DA, and e-SIMCA were 95.3 ± 0.5 %, 100 %, 100 %, and 91.8 ± 0.2 %, respectively. The established economic classifiers were used to classify a new validation set collected 2.5 months later with no parametric change to experimental procedure. Classification rates with e-FOAM, e-FuRES, e-PLS-DA, and e-SIMCA were 96.7 %, 100 %, 100 %, and 96.7 %, respectively. Characteristic components in basil extracts corresponding to highest-ranked useful features were putatively identified. The feature subset may prove valuable as a rapid approach for organic basil authentication.

Description: A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-μm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 μL min −1 flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.

Description: We have recently developed a novel portable NIR imaging device (D-NIRs), which has a high speed and high wavelength resolution. This NIR imaging approach has been developed by utilizing D-NIRs for studying the dissolution of a model tablet containing 20 % ascorbic acid (AsA) as an active pharmaceutical ingredient and 80 % hydroxypropyl methylcellulose, where the tablet is sealed by a special cell. Diffuse reflectance NIR spectra in the 1,000 to 1,600 nm region were measured during the dissolution of the tablet. A unique band at around 1,361 nm of AsA was identified by the second derivative spectra of tablet and used for AsA distribution NIR imaging. Two-dimensional change of AsA concentration of the tablet due to water penetration is clearly shown by using the band-based image at 1,361 nm in NIR spectra obtained with high speed. Moreover, it is significantly enhanced by using the intensity ratio of two bands at 1,361 and 1,354 nm corresponding to AsA and water absorption, respectively, showing the dissolution process. The imaging results suggest that the amount of AsA in the imaged area decreases with increasing water penetration. The proposed NIR imaging approach using the intensity of a specific band or the ratio of two bands combined with the developed portable NIR imaging instrument, is a potentially useful practical way to evaluate the tablet at every moment during dissolution and to monitor the concentration distribution of each drug component in the tablet. Figure Visible photo and NIR image for tablet dissolution obtained by using a newly developed portable NIR imaging device: D-NIRs

Description: Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.

Description: The use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability. Figure Concept of whole-cell bioluminescent biosensor

Description: A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography–tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100. The priciple of the direct inhibition microbead immunoassay using fluorescent mycotoxin-reporter conjugates

Description: Molecularly imprinted polymers (MIPs) are synthetic receptors that are able to specifically bind their target molecules in complex samples, making them a versatile tool in biosensor technology. The combination of MIPs as a recognition element with quartz crystal microbalances (QCM-D with dissipation monitoring) gives a straightforward and sensitive device, which can simultaneously measure frequency and dissipation changes. In this work, bulk-polymerized l -nicotine MIPs were used to test the feasibility of l -nicotine detection in saliva and urine samples. First, l -nicotine-spiked saliva and urine were measured after dilution in demineralized water and 0.1× phosphate-buffered saline solution for proof-of-concept purposes. l -nicotine could indeed be detected specifically in the biologically relevant micromolar concentration range. After successfully testing on spiked samples, saliva was analyzed, which was collected during chewing of either nicotine tablets with different concentrations or of smokeless tobacco. The MIPs in combination with QCM-D were able to distinguish clearly between these samples: This proves the functioning of the concept with saliva, which mediates the oral uptake of nicotine as an alternative to the consumption of cigarettes. Figure Schematics of the sample-preparation procedure for l -nicotine spiked saliva- and urine samples with various concentration levels

Description: The generation of key drug metabolites for the purpose of their complete structural characterization, toxicity testing, as well as to serve as standards for quantitative studies, is a critical step in the pharmaceutical discovery and development cycle. Here, we utilized electrochemistry/mass spectrometry for the detection and subsequent generation of six phase I metabolites of simvastatin and lovastatin. Both simvastatin and lovastatin are widely used for the treatment of hypercholesterolemia. There are known drug–drug interaction issues of statin therapy, and it has been suggested that the oxidative metabolites may contribute to the cholesterol-lowering effect of both statins. Of the known phase I metabolites of simvastatin and lovastatin, none are commercially available, and chemical means for the synthesis of a very few of them have been previously reported. Here, we report that electrochemical oxidation of less than 1 mg each of simvastatin and lovastatin led to the generation of three oxidative metabolites of each parent to allow complete nuclear magnetic resonance characterization of all six metabolites. The yields obtained by the electrochemical approach were also compared with incubation of parent drug with commercially available bacterial mutant CYP102A1 enzymes, and it was found that the electrochemical approach gave higher yields than the enzymatic oxidations for the generation of most of the observed oxidative metabolites in this study. Figure Generation of statin drug metabolites by EC/MS (representative mass voltammogram shown), and recombinant CYP enzymes

Description: The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356–364 and 395–432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.

Description: A monodisperse molecularly imprinted polymer (MIP) for curcumin was first prepared by precipitation polymerization using methacrylamide (MAM) and 4-vinylpyridine as functional co-monomers, divinylbenzene as a crosslinker, and a mixture of acetonitrile and toluene as a porogen. The use of MAM as the co-monomer resulted in the formation of a monodisperse MIP and non-imprinted polymer (NIP). MIP and NIP, respectively, were monodispersed with a narrow particle size distribution (3.3 ± 0.09 and 3.5 ± 0.10 μm). In addition to shape recognition, hydrophobic and hydrogen-bonding interactions affected the retention and molecular-recognition of curcumin on the MIP. The MIP for curcumin could extract curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in Curcuma longa L . Figure MIPs prepared with 4-VPY ( left hand ) and 4-VPY and MAM ( right hand ) as the functional monomers were polydispersed and monodispersed, respectively

Description: An application of plasma-induced grafting of polyethylene membranes with a thin layer of molecularly imprinted polymer (MIP) was presented. High-density polyethylene (HDPE) membranes, “Vyon,” were used as a substrate for plasma grafting modification. The herbicide atrazine, one of the most popular targets of the molecular imprinting, was chosen as a template. The parameters of the plasma treatment were optimized in order to achieve a good balance between polymerization and ablation processes. Modified HDPE membranes were characterized, and the presence of the grafted polymeric layer was confirmed based on the observed weight gain, pore size measurements, and infrared spectrometry. Since there was no significant change in the porosity of the modified membranes, it was assumed that only a thin layer of the polymer was introduced on the surface. The experiments on the re-binding of the template atrazine to the membranes modified with MIP and blank polymers were performed. HDPE membranes which were grafted with polymer using continuous plasma polymerization demonstrated the best result which was expressed in an imprinted factor equal to 3, suggesting that molecular imprinting was successfully achieved. Figure Atrazine and simazine adsorption by untreated HDPE membranes and membranes plasmagrafted with molecular imprinted polymer

Description: We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase. Figure New zwitterionic ion-exchangers can separate free amino phosphonic acids and a change from Chiralpak ZWIX(+) to ZWIX(-) allows reversal of enantiomer elution order

Description: Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z 31 , with a dissociation constant ( K d ) of 41 ± 5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14 × 10 −9 to 3.14 × 10 −5  M for ZEN, and the detection limit was 7.85 × 10 −10  M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products. Figure Determination of dissociation constant ( K d ) and specificity of aptamers recognizing zearalenone

Description: Countercurrent chromatography (CCC) is an attractive separation method because the analytes are partitioned between two immiscible liquid phases avoiding problems related to solid stationary phase. In recent years, this technique has made great progress in separation power and detection potential. This review describes coupling strategies involving high speed CCC (HSCCC) or centrifugal partition chromatography (CPC). It includes on-line extraction–isolation, hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detectors, multidimensional CCC (MDCCC), two-dimensional CCC (2D-CCC), on-line coupling with liquid chromatography (LC), and biological tests, and innovative off-line developments. The basic principles of each method are presented and applications are summarized.

Description: Comprehensive two-dimensional liquid chromatography (LC × LC) has received much attention because it offers much higher peak capacities than separation in a single dimension. The advantageous peak capacity makes it attractive for the separation of complex samples. Various gradient methods have been used in LC × LC systems. The use of continuous shift gradient is advantageous because it combines the peak compression effect of full gradient mode and the tailed gradient program in parallel gradient mode. Here, a comparison of LC × LC analysis of Chinese herbal medicine with full gradient mode and shift gradient mode in the second dimension was performed. A correlation between the first and second dimensions was found in full gradient mode, and this was significantly reduced with shift gradient mode. The orthogonality increased by 43.7 %. The effective peak distribution area increased significantly, which produced better separation.

Description: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders resulting from multiple factors. Diagnosis is based on behavioural and developmental signs detected before 3 years of age, and there is no reliable biological marker. The purpose of this study was to evaluate the value of gas chromatography combined with mass spectroscopy (GC-MS) associated with multivariate statistical modeling to capture the global biochemical signature of autistic individuals. GC-MS urinary metabolic profiles of 26 autistic and 24 healthy children were obtained by liq/liq extraction, and were or were not subjected to an oximation step, and then were subjected to a persilylation step. These metabolic profiles were then processed by multivariate analysis, in particular orthogonal partial least-squares discriminant analysis (OPLS-DA, R 2 Y(cum) = 0.97, Q 2 (cum) = 0.88). Discriminating metabolites were identified. The relative concentrations of the succinate and glycolate were higher for autistic than healthy children, whereas those of hippurate, 3-hydroxyphenylacetate, vanillylhydracrylate, 3-hydroxyhippurate, 4-hydroxyphenyl-2-hydroxyacetate, 1 H -indole-3-acetate, phosphate, palmitate, stearate, and 3-methyladipate were lower. Eight other metabolites, which were not identified but characterized by a retention time plus a quantifier and its qualifier ion masses, were found to differ between the two groups. Comparison of statistical models leads to the conclusion that the combination of data obtained from both derivatization techniques leads to the model best discriminating between autistic and healthy groups of children.

Description: Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme , shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination. Figure The Nimzyme high-throughput enzyme activity assay allows for the detection of enzyme activity in cell lysate. Fluorous interactions between a specialized substrate probe and a nanostructure-initiator mass spectrometry surface allow for in situ cleanup and the subsequent collection of unambiguous mass spectra. One of the main hurdles that prevents the widespread adoption of this technology is the need to chemically synthesize the required probes. Here, we present a simplified route to derive Nimzyme probes from a wide variety of biologically interesting substrates.

Description: Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n  = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

Description: A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C 18 stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae. Figure LC-MS/MS MRM chromatogram of mixed nucleoside and nucleotide standard

Description: Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI–MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.

Description: β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC 50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L −1 . Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg −1 , respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits. Figure The receptor-based ELISA for blank sample (negative samples, left ) and sample containing β-lactam antibiotics (positive samples, right )

Description: In recent years, near-infrared (NIR) hyperspectral imaging has proved its suitability for quality and safety control in the cereal sector by allowing spectroscopic images to be collected at single-kernel level, which is of great interest to cereal control laboratories. Contaminants in cereals include, inter alia , impurities such as straw, grains from other crops, and insects, as well as undesirable substances such as ergot (sclerotium of Claviceps purpurea ). For the cereal sector, the presence of ergot creates a high toxicity risk for animals and humans because of its alkaloid content. A study was undertaken, in which a complete procedure for detecting ergot bodies in cereals was developed, based on their NIR spectral characteristics. These were used to build relevant decision rules based on chemometric tools and on the morphological information obtained from the NIR images. The study sought to transfer this procedure from a pilot online NIR hyperspectral imaging system at laboratory level to a NIR hyperspectral imaging system at industrial level and to validate the latter. All the analyses performed showed that the results obtained using both NIR hyperspectral imaging cameras were quite stable and repeatable. In addition, a correlation higher than 0.94 was obtained between the predicted values obtained by NIR hyperspectral imaging and those supplied by the stereo-microscopic method which is the reference method. The validation of the transferred protocol on blind samples showed that the method could identify and quantify ergot contamination, demonstrating the transferability of the method. These results were obtained on samples with an ergot concentration of 0.02 % which is less than the EC limit for cereals (intervention grains) destined for humans fixed at 0.05 %. Online Abstract Figure Pictures showing a the manual removal of ergot bodies and b the observation by the stereo-microscopic method (official method); c the metallic holder with the reference material, and d the NIR hyperspectral SisuCHEMA instrument

Description: Nuclear magnetic resonance (NMR) spectroscopy-based metabonomics is of growing importance for discovery of human disease biomarkers. Identification and validation of disease biomarkers using statistical significance analysis (SSA) is critical for translation to clinical practice. SSA is performed by assessing a null hypothesis test using a derivative of the Student's t test, e.g., a Welch's t test. Choosing how to correct the significance level for rejecting null hypotheses in the case of multiple testing to maintain a constant family-wise type I error rate is a common problem in such tests. The multiple testing problem arises because the likelihood of falsely rejecting the null hypothesis, i.e., a false positive, grows as the number of tests applied to the same data set increases. Several methods have been introduced to address this problem. Bonferroni correction (BC) assumes all variables are independent and therefore sacrifices sensitivity for detecting true positives in partially dependent data sets. False discovery rate (FDR) methods are more sensitive than BC but uniformly ascribe highest stringency to lowest p value variables. Here, we introduce standard deviation step down (SDSD), which is more sensitive and appropriate than BC for partially dependent data sets. Sensitivity and type I error rate of SDSD can be adjusted based on the degree of variable dependency. SDSD generates fundamentally different profiles of critical p values compared with FDR methods potentially leading to reduced type II error rates. SDSD is increasingly sensitive for more concentrated metabolites. SDSD is demonstrated using NMR-based metabonomics data collected on three different breast cancer cell line extracts.

Description: Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration. Figure Illustration of the photometry of tyrosine and nitrotyrosine

Description: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a well-established technique in materials science, but is now increasingly applied also in the life sciences. Here, we demonstrate the potential of this analytical technique for use in the development of new bone implant materials. We tracked strontium-enriched calcium phosphate cements, which were developed for the treatment of osteoporotic bone, from in vitro to in vivo. Essentially, the spatial distribution of strontium in two different types of strontium-modified calcium phosphate cements is analysed by SIMS depth profiling. To gain information about the strontium release kinetics, the cements were immersed for 3, 7, 14 and 21 days in α-MEM and tris(hydroxymethyl)-aminomethane solution and analysed afterwards by ToF-SIMS depth profiling. For cements stored in α-MEM solution an inhibited strontium release was observed. By using principal component analysis to evaluate TOF-SIMS surface spectra, we are able to prove the adsorption of proteins on the cement surface, which inhibit the release kinetics. Cell experiments with human osteoblast-like cells cultured on the strontium-modified cements and subsequent mass spectrometric analysis of the mineralised extracellular matrix (mECM) prove clearly that strontium is incorporated into the mECM of the osteoblast-like cells. Finally, in an animal experiment, the strontium-doped cements are implanted into the femur of osteoporotic rats. After 6 weeks, only a slight release of strontium was found in the vicinity of the implant material. By using ToF-SIMS, it is proven that strontium is localised in regions of newly formed bone but also within the pre-existing tissue. Figure Schematic illustration of the performed measurements.

Description: Prompt gamma-ray neutron activation analysis (PGNAA) using the internal mono-standard method was tested for its applicability to analyzing large solid samples including irregularly shaped meteorite samples. For evaluating the accuracy and precision of the method, large quantities of the Geological Survey of Japan standardized rock powders (JB-1a, JG-1a, and JP-1) were analyzed and 12 elements (B, Na, Mg, Al, Cl, K, Ca, Ti, Mn, Fe, Sm, and Gd) were determined by using Si as an internal standard element. Analytical results were mostly in agreement with literature values within 10 %. The precision of the method was also shown to be within 10 % (1σ) for most of these elements. The analytical procedure was then applied to four stony meteorites (Allende, Kimble County, Leedey, Lake Labyrinth) and four iron meteorites (Canyon Diablo, Toluca (Mexico), Toluca (Xiquipilco), Squaw Creek) consisting of large chunks or single slabs. For stony meteorites, major elements (Mg, Al, Si, S, Ca, and Ni), minor elements (Na and Mn) and trace element (B, Cl, K, Ti, Co, and Sm) were determined with adequate accuracy. For iron meteorites, results for the Co and Ni mass fractions determined are all consistent with corresponding literature values. After the analysis, it was confirmed that the residual radioactivity remaining in the sample after PGNAA was very low and decreased down to the background level. This study shows that PGNAA with the internal mono-standard method is highly practical for determining the elemental composition of large, irregularly shaped solid samples including meteorites.

Description: Many research efforts over the last few decades have been devoted to sensing lactate as an important analytical target in clinical care, sport medicine, and food processing. Therefore, research in designing lactate sensors is no longer in its infancy and now is more directed toward viable sensors for direct applications. In this review, we provide an overview of the most immediate and relevant developments toward this end, and we discuss and assess common transduction approaches. Further, we critically describe the pros and cons of current commercial lactate sensors and envision how future sensing design may benefit from emerging new technologies.

Description: Currently, prostate-specific antigen (PSA) is considered to be the most sensitive marker available for prostate cancer detection and for monitoring of disease progression. In addition to its importance as a tumor marker, PSA has a role in the biological activity of cancer growth and proliferation. Therefore, the inhibition or activation of its biological activity may be used in prostate cancer therapy. Here, we describe the isolation and characterization of new 2′F-modified RNA aptamers directed against PSA. Binding studies demonstrate the ability of these new aptamers to specifically recognize their target with dissociation constants in the nanomolar range. In order to demonstrate the functionality of the selected aptamers, an apta-PCR approach was used for the quantitative detection of PSA, achieving a limit of detection of 11 nM. Furthermore, the potential use of the selected aptamers in therapeutics was demonstrated with the 2′F-modified aptamers being highly stable in human serum and having the ability to moderate the activity of PSA, which will be explored for the treatment of prostate cancer.

Description: Although metabolic study of individual active compounds isolated from herbal plants has been intensive, it cannot truly reflect the fate of herbs because the herbal extracts in use have many constituents. To address this problem, whole extracts of herbs should be investigated. Microsomes have been heavily used in the in vitro metabolic study of drugs, and various materials have been used to immobilize microsomes to develop highly effective and reusable bioreactors in this field. In this work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) to develop a highly active and recoverable nanoparticle bioreactor. Using this bioreactor, we investigated the in vitro metabolism of Rhizoma coptidis extract. Incubation of berberine, a major active ingredient of R. coptidis , with LMMNPs for 20 min produced two metabolites, i.e., demethyleneberberine and thalifendine, at high levels. From a comparison of the time courses of thalifendine formation obtained by ultraperformance liquid chromatography–mass spectrometry analysis, it was found that LMMNPs had a higher biological activity than free liver microsomes in metabolizing berberine. Further, the activity of LMMNPs remained almost unchanged after six consecutive uses in the incubation tests. Metabolism of R. coptidis extracts by LMMNPs was studied. The same two metabolites of berberine, i.e., demethyleneberberine and thalifendine, were detected. After a thorough study seeking support for this observation, it was found that demethyleneberberine was the common metabolite of five protoberberine-type alkaloids present in R. coptidis extract, including palmatine, jatrorrhizine, columbanine, epiberberine, and berberine. Figure A highly effective and reusable bioreactor was developed by immobilizing liver microsomes on magnetic nanoparticles, and it was used to investigate the metabolism of the whole extract of a Chinese herb Rhizoma coptidis

Description: A better understanding of Se fate in soils is required for different environmental issues, such as radioactive waste management or soil fertilization procedures. In these contexts, the mobility and speciation of Se have to be studied at both short and long terms after Se inputs. Here, we present a new methodology to monitor simultaneously the reactivity of added (isotopic enriched tracers) and ambient Se at trace level in soils by high-performance liquid chromatography inductively coupled plasma mass spectrometry (ICP-MS) following specific extractions. To do so, the collision/reaction cell of the ICP-MS instrument and the interference corrections were optimized to measure reliably the four major Se isotopes. To exemplify the method capabilities, the behaviors of added 77 Se(IV) and ambient Se were followed up in two soils submitted to an ageing process during 3 months. The solid/liquid distribution of added Se reached a steady state after 1 month while its speciation and distribution among soil solid phases were still changing after 3 months. The results clearly demonstrate that slow processes are involved in Se retention and transformation in soils. The usual short-term experiments (〈1 month) performed after Se addition are thus not suitable for long-term risk assessment. Interestingly, the behavior of added Se tended to that of ambient Se, suggesting that ambient Se would be useful to infer the fate of Se input over long time scales. Figure ᅟ

Description: A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence ( λ Ex 250 nm/ λ Em 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (〉35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200–600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.

Description: The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium ( 1a ) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8- bis [3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d 6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a ).

Description: A new approach for the implementation of liquid–liquid extraction in a sequential injection manifold is presented. The manifold consists of two syringe pumps and one multi-position valve. The use of a 30 % MeOH/H 2 O (v/v) solution as the carrier, with isobutanol as the extractant, made it possible to avoid the problems associated with the different affinities of the organic and aqueous phases for Teflon tubing, and the formation of bubbles. The suitability of the proposed method was demonstrated by the determination of thiamine in pharmaceutical preparations and dietary supplements. Detection and quantification limits were estimated as 9 and 30 μg L –1 , respectively. The repeatability was lower than 3 % and the intermediate precision (3 d) was lower than 7 %. The sample throughput was 14 samples per h. The results were in agreement with a high-performance liquid chromatography-electrospray/mass spectrometry reference method.

Description: Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C 18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12 and 14 μg/L for 19 of the 26 analyzed polyphenols), linearity ( r 2  〉 0.991), run-to-run and day-to-day precisions (relative standard deviation (RSD) values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone/water/hydrochloric acid (70:29.9:0.1 v / v / v ) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods ( p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin.

Description: This paper reports the development of a method based on high-performance liquid chromatography (HPLC) coupled to second-order data modeling with multivariate curve resolution-alternating least-squares (MCR–ALS) for quantification of retinoic acid and its main isomers in plasma in only 5.5 min. The compounds retinoic acid (RA), 13- cis -retinoic acid, 9- cis -retinoic acid, and 9,13-di- cis -retinoic acid were partially separated by use of a Poroshell 120 EC–C18 (3.0 mm × 30 mm, 2.7 μm particle size) column. Overlapping not only among the target analytes but also with the plasma interferents was resolved by exploiting the second-order advantage of the multi-way calibration. A validation study led to the following results: trueness with recoveries of 98.5–105.9 % for RA, 95.7–110.1 % for 13- cis -RA, 97.1–110.8 % for 9- cis -RA, and 99.5–110.9 % for 9,13-di- cis -RA; repeatability with RSD of 3.5–3.1 % for RA, 3.5–1.5 % for 13- cis -RA, 4.6–2.7 % for 9- cis -RA, and 5.2–2.7 % for 9,13-di- cis -RA (low and high levels); and intermediate precision (inter-day precision) with RSD of 3.8–3.0 % for RA, 2.9–2.4 % for 13- cis -RA, 3.6–3.2 % for 9,13-di- cis -RA, and 3.2–2.9 % for 9- cis -RA (low and high levels). In addition, a robustness study revealed the method was suitable for monitoring patients with dermatological diseases treated with pharmaceutical products containing RA and 13- cis -RA. Graphical Abstract Rapid determination of retinoic acid and its main isomers

Description: Salmon is a popular food but it is easily susceptible to spoilage by contamination with microorganisms. In this study, a method using hydrophilic interaction chromatography (HILIC)-based solid-phase extraction (SPE) and matrix-assisted laser desorption and ionization time-of-flight/time-of-flight mass spectrometry was developed and applied to reveal the effect of Pseudomonas fluorescens on salmon fillet during the shelf-life period by measuring the changes in the levels of phosphatidylcholine and phosphatidylethanolamine. Fresh samples were inoculated with P. fluorescens (10 6  cfu g -1 ) for 30 s, and lipids were extracted at 0, 24, 48, and 72 h. A homemade SPE cartridge packed with HILIC sorbent (silica derivatized with 1,2-dihydroxypropane) was used for matrix cleanup prior to analysis by mass spectrometry. In total, 30 phospholipids and 16 lysophospholipids were detected and elucidated. The results revealed that the content of phospholipids decreased significantly, whereas that of lysophospholipids increased initially, followed by a gradual reduction as the cold storage time increased. The contamination by P. fluorescens negatively affected the quality of fresh salmon without obvious physical changes, but it posed a potential threat to human health. This study suggests that the well-established method could be used for detecting phospholipids in salmon fillet and perhaps other foods as well. Graphical Abstract Freshness of salmon on sale in supermarket could be monitored by means of MALDI-TOF/MS

Description: Quantification by mass spectrometry imaging (Q-MSI) is one of the hottest topics of the current discussions among the experts of the MS imaging community. If MSI is established as a powerful qualitative tool in drug and biomarker discovery, its reliability for absolute and accurate quantification (QUAN) is still controversial. Indeed, Q-MSI has to deal with several fundamental aspects that are difficult to control, and to account for absolute quantification. The first objective of this manuscript is to review the state-of-the-art of Q-MSI and the current strategies developed for absolute quantification by direct surface sampling from tissue sections. This includes comments on the quest for the perfect matrix-matched standards and signal normalization approaches. Furthermore, this work investigates quantification at a pixel level to determine how many pixels must be considered for accurate quantification by ultraviolet matrix-assisted laser desorption/ionization (MALDI), the most widely used technique for MSI. Particularly, this study focuses on the MALDI-selected reaction monitoring (SRM) in rastering mode, previously demonstrated as a quantitative and robust approach for small analyte and peptide-targeted analyses. The importance of designing experiments of good quality and the use of a labeled compound for signal normalization is emphasized to minimize the signal variability. This is exemplified by measuring the signal for cocaine and a tryptic peptide (i.e., obtained after digestion of a monoclonal antibody) upon different experimental conditions, such as sample stage velocity, laser power and frequency, or distance between two raster lines. Our findings show that accurate quantification cannot be performed on a single pixel but requires averaging of at least 4–5 pixels. The present work demonstrates that MALDI-SRM/MSI is quantitative with precision better than 10–15 %, which meets the requirements of most guidelines (i.e., in bioanalysis or toxicology) for quantification of drugs or peptides from tissue homogenates. Graphical Abstract MALDI-SRM/MSI is quantitative with precision better than 10–15 % when instrumental parameters are correctly set and after pixel-by-pixel normalization

Description: Mass spectrometry imaging (MSI) allows for the direct and simultaneous analysis of the spatial distribution of molecular species from sample surfaces such as tissue sections. One of the goals of MSI is monitoring the distribution of compounds at the cellular resolution in order to gain insights about the biology that occurs at this spatial level. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging of cervical tissue sections was performed using a spot-to-spot distance of 10 μm by utilizing the method of oversampling, where the target plate is moved by a distance that is less than the desorption radius of the laser. In addition to high spatial resolution, high mass accuracy (±1 ppm) and high mass resolving power (140,000 at m /z = 200) were achieved by coupling the IR-MALDESI imaging source to a hybrid quadrupole Orbitrap mass spectrometer. Ion maps of cholesterol in tissues were generated from voxels containing 〈1 cell, on average. Additionally, the challenges of imaging at the cellular level in terms of loss of sensitivity and longer analysis time are discussed. Graphical abstract Cellular-level mass spectrometry imaging using IR-MALDESI and oversampling

Description: Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC–HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14–1000 ng mL −1 in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC–HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 μg kg −1 in crude mussel extracts and 15 μg kg −1 after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this.

Description: Proteins are separated in field flow fractionation (FFF) according to a well-established mechanism described as the “Normal or Brownian” mode. In the case of the sub-technique using a hollow fiber, the focalization/relaxation position can be observed visually only with a transparent holder and using dyes as samples. Whatever the choice of instrumentation, a dye-free method is proposed to determine the center of the zone from experimental fractograms by means of only two sample elutions. It is also possible to determine and model the kinematics of the sample toward the equilibrium focalization/relaxation position as well as the real dimensions of the fiber during the separation process.

Description: Tyrosine phosphorylation is an important regulator of signaling in cellular pathways, and dysregulated tyrosine phosphorylation causes several diseases. Mass spectrometry has revealed the importance of global phosphoproteomic characterization. Analysis of tyrosine phosphorylation by studying the mass-spectrometry (MS)-determined phosphoproteome remains difficult because of the relatively low abundance of tyrosine phosphoproteins. To effectively evaluate tyrosine-phosphopeptide enrichment and reduce ion suppression from non-phosphorylated peptides in MS analysis, three trypsin-digested BSA peptides and 14 standard phosphopeptides, including six tyrosine phosphopeptides, four serine phosphopeptides, and four threonine phosphopeptides, were subjected to titanium dioxide immunoaffinity-based enrichment and also to combined enrichment using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography–mass spectrometry (LC–MS) analyses. The enrichment factors were evaluated to determine the efficiency of each enrichment procedure. Comparison of five optimized enrichment methods, including TiO 2 -based immunoaffinity purification in Tris and MOPS buffer systems, TiO 2 –immunoaffinity enrichment, and immunoaffinity–TiO 2 enrichment for total tyrosine, serine and threonine phosphopeptides, revealed that the order of the enrichment factors for total tyrosine phosphopeptides is: (i) immunoaffinity–TiO 2 (enrichment factor = 38,244), (ii) TiO 2 –immunoaffinity (enrichment factor = 24,987), (iii) TiO 2 micro-column (enrichment factor = 10,305), (iv) immunoaffinity in Tris buffer system (enrichment factor = 1450), and (v) immunoaffinity in the MOPS buffer system (enrichment factor = 32). These results reveal that an alternative enrichment scheme before use of a TiO 2 micro-column, using immunoaffinity 4G10 and PY99 antibody enrichment under optimized conditions, can provide greater selectivity for tyrosine-phosphopeptide enrichment.

Description: One of the unresolved issues of the European Water Framework Directive is the unavailability of realistic water reference materials for the organic priority pollutants at low nanogram-per-liter concentrations. In the present study, three different types of ready-to-use water test materials were developed for polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs) and tributyltin (TBT) at nanogram-per-liter levels. The first type simulated the dissolved phase in the water and comprised of a solution of humic acids (HA) at 5 mg L −1 dissolved organic carbon (DOC) and a spike of the target compounds. The second type of water sample incorporated the particulate phase in water. To this end, model suspended particulate matter (SPM) with a realistic particle size was produced by jet milling soil and sediments containing known amounts of PAHs, PBDEs and TBT and added as slurry to mineral water. The most complex test materials mimicked “whole water” consequently containing both phases, the model SPM and the HA solution with the target analytes strongly bound to the SPM. In this paper, the development of concepts, processing of the starting materials, characterisation of the HA and model SPMs as well as results for homogeneity and stability testing of the ready-to-use test materials are described in detail. Graphical Abstract Vials containing 0.5 g of model SPM, black caps for TBT, silver caps for PAH and red caps for PBDEs, respectively. Graphical Abstract Petri dishes with dried model SPMs; to the left 95.7 ± 0.9 mg of SPM containing PBDEs; in the middle 95.8 ± 0.7 mg of SPM containing TBT and to the right 93.7 mg ± 0.7 mg of SPM containing PAHs

Description:    Nanomaterials have emerging importance in laser desorption ionization mass spectrometry (LDI–MS) with the ultimate objective being to overcome some of the most important limitations intrinsically related to the use of conventional organic matrices in matrix-assisted (MA) LDI–MS. This review provides a critical overview of the most recent literature on the use of gold nanomaterials as non-conventional desorption ionization promoters in LDI–MS, with particular emphasis on bioanalytical applications. Old seminal papers will also be discussed to provide a timeline of the most significant achievements in the field. Future prospects and research needs are also briefly discussed. Content Type Journal Article Pages 1-23 DOI 10.1007/s00216-011-5120-2 Authors Rosa Pilolli, Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via Orabona, 4, 70126 Bari, Italy Francesco Palmisano, Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via Orabona, 4, 70126 Bari, Italy Nicola Cioffi, Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via Orabona, 4, 70126 Bari, Italy Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    In this work, we have characterized a diamine oxidase (DAO) from Lathyrus sativus and evaluated its use, for the first time, as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples. Firstly, DAO was electrokinetically characterized free in solution by means of a platinum electrode and then immobilized by using polyazetidine prepolimer on the surface of screen-printed electrodes constituted of two gold working electrodes. The amperometric measurements were carried out by using a flow system at a fixed potential of +600 mV vs the internal silver pseudo reference in phosphate buffer solution (0.1 mol l -1 , pH = 7.4). The analysis of wine and beer samples were performed in flow injection system using the dual channel transducer providing simultaneous detection of sample and blank signal, and the resulting signal (after subtraction of the blank signal) was referred to that of putrescine. The results were compared with those obtained using a modified reference method based on gas chromatography-mass spectrometry analysis on the same samples. The results obtained in the analysis of Italian wines shows the better suitability of DAO-based biosensor in the determination of the biogenic amines (BAs) index expressed as putrescine equivalent in both red and white wines, being less efficient in beer samples where it underestimates by about 50% the BAs content. Content Type Journal Article Pages 1-10 DOI 10.1007/s00216-011-5131-z Authors Massimo Di Fusco, Department of Chemistry and Drugs Technologies, University of Rome “La Sapienza”, Piazzale A. Moro, 5, 00185 Rome, Italy Rodolfo Federico, Department of Biology, University ‘Roma Tre’, Viale Marconi, 446, 00146 Rome, Italy Alberto Boffi, Department of Biochemical Sciences, University of Rome “La Sapienza”, Piazzale A. Moro, 5, 00185 Rome, Italy Alberto Macone, Department of Biochemical Sciences, University of Rome “La Sapienza”, Piazzale A. Moro, 5, 00185 Rome, Italy Gabriele Favero, Department of Chemistry and Drugs Technologies, University of Rome “La Sapienza”, Piazzale A. Moro, 5, 00185 Rome, Italy Franco Mazzei, Department of Chemistry and Drugs Technologies, University of Rome “La Sapienza”, Piazzale A. Moro, 5, 00185 Rome, Italy Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description: ANAKON 2011 – German thoroughness meets Swiss precision Content Type Journal Article Pages 1-2 DOI 10.1007/s00216-011-5096-y Authors Oliver Bleher, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany Melanie Ewald, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany Felix Kolarov, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany A. Katrin Krieg, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany Alexander F. Le Blanc, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany Sabrina Rau, Institute of Physical and Theoretical Chemistry, University of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    This paper describes a novel method of sample preparation for the determination of trace concentrations of polycyclic aromatic hydrocarbons (PAHs) in high-boiling petroleum products. Limits of quantitation of the investigated PAHs in materials of this type range from tens of nanograms per kilogram to 〈20 μg/kg. The studies revealed that in order to separate most of interferences from the analytes without a significant loss of PAHs, it is necessary to use size exclusion chromatography as the first step of sample preparation, followed by adsorption using normal-phase liquid chromatography. The use of orthogonal separation procedure described in the paper allows the isolation of only a group of unsubstituted and substituted aromatic hydrocarbons with a specific range of molar mass. The lower the required limit of quantitation of PAHs, the larger is the scale of preparative liquid chromatography in both steps of sample preparation needed. The use of internal standard allows quantitative results to be corrected for the degree of recovery of PAHs during the sample preparation step. Final determination can be carried out using HPLC-FLD, GC-MS, or HPLC-UV–VIS/DAD. The last technique provides a degree of identification through the acquired UV–VIS spectra. Figure  Chromatograms obtained using UV-DAD detection with wavelength programming (A) and fluorimetric detection (B) for the separation of 18 PAH standards ((A) and (B)) and the fraction containing PAHs from road asphalt 50/70 prepared according to the procedure described in this work (C) . Peak designation: 1 naphthalene, 2 acenaphthylene, 3 acenaphthene, 4 fluorene, 5 phenanthrene, 6 anthracene, 7 fluoranthene, 8 pyrene, 9 benzo[a]anthracene, 10 chrysene, 11 benzo[b]fluoranthene, 12 benzo[k]fluoranthene, 13 benzo[a]pyrene, 14 dibenzo[a,h]anthracene, 15 indeno[1,2,3-cd]pyrene, 16 benzo[ghi]perylene, 17 benzo[j]fluoranthene, 18 benzo[e]pyrene,19 highly polar components of road asphalt 50/70 eluted during backflush. BF backflush point Content Type Journal Article Pages 1-11 DOI 10.1007/s00216-011-5134-9 Authors Ewelina Gilgenast, Chemical Faculty, Department of Chemical and Process Engineering, Gdansk University of Technology, Narutowicza St. 11/12, 80-233 Gdansk, Poland Grzegorz Boczkaj, Chemical Faculty, Department of Chemical and Process Engineering, Gdansk University of Technology, Narutowicza St. 11/12, 80-233 Gdansk, Poland Andrzej Przyjazny, Chemistry & Biochemistry Department, Kettering University, 1700 West Third Avenue, Flint, MI 48504, USA Marian Kamiński, Chemical Faculty, Department of Chemical and Process Engineering, Gdansk University of Technology, Narutowicza St. 11/12, 80-233 Gdansk, Poland Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference–nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid–nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to l -arginine–bisoxyran–Sepharose and l -lysine–Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid–nucleotide interactions and determine the binding affinities of the complexes. Content Type Journal Article Pages 1-11 DOI 10.1007/s00216-011-5124-y Authors Carla Cruz, CICS-UBI–Centro de Investigação em Ciências da Saúde, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal Eurico J. Cabrita, REQUIMTE, CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova Lisboa, 2829-516 Caparica, Portugal João A. Queiroz, CICS-UBI–Centro de Investigação em Ciências da Saúde, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    In this work, an optimization study was conducted to investigate the performance of a custom-designed miniaturized dielectric barrier discharge (DBD) microplasma chip to be utilized as a radiation source for mercury determination in water samples. The experimental work was implemented by using experimental design, and the results were assessed by applying statistical techniques. The proposed DBD chip was designed and fabricated in a simple way by using a few microscope glass slides aligned together and held by a Perspex chip holder, which proved useful for miniaturization purposes. Argon gas at 75–180 mL/min was used in the experiments as a discharge gas, while AC power in the range 75–175 W at 38 kHz was supplied to the load from a custom-made power source. A UV-visible spectrometer was used, and the spectroscopic parameters were optimized thoroughly and applied in the later analysis. Plasma characteristics were determined theoretically by analysing the recorded spectroscopic data. The estimated electron temperature ( T e  = 0.849 eV) was found to be higher than the excitation temperature ( T exc  = 0.55 eV) and the rotational temperature ( T rot  = 0.064 eV), which indicates non-thermal plasma is generated in the proposed chip. Mercury cold vapour generation experiments were conducted according to experimental plan by examining four parameters (HCl and SnCl 2 concentrations, argon flow rate, and the applied power) and considering the recorded intensity for the mercury line (253.65 nm) as the objective function. Furthermore, an optimization technique and statistical approaches were applied to investigate the individual and interaction effects of the tested parameters on the system performance. The calculated analytical figures of merit (LOD = 2.8 μg/L and RSD = 3.5%) indicates a reasonable precision system to be adopted as a basis for a miniaturized portable device for mercury detection in water samples. Content Type Journal Article Pages 1-10 DOI 10.1007/s00216-011-5118-9 Authors Wameath S. Abdul-Majeed, Department of Chemical and Biological Engineering, The University of Sheffield, Newcastle Street, Sheffield, S1 3JD UK Jaime H. Lozano Parada, Department of Chemical and Biological Engineering, The University of Sheffield, Newcastle Street, Sheffield, S1 3JD UK William B. Zimmerman, Department of Chemical and Biological Engineering, The University of Sheffield, Newcastle Street, Sheffield, S1 3JD UK Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min -1 , with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [ r 2  = 0.998 ± 0.002 for plasma ( n  = 10) and r 2  = 0.999 ± 0.001 for saliva ( n  = 9)] over the range of 0.25–20.0 μg ml -1 , with a limit of quantification at 0.25 μg ml -1 . Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring. Content Type Journal Article Pages 1-9 DOI 10.1007/s00216-011-5126-9 Authors Iolanda Mazzucchelli, Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy Manuela Rapetti, Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy Cinzia Fattore, Clinical Trial Center, IRCCS National Neurological Institute C. Mondino Foundation, 27100 Pavia, Italy Valentina Franco, Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy Giuliana Gatti, Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy Emilio Perucca, Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T m values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T m values were separated and detected quantitatively by using a packed column carrying two different DNA probes. Content Type Journal Article Pages 1-10 DOI 10.1007/s00216-011-5129-6 Authors Seung Pil Pack, Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Chungnam 339-700, South Korea Tae-Hwe Heo, College of Pharmacy, The Catholic University of Korea, Bucheon, 420-743 South Korea Kamakshaiah Charyulu Devarayapalli, Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan Keisuke Makino, Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Breath analysis could offer a non-invasive means of intravenous drug monitoring if robust correlations between drug concentrations in breath and blood can be established. In this study, propofol blood and breath concentrations were determined in an animal model under varying physiological conditions. Propofol concentrations in breath were determined by means of two independently calibrated analytical methods: continuous, real-time proton transfer reaction mass spectrometry (PTR-MS) and discontinuous solid-phase micro-extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS). Blood concentrations were determined by means of SPME-GC-MS. Effects of changes in pulmonary blood flow resulting in a decreased cardiac output (CO) and effects of dobutamine administration resulting in an increased CO on propofol breath concentrations and on the correlation between propofol blood and breath concentrations were investigated in seven acutely instrumented pigs. Discontinuous propofol determination in breath by means of alveolar sampling and SPME-GC-MS showed good agreement ( R 2  = 0.959) with continuous alveolar real-time measurement by means of PTR-MS. In all investigated animals, increasing cardiac output led to a deterioration of the relationship between breath and blood propofol concentrations ( R 2  = 0.783 for gas chromatography-mass spectrometry and R 2  = 0.795 for PTR-MS). Decreasing pulmonary blood flow and cardiac output through banding of the pulmonary artery did not significantly affect the relationship between propofol breath and blood concentrations ( R 2  〉 0.90). Estimation of propofol blood concentrations from exhaled alveolar concentrations seems possible by means of different analytical methods even when cardiac output is decreased. Increases in cardiac output preclude prediction of blood propofol concentration from exhaled concentrations. Figure  Experimental setup for simultaneous real-time (PTR-MS) and discontinuous (SPME-GC-MS) drug determination in the breath of acutely instrumented pigs ( A ). In order to assess the influence of hemodynamic variables pulmonary artery blood flow was determined by means of Doppler-measurement ( B ). Content Type Journal Article Pages 1-10 DOI 10.1007/s00216-011-5099-8 Authors Svend Kamysek, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Patricia Fuchs, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Henny Schwoebel, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Jan P. Roesner, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Sabine Kischkel, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Kathi Wolter, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Christian Loeseken, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Jochen K. Schubert, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Wolfram Miekisch, Department of Anesthesiology and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock, Germany Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Predicting the amount of time that a petroleum mixture has been exposed to weathering effects has applications in areas of environmental and other forensic investigations, such as aiding in determining the cause and intent of a fire. Historically, research on the evaporation rates of hydrocarbon mixtures has focused on forensic oil spill identification and predicting if a fresh sample could be weathered to give an observed composition in an aged sample. Relatively little attention has focused on approaching the problem from the other direction: estimating exposure time based on the observed composition of a weathered sample at a given time and assuming a prior composition. Here, we build upon our previous research into the weathering of model mixtures by extending our work to gasoline. Samples of gasoline with varying octane ratings and from several vendors were weathered under controlled conditions and their composition monitored over time by two-dimensional gas chromatography (GC × GC). A variety of chemometric models were explored, including partial least squares (PLS), nonlinear PLS (PolyPLS) and locally weighted regression (LWR). A hierarchical application of multivariate techniques was able to predict the time for which a sample had been exposed to evaporative weathering. Partial least squares discriminant analysis could predict whether a sample was relatively fresh (〈12 h exposure time) or highly weathered (〉20 h exposure time). Subsequent regression models for these classes were evaluated for accuracy using the root mean square error of prediction. LWR was the most successful, whereby fresh and highly weathered samples were predicted to within 30 min and 5 h of exposure, respectively. Content Type Journal Article Pages 1-9 DOI 10.1007/s00216-011-5130-0 Authors Brianne M. Zorzetti, Department of Chemistry, University of Alberta, Edmonton, AB T6G 2 G2, Canada James J. Harynuk, Department of Chemistry, University of Alberta, Edmonton, AB T6G 2 G2, Canada Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Vibrational spectroscopy techniques can be applied to identify a susceptibility-to-adenocarcinoma biochemical signature. A sevenfold difference in incidence of prostate adenocarcinoma (CaP) remains apparent amongst populations of low- (e.g. India) compared with high-risk (e.g. UK) regions, with migrant studies implicating environmental and/or lifestyle/dietary causative factors. This study set out to determine the biospectroscopy-derived spectral differences between risk-associated cohorts to CaP. Benign prostate tissues were obtained using transurethral resection from high-risk ( n  = 11, UK) and low-risk ( n  = 14, India) cohorts. Samples were analysed using attenuated total reflection Fourier-transform infrared (FTIR) spectroscopy, FTIR microspectroscopy and Raman microspectroscopy. Spectra were subsequently processed within the biochemical cell region (1,800 −1 –500 cm –1 ) employing principal component analysis (PCA) and linear discriminant analysis (LDA) to determine whether wavenumber–absorbance/intensity relationships might reveal biochemical differences associated with region-specific susceptibility to CaP. PCA-LDA scores and corresponding cluster vector plots identified pivotal segregating biomarkers as 1,582 cm −1 (Amide I/II trough); 1,551 cm −1 (Amide II); 1,667 cm −1 (Amide I); 1,080 cm −1 (DNA/RNA); 1,541 cm −1 (Amide II); 1,468 cm −1 (protein); 1,232 cm −1 (DNA); 1,003 cm −1 (phenylalanine); 1,632 cm −1 [right-hand side (RHS) Amide I] for glandular epithelium ( P  〈 0.0001) and 1,663 cm −1 (Amide I); 1,624 cm −1 (RHS Amide I); 1,126 cm −1 (RNA); 1,761, 1,782, 1,497 cm −1 (RHS Amide II); 1,003 cm −1 (phenylalanine); and 1,624 cm −1 (RHS Amide I) for adjacent stroma ( P  〈 0.0001). Primarily protein secondary structure variations were biomolecular markers responsible for cohort segregation with DNA alterations exclusively located in the glandular epithelial layers. These biochemical differences may lend vital insights into the aetiology of CaP. Figure  The first study to apply biospectroscopy techniques to identify the underlying differences in the aetiology of prostate cancer between low- (India) compared to high-risk (UK) cohorts Content Type Journal Article Pages 1-14 DOI 10.1007/s00216-011-5123-z Authors Imran I. Patel, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Júlio Trevisan, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Paras B. Singh, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Caroline M. Nicholson, Lancashire Teaching Hospitals NHS Trust, Fulwood, Preston, PR2 9HT UK R. K. Gopala Krishnan, Workhardt Hospital, Kolkata, 700020 India Shyam S. Matanhelia, Lancashire Teaching Hospitals NHS Trust, Fulwood, Preston, PR2 9HT UK Francis L. Martin, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N -glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N -glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N -glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O -glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis. Figure  Overlaid chromatograms and associated structural assignments of glycopeptides from bovine κ-casein. Color denotes the site(s) of glycosylation from which the glycopeptide originated Content Type Journal Article Pages 1-12 DOI 10.1007/s00216-011-5109-x Authors Serenus Hua, Department of Chemistry, University of California, Davis, CA 95616, USA Charles C. Nwosu, Department of Chemistry, University of California, Davis, CA 95616, USA John S. Strum, Department of Chemistry, University of California, Davis, CA 95616, USA Richard R. Seipert, Department of Chemistry, University of California, Davis, CA 95616, USA Hyun Joo An, Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 South Korea Angela M. Zivkovic, Department of Food Science and Technology, University of California, Davis, CA 95616, USA J. Bruce German, Department of Food Science and Technology, University of California, Davis, CA 95616, USA Carlito B. Lebrilla, Department of Chemistry, University of California, Davis, CA 95616, USA Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    The American visceral leishmaniasis is an important cause of morbidity and mortality in Brazil for both humans and dogs. Attempts to make a diagnosis of this disease need to be improved, especially in endemic areas, and in the tracking and screening of asymptomatic dogs, which are their main host in urban areas. A quartz crystal microbalance immunosensor for the diagnosis of the canine visceral leishmaniasis using a recombinant antigen of Leishmania chagasi (rLci2B-NH6) was developed. The rLci2B-NH6 was tightly immobilized on a quartz crystal gold electrode by self-assembled monolayer based on short-chain length thiol. The strategy was the use of the antigen-histidine tail covalently linked to glutaraldehyde performing a Schift base which permits a major exposure of epitopes and a reduced steric hindrance. The immunosensor showed good results regarding sensitivity and reproducibility, being able to distinguish positive and negative canine serum for L. chagasi. Furthermore, the immunosensor can be reused through exposure to sodium dodecyl sulfate solution, which promotes the dissociation of antigen–antibody binding, restoring the sensor surface with immobilized biologically active antigens for further analysis. Content Type Journal Article Pages 1-9 DOI 10.1007/s00216-011-5136-7 Authors Joilson Ramos-Jesus, Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235-Cidade Universitária, Recife, Pernambuco 50670-901, Brazil Kellyanne A. Carvalho, Departamento de Biointeração, Universidade Federal da Bahia, Salvador, Bahia 40110-060, Brazil Rosana A. S. Fonseca, Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235-Cidade Universitária, Recife, Pernambuco 50670-901, Brazil Geraldo G. S. Oliveira, Centro de Pesquisa Gonçalo Muniz, Fundação Oswaldo Cruz, Bahia 40296-710, Brazil Stella M. Barrouin Melo, Departamento de Patologia e Clínicas, Universidade Federal da Bahia, Salvador, Bahia 40110-060, Brazil Neuza M. Alcântara-Neves, Departamento de Biointeração, Universidade Federal da Bahia, Salvador, Bahia 40110-060, Brazil Rosa F. Dutra, Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235-Cidade Universitária, Recife, Pernambuco 50670-901, Brazil Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Residues from medicine containers in the collections of the British Museum have been investigated as part of a wider programme of scientific work on Roman surgical instruments. The cylindrical bronze containers are often described as instrument cases, but some contain materia medica , ranging from extensive extant remains of ancient preparations to possible minor deposits on the interior surfaces of the containers. Samples from seven residues have been analysed by gas chromatography–mass spectrometry (GC-MS) to identify lipid, resin and carbohydrate components and by X-ray fluorescence and Raman spectroscopy to characterise inorganic materials. The results have provided evidence for ointments and powders or pills consistent with a medical purpose. The ingredients identified include beeswax, fat, conifer resin and gum-derived sugars, plus elemental carbon and lead and zinc salts. Particularly significant were the varied compositions of residues from four sections of a multi-compartment container. In one of these compartments, the beeswax seems to have been prepared as the ‘Punic wax’ described by Pliny. Experimental preparation of Punic wax following Pliny’s method was undertaken in the laboratory and the product analysed to compare with the ointment residues. This paper discusses the GC-MS results of both the experimental material and the archaeological residues and their significance for the interpretation of the past intended applications of the medicines and the use of the containers. Content Type Journal Article Pages 1-11 DOI 10.1007/s00216-011-5160-7 Authors R. J. Stacey, Department of Conservation and Scientific Research, The British Museum, Great Russell Street, London, WC1B 3DG UK Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide 〉〉 corticosterone 〉 dexamethasone 〉 cortisol = betamethasone 〉 prednisolone 〉 aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity. Content Type Journal Article Pages 1-10 DOI 10.1007/s00216-011-5162-5 Authors Toine F. H. Bovee, RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen, the Netherlands Richard J. R. Helsdingen, RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen, the Netherlands Astrid R. M. Hamers, RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen, the Netherlands Bram A. Brouwer, BioDetection Systems B.V. (BDS), Kruislaan 406, 1098 SM Amsterdam, the Netherlands Michel W. F. Nielen, RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen, the Netherlands Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description: The emergence of multimodal imaging methods for real-time nanoscopy Content Type Journal Article Pages 1-2 DOI 10.1007/s00216-011-5125-x Authors Cyril Petibois, University of Bordeaux, CNRS UMR 5248 CBMN Allée de Saint-Hillaire, F33600 Pessac-Cedex, France Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Chemometric methods have critical importance for the discovery of the information/knowledge buried or concealed in high-dimensional datasets acquired from comprehensive multidimensional separations (CMDS), and for interpretation of experiments or chemical processes. In this work, employment of new developments in chemometrics making full use of the data to maximize the potential of CMDS to resolve mathematically a variety of practical problems is reviewed whilst providing the authors' point of view. During the past several years, chemometrics has been successfully applied to many areas of concern to CMDS investigation, including experimental parameter optimization, data quality improvement, identification and quantification of target chemical components, pattern recognition technique for clustering and classification, multivariate model establishment to correlate chromatographic properties and molecular descriptors. On the basis of the high-dimensionality characteristics of CMDS, some special aspects such as evaluation of orthogonality and image processing have also been included in this review. It is expected that an overview of the diverse ways in which chemometrics can aid CMDS investigations will prove valuable to interested users in this area through a comprehensive survey of previous research contributions. Chemometrics lends itself well to the powerful separation capability of CMDS. Content Type Journal Article Pages 1-14 DOI 10.1007/s00216-011-5139-4 Authors Zhong-Da Zeng, Centre for Green Chemistry, School of Chemistry, Monash University, Wellington Rd, Clayton, 3800 Australia Helmut M. Hugel, School of Applied Sciences, RMIT University, G.P.O. Box 2476, Melbourne, 3001 Australia Philip J. Marriott, Centre for Green Chemistry, School of Chemistry, Monash University, Wellington Rd, Clayton, 3800 Australia Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Chemical analysis of ancient residues of pharmaceutical or cosmetic preparations such as balms or ointments is made problematic by the high complexity of these mixtures, composed of organic and inorganic materials. Consequently, a multi-analytical approach and special caution in the interpretation of the results are necessary. In order to contribute to the improvement of analytical strategies for the characterization of complex residues and to reconstruct ancient medical practices, a replica of a pharmaceutical formulation of the seventeenth century was prepared in the laboratory according to a historically documented recipe. In a round robin exercise, a portion of the preparation was analysed as a blind sample by 11 laboratories using various analytical techniques. These included spectroscopic, chromatographic and mass spectrometric methods. None of the laboratories was able to completely reconstruct the complex formulation, but each of them gave partial positive results. The round robin exercise has demonstrated that the application of a multi-analytical approach can permit a complete and reliable reconstruction of the composition. Finally, on the basis of the results, an analytical protocol for the study of residues of ancient medical and pharmaceutical preparations has been outlined. Content Type Journal Article Pages 1-14 DOI 10.1007/s00216-011-5105-1 Authors M. P. Colombini, Dipartimento di Chimica, SCIBEC, Università di Pisa, 56126 Pisa, Italy F. Modugno, Dipartimento di Chimica, SCIBEC, Università di Pisa, 56126 Pisa, Italy M. C. Gamberini, Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy M. Rocchi, Dipartimento di Chimica, SCIBEC, Università di Pisa, 56126 Pisa, Italy C. Baraldi, Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy T. Deviese, British Museum, Department of Conservation and Scientific Research, London, WC1B 3DG UK R. J. Stacey, British Museum, Department of Conservation and Scientific Research, London, WC1B 3DG UK M. Orlandi, Dipartimento di Scienze dell’Ambiente e del Territorio, Università di Milano Bicocca, 20126 Milan, Italy F. Saliu, Dipartimento di Scienze dell’Ambiente e del Territorio, Università di Milano Bicocca, 20126 Milan, Italy C. Riedo, Dipartimentodi Chimica IFM e NIS Centro di Eccellenza, Università degli Studi di Torino, 10124 Turin, Italy O. Chiantore, Dipartimentodi Chimica IFM e NIS Centro di Eccellenza, Università degli Studi di Torino, 10124 Turin, Italy G. Sciutto, Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Microchemistry and Microscopy Art Diagnostic Laboratory (M2ADL), Università di Bologna, 40126 Bologna, Italy E. Catelli, Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Microchemistry and Microscopy Art Diagnostic Laboratory (M2ADL), Università di Bologna, 40126 Bologna, Italy L. Brambilla, Dipartimento di Chimica, Materiali e Ingegneria Chimica, Politecnico di Milano, 20133 Milan, Italy L. Toniolo, Dipartimento di Chimica, Materiali e Ingegneria Chimica, Politecnico di Milano, 20133 Milan, Italy C. Miliani, Istituto CNR-ISTM Perugia, 06123 Perugia, Italy P. Rocchi, Istituto CNR-ISTM Perugia, 06123 Perugia, Italy J. Bleton, LETIAM, IUT d’Orsay, 91400 Orsay, France U. Baumer, Doerner Institut, 80799 Munich, Germany P. Dietemann, Doerner Institut, 80799 Munich, Germany G. Pojana, Università Ca‘Foscari Venezia, 30123 Venetia, Italy S. Marras, Università Ca‘Foscari Venezia, 30123 Venetia, Italy Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Bisphenol A (BPA) is a synthetic industrial reactant used in the production of polycarbonate plastics, and genistein is a natural phytoestrogen abundant in the soybean. Current studies investigating the endocrine-disrupting effects of concomitant exposures to BPA and genistein have warranted the development of an analytical method for the simultaneous measurement of BPA and genistein, as well as their primary metabolites, bisphenol A ß- d -glucuronide (BPA gluc) and genistein 4′-ß- d -glucuronide (genistein gluc), respectively. All four analytes were extracted from rat plasma via solid phase extraction (SPE). Three SPE cartridges and four elution schemes were tested. Plasma extraction using Bond Elut Plexa cartridges with sequential addition of ethyl acetate, methanol, and acetonitrile yielded optimal average recoveries of 98.1 ± 1.8% BPA, 94.9 ± 8.0% genistein, 91.4 ± 6.1% BPA gluc, and 103 ± 6.1% genistein gluc. Identification and quantification of the four analytes were performed by a validated HPLC-MS/MS method using electrospray ionization and selective reaction monitoring. This novel analytical method should be applicable to the measurement of BPA, genistein, BPA gluc, and genistein gluc in urine, cultures, and tissue following in vivo exposures. While reports of the determination of BPA and genistein independently exist, the simultaneous optimized extraction and detection of BPA, genistein, BPA gluc, and genistein gluc have not previously been reported. Figure  BPA and genistein co-exposure scenario. BPA-laden polycarbonate plastic baby bottle filled with soymilk, a rich source of genistein, provides a classic exposure scenario to young children—a population that is particularly vulnerable to the effects of endocrine-disrupting compounds Content Type Journal Article Pages 1-8 DOI 10.1007/s00216-011-5151-8 Authors Janis L. Coughlin, Environmental and Occupational Health Sciences Institute, A Joint Institute of Rutgers University and the University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, NJ 08854, USA Bozena Winnik, Environmental and Occupational Health Sciences Institute, A Joint Institute of Rutgers University and the University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, NJ 08854, USA Brian Buckley, Environmental and Occupational Health Sciences Institute, A Joint Institute of Rutgers University and the University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, NJ 08854, USA Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    The recent use of a one-dimensional (1D) X-ray Talbot interferometer has triggered great interest in X-ray differential phase contrast imaging. As an improved version of a 1D interferometer, the development of two-dimensional (2D) grating interferometry strongly stimulated applications of grating-based imaging. In the framework of Fresnel diffraction theory, we investigated the self-image of 2D-phase gratings under partially coherent illumination. The fringe visibility of the self-image has been analyzed as a function of the spatial coherence length. From the viewpoint of self-image visibility, it is possible to find the optimal 2D grid for 2D X-ray grating interferometer imaging. Numerical simulations have been also carried out for quantitative evaluation. Results, in good agreement with theoretical analysis, indicate the spatial coherence requirements of the radiation illuminating a 2D grating interferometer. Moreover, our results can be used to optimize performances of a 2D grating interferometer and for further theoretical and experimental research on grating-based imaging systems. Content Type Journal Article Pages 1-6 DOI 10.1007/s00216-011-5146-5 Authors Xin Ge, National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230026 China Zhili Wang, National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230026 China Kun Gao, National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230026 China Kai Zhang, Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049 China Youli Hong, Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049 China Dajiang Wang, National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230026 China Zhongzhu Zhu, Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049 China Peiping Zhu, Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049 China Ziyu Wu, National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230026 China Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl- l -glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of 13 C 5 labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018–14 μg FA/g of fresh bread ( r 2  = 0.997) and 9.3–900 ng 5-MTHF/g of fresh bread ( r 2  = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79–110 μg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200–300 μg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors. Figure  Chromatogram of labelled folic acid using UPLC-MS/MS Content Type Journal Article Pages 1-8 DOI 10.1007/s00216-011-5156-3 Authors Maria V. Chandra-Hioe, Food Science and Technology, School of Chemical Engineering, University of New South Wales, Sydney, 2052 Australia Martin P. Bucknall, Mark Wainwright Analytical Centre, Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, 2052 Australia Jayashree Arcot, Food Science and Technology, School of Chemical Engineering, University of New South Wales, Sydney, 2052 Australia Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Multi-walled carbon nanotubes (MWCNTs) were evaluated as potential adsorbents for miniaturized solid-phase extraction coupled to liquid chromatography. The adsorption capacity of this sorbent was applied to assess the speciation of four cobalamins representing the various forms of vitamin B 12 . The preconcentration on the MWCNTs was based on the retention of analytes by introducing the sample online into the mini-column system. Dimethyl sulfoxide was used to elute the retained vitamins for liquid chromatographic analysis. The experimental conditions of the continuous flow device, which affect the enrichment procedure, such as the type and amount of nanotubes, the volume, pH and flow rate of the sample solution, and the eluent and its volume, were optimized. For detection purposes, a diode array device was used and good resolution was obtained with a mobile-phase acetonitrile–phosphate buffer and gradient elution. Specificity was demonstrated by the retention characteristics and UV spectra and by comparing the peak purity index with commercial standards. Linearity, precision, recovery, and sensitivity were satisfactory. Detection limits ranged from 0.35 to 30 ng mL −1 . The method was successfully applied to the determination of cobalamins in seafoods, which were extracted from the sample with a buffer solution using an ultrasonic probe. The reliability of the procedure was checked by analyzing a certified reference material. Content Type Journal Article Pages 1-7 DOI 10.1007/s00216-011-5158-1 Authors Pilar Viñas, Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain Ignacio López-García, Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain María Bravo Bravo, Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain Manuel Hernández-Córdoba, Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Human remains detection canines are used in locating deceased humans in diverse scenarios and environments based on odor produced during the decay process of the human body. It has been established that human remains detection canines are capable of locating human remains specifically, as opposed to living humans or animal remains, thus suggesting a difference in odor between the different sources. This work explores the collection and determination of such odors using a dynamic headspace concentration device. The airflow rate and three sorbent materials—Dukal cotton gauze, Johnson & Johnson cotton-blend gauze, and polyester material—used for odor collection were evaluated using standard compounds. It was determined that higher airflow rates and openly woven material, e.g., Dukal cotton gauze, yielded significantly less total volatile compounds due to compound breakthrough through the sorbent material. Collection from polymer- and cellulose-based materials demonstrated that the molecular backbone of the material is a factor in compound collection as well. Volatiles, including cyclic and straight-chain hydrocarbons, organic acids, sulfides, aldehydes, ketones, and alcohols, were collected from a population of 27 deceased bodies from two collection locations. The common compounds between the subjects were compared and the odor profiles were determined. These odor profiles were compared with those of animal remains and living human subjects collected in the same manner. Principal component analysis showed that the odor profiles of the three sample types were distinct. Content Type Journal Article Pages 1-13 DOI 10.1007/s00216-011-5167-0 Authors Lauryn E. DeGreeff, International Forensic Research Institute, Department of Chemistry and Biochemistry, Florida International University, 11200 S.W. 8th Street, Miami, FL 33199, USA Kenneth G. Furton, International Forensic Research Institute, Department of Chemistry and Biochemistry, Florida International University, 11200 S.W. 8th Street, Miami, FL 33199, USA Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80–120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C max of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled. Content Type Journal Article Pages 1-12 DOI 10.1007/s00216-011-5147-4 Authors Luigi Silvestro, 3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany Mihaela Gheorghe, Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania Adriana Iordachescu, Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania Valentin Ciuca, Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania Ariana Tudoroniu, Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania Simona Rizea Savu, 3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany Isabela Tarcomnicu, Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    In this study, we employed laser ablation/inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the spatial distribution of Gd-doped iron oxide nanoparticles (IONPs) in one tumor slice that had been subjected to magnetic fluid hyperthermia (MFH). The mapping results revealed the high resolution of the elemental analysis, with the distribution of Gd atoms highly correlated with that of the Fe atoms. The spatial distributions of C, P, S, and Zn atoms revealed that the effect of MFH treatment was significantly dependent on the diffusion of the magnetic fluid in the tissue. An observed enrichment of Cu atoms after MFH treatment was probably due to inflammation in the tumor. The abnormal distribution of Ni atoms suggests a probable biochemical reaction in the tumor. Therefore, this LA-ICP-MS mapping technique can provide novel information regarding the spatial distribution of elements in tumors after cancer therapy. Figure  Mapping and ion intensities of a 56 Fe and b 158 Gd atoms. The red line indicates the path taken during the time-resolved analyses of Fe and Gd atoms Content Type Journal Article Pages 1-7 DOI 10.1007/s00216-011-5144-7 Authors Yi-Kong Hsieh, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013 Taiwan Pei-Shin Jiang, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013 Taiwan Bing-Shen Yang, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013 Taiwan Tian-Ye Sun, Department of Environmental Science and Engineering, Tsinghua University, Beijing, 100084 China Hsu-Hsia Peng, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013 Taiwan Chu-Fang Wang, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013 Taiwan Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    The new analytical method using Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) procedure for simultaneous determination of diacylhydrazine insecticide residues in fruits and vegetables was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The four insecticides (tebufenozide, methoxfenozide, chromafenozide, and halofenozide) were extracted from six fruit and vegetable matrices using acetonitrile and subsequently cleaned up using primary secondary amine (PSA) or octadecylsilane (C18) as sorbent prior to UPLC-MS/MS analysis. The determination of the target compounds was achieved in less than 3.0 min using an electrospray ionization source in positive mode (ESI+) for tebufenozide, methoxfenozide, and halofenozide and in negative mode (ESI−) for chromafenozide. The limits of detection were below 0.6 μg kg −1 , while the limit of quantification did not exceed 2 μg kg −1 in different matrices. The QuEChERS procedure by using two sorbents (PSA and C18) and the matrix-matched standards gave satisfactory recoveries and relative standard deviation (RSD) values in different matrices at four spiked levels (0.01, 0.05, 0.1, and 1 mg kg −1 ). The overall average recoveries for this method in apple, grape, cucumber, tomato, cabbage, and spinach at four levels ranged from 74.2% to 112.5% with RSDs in the range of 1.4–13.8% ( n  = 5) for all analytes. This study provides a theoretical basis for China to draw up maximum residue limits and analytical method for diacylhydrazine insecticide in vegetables and fruits. Content Type Journal Article Pages 1-8 DOI 10.1007/s00216-011-5148-3 Authors Xingang Liu, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193 China Jun Xu, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193 China Fengshou Dong, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193 China Yuanbo Li, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193 China Wenchen Song, Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing, 100125 China Yongquan Zheng, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193 China Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Metabolites of synthetic pyrethroids such as cis -3-(2,2-dibromovinyl)-2,2-di-methylcyclo-propane-1-carboxylic acid, cis - and trans -3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid), 3-phenoxybenzoic acid (3-PBA), and 4-fluoro-3-PBA are biomarkers for exposure to phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin, and permethrin. In this study, the pyrethroid metabolites in workers’ urine samples were monitored for the first time with a novel sample pretreatment process combining hollow fiber liquid phase microextraction (HF-LPME) and in-syringe derivatization (ISD) followed by gas chromatography–electron capture detector (GC-ECD) analysis. A micro-syringe pre-filled with derivatizing agents and syringe needle connected to an extracting solvent impregnated hollow fiber segment was used as the LPME probe. Pyrethroid metabolites were extracted and enriched simultaneously from urine samples by HF-LPME sampling and acid hydrolysis at 70 °C for 10 min. After sampling, the ISD was performed by mixing the extracting solution and derivatizing agents through plunger movements, followed by GC-ECD analysis. Parameters influencing the HF-LPME efficiency and ISD were investigated and optimized. Under optimum conditions, the method provided enrichment factors of 69.8–154.6, repeatability from 5.0 to 12% ( n  = 5), and good linearity ( R 2  = 0.9980–0.9998) for interested analytes spiked in urine samples. The method detection limits ranged from 1.6 to 17 ng/mL. A comparison was performed between the proposed method and conventional methods. The proposed method was applied to analyze pyrethroid metabolites in the urine samples collected from workers of pesticide formulation plants. The results suggested that the proposed HF-LPME coupled ISD method was a rapid, simple, efficient, and eco-friendly technique in the biomonitoring of metabolites of pyrethroids in workers’ urine. Content Type Journal Article Pages 1-11 DOI 10.1007/s00216-011-5122-0 Authors Chiu-Hwa Lin, Department of Chemistry, National Chung Hsing University, Taichung City, 402 Taiwan Cheing-Tong Yan, Department of Occupational Safety and Health, Chung-Shan Medical University, Taichung, 402 Taiwan Ponnusamy Vinoth Kumar, Department of Chemistry, National Chung Hsing University, Taichung City, 402 Taiwan Hong-Ping Li, Residue Control Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture (TACTRI/COA), Wu-Fung, Taichung City, 413 Taiwan Jen-Fon Jen, Department of Chemistry, National Chung Hsing University, Taichung City, 402 Taiwan Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    An integrated approach based on the use of inductively coupled plasma mass spectrometry (ICP-MS) and scanning electron microscopy (SEM) for the qualitative and quantitative analyses of metal particles in foods was devised and validated. Different raw materials and food products, like wheat, durum wheat, wheat flour, semolina, cookies, and pasta were considered. Attention was paid to the development of sample treatment protocols for each type of sample to avoid potential artifacts such as aggregation or agglomeration. The analytical protocols developed followed by ICP-MS and SEM investigations allowed us the quantitative determination and the morphological and dimensional characterization of metal nano- and microparticles isolated from the raw materials and finished food products considered. The ICP-MS method was validated in terms of linearity (0.8–80 μg/g and 0.09–9 μg/g for Fe and Ti, respectively), quantification limits (0.73 μg/g for Fe and 0.09 μg/g for Ti), repeatability (relative standard deviation (RSD) % equal to 10% for Fe and 20% in a wheat matrix as an example), and extraction recoveries (93 ± 2–101 ± 2%). Validation of the scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDS) measurements was performed working in a dimensional range from 1 to 100 μm with an estimated error in the size determination equal to 0.5 μm. ICP-MS data as well as SEM measurements showed a decrease in the concentration of metal particles from wheat to flour and from durum wheat to semolina samples, thus indicating an external contamination of grains by metal particles. These findings were confirmed by environmental SEM analysis, which allowed investigation of particles of lower dimensions. Generally, the largest number of particles was found in the case of iron and titanium, whereas particles of copper and zinc were only occasionally found without any possibility of quantifying their number. Content Type Journal Article Pages 1-9 DOI 10.1007/s00216-011-5149-2 Authors D. Beltrami, Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy D. Calestani, IMEM-CNR, Parco Area delle Scienze 37/A, 43124 Parma, Italy M. Maffini, Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy M. Suman, Barilla Food Research Labs, via Mantova 166, 43100 Parma, Italy B. Melegari, Barilla Food Research Labs, via Mantova 166, 43100 Parma, Italy A. Zappettini, IMEM-CNR, Parco Area delle Scienze 37/A, 43124 Parma, Italy L. Zanotti, IMEM-CNR, Parco Area delle Scienze 37/A, 43124 Parma, Italy U. Casellato, ICIS-CNR, Area della Ricerca, C.so Stati Uniti 4, 35127 Padova, Italy M. Careri, Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy A. Mangia, Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective “ESR-silent” hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified. Figure  Comparison of the ESR and MS results concerning the distribution of the nitroxide radical CAT-1 and CAT-1-H in a human skin biopsy Content Type Journal Article Pages 1-7 DOI 10.1007/s00216-011-5150-9 Authors U. Hochkirch, Institute of Chemistry, Humboldt-Universität zu Berlin, 10117 Berlin, Germany W. Herrmann, Institute of Pharmacy, Free University Berlin, Kelchstr. 31, 12169 Berlin, Germany R. Stößer, Institute of Chemistry, Humboldt-Universität zu Berlin, 10117 Berlin, Germany M. Linscheid, Institute of Chemistry, Humboldt-Universität zu Berlin, 10117 Berlin, Germany H.-H. Borchert, Institute of Pharmacy, Free University Berlin, Kelchstr. 31, 12169 Berlin, Germany Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Fine needle aspirates (FNAs) of suspicious breast lesions are often used to aid the diagnosis of female breast cancer. Biospectroscopy tools facilitate the acquisition of a biochemical cell fingerprint representative of chemical bonds present in a biological sample. The mid-infrared (IR; 4,000–400 cm −1 ) is absorbed by the chemical bonds present, allowing one to derive an absorbance spectrum. Complementary to IR spectroscopy, Raman spectroscopy measures the scattering by chemical bonds following excitation by a laser to generate an intensity spectrum. Our objective was to apply these methods to determine whether a biospectroscopy approach could objectively segregate different categories of FNAs. FNAs of breast tissue were collected ( n  = 48) in a preservative solution and graded into categories by a cytologist as C1 (non-diagnostic), C2 (benign), C3 (suspicious, probably benign) or C5 (malignant) [or C4 (suspicious, probably malignant); no samples falling within this category were identified during the collection period of the study]. Following washing, the cellular material was transferred onto BaF 2 (IR-transparent) slides for interrogation by Raman or Fourier-transform IR (FTIR) microspectroscopy. In some cases where sufficient material was obtained, this was transferred to low-E (IR-reflective) glass slides for attenuated total reflection–FTIR spectroscopy. The spectral datasets produced from these techniques required multivariate analysis for data handling. Principal component analysis followed by linear discriminant analysis was performed independently on each of the spectral datasets for only C2, C3 and C5. The resulting scores plots revealed a marked overlap of C2 with C3 and C5, although the latter pair were both significantly segregated ( P  〈 0.001) in the Raman spectra. Good separation was observed between C3 and C5 in all three spectral datasets. Analysis performed on the average spectra showed the presence of three distinct cytological groups. Our findings suggest that biospectroscopy tools coupled with multivariate analysis may support the current FNA tests whilst increasing the sensitivity and associated reliability for improved diagnostics. Figure  Average IR spectra derived from different categories of FNA specimens Content Type Journal Article Pages 1-11 DOI 10.1007/s00216-011-5137-6 Authors Jemma G. Kelly, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Abdullah A. Ahmadzai, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Paul Hermansen, Lancashire Teaching Hospitals NHS Trust, Sharoe Green Lane, Fulwood, Preston, PR2 9HT UK Mark A. Pitt, Lancashire Teaching Hospitals NHS Trust, Sharoe Green Lane, Fulwood, Preston, PR2 9HT UK Zuhair Saidan, Lancashire Teaching Hospitals NHS Trust, Sharoe Green Lane, Fulwood, Preston, PR2 9HT UK Pierre L. Martin-Hirsch, Lancashire Teaching Hospitals NHS Trust, Sharoe Green Lane, Fulwood, Preston, PR2 9HT UK Francis L. Martin, Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ UK Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    A new resistance-type sensor based on Prussian blue film has been fabricated for the detection of chlorobenzene vapor. The effect of Prussian blue preparation conditions on the response of sensor was studied. The sensor exhibited good response and selectivity to chlorobenzene vapor. The sensor prepared with Fe 2 (SO 4 ) 3 at 298 K has response 8.5 at operating voltage of 10 V. The selectivity of the sensor to chlorobenzene against all other tested gases is exceeding almost by 5.6 times. The sensor showed linear response to chlorobenzene vapor in the concentration range of 24–169 ppm at room temperature and at a 10 V operating voltage. The response and recovery time of the sensor was about 18 and 12 s, respectively. Sensor stability test indicated the sensor had a good stability. Furthermore, seven real samples of chlorobenzene vapor was measured using the sensor. The relative error was in the range of about ±1.3%. Figure  Response to different gases of the sensor based on Prussian blue prepared with Fe 2 (SO 4 ) 3 at 298 K Content Type Journal Article Pages 1-6 DOI 10.1007/s00216-011-5143-8 Authors Tiexiang Fu, Department of Chemistry, Changsha University of Science and Technology, Wanjiali Road, Changsha, 410004 Hunan, China Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642

Description:    Egyptian blue has been identified in a painting from 1524 by the Italian artist Ortolano Ferrarese (Giovanni Battista Benvenuto). Egyptian blue is the oldest known synthetic pigment, invented by the Egyptians in the fourth dynasty (2613–2494  bc ) of the Old Kingdom and extensively used throughout Antiquity. From about 1000  a.d ., it disappeared from the historical record and was only reinvented in the late nineteenth and early twentieth century. The discovery of Egyptian blue in Ortolano Ferrarese’s painting from 1524 shows that Egyptian blue was in fact available in the period from which it is normally considered not to exist. The identification of Egyptian blue is based on optical microscopy supported by energy-dispersive spectroscopy and visual light photon-induced spectroscopy, and finally confirmed by Raman microspectroscopy. Figure  St. Margaret by Giovanni Batista Benvenuto. National Gallery of Denmark. Content Type Journal Article Pages 1-7 DOI 10.1007/s00216-011-5140-y Authors Jørn Bredal-Jørgensen, School of Conservation, Esplanaden 34, 1263 Copenhagen K, Denmark Jana Sanyova, Laboratories of IRPA/KIK, Parc du Cinquantenaire, 1, 1000 Brussels, Belgium Vibeke Rask, School of Conservation, Esplanaden 34, 1263 Copenhagen K, Denmark Maria Louise Sargent, Ny Carlsberg Glyptotek, Dantes Plads 7, 1556 Copenhagen V, Denmark Rikke Hoberg Therkildsen, Ny Carlsberg Glyptotek, Dantes Plads 7, 1556 Copenhagen V, Denmark Journal Analytical and Bioanalytical Chemistry Online ISSN 1618-2650 Print ISSN 1618-2642