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: This review summarizes various approaches for the analysis of low molecular weight (LMW) compounds by different laser desorption/ionization mass spectrometry techniques (LDI-MS). It is common to use an agent to assist the ionization, and small molecules are normally difficult to analyze by, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the common matrices available today, because the latter are generally small organic compounds themselves. This often results in severe suppression of analyte peaks, or interference of the matrix and analyte signals in the low mass region. However, intrinsic properties of several LDI techniques such as high sensitivity, low sample consumption, high tolerance towards salts and solid particles, and rapid analysis have stimulated scientists to develop methods to circumvent matrix-related issues in the analysis of LMW molecules. Recent developments within this field as well as historical considerations and future prospects are presented in this review.

Description: An analytical procedure for the simultaneous determination of cyanide and thiocyanate in swine plasma was developed and validated. Cyanide and thiocyanate were simultaneously analyzed by high-performance liquid chromatography tandem mass spectrometry in negative ionization mode after rapid and simple sample preparation. Isotopically labeled internal standards, Na 13 C 15 N and NaS 13 C 15 N, were mixed with swine plasma (spiked and nonspiked), proteins were precipitated with acetone, the samples were centrifuged, and the supernatant was removed and dried. The dried samples were reconstituted in 10 mM ammonium formate. Cyanide was reacted with naphthalene-2,3-dicarboxaldehyde and taurine to form N-substituted 1-cyano[f]benzoisoindole, while thiocyanate was chemically modified with monobromobimane to form an SCN-bimane product. The method produced dynamic ranges of 0.1–50 and 0.2–50 μM for cyanide and thiocyanate, respectively, with limits of detection of 10 nM for cyanide and 50 nM for thiocyanate. For quality control standards, the precision, as measured by percent relative standard deviation, was below 8 %, and the accuracy was within ±10 % of the nominal concentration. Following validation, the analytical procedure successfully detected cyanide and thiocyanate simultaneously from the plasma of cyanide-exposed swine.

Description: The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium , but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.

Description: This work examines the new possibilities introduced with the arrival of commercially available high-resolution continuum source atomic absorption spectrometers for the determination of metalloids (B, Si, Ge, As, Se, Sb and Te) and non-metals (P, S, F, Cl, Br, I and N-based species), such as the improved potential to detect and correct for spectral overlaps and the strategies available to correct for matrix effects. In particular, and considering the increasing number of papers reporting on the use of molecular absorption spectrometry using graphite furnaces and flames as vaporizers, the work discusses in detail the advantages and limitations derived from the monitoring of molecular spectra from a practical point of view, in an attempt to guide future users of the technique. Figure Heating of a graphite furnace with platform

Description: This study describes a simple method for analyzing the abundance of mRNA molecules in a total DNA sample. Due to the dependence on the near-infrared fluorescence technique, this method is named near-infrared fluorescence gene expression detection (NIRF-GED). The procedure has three steps: (1) isolating total RNA from detected samples and reverse-transcription into cDNA with a biotin-labeled oligo dT; (2) hybridizing cDNA to oligonucleotide probes coupled to a 96-well microplate; and (3) detecting biotins with NIRF-labeled streptavidin. The method was evaluated by performing proof-in-concept detections of absolute and relative expressions of housekeeping and NF-κB target genes in HeLa cells. As a result, the absolute expression of three genes, Ccl20 , Cxcl2 , and Gapdh , in TNF-α-uninduced HeLa cells was determined with a standard curve constructed on the same microplate, and the relative expression of five genes, Ccl20 , Cxcl2 , Il-6 , STAT5A , and Gapdh , in TNF-α-induced and -uninduced HeLa cells was measured by using NIRF-GED. The results were verified by quantitative PCR (qPCR) and DNA microarray detections. The biggest advantage of NIRF-GED over the current techniques lies in its independence of exponential or linear amplification of nucleic acids. Moreover, NIRF-GED also has several other benefits, including high sensitivity as low as several fmols, absolute quantification in the range of 9 to 147 fmols, low cDNA consumption similar to qPCR template, and the current medium throughput in 96-well microplate format and future high throughput in DNA microarray format. NIRF-GED thus provides a new tool for analyzing gene transcripts and other nucleic acid molecules. Figure ᅟ

Description: In the present work, a novel flow-injection chemiluminescence method based on CdTe quantum dots (QDs) was developed for the determination of nitrite. Weak chemiluminescence (CL) signals were observed from a CdTe QDs–H 2 O 2 system under basic conditions. The addition of a trace amount of hemoglobin (Hb) caused the CL from the CdTe QDs–H 2 O 2 system to increase substantially. In the presence of nitrite, the ferrous Hb reacted with the nitrate to form ferric Hb and NO. The NO then bound to ferrous Hb to generate iron nitrosyl Hb. As a result, the CL signal from the CdTe QDs–H 2 O 2 –Hb system was quenched. Thus, a flow-injection CL analytical system for the determination of trace nitrite was established. Under optimum conditions, there was a good linear relationship between CL intensity and the concentration of nitrite in the range 1.0 × 10 −9 to 8.0 × 10 −7  mol L −1 ( R 2  = 0.9957). The limit of detection for nitrite using this system was 3.0 × 10 −10  mol L −1 (S/N = 3). This method was successfully applied to detect nitrite in water samples. Figure The scheme of the mechanism of the CL system

Description: A new carbon nanotube porous silica poroshell stationary phase was developed. The chromatographic support was coated with ultrashort single-wall carbon nanotubes (SWCNTs) in a noncovalent way. It was demonstrated that the porous amino silica surface of the 300 NH 2 poroshell column stabilized with 1-methyl-2-pyrrolidinone efficiently and stably adsorbed SWCNTs onto the chromatographic support. It was shown that this novel poroshell carbon nanotube (CNT) stationary phase was very useful for the HPLC separation of a series of monoclonal antibodies (mAbs) in a short analysis time (〈3 min). The high-performance liquid chromatography (HPLC) method was validated and was successfully tested for the fast quantitative and qualitative control of chemotherapeutic bags fabricated in a hospital pharmacy.

Description: High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5 × 10 −8 to 2.5 × 10 −7  M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations. Figure 2D-HPLC separation and permanganate chemiluminescence detection of neurotransmitter metabolites

Description: This study reports on the development and application of a piecewise linear model for the determination of copper-binding parameters at concentrations in the nanomolar range using fluorescence quenching. l -Tyrosine, Suwannee River natural organic matter, and two leaf leachates with similar fluorescence signatures were used as test compounds, and results were compared with those of the standard Ryan–Weber model. The piecewise model was also applied to and compared with data from an earlier study. Parallel factor analysis (PARAFAC) was used to identify three to five independent fluorophores in each test compound, and copper-binding parameters were estimated for one to three binding sites for each fluorophore. The binding properties of similar and different fluorophores were also compared. The conditional binding strengths (log K ′) estimated using the piecewise approach were similar to those obtained using the Ryan–Weber approach ( p  〉 0.05); however, the piecewise linear model provided superior results compared to models based on the Ryan–Weber equation in several ways, including (1) capable of distinguishing more binding sites for a single fluorophore, (2) capable of extracting binding parameters at environmentally relevant, nanomolar concentrations of copper, where fluorescence changes are often observed as enhancement, (3) greater precision over repeated titrations, and (4) no severe underestimation of complexing capacities. Finally, the copper-binding properties of PARAFAC components with similar optical signatures were found to be similar, both in sources with dramatically different and similar total fluorescence signatures. Figure Changes in copper binding behaviour of single fluorophore in dissolved organic matter estimated using piecewise linear fitting of fluorescence quenching

Description: The increase in the incidence of neurodegenerative diseases linked to aging or injury needs to be addressed in research into neuroprotective or neuroregenerative therapies, and requires the development of specific biological models. To achieve this goal we propose (1) the use of the mouse olfactory epithelium as a biological support which specifically exhibits a regenerative or a self-renewing capacity and during the lifetime necessitates the presence of neural stem cells, and (2) the use of an intraperitoneal injection of 2,6-dichlorobenzonitrile (diclobenil) as a chemical inducer of neurodegeneration in olfactory epithelium by selectively killing mature cells. We developed a biological model to follow the processes of neurodegeneration (chemically induced) and neuroregeneration (self-renewal of olfactory epithelium). The purpose of this study was to develop a method to monitor quickly neurodegeneration/neuroregeneration processes in order to further screen protective and regenerative therapies. For this purpose, we used the sedimentation field flow fractionation elution of olfactory epithelium. We obtained specific elution profiles and retention parameters allowing the monitoring of the induction and kinetics of biological processes. The use of insulin-like growth factor 1α as a neuroprotective agent in an innovative nebulization protocol showed sedimentation field flow fractionation to be a simple, fast and low-cost method to monitor such a biological event on the scale of an entire organism. Figure ᅟ

Description: A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-μm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly 13 C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % ( n  = 5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography–tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).

Description: Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N -oxides of PAs. This first multiplex immunoassay for PA detection with N -oxide reduction can be used for the simultaneous screening of 21 samples for 〉12 PA analytes. Honey samples ( n  = 146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs 〉25 μg/kg by ELISA which correlated to 〉10 μg/kg by LC-MS/MS.

Description: This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH = 8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time = 5.0 s, effective capillary length = 55 cm, and run temperature = 20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure. Figure Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds. Establishing direct pathways and developing sensitive/selective analytical tools to measure and characterize xenobiotics provides a defined link between potential hazards and suspected health effects.

Description: A gel-free, shotgun proteomics approach was used to characterize pomegranate aril proteome by nanoliquid chromatography–high-resolution tandem mass spectrometry. To identify both high-abundance and low-abundance proteins, we applied two distinct sample preparation protocols, i.e., a classical one widely applied in literature and a second one able to reduce the dynamic range of protein concentration of the sample, based on combinatorial hexapeptide ligand library technology. However, the proteins identified with the latter protocol were only a small minority. Because pomegranate is a non-model plant species, i.e., information of its genome sequence are lacking, only a few protein sequences are included in the most widely known protein sequence databases. To improve both the number of identified proteins and data reliability, identification was performed integrating the results obtained with three distinct plant protein databases, since the majority of proteins could only be attributed by homology with other plant species. Nevertheless, many proteins had assigned only one unique peptide, because of the phylogenetic distance of pomegranate from the main model plants. After manual revision of the identified proteins to eliminate the redundant or ambiguous identifications, a list of 1,488 proteins was obtained, only six of which belonging to pomegranate species. To the author's best knowledge, this is the first work aimed at the proteomic characterization of Punica granatum .

Description: 5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg −1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg −1 . Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg −1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 ( n  = 6), mean intra-day precision 3.2 % ( n  = 3 within a plate; n  = 2 repeated within a day), mean inter-day precision 3.7 % ( n  = 3), mean reproducibility over the whole procedure including sample preparation 4.1 % ( n  = 2), and mean recovery 106.9 % ( n  = 5 different concentrations; n  = 4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved. Figure HPTLC separation of HMF from honey matrix for honey with very low HMF content

Description: Reproducible and quantitative gas chromatography–mass spectrometry (GC-MS)-based metabolomics analysis of complex biological mixtures requires robust and broad-spectrum derivatization. We have evaluated derivatization of complex metabolite mixtures using trimethylsilyl cyanide (TMSCN) and the most commonly used silylation reagent N -methyl- N -(trimethylsilyl)trifluoroacetamide (MSTFA). For the comparative analysis, two metabolite mixtures, a standard complex mixture of 35 metabolites covering a range of amino acids, carbohydrates, small organic acids, phenolic acids, flavonoids and triterpenoids, and a phenolic extract of blueberry fruits were used. Four different derivatization methods, (1) direct silylation using TMSCN, (2) methoximation followed by TMSCN (M-TMSCN), (3) direct silylation using MSTFA, and (4) methoximation followed by MSTFA (M-MSTFA) were compared in terms of method sensitivity, repeatability, and derivatization reaction time. The derivatization methods were observed at 13 different derivatization times, 5 min to 60 h, for both metabolite mixtures. Fully automated sample derivatization and injection enabled excellent repeatability and precise method comparisons. At the optimal silylation times, peak intensities of 34 out of 35 metabolites of the standard mixture were up to five times higher using M-TMSCN compared with M-MSTFA. For direct silylation of the complex standard mixture, the TMSCN method was up to 54 times more sensitive than MSTFA. Similarly, all the metabolites detected from the blueberry extract showed up to 8.8 times higher intensities when derivatized using TMSCN than with MSTFA. Moreover, TMSCN-based silylation showed fewer artifact peaks, robust profiles, and higher reaction speed as compared with MSTFA. A method repeatability test revealed the following robustness of the four methods: TMSCN 〉 M-TMSCN 〉 M-MSTFA 〉 MSTFA. Figure Improved GC-MS profiling of Complex Biological Mixtures by TMSCN based Derivatization

Description: Fourier transform infrared microspectroscopy is a powerful tool to obtain knowledge about the spatial and/or temporal distributions of the chemical compositions of plants for better understanding of their biological properties. However, the chemical morphologies of plant leaves in the plane of the blade are barely studied, because sections in this plane for mid-infrared transmission measurements are difficult to obtain. Besides, native compositions may be changed by chemical reagents used when plant samples are microtomed. To improve methods for direct infrared microspectroscopic imaging of plant leaves in the plane of the blade, the bulk and surface chemical morphologies of nonmicrotomed Ginkgo biloba leaves were characterized by near-infrared transmission and mid-infrared attenuated total reflection microspectroscopic imaging. A new self-modeling curve resolution procedure was proposed to extract the spectral and concentration information of pure compounds. Primary and secondary metabolites of secretory cavities, veins, and mesophylls of Ginkgo biloba leaf blades were analyzed, and the distributions of cuticle, protein, calcium oxalate, cellulose, and ginkgolic acids on the adaxial surface were determined. By the integration of multiple infrared microspectroscopic imaging and chemometrics methods, it is possible to analyze nonmicrotomed leaves and other plant samples directly to understand their native chemical morphologies in detail. Graphical abstract Visible and infrared microspectroscopic images of a Ginkgo biloba leaf blade. PC-1 score image shows the physical morphology, while the positive and negative part of PC-2 score image shows the distribution of dichotomous branching veins and secretory cavities, respectively

Description: Therapeutic drug monitoring (TDM) requires timely results in order to be clinically helpful. Such assays, when carried out using mass spectrometry-based methods, typically involve a batched sample approach with multipoint calibration. Isotopic internal calibration offers the possibility of open-access mass spectrometric analysis with consequent shortening of turnaround times. We measured plasma clozapine and N -desmethylclozapine (norclozapine) concentrations in (1) external quality assessment (EQA) samples ( N  = 22) and (2) patient samples ( N  = 100) using liquid chromatography-tandem mass spectrometry with isotopic internal calibration (ICAL-LC-MS/MS). Analyte concentrations were calculated from graphs of the response of three internal calibrators (clozapine-D 4 , norclozapine-D 8 , and clozapine-D 8 ) against concentration. Precision (% RSD) and accuracy (% nominal concentrations) for the ICAL-LC-MS/MS method were 〈5 % and 104–112 %, respectively for both analytes. There was excellent agreement with consensus mean and with ‘spiked’ values on analysis of the EQA samples ( R 2  = 0.98 and 0.97, respectively, inclusive of clozapine and norclozapine results). In the patient samples, comparison against traditionally calibrated HPLC-UV and LC-MS/MS methods showed excellent agreement ( R 2  = 0.97 or better) with small albeit significant mean differences (〈0.041 and 〈0.042 mg/L for clozapine and norclozapine, respectively). These differences probably reflect discrepancies in the in-house preparation of calibrators and/or interference in the UV method. Internal calibration offers a novel and attractive alternative to traditionally calibrated batch analysis in analytical toxicology. The method described has been validated for use in the high-throughput TDM of clozapine and norclozapine, and allows for (1) same-day reporting of results and (2) significant cost savings. Figure A typical internal calibration curve produced using three different isotopically-labelled analogues of clozapine and norclozapine (clozapine-D 4 , norclozapine-D 8 and clozapine-D 8 ) and interpolation of the clozapine and norclozapine concentrations from an extracted patient sample (calculated clozapine and norclozapine 0.47 and 0.31 mg/L, respectively)

Description: Because norcantharidin (NCTD) is unstable and subject to ring opening and hydrolysis, the diacid metabolite of norcantharidin (DM-NCTD) is the stable form of NCTD found in normal saline solution. Conversion of NCTD to DM-NCTD is almost 100 %, making it possible to determine and investigate the pharmacokinetics of DM-NCTD converted from NCTD. In this paper, a sensitive, simple and selective liquid chromatographic–tandem mass spectrometric method was developed and validated for determination of DM-NCTD in beagle plasma. DM-NCTD was detected in multiple-reaction monitoring (MRM) mode by using the dehydrated ion 169.3 as precursor ion and its product ion 123.1 as the detected ion. Ribavirin was used as internal standard and detected in MRM mode by use of precursor ions, resulting in a product ion transition of m /z 267.1 → 135.1. This method was successfully used for a pharmacokinetic study of DM-NCTD in beagles after intravenous administration of DM-NCTD in normal saline solution at doses of 0.39, 0.78, and 1.6 mg kg −1 . DM-NCTD had dose-dependent kinetics across the dosage range investigated, with enhanced T 1/2α and AUC 0-12 and apparently decreasing V d and CL with increasing dosage. After single-dose administration, T 1/2α ranged from 0.20 to 0.55 h, AUC 0-12 from 1.81 to 43.6 μg mL −1  h −1 , V d from 228 to 55.9 mL kg −1 , and CL from 220 to 36.5 mL kg −1  h −1 ( P  〈 0.01). The results indicated nonlinear pharmacokinetic behavior of DM-NCTD in beagles, suggesting that the risk of DM-NCTD in normal saline solution intoxication may be non-proportionally increased at higher doses. Figure Determination and pharmacokinetic study of the diacid metabolite of norcantharidin

Description: This work demonstrates the application of silica supported Fe 3 O 4 nanoparticles as sorbent phase for magnetic solid-phase extraction (MSPE) and magnetic on-line in-tube solid-phase microextraction (Magnetic-IT-SPME) combined with capillary liquid chromatography–diode array detection (CapLC-DAD) to determine organophosphorous compounds (OPs) at trace level. In MSPE, magnetism is used as separation tool while in Magnetic-IT-SPME, the application of an external magnetic field gave rise to a significant improvement of the adsorption of OPs on the sorbent phase. Extraction efficiency, analysis time, reproducibility and sensitivity have been compared. This work showed that Magnetic-IT-SPME can be extended to OPs with successful results in terms of simplicity, speed, extraction efficiency and limit of detection. Finally, wastewater samples were analysed to determine OPs at nanograms per litre.

Description: We describe here the preparation of carbon-coated Fe 3 O 4 magnetic nanoparticles that were further fabricated into multifunctional core/shell nanoparticles (Fe 3 O 4 @C@CNCs) through a layer-by-layer self-assembly process of carbon nanocrystals (CNCs). The nanoparticles were applied in a photoluminescence (PL) immunosensor to detect the carcinoembryonic antigen (CEA), and CEA primary antibody was immobilized onto the surface of the nanoparticles. In addition, CEA secondary antibody and glucose oxidase were covalently bonded to silica nanoparticles. After stepwise immunoreactions, the immunoreagent was injected into the PL cell using a flow-injection PL system. When glucose was injected, hydrogen peroxide was obtained because of glucose oxidase catalysis and quenched the PL of the Fe 3 O 4 @C@CNC nanoparticles. The here proposed PL immunosensor allowed us to determine CEA concentrations in the 0.005–50 ng · mL -1 concentration range, with a detection limit of 1.8 pg · mL -1 . Figure The Fe 3 O 4 @C@CNC was prepared and applied in a CEA immunosensor with the help of a flow-injection photoluminescence system.

Description: A fully automated method has been developed for determining eight macrocyclic musk fragrances in wastewater samples. The method is based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC-MS). Five different fibres (PDMS 7 μm, PDMS 30 μm, PDMS 100 μm, PDMS/DVB 65 μm and PA 85 μm) were tested. The best conditions were achieved when a PDMS/DVB 65 μm fibre was exposed for 45 min in the headspace of 10 mL water samples at 100 °C. Method detection limits were found in the low ng L −1 range between 0.75 and 5 ng L −1 depending on the target analytes. Moreover, under optimized conditions, the method gave good levels of intra-day and inter-day repeatabilities in wastewater samples with relative standard deviations ( n  = 5, 1,000 ng L −1 ) less than 9 and 14 %, respectively. The applicability of the method was tested with influent and effluent urban wastewater samples from different wastewater treatment plants (WWTPs). The analysis of influent urban wastewater revealed the presence of most of the target macrocyclic musks with, most notably, the maximum concentration of ambrettolide being obtained in WWTP A (4.36 μg L −1 ) and WWTP B (12.29 μg L −1 ), respectively. The analysis of effluent urban wastewater showed a decrease in target analyte concentrations, with exaltone and ambrettolide being the most abundant compounds with concentrations varying between below method quantification limit (〈MQL) and 2.46 μg L −1 . Figure Scheme of a HS-SPME followed by GC-MS to determine macrocyclic musk fragrances in wastewater samples

Description: The purpose of this study was to assess the stability of the polyfructan levan under different pH solution conditions by monitoring changes in the levan physicochemical properties, such as molar mass ( M ), root mean square radius ( r rms ), hydrodynamic radius ( r h ), structure factor ( r rms /r h ), and aggregation state with respect to solution pH and hydrolysis time. A commercial levan produced from Z. Mobilis was characterized using asymmetric flow field-flow fractionation (AF4) in combination with online multiangle light scattering (MALS) and differential refractive index (dRI) detection. Under neutral pH solution conditions the levan was found to have a M ranging from 10 5 to 5 × 10 7  g/mol, a r rms ranging from ~25 to 100 nm and a r h from ~3 to 151 nm. Two populations were observed in the sample. One population with a M less than 10 6  g/mol which represented ~60 % of the sample and a second population with an ultrahigh M up to 5 × 10 7  g/mol, which comprised ~40 % of the sample. The measured r rms /r h structure factor decreased from 1.8 to 0.65 across the AF4 fractogram indicating that early eluting low M levan species had a random coil configuration and late eluting high M species had more homogeneous spherical structures. The measured apparent density values decreased from 80 to 10 kg/m 3 across the elution profile and suggest that the observed second population also contains aggregates. The stability of levan in different pH conditions ranging from 1.3 to 8.5 was assessed by tracking changes in the average M and r h , and monitoring the formation of fructose over 1 week. The onset of levan acid hydrolysis was observed to occur sooner at lower pH conditions and no hydrolysis was observed for pH 5.5 and higher.

Description: The National Institute of Standards and Technology (NIST) has a wide range of Standard Reference Materials (SRMs) which have values assigned for legacy organic pollutants and toxic elements. Existing SRMs serve as homogenous materials that can be used for method development, method validation, and measurement for contaminants that are now of concern. NIST and multiple groups have been measuring the mass fraction of a group of emerging contaminants, polyfluorinated substances (PFASs), in a variety of SRMs. Here we report levels determined in an interlaboratory comparison of up to 23 PFASs determined in five SRMs: sediment (SRMs 1941b and 1944), house dust (SRM 2585), soil (SRM 2586), and sludge (SRM 2781). Measurements presented show an array of PFASs, with perfluorooctane sulfonate being the most frequently detected. SRMs 1941b, 1944, and 2586 had relatively low concentrations of most PFASs measured while 23 PFASs were at detectable levels in SRM 2585 and most of the PFASs measured were at detectable levels in SRM 2781. The measurements made in this study were used to add values to the Certificates of Analysis for SRMs 2585 and 2781.

Description: A sensitive and selective method was developed to quantitate allopregnanolone and its 5β isomer pregnanolone in human plasma using liquid chromatography-differential mobility separation combined with MS/MS detection. The method employed a simple liquid–liquid extraction of 100 μL plasma with hexane/ethyl acetate. After extraction, the sample was derivatized using a quaternary aminooxy reagent. Separation of allopregnanolone, pregnanolone, and their 3β epimers (epiallopregnanolone and epipregnanolone) was achieved using a Phenomenex Kinetex C18 2.1 × 100-mm 2.6-μm column. A linear calibration curve was obtained over the concentration range from 10 to 25,000 pg/mL, and the inter- and intra-day accuracy of the quality control samples were between 90 and 110 % with the inter- and intra-day precision less than 10 %. The lower limit of quantitation is 50 fg (157 amol) on column for both allopregnanolone and pregnanolone which is 100-fold less than the underivatized compounds. The recovery is above 95 %, and the extracted samples are stable for at least 6 days when stored at 4 °C. Plasma samples from normal, pregnant, and postpartum women were analyzed using this method.

Description: We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reverse-phase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm × 4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ  = 214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.

Description: An in vitro steroidogenesis assay using H295R human adenocarcinoma cells is a useful tool for the fast identification of compounds that affect the production of testosterone and 17β-estradiol. Selective and sensitive hormone measurement by liquid chromatography–tandem mass spectrometry (LC–MS/MS) can make this assay more reliable. Therefore, in the present study, a sensitive and selective method for the quantification of testosterone and 17β-estradiol in the H295R steroidogenesis assay was developed and fully validated using LC–MS/MS coupled with an online sample enrichment technique. To prove its usefulness, the method developed was applied to investigate the effect of sildenafil on steroidogenesis. Cell medium samples were diluted and prepared using solid-phase extraction. The samples were prepared on ice and were not kept for more than 30 min to prevent degradation of hormones. The extracts were dried, reconstituted, filtered, and analyzed by LC–MS/MS with polarity switching electrospray ionization. The validation results for selectivity, matrix effect, recovery, linearity, precision, and accuracy were satisfactory. The limits of detection for testosterone and 17β-estradiol were 5 and 10 pg/mL, respectively, and the limit of quantification for both testosterone and 17β-estradiol was 10 pg/mL, which was in accordance with the OECD guideline. No degradation was observed under the storage conditions for 7 and 14 days at -80 °C as well as after three freeze–thaw cycles, whereas 17β-estradiol was degraded after 1 h on ice during sample processing. The method developed was successfully used for the investigation of the effect of sildenafil on steroidogenesis. This method can be very useful for the initial selection of drugs with androgenic and/or estrogenic effects for specific purposes, e.g., in the selection of drugs that are used to reverse the effects of chemical castration.

Description: In tandem mass spectrometry the multiple reaction monitoring (MRM) mode is normally used for targeted analysis but this mode also has the potential to screen for structural similarities of analytes. On the basis of the fact that in general similar molecular structures result in similar fragments or losses of neutrals, this approach was used for pyrrolizidine alkaloid (PA) screening but could also be easily adapted to screen for other compound classes. PA are plant toxins of which several hundred individual compounds have been identified. Our MRM screening approach uses the structural relation and similar core structure of all PA which results in a common and thus predictable mass spectrometric fragmentation behaviour. On this basis a method was developed which screens for PA structures by MRM transitions and allows the detection of each individual PA down to a low microgram per kilogram concentration range. The approach was applied to investigate plants from the families of Asteraceae (several species of Senecio and Eupatorium ), Boraginaceae ( Echium , Cynoglossum , Borago and Anchusa officinalis as well as Heliotropium europaeum ) and Fabaceae ( Crotalaria incana ) for a complete qualitative and quantitative PA characterisation. All analytes that were detected as possible PA by MRM screening were further investigated by recording product ion spectra. Analytes which exhibited a typical PA fragmentation pattern were either confirmed as PA or otherwise deleted as false positive signals (false positive rate was below 10 %). Sum formulas of confirmed PA were determined by additional measurements applying high resolution mass spectrometry. In that way 121 unknown PA were identified and for the first time complete PA profiles of different PA plants were delivered.

Description: This paper describes the retention behavior of oligolysine and oligoarginine peptides of different lengths as a function of heptafluorobutyric acid (HFBA) concentration in ion-pairing reversed-phase chromatography in isocratic elution. A mixture of oligolysine and a mixture of oligoarginine with number of amino acid residues (dp) from two to eight were conveniently prepared by one-pot protease-catalyzed synthesis. Analysis of the logarithm of the retention factor k as a function of [HFBA] for each oligopeptide component, using a closed pairing model, provided values for (1) number ( n ) of paired HFBA anions per peptide molecule, (2) equilibrium constant ( K ip,m ) for ion pairing between oligopeptides and HFBA anions, and (3) product of the phase ratio and the distribution constant of the paired oligopeptide between the mobile and stationary phases ( βK d,ip ). We found that βK d,ip of oligoarginine is larger compared with oligolysine having the same dp. A linear relationship was obtained for ln βK d,ip as a function of n  +  g  · dp. By optimizing constant g separately for oligolysine and oligoarginine, we determined that g is larger for oligoarginine, in agreement with the higher hydrophobicity of arginine residues. Plotting the fraction of paired oligoarginine and oligolysine as a function of [HFBA] shows that the cooperative effect in forming ion pairs is greater for oligoarginine than oligolysine. Figure Fraction Φ of paired oligolysine (dp = 3 to 6, solid symbols and solid lines ) and oligoarginine (dp = 3 to 6, open symbols and dashed lines ) in the mobile phase, plotted as a function of the HFBA concentration

Description: Matrix-assisted laser desorption/ionization mass spectrometry imaging is an emerging powerful technique in drug metabolism and pharmacokinetics research. Despite recent progress in mass-spectrometry-based localization and relative quantification of small-molecule drugs and their metabolites in tissue, improved methods for drug extraction/ionization are required. Furthermore, relative quantification of drugs by mass spectrometry imaging in larger rodent cohorts is a necessary proof-of-concept study to demonstrate the utility of such a workflow in an industrial setting. Using as an example the tyrosine kinase inhibitor dasatinib, a leukemia drug, we demonstrate that inclusion of dimethyl sulfoxide in standard matrix solutions significantly improves ion intensity in mass spectrometry images and reveals enrichment of the drug in mouse kidney medulla. We furthermore show in a time-course study in multiple mice that normalization against a deuterated internal standard, dasatinib- D 8 , which is applied together with the matrix, makes possible relative quantification of the drug that correlates well with canonical liquid chromatography–tandem mass spectrometry based drug quantification.

Description: Breast cancer has become one of the most threatening diseases to women throughout the world. Emerging evidence implies that estrogen receptor (ER) and phosphatidylinositol 3-kinase (PI3K) pathways play central roles in both breast cancer progression and response to therapy. In this work, we have probed into ER expression related to the PI3K pathway at the protein level with an electrochemical technique based on the detection of ER proteins in nuclear extracts with an Exonuclease III protection-based strategy. Experimental results show that an increased number of ER proteins can be detected upon PI3K inhibition, demonstrating the reversal effect of the PI3K inhibitor on ER expression. Moreover, treatment with different concentrations of the PI3K inhibitor NVP-BEZ235 can result in a dose-dependent alteration of ER protein levels, implying an intimate link between ER and PI3K pathways. This work may be a great help to understand the mysteries underlying PI3K-related endocrine resistance and to evaluate the effect of therapeutic interventions in the future.

Description: We present for the first time the Raman microspectroscopic identification and characterization of individual airborne volcanic ash (VA) particles. The particles were collected in April/May 2010 during research aircraft flights, which were performed by Deutsches Zentrum für Luft- und Raumfahrt in the airspace near the Eyjafjallajökull volcano eruption and over Europe (between Iceland and Southern Germany). In addition, aerosol particles were sampled by an Electrical Low Pressure Impactor in Munich, Germany. As references for the Raman analysis, we used the spectra of VA collected at the ground near the place of eruption, of mineral basaltic rock, and of different minerals from a database. We found significant differences in the spectra of VA and other aerosol particles (e.g., soot, nitrates, sulfates, and clay minerals), which allowed us to identify VA among other atmospheric particulate matter. Furthermore, while the airborne VA shows a characteristic Raman pattern (with broad band from ca. 200 to ca. 700 cm −1 typical for SiO 2 glasses and additional bands of ferric minerals), the differences between the spectra of aged and fresh particles were observed, suggesting differences in their chemical composition and/or structure. We also analyzed similarities between Eyjafjallajökull VA particles collected at different sampling sites and compared the particles with a large variety of glassy and crystalline minerals. This was done by applying cluster analysis, in order to get information on the composition and structure of volcanic ash. Figure Images and Raman spectra of airborne volcanic ash

Description: An optimised and validated method for the determination of pharmaceutical residues in blue mussels ( Mytilus spp.) is presented herein, as well as an investigation of the effect of cooking (by steaming) on any potential difference in human exposure risk. Selected pharmaceuticals included two non-steroidal anti-inflammatory drugs (diclofenac and mefenamic acid), an antibiotic (trimethoprim), an anti-epileptic (carbamazepine) and a lipid regulator (gemfibrozil). An in vivo exposure experiment was set up in the laboratory in which mussels were exposed either directly by injection (10 ng) or daily through spiked artificial seawater (ASW) over 96 h. In liquid matrices, pharmaceutical residues were either determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS) directly, or in combination with solid-phase extraction (SPE) for analyte concentration purposes. The extraction of pharmaceuticals from mussel tissues used an additional pressurised liquid extraction step prior to SPE and LC-MS/MS. Limits of quantification of between 2 and 46 ng L −1 were achieved for extracted cooking water and ASW, between 2 and 64 μg L −1 for ASW in exposure tanks, and between 4 and 29 ng g −1 for mussel tissue. Method linearities were achieved for pharmaceuticals in each matrix with correlation coefficients of R 2  〉 0.975. A selection of exposed mussels was also cooked (via steaming) and analysed using the optimised method to observe any effect on detectable concentrations of parent pharmaceuticals present. An overall increase in pharmaceutical residues in the contaminated mussel tissue and cooking water was observed after cooking. Figure Pharmaceutical residues in cooked and uncooked marine bivalves

Description: Extraction and analysis of labile compounds in complex sample matrices, such as plants, is often a big analytical challenge. In this work, the use of a “green and clean” pressurised hot water extraction (PHWE) approach performed in continuous flow mode is explored. Experimental data for extraction and degradation kinetics of selected compounds were utilised to develop a continuous flow extraction (CFE) method targeting thermolabile polyphenols in red onions, with detection by high-performance liquid chromatography (HPLC)–diode array detection (DAD)–mass spectrometry (MS). Water containing ethanol and formic acid was used as extraction solvent. Method performance was focused on extraction yield with minimal analyte degradation. By adjusting the flow rate of the extraction solvent, degradation effects were minimised, and complete extraction could be achieved within 60 min. The CFE extraction yields of the polyphenols investigated were 80–90 % of the theoretically calculated quantitative yields and were significantly higher than the yields obtained by conventional methanol extraction and static batch extraction (70–79 and 58–67 % of the theoretical yields, respectively). The precision of the developed method was lower than 8 % expressed as relative standard deviation. Figure Scheme of pressurised hot water extraction of polyphenols in continuous flow mode

Description: Twenty-five phages that selectively bind to a monoclonal antibody (Mab) 1H2 specific to 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) in the absence or presence of BDE47 have been selected from phage‐display libraries containing cyclic 7-mer, linear 7-mer, and linear 12-mer randomized peptides. Competitive and noncompetitive enzyme-linked immunosorbent assays (ELISA) for BDE47 were developed by using a clone C7-1 specific to the BDE47-free Mab 1H2 and a clone XC7-8 specific to the BDE47-bound Mab 1H2, respectively. The half-maximum signal inhibition concentration (IC 50 ) of the competitive phage ELISA and the half-maximum signal enhancement concentration (EC 50 ) of the noncompetitive phage ELISA for BDE47 were 6.8 ng mL −1 and 4.2 ng mL −1 , respectively. The noncompetitive phage ELISA showed higher cross-reactivity with BDE28, BDE99, and BDE100 than the competitive one, ranging between 1.3 and 6.5 % versus 0.3 and 0.8 %. Recoveries of the competitive and the noncompetitive phage ELISAs for BDE47 in sewage sludge and fillet samples were 96–124 % and 97–120 %, respectively. The results of the two types of phage ELISAs for BDE47 in the real-world samples agreed well with a gas chromatography/electron capture detector-ion trap mass spectrometer method.

Description: Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3′-O-glucuronide, hydroxytyrosol-4′-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4′-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80 %, and the matrix effect (%ME) was less than 8 %. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70 % and lower than 17 %, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.

Description: Fenoterol, a fast-acting β 2 -adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for β 2 -agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono -sulfoconjugates and one bis -sulfoconjugate were synthesised and their structures confirmed by liquid chromatography–high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two mono -sulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono -sulfoconjugates and a fenoterol bis -sulfoconjugate were detected in urine. This is the first report of the bis -sulfoconjugate.

Description: Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8–11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12–15; 9/10 of tumors; 94 % of variance) and β-turns (day 18–21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced ( P  〈 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.

Description: The results of validation of a method for the analysis of free and bound phenolics in wine and grapes are presented. Wine and grape extracts are fractionated by automated solid-phase extraction on Bond Elut PPL cartridges to give free and bound phenolic fractions. Bound fractions are subjected to acid hydrolysis, and the phenolics released are recovered by solid-phase extraction on Bond Elut PPL cartridges. The fractions are further purified by automated solid-phase extraction on Bond Elut silica cartridges. After derivatisation to form trimethylsilyl ethers, the phenolics are determined by gas chromatography–mass spectrometry with selected ion monitoring. The method is suitable for robust, high-throughput monitoring of the concentrations of phenolics that can affect the palatability of wine.

Description: There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip–triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters—selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze–thaw, autosampler stability). The calibration curves (weight 1/ x 2 ) were linear for the range 50–10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350–400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities. Figure ᅟ

Description: Poly( N -isopropylacrylamide) (PNiPAM) compounds with various diad tacticities were prepared, and the molecular interaction properties in a thin film deposited on a gold surface were analyzed using infrared spectroscopy. The intramolecular and intermolecular interactions were found to depend on the tacticity, and only atactic (diad ratio 46 %) PNiPAM exhibits poor molecular interaction even in the bulk sample. On the other hand, the same series of compounds dissolved in an acetone solution were spread on a gold surface to form a thin film. In the dissolution process, the polymer molecules are relaxed via solvation, and they are bound to the gold surface by a molecular interaction to form a submonolayer thin film. In the thin film, the molecular interaction with the gold surface via the N–H group was monitored in the infrared spectra only for a nearly isotactic ( m  = 90) PNiPAM by an apparent shift of the N–H stretching vibration band. This shift was confirmed by changing the degree of hydrophilicity of the gold surface: a larger shift is found on a gold surface with stronger hydrophilicity. As a result, the conformation of a nearly isotactic molecule is found to be extended by the interaction with the gold surface, which works to immobilize the molecule.