Analytical and Bioanalytical Chemistry (v.407, #5)

Using MOOCs for teaching analytical chemistry: experience at University of Tartu by Ivo Leito; Irja Helm; Lauri Jalukse (1277-1281).
works as professor of analytical chemistry at University of Tartu. His research directions are on the borderline of analytical chemistry with other disciplines: chemistry of superacids and superbases; metrology in chemistry (MiC); liquid chromatography and mass spectrometry; sensors and their metrological characterization; applications of instrumental methods in analysis of historical objects. He teaches analytical chemistry and its metrological aspects at UT and has been involved in setting up several international MiC-related educational activities. is an analytical chemistry research fellow at University of Tartu. She teaches practical classes of analytical chemistry. She takes care that metrological concepts and approaches are introduced to students at as early stage of analytical chemistry studies as possible. Her research is focused on high-accuracy reference measurements of dissolved oxygen concentration. is analytical chemistry research fellow at University of Tartu. He teaches analytical chemistry and metrology in chemistry at all study levels. He is continuously introducing innovative and active learning approaches into teaching. His research work is focused on metrological studies of electrochemical and optical sensors, measurements of dissolved oxygen concentration and moisture content, as well as organization of interlaboratory comparisons.

Biomarkers probed in saliva by surface plasmon resonance imaging coupled to matrix-assisted laser desorption/ionization mass spectrometry in array format by Johana Musso; William Buchmann; Florence Gonnet; Nathalie Jarroux; Sophie Bellon; Chiraz Frydman; Didier-Luc Brunet; Regis Daniel (1285-1294).
completed a Master’s degree in Chemistry and holds a PhD in Analytical Chemistry. Her research, performed at the University of Evry-Val-d’Essonne in the Laboratory for Analysis and Modelling for Biology and Environment and in partnership with HORIBA Jobin Yvon, focussed on surface plasmon resonance and mass spectrometry for biodiagnostic applications. is Assistant Professor and Head of the Department of Chemistry at the University of Evry-Val-d’Essonne. His research works mainly concern the characterization of synthetic polymers and the study of molecular interactions by mass spectrometry. is Professor in Analytical Chemistry at the University of Evry-Val-d’Essonne, Evry, France. She is co-leader of the CNRS team “Structure and Reactivity of Biomolecules” at Evry-Val-d’Essonne University, and co-director of the doctoral school “from genomes to organisms”. She is currently focusing in the development of Mass Spectrometry-based methods and hyphenated methods for the characterization of bio-molecular interactions, essentially between proteins, and between proteins and carbohydrates Assistant Professor in Macromolecular and Supramolecular Chemistry at the University of Evry-Val-d’Essonne, is team co-leader of the Polymer Materials at Interfaces group. She has made substantial contributions to the formation of supramolecular assemblies including polyrotaxane architectures and cyclodextrin-based polymers for biomaterials and electronic applications. has been working for several years on surface plasmon resonance imaging application systems development as head of the application lab at Horiba Scientific in Paris, France. is currently product manager of SPRi and life-sciences instruments at HORIBA Jobin Yvon. In 1996 she received her engineering diploma in Biology and in 1997 her MSc in Applied Chemistry and Industrial Process Engineering. After her PhD in Enzymatic Engineering, Bioconversation, and Microbiology she set up her own company dedicated to developing immunoaffinity kit, and later worked as a Biophotonic Project Manager at Opticsvalley. Frydman’s expertise covers molecular interaction, surface plasmon resonance analysis, surface chemistry, and biophotonics. is currently senior Director of EMEA operations at HORIBA Jobin Yvon. In 1979 he graduated in electronic engineering, and in 1985 he obtained his MBA degree specializing in international high technology trade administration. From 2007 to 2011 he was in charge of the biophotonic initiative at HORIBA Scientific. For some of this period he was the managing Director of a startup company dedicated to high-throughput biomolecular-interactions monitoring based on advanced optical design. Brunet’s expertise covers biophotonic instrumentation design based on optical techniques including fluorescence, Raman, and surface plasmon resonance. PhD, is senior researcher at the National Center for Scientific Research (CNRS, France) and co-leader of the CNRS team “Structure and Reactivity of Biomolecules” at Evry-Val-d’Essonne University. He is researching bioactive carbohydrates with the objective of better understanding their involvement in biological cascades. Their structure–activity relationships and interaction properties with biomolecular partners are addressed through modern hyphenated techniques based on mass spectrometry, including capillary electrophoresis MS and surface plasmon resonance MS. Detection of protein biomarkers is of major interest in proteomics. This work reports the analysis of protein biomarkers directly from a biological fluid, human saliva, by surface plasmon resonance imaging coupled to mass spectrometry (SPRi-MS), using a functionalized biochip in an array format enabling multiplex SPR-MS analysis. The SPR biochip presented a gold surface functionalized by a self-assembled monolayer of short poly(ethylene oxide) chains carrying an N-hydroxysuccinimide end-group for the immobilization of antibodies. The experiments were accomplished without any sample pre-purification or spiking with the targeted biomarkers. SPRi monitoring of the interactions, immune capture from the biochip surface, and finally on-chip matrix-assisted laser desorption/ionization-MS structural identification of two protein biomarkers, salivary α-amylase and lysozyme, were successively achieved directly from saliva at the femtomole level. For lysozyme, the on-chip MS identification was completed by a proteomic analysis based on an on-chip proteolysis procedure and a peptide mass fingerprint.
Keywords: SPR-MS; Biomolecular interaction analysis; Surface plasmon resonance; Mass spectrometry; Biological fluid; α-amylase; Lysozyme

A rapid microfluidic technique for integrated viability determination of adherent single cells by Shijun Xu; Anna Kim; Gavin D. M. Jeffries; Aldo Jesorka (1295-1301).
Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.
Keywords: Adherent single cells; Viability; Multifunctional Pipette; FDA and PI

Algal toxins can cause neurovirulence, hepatotoxicity, and cytotoxicity in humans through the consumption of contaminated water and food. In this work, we presented a novel aptasensor for the simultaneous detection of two algal toxins, microcysin-LR (MC-LR) and okadaic acid (OA). This system employed green and red upconversion nanoparticle (UCNP) luminescence as the donors and two quenchers (BHQ1 and BHQ3) as the corresponding acceptors. The two donor–acceptor couples were fabricated by hybridizing the aptamers with their corresponding complementary DNA. The results indicated that the green and red upconversion luminescence could be quenched by the quencher probes because of their highly overlapping spectrum. In the presence of MC-LR and OA, the aptamers preferred to bind to their corresponding analytes and de-hybridize with the complementary DNA. This effect became sufficiently large to prevent green and red luminescence quenching. Under the optimized experimental conditions, the relative luminescence intensity increased as the algal toxin concentrations increased, allowing for the quantification of MC-LR and OA. The relationships between the luminescence intensity and plotting logarithms of algal toxin concentrations were linear in the range from 0.1 to 50 ng mL−1 for MC-LR and OA. As a practical application, this type of dual fluorescence resonance energy transfer (FRET) aptasensor was used to monitor the MC-LR and OA levels in naturally contaminated food samples such as fish and shrimps. Graphical Abstract Schematic illustration of dual FRET aptasensor between the upconversion nanoparticles and quenching agent for the simultaneous detection of MC-LR and OA
Keywords: Upconversion nanoparticles; Microcysin-LR; Okadaic acid; Aptamer; Simultaneous detection

Rapid on-site detection of ephedrine and its analogues used as adulterants in slimming dietary supplements by TLC-SERS by Diya Lv; Yan Cao; Ziyang Lou; Shujin Li; Xiaofei Chen; Yifeng Chai; Feng Lu (1313-1325).
Ephedrine and its analogues are in the list of prohibited substance in adulteration to botanical dietary supplements (BDS) for their uncontrollable stimulating side effects. However, they were always adulterated illegally in BDS to promote losing weight. In order to avoid detection, various kinds of ephedrine analogues were added rather than ephedrine itself. This has brought about great difficulties in authentication of BDS. In this study, we put forward for the first time a method which combined thin-layer chromatography (TLC) and surface-enhanced Raman scattering (SERS) to directly identify trace adulterant. Ephedrine, pseudoephedrine, methylephedrine, and norephedrine were mixed and used in this method to develop an analytical model. As a result, the four analogues were separated efficiently in TLC analysis, and trace-components and low-background SERS detection was realized. The limit of detection (LOD) of the four analogues was 0.01 mg/mL. Eight common Raman peaks (△υ = 620, 1003, 1030, 1159, 1181, 1205, 1454, 1603 cm−1) were extracted experimentally and statistically to characterize the common feature of ephedrine analogues. A TLC-SERS method coupled with common-peak model was adopted to examine nine practical samples, two of which were found to be adulterated with ephedrine analogues. Identification results were then confirmed by UPLC-QTOF/MS analysis. The proposed method was simple, rapid, and accurate and can also be employed to trace adulterant identification even when there are no available reference derivatives on-site or unknown types of ephedrine analogues are adulterated. Graphical Abstract Common and characteristic peaks to identify trace amounts of EP, PSE, NEP, MEP adulterated in BDS by TLC-SERS
Keywords: Thin-layer chromatography; Surface-enhanced Raman spectroscopy; Ephedrine; Analogues; Adulteration; Botanical dietary supplements

This study describes the effect of ionic strength on the molecular structure of hyaluronic acid (HA) in an aqueous solution using flow field-flow fractionation and multiangle light scattering (FlFFF-MALS). Sodium salts of HA (NaHA) raw materials (∼2 × 106 Da) dispersed in different concentrations of NaCl prepared by repeated dilution/ultrafiltration procedures were examined in order to study conformational changes in terms of the relationship between the radius of gyration and molecular weight (MW) and molecular weight distribution (MWD) of NaHA in solution. This was achieved by varying the ionic strength of the carrier solution used in a frit-inlet asymmetrical FlFFF (FIAF4) channel. Experiments showed that the average MW of NaHA increased as the ionic strength of the NaHA solution decreased due to enhanced entanglement or aggregation of HA molecules. Relatively large molecules (greater than ∼5 MDa) did not show a large increase in RMS radius value as the NaCl concentration decreased. Conversely, smaller species showed larger changes, suggesting molecular expansion at lower ionic strengths. When the ionic strength of the FlFFF carrier solution was decreased, the HA species in a salt-rich solution (0.2 M NaCl) underwent rapid molecular aggregation during FlFFF separation. However, when salt-depleted HA samples (I = 4.66∼0.38 mM) were analyzed with FFF carrier solutions of a high ionic strength, the changes in both molecular structure and size were somewhat reversible, although there was a delay in correction of the molecular structure.
Keywords: Sodium hyaluronate; Flow field-flow fractionation; Multiangle light scattering; FlFFF-MALS; Ionic strength effect

The minimizing of fluorescence background in Raman optical activity and Raman spectra of human blood plasma by Michal Tatarkovič; Alla Synytsya; Lucie Šťovíčková; Bohuš Bunganič; Michaela Miškovičová; Luboš Petruželka; Vladimír Setnička (1335-1342).
Raman optical activity (ROA) is inherently sensitive to the secondary structure of biomolecules, which makes it a method of interest for finding new approaches to clinical applications based on blood plasma analysis, for instance the diagnostics of several protein-misfolding diseases. Unfortunately, real blood plasma exhibits strong background fluorescence when excited at 532 nm; hence, measuring the ROA spectra appears to be impossible. Therefore, we established a suitable method using a combination of kinetic quenchers, filtering, photobleaching, and a mathematical correction of residual fluorescence. Our method reduced the background fluorescence approximately by 90 %, which allowed speedup for each measurement by an average of 50 %. In addition, the signal-to-noise ratio was significantly increased, while the baseline distortion remained low. We assume that our method is suitable for the investigation of human blood plasma by ROA and may lead to the development of a new tool for clinical diagnostics. Graphical abstract The effect of a newly developed fluorescence quenching method for the ROA measurements of human blood plasma
Keywords: Human blood plasma; Raman optical activity; Protein; Fluorescence; Spectroscopy

Tyrosine phosphorylation is an important regulator of signaling in cellular pathways, and dysregulated tyrosine phosphorylation causes several diseases. Mass spectrometry has revealed the importance of global phosphoproteomic characterization. Analysis of tyrosine phosphorylation by studying the mass-spectrometry (MS)-determined phosphoproteome remains difficult because of the relatively low abundance of tyrosine phosphoproteins. To effectively evaluate tyrosine-phosphopeptide enrichment and reduce ion suppression from non-phosphorylated peptides in MS analysis, three trypsin-digested BSA peptides and 14 standard phosphopeptides, including six tyrosine phosphopeptides, four serine phosphopeptides, and four threonine phosphopeptides, were subjected to titanium dioxide immunoaffinity-based enrichment and also to combined enrichment using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography–mass spectrometry (LC–MS) analyses. The enrichment factors were evaluated to determine the efficiency of each enrichment procedure. Comparison of five optimized enrichment methods, including TiO2-based immunoaffinity purification in Tris and MOPS buffer systems, TiO2–immunoaffinity enrichment, and immunoaffinity–TiO2 enrichment for total tyrosine, serine and threonine phosphopeptides, revealed that the order of the enrichment factors for total tyrosine phosphopeptides is: (i) immunoaffinity–TiO2 (enrichment factor = 38,244), (ii) TiO2–immunoaffinity (enrichment factor = 24,987), (iii) TiO2 micro-column (enrichment factor = 10,305), (iv) immunoaffinity in Tris buffer system (enrichment factor = 1450), and (v) immunoaffinity in the MOPS buffer system (enrichment factor = 32). These results reveal that an alternative enrichment scheme before use of a TiO2 micro-column, using immunoaffinity 4G10 and PY99 antibody enrichment under optimized conditions, can provide greater selectivity for tyrosine-phosphopeptide enrichment.
Keywords: Mass spectrometry; Phosphoproteome; Tyrosine phosphopeptide; Enrichment factor

An unprecedented stable isotope dilution assay for the genotoxic altertoxins along with exposure data of consumers is presented to enable a first risk assessment of these Alternaria toxins in foods. Altertoxins were produced as the most abundant Alternaria toxins in a modified Czapek–Dox medium with a low level of glucose as the carbon source and ammonium sulfate as the sole nitrogen source. Labeled altertoxins were synthesized in the same way using [13C6]glucose. Moreover, labeled alternariol, alternariol methyl ether, altenuene, and alternuisol were biosynthesized in another modified medium containing [13C6]glucose and sodium [13C2]acetate. A stable isotope dilution LC–MS/MS method was developed and used for food analysis. For altertoxin I, altertoxin II, alterperylenol, alternariol, and alternariol methyl ether, the limits of detection ranged from 0.09 to 0.53 μg kg−1. The inter-/intra-day (n = 3 × 6) relative standard deviations of the method were below 13 %, and the recoveries ranged between 96 and 109 %. Among the various commercial food samples, some of the organic whole grains revealed low-level contamination with altertoxin I and alterperylenol, and paprika powder, which was heavily loaded with alternariol, alternariol methyl ether, and tentoxin, showed higher contamination level of altertoxin I and alterperylenol. Altertoxin II and III and stemphyltoxin III were not detectable. In addition, if the food was contaminated with altertoxins, it was likely to be co-contaminated with the other Alternaria toxins, but not necessarily vice versa. Maximum concentrations of altertoxin I and alterperylenol were detected in sorghum feed samples containing 43 and 58 μg kg−1, respectively. This was significantly higher than that in the measured food samples.
Keywords: Altertoxin; Alternariol; Alternariol methyl ether; Alternaria ; Mycotoxin; Stable isotope dilution assay

5-APB (5-(2-aminopropyl)benzofuran) and its N-methyl derivative 5-MAPB (N-methyl-5-(2-aminopropyl)benzofuran) are analogues of amphetamine and methamphetamine, respectively, and belong to the so-called novel psychoactive substances (NPS). They were consumed as stimulants or entactogens with euphoric and empathogenic effects. Being controlled in some countries, both compounds should be covered by drug testing in clinical and forensic toxicology. Therefore, metabolism studies have been performed by working up rat urine samples after a high single dose of the corresponding NPS with solid-phase extraction without and after enzymatic conjugates cleavage. The phase I metabolites were separated and identified after acetylation by GC-MS and/or LC-HR-MSn and the phase II metabolites by LC-HR-MSn. The main metabolite of 5-APB was 3-carboxymethyl-4-hydroxy amphetamine and the main metabolites of 5-MAPB were 5-APB (N-demethyl metabolite) and 3-carboxymethyl-4-hydroxy methamphetamine. The cytochrome P450 (CYP) isoenzymes involved in the 5-MAPB N-demethylation were CYP1A2, CYP2B6, CYP2C19, and CYP2D6, and according to the kinetic parameters, CYP2B6 was responsible for the main part of the total CYP-dependent clearance. An intake of a common users’ dose of 5-APB or 5-MAPB could be confirmed in rat urine using the authors’ GC-MS and the LC-MSn standard urine screening approaches with the corresponding parent drugs as major target. In authentic human urine samples after ingestion of unknown doses of 5-MAPB, both metabolites could also be detected besides the parent drug. The plasma concentrations determined in six clinical cases ranged from 5 to 124 μg/L for 5-MAPB and from 1 to 38 μg/L for its N-demethyl metabolite 5-APB. Graphical abstract Contribution of the given CYPs to the 5-MAPB N-demethylation
Keywords: 5-APB; 5-MAPB; Metabolism; Plasma concentration; GC-MS; LC-(HR)-MSn

In this study, unsupervised and supervised classification methods were compared for comprehensive analysis of the fingerprints of 26 Phyllanthus samples from different geographical regions and species. A total of 63 compounds were identified and tentatively assigned structures for the establishment of fingerprints using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC/TOFMS). Unsupervised and supervised pattern recognition technologies including principal component analysis (PCA), nearest neighbors algorithm (NN), partial least squares discriminant analysis (PLS-DA), and artificial neural network (ANN) were employed. Results showed that Phyllanthus could be correctly classified according to their geographical locations and species through ANN and PLS-DA. Important variables for clusters discrimination were also identified by PCA. Although unsupervised and supervised pattern recognitions have their own disadvantage and application scope, they are effective and reliable for studying fingerprints of traditional Chinese medicines (TCM). These two technologies are complementary and can be superimposed. Our study is the first holistic comparison of supervised and unsupervised pattern recognition technologies in the TCM chemical fingerprinting. They showed advantages in sample classification and data mining, respectively.
Keywords: Phyllanthus ; Unsupervised; Supervised; Pattern recognition; High-performance liquid chromatography time-of-flight mass spectrometry

Oxylipins are potent lipid mediators. For the evaluation of their biological roles, several LC-MS based methods have been developed. While these methods are similar, the described sample preparation procedures for the extraction of oxylipins differ considerably. In order to deduce the most appropriate method for the analysis of non-esterified oxylipins in human plasma, we evaluated the performance of seven established sample preparation procedures. Six commonly used solid phase extraction (SPE) and one liquid-liquid extraction (LLE) protocol were compared based on the recovery of 13 added internal standards, extraction efficacy of oxylipins from plasma and reduction of ion-suppressing matrix. Dramatic differences in the performance in all three parameters were found. LLE with ethyl acetate was overall not a sufficient sample preparation strategy. The protocols using Oasis- and StrataX-material insufficiently removed interfering matrix compounds. Extraction efficacy of oxylipins on anion-exchanging BondElut cartridges was low, while removal of matrix was nearly perfect. None of the protocols led to a high extraction efficacy of analytes while removing all interfering matrix components. However, SPE on a C18-material with removal of matrix by water and n-hexane prior elution with methyl formate showed the best performance for the analysis of a broad spectrum of oxylipins in plasma. Graphical Abstract TOC art: Scheme of oxylipin extraction from plasma and ion suppression analysis of PGE2
Keywords: Eicosanoids; Lipidomics; Liquid-liquid extraction; Solid phase extraction; Oxylipins; LC-MS

Py-GC/MS applied to the analysis of synthetic organic pigments: characterization and identification in paint samples by Elisa Ghelardi; Ilaria Degano; Maria Perla Colombini; Joy Mazurek; Michael Schilling; Tom Learner (1415-1431).
A collection of 76 synthetic organic pigments was analysed using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS). The purpose of this work was to expand the knowledge on synthetic pigments and to assess characteristic pyrolysis products that could help in the identification of these pigments in paint samples. We analysed several classes of synthetic pigments not previously reported as being analysed by this technique: some metal complexes, β-naphthol pigment lakes, BONA pigment lakes, disazopyrazolone, triarylcarbonium, dioxazine, anthraquinone, indanthrone, isoindoline and thioindigo classes. We also report for the first time the Py-GC/MS analysis of a number of naphthol AS, benzimidazolone, phthalocyanine and perylene pigments and other miscellaneous pigments including pigments with unpublished chemical structure. We successfully used the Py-GC/MS technique for the analysis of paints by artists Clyfford Still and Jackson Pollock to identify the synthetic organic pigments and the binding media. Graphical Abstract Pyrogram of PR49, with fragments produced by pyrolysis
Keywords: Py-GC/MS; Synthetic organic pigments; Clyfford Still; Jackson Pollock; Contemporary art; Mass spectrometry

Analytical markers for silk degradation: comparing historic silk and silk artificially aged in different environments by Francisco Vilaplana; Johanna Nilsson; Dorte V. P. Sommer; Sigbritt Karlsson (1433-1449).
Suitable analytical markers to assess the degree of degradation of historic silk textiles at molecular and macroscopic levels have been identified and compared with silk textiles aged artificially in different environments, namely (i) ultraviolet (UV) exposure, (ii) thermo-oxidation, (iii) controlled humidity and (iv) pH. The changes at the molecular level in the amino acid composition, the formation of oxidative moieties, crystallinity and molecular weight correlate well with the changes in the macroscopic properties such as brightness, pH and mechanical properties. These analytical markers are useful to understand the degradation mechanisms that silk textiles undergo under different degradation environments, involving oxidation processes, hydrolysis, chain scission and physical arrangements. Thermo-oxidation at high temperatures proves to be the accelerated ageing procedure producing silk samples that most resembled the degree of degradation of early seventeenth-century silk. These analytical markers will be valuable to support the textile conservation tasks currently being performed in museums to preserve our heritage.
Keywords: Silk; Conservation; Multivariate analysis; Amino acid composition; Infrared spectroscopy; Mechanical properties

Microcystins, anatoxins and okadaic acid are toxins produced by freshwater cyanobacteria and marine dinoflagellates. These toxins have been the responsible for the illness and death of biota and humans. To determine their presence in water during blooms, sensitive analytical methods are needed. In this study, we have developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method for fast multiresidue determination of five toxins in suspended material and sediment samples. For each target compound, two selected reaction monitoring (SRM) transitions were optimised. Chromatographic conditions were optimised considering that the compounds analysed had different chemical structure and chromatographic behaviour. Using a Luna C18 column and specific SRM transitions, five phytotoxins were resolved. Method detection limits (MDL) for anatoxin-a, microcystins RR, LR and YR and okadaic acid were 7.1, 3.3, 81.7, 102.8 and 28.8 ng g−1 dry weight in sediment, respectively. The developed analytical method was successfully applied to analyse the presence of toxins in suspended solids and sediment from Ebro River (NE Spain) and Ebro delta-associated lagoons. Anatoxin-a was detected downstream of the Riba-Roja reservoir with levels ranging from 20 to 1120 ng g−1 dry weight of suspended solids. Okadaic acid was only detected in three samples collected in the Alfacs Bay (Ebro delta, Spain) affected by Dinophysis blooms in 2012.
Keywords: Cyanotoxins; Okadaic acid; LC-MS/MS; Sediment; Ebro

Liquid chromatography–high-resolution mass spectrometry for palytoxins in mussels by Patrizia Ciminiello; Carmela Dell’Aversano; Emma Dello Iacovo; Martino Forino; Luciana Tartaglione (1463-1473).
Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC–HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14–1000 ng mL−1 in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC–HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 μg kg−1 in crude mussel extracts and 15 μg kg−1 after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this.
Keywords: Palytoxin; Putative palytoxin; Isobaric palytoxin; Ovatoxins; LC–HRMS; Mussels

Salmon is a popular food but it is easily susceptible to spoilage by contamination with microorganisms. In this study, a method using hydrophilic interaction chromatography (HILIC)-based solid-phase extraction (SPE) and matrix-assisted laser desorption and ionization time-of-flight/time-of-flight mass spectrometry was developed and applied to reveal the effect of Pseudomonas fluorescens on salmon fillet during the shelf-life period by measuring the changes in the levels of phosphatidylcholine and phosphatidylethanolamine. Fresh samples were inoculated with P. fluorescens (106 cfu g-1) for 30 s, and lipids were extracted at 0, 24, 48, and 72 h. A homemade SPE cartridge packed with HILIC sorbent (silica derivatized with 1,2-dihydroxypropane) was used for matrix cleanup prior to analysis by mass spectrometry. In total, 30 phospholipids and 16 lysophospholipids were detected and elucidated. The results revealed that the content of phospholipids decreased significantly, whereas that of lysophospholipids increased initially, followed by a gradual reduction as the cold storage time increased. The contamination by P. fluorescens negatively affected the quality of fresh salmon without obvious physical changes, but it posed a potential threat to human health. This study suggests that the well-established method could be used for detecting phospholipids in salmon fillet and perhaps other foods as well. Graphical Abstract Freshness of salmon on sale in supermarket could be monitored by means of MALDI-TOF/MS
Keywords: Hydrophilic interaction chromatography; Lipidomics; Salmon; Pseudomonas fluorescens ; Shelf life; Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry

Analysis of polar triazines and degradation products in waters by in-tube solid-phase microextraction and capillary chromatography: an environmentally friendly method by Yolanda Moliner-Martínez; Pascual Serra-Mora; Jorge Verdú-Andrés; Rosa Herráez-Hernández; Pilar Campíns-Falcó (1485-1497).
This paper describes a new method for the determination of polar triazines, including some degradation products, which combines online in-tube solid-phase microextraction (IT-SPME) and capillary liquid chromatography with UV-diode array detection (DAD). Different extractive coatings have been evaluated for IT-SPME, a capillary column with a polydimethylsiloxane (PDMS) coating, and the same coating modified with carboxylated single-wall carbon nanotubes (c-SWCNTs) and carboxylated multiwall carbon nanotubes (c-MWCNTs). On the basis of the results obtained, a new method is presented for the identification and determination of triazines in water samples. A careful selection of the eluent composition provides the required selectivity and sensitivity for the quantification of the target analytes, even those highly polar (log K ow ≤ 2.3). The proposed conditions have been successfully used for the quantitation of the analytes in the 0.25–50.0 μg L−1 range. The limits of detection (LODs) are in the 0.02–0.1 μg L−1 range, and the intraday and interday relative standard deviation (RSD) coefficients are ≤9 and ≤17 %, respectively. The reliability of the described method has been tested by analyzing several real water samples. The proposed method can be considered an environmentally friendly and cost-effective alternative for routine monitoring of triazines and their degradation products in waters. Graphical Abstract Schematic diagram of the system used for the analysis of triazines
Keywords: Triazines; In-tube solid-phase microextraction (IT-SPME); Capillary chromatography; Water analysis; Single-wall carbon nanotubes (SWCNTs); Multiwall carbon nanotubes (MWCNTs)

A rapid and highly sensitive microchip electrophoresis of mono- and mucin-type oligosaccharides labeled with 7-amino-4-methylcoumarin by Sachio Yamamoto; Eriko Nagai; Yuki Asada; Mitsuhiro Kinoshita; Shigeo Suzuki (1499-1503).
A selective separation method using a poly(methylmethacrylate) microchip was developed for 7-amino-4-methylcoumarin-labeled saccharides in a crude reaction mixture. In an alkaline borate buffer, saccharide derivatives formed strong anionic borate complexes. These complexes moved from the cathode to the anode in an electric field and were detected near the anode. Excess labeling reagents and other foreign substances remained at the inlet reservoir. A confocal fluorimetric detection system enabled the determination of monosaccharide derivatives with good linearity between at least 5 and 100 nM, corresponding to 50 fmol to 1 pmol per injection. The lower limit of detection (signal-to-noise = 5) was 2 nM. The sensitivity and linear quantitation range were comparable to reported values using fluorometric detection, capillary electrophoresis, or liquid chromatography. Application of the method showed excellent resolution in the analysis of O-linked glycans chemically released from glycoproteins.
Keywords: Microchip electrophoresis; 7-Amino-4-methylcoumarin; Borate complex; Monosaccharide analysis; O-linked glycan

The method for the determination of acesulfame-K, saccharine, cyclamate, aspartame, sucralose, alitame, neohesperidin dihydrochalcone, neotame and five common steviol glycosides (rebaudioside A, rebaudioside C, steviol, steviolbioside and stevioside) in soft and alcoholic beverages was developed using high-performance liquid chromatography and tandem mass spectrometry with electrospray ionisation (HPLC-ESI-MS/MS). To the best of our knowledge, this is the first work that presents an HPLC-ESI-MS/MS method which allows for the simultaneous determination of all EU-authorised high-potency sweeteners (thaumatin being the only exception) in one analytical run. The minimalistic sample preparation procedure consisted of only two operations; dilution and centrifugation. Linearity, limits of detection and quantitation, repeatability, and trueness of the method were evaluated. The obtained recoveries at three tested concentration levels varied from 97.0 to 105.7 %, with relative standard deviations lower than 4.1 %. The proposed method was successfully applied for the determination of sweeteners in 24 samples of different soft and alcoholic drinks.
Keywords: Artificial sweeteners; Steviol glycosides; Stevia rebaudiana ; Tandem mass spectrometry; Liquid chromatography

Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library by Mohamed Amine Ben Mlouka; Arbia Khemiri; Damien Seyer; Julie Hardouin; Philippe Chan Tchi Song; Emmanuelle Dé; Thierry Jouenne; Pascal Cosette (1513-1518).
Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample.
Keywords: Pseudomonas aeruginosa ; Outer membrane proteins; Protein normalization; Combinatorial peptide ligand library

Agar films containing silver nanoparticles as new supports for electromembrane extraction by Cristina Román Hidalgo; María Ramos-Payán; Juan Antonio Ocaña-González; María Jesús Martín-Valero; Miguel Ángel Bello-López (1519-1525).
A new support containing silver nanoparticles to assist electromembrane extraction (EME) procedures is proposed. For the first time, synthesized agar films containing silver nanoparticles (AgNPs) have been used as a support for liquid membranes in EME. Agarose films of 20 μm thickness containing 107.9 mg Ag/g agar were synthesized and characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), showing isolated spherical silver nanoparticles of 20–30 nm diameter with homogeneous distribution. Nanometallic films were cut and glued to narrow bore glass tubes and used as supports for a dihexyl ether liquid membrane for use in an EME procedure. EME conditions were optimized and applied to the extraction of selected non-steroidal anti-inflammatory drugs (NSAIDs). The results were compared to those using polypropylene membranes (450 μm and 100 μm thickness), achieving 10- to 70-fold higher extraction efficiency. This article opens a new line of research into electrically assisted microextraction systems by combining other possible kinds of nanometallic films, including different metals, film functionalization through metallic NPs, and the use of low polarity solvents. Also, very low currents are obtained during the extraction process, which lead to high electromigration of the analytes. Graphical Abstract Agar films-silver nanoparticles for electromembrane extraction
Keywords: Silver nanoparticles; Electromembrane extraction; Agar-nanometallic films; Non-steroidal anti-inflammatory drugs