Analytical and Bioanalytical Chemistry (v.399, #3)

News from ABC: changes and challenges by Steffen Pauly (1003-1004).
is Senior Editor, Chemistry, for Springer and now also Managing Editor of Analytical and Bioanalytical Chemistry. He has been active in scientific publishing since 1992, particularly in analytical science. He received his PhD from the Technische Universität Berlin in 1991 after studying Chemistry in Göttingen and Berlin.

Jiří Janata: Principles of chemical sensors, 2nd ed. by María Cruz Moreno-Bondi (1005-1006).

Characterisation of workplace aerosols in the manganese alloy production industry by electron microscopy by Kjersti Gjønnes; Asbjørn Skogstad; Siri Hetland; Dag G. Ellingsen; Yngvar Thomassen; Stephan Weinbruch (1011-1020).
Workplace aerosols in a combined FeMn and SiMn alloy smelter were studied by scanning and transmission electron microscopy. Special emphasis was placed on the characterisation of individual particles with diameters below 500 nm and on identification of the different manganese phases present in the workroom air. In high-carbon FeMn production, the submicron size fraction is dominated by MnO particles forming chain-like or compact agglomerates. Minor amounts of MnO2, Mn3O4, Mn2O3 and Fe3O4 are also observed. During production of SiMn, the submicron size fraction consists predominantly of MnSi particles, but small amounts of Mn3Si, Mn6Si and Mn5Si2 are also found. Workplace aerosols from the manganese oxide refinement (MOR) process consist mostly of Mn oxides. Minor amounts of carbonaceous particles occurring as sheets, ribbons and as hollow carbon structures are observed along the whole production line. Carbonaceous particles are either amorphous or consist of poorly crystallised graphite. Particles with fibre morphology were encountered at all sampling locations but most prominently during tapping of FeMn with fibre concentrations between 0.1 and 0.7 per cm3. The pronounced differences in particle composition along the production line clearly show that workers are exposed to a variety of Mn-containing species. MnO particles have a higher solubility than MnSi particles and are thus more bioaccessible, suggesting a higher risk of adverse health effects in the FeMn production than in the SiMn production. Figure TEM bright field image shows chain-like agglomerates of SiMn primary nanoparticles found in workroom air during casting of SiMn alloy
Keywords: Workplace; Aerosol; Manganese; Electron microscopy; Speciation

Multifunctional nanoparticles as simulants for a gravimetric immunoassay by Scott A. Miller; Leslie A. Hiatt; Robert G. Keil; David W. Wright; David E. Cliffel (1021-1029).
Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and 1H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.
Keywords: Immunosensor; Nanoparticle; Quartz crystal microbalance; Viral simulant; ELISA; Influenza simulant; FLAG; Hemagglutinin; Epitope; PEG linkage; H5N1

Applicability of a yeast bioassay in the detection of steroid esters in hair by Ilse Becue; Toine F. H. Bovee; Christof Van Poucke; Maria J. Groot; Michel W. F. Nielen; Carlos Van Peteghem (1031-1039).
The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.
Keywords: Testosterone ester; Estradiol benzoate; Yeast bioassay; Untargeted analysis; Hair

Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4 × 105 cells mL−1. By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.
Keywords: Biochips/high-throughput screening; Bioanalytical methods; Flow injection; Fluorescence/luminescence; Water; Immunoassays/ELISA

Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment by Johannes Thal; Monika Steffen; Bianca Meier; Elisabeth Schneider; Ansgar Adriany; Ewald Usleber (1051-1059).
The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml−1, recoveries were 80–128% at 4–40 ng ml−1. Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ≈ 20 ng ml−1) and a receptor assay (ßeta-s.t.a.r., detection limit ≈ 15 ng ml−1) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10–27 μg ml−1) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml−1). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 μg kg−1) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material. Figure Cefquinome residue levels in milk from intramammary treated udder quarters of cows suffering from clinical mastitis, after repeated intramammary (2 x per day) and intramuscular (1 x per day) dosing of cefquinome for 3 days.
Keywords: Beta-lactam; Antibody; Geobacillus stearothermophilus ; Mastitis; Food

A rapid and reversible colorimetric assay for the characterization of aminated solid surfaces by Gaëlle Coussot; Catherine Perrin; Thomas Moreau; Michel Dobrijevic; Aurélie Le Postollec; Odile Vandenabeele-Trambouze (1061-1069).
The covalent immobilization of synthetic or natural macromolecular compounds containing amino groups onto polystyrene (PS) solid surfaces is of great interest in diagnostic applications. A sensitive assay allowing the determination of reactive end groups is therefore a powerful tool for predicting the performance of the active surface. Recently, we reported the use of the Coomassie brilliant blue (CBB) colorimetric reagent to quantify protonated groups (N+) in linear and dendritic structures in solution (Coussot et al., Polym Int 58(5):511–518, 2009). In this work, a simple method using CBB dye for the characterization of PS aminated solid surfaces is developed. The proposed amino density estimation by colorimetric assay (ADECA) method is based on the reversible complexation of the dye with the N+ groups on solid surfaces. The assay measures the released dye thanks to the use of a unique sodium carbonate–methanol buffer. Thereby, for the first time, the same surface can be used for characterization and for further coupling applications. A surface density of four N+ groups per square nanometer can be measured in PS microwell format, the whole characterization being done within 30 min. Performances of this new colorimetric-based method are detailed. The ADECA method is further demonstrated to be useful for the characterization of aminated polypropylene and glass materials with various sizes and shapes. Figure “ADECA principle” Adeca priciple for the characterization of aminated surfaces
Keywords: Coomassie blue; Quantification; Amine density; Solid support characterization; ADECA

In-vitro oxidation of bisphenol A: Is bisphenol A catechol a suitable biomarker for human exposure to bisphenol A? by Xiaoyun Ye; Xiaoliu Zhou; Larry L. Needham; Antonia M. Calafat (1071-1079).
The extensive use of bisphenol A (BPA) in the manufacture of consumer products results in widespread human exposure to the chemical. In the body, BPA undergoes first-pass metabolism to form BPA glucuronide, considered to be a major BPA byproduct. Concentrations of total (free plus conjugated) urinary species of BPA are used to assess human exposure to BPA. However, because BPA can be present in numerous consumer and household products, potential contamination with parent BPA during collection and handling may pose a challenge when measuring BPA in such biological samples as blood or urine. In this study we investigated the in-vitro phase I metabolism of BPA in rat and human liver microsomes by using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry to identify phase I metabolites (e.g., BPA oxidation products) that could be used as potential alternative biomarkers of BPA exposure. We unambiguously identified 5-hydroxy BPA (BPA catechol) as an in-vitro oxidative metabolite of BPA, but human microsomes oxidized only about 10% of BPA to BPA catechol. We evaluated the usefulness of BPA catechol as a potential biomarker of human exposure to BPA by measuring total concentrations of BPA catechol and BPA in 20 urine samples. We detected BPA catechol at much lower concentrations and frequency than those of BPA. Furthermore, we found that free BPA catechol was rather unstable in urine, which highlights the importance of sampling techniques to adequate interpretation of biomonitoring data. Together, these findings suggest that BPA catechol may not be a suitable biomarker of environmental exposure to BPA, but could be used to confirm BPA exposure in special populations or in situations when urine specimens were potentially contaminated with BPA.
Keywords: Bisphenol A; Bisphenol A catechol; In-vitro metabolism; In-vivo metabolism; Exposure

An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions by Lennard J. M. Dekker; Lona Zeneyedpour; Eric Brouwer; Martijn M. van Duijn; Peter A. E. Sillevis Smitt; Theo M. Luider (1081-1091).
Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.
Keywords: Mass spectrometry; Antibodies; CDRs; de novo sequencing

Automated targeting analysis of eicosanoid inflammation biomarkers in human serum and in the exometabolome of stem cells by SPE–LC–MS/MS by Carlos Ferreiro-Vera; José María Mata-Granados; Feliciano Priego-Capote; José Manuel Quesada-Gómez; María Dolores Luque de Castro (1093-1103).
Inflammation is a complex cascade process involved in the pathogenesis of a number of diseases or generated as response to external or internal stimuli. Current research is focused on the development of assays for fast identification and quantitation of inflammation biomarkers. Eicosanoids are the oxidation metabolites of polyunsaturated fatty acids (mainly 20-carbon fatty acids) that play a regulation role in inflammation and, therefore, they have proved to be involved in different pathological states such as cancer, atherosclerosis, arthritis and cardiovascular or immunological diseases. Eicosanoids can be metabolized by different oxygenase enzymes to prostanoids such as prostaglandins and thromboxanes or hydroxyl fatty acids such as hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids. A high-throughput automated approach is here presented for direct eicosanoid analysis in biofluids such as human serum and cells culture media. The approach is based on a hyphenated system composed by a solid-phase extraction workstation (Prospekt 2 unit) on-line coupled to a liquid chromatograph–triple quadrupole-tandem mass spectrometer. The detection limits for the target analytes ranged from 0.009 to 204 pg on-column, with precision between 2.65% and 7.33%, expressed as relative standard deviation. Accuracy studies with a dual-cartridge configuration resulted in recoveries between 78.6% and 100%, which validated internally the proposed approach ensuring highly efficient cleanup of proteins and salts. The method is reliable, robust and endowed with a great potential for implementation in clinical and routine laboratories. Analysis of culture media of stem cells stimulated with arachidonic acid was carried out to evaluate its incidence on the eicosanoid profile of the exometabolome. Figure Main biochemical pathways involved in eicosanoids metabolism
Keywords: Eicosanoids; Prostaglandins; Hydroxyeicosatetraenoic acids; Hydroxyoctadecadienoic acids

Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry by Parisa Ansari; Norbert Stoppacher; Judith Rudolf; Rainer Schuhmacher; Sabine Baumgartner (1105-1115).
The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α- and β-casein) and two whey proteins (α-lactalbumin and β-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from β-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPFPEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from β-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%). Figure Possible marker peptides from four milk allergens for the detection of ruminant milk proteins
Keywords: Milk allergens; α-Casein; β-Casein; α-Lactalbumin; β-Lactoglobulin; Liquid chromatography-tandem mass spectrometry

Compact, cost-efficient microfluidics-based stopped-flow device by Regina Bleul; Marion Ritzi-Lehnert; Julian Höth; Nico Scharpfenecker; Ines Frese; Dominik Düchs; Sabine Brunklaus; Thomas E. Hansen-Hagge; Franz-Josef Meyer-Almes; Klaus S. Drese (1117-1125).
Stopped-flow technology is frequently used to monitor rapid (bio)chemical reactions with high temporal resolution, e.g., in dynamic investigations of enzyme reactions, protein interactions, or molecular transport mechanisms. However, conventional stopped-flow devices are often overly complex, voluminous, or costly. Moreover, excessive amounts of sample are often wasted owing to inefficient designs. To address these shortcomings, we propose a stopped-flow system based on microfluidic design principles. Our simple and cost-efficient approach offers distinct advantages over existing technology. In particular, the use of injection-molded disposable microfluidic chips minimizes required sample volumes and associated costs, simplifies handling, and prevents adverse cross-contamination effects. The cost of the system developed is reduced by an order of magnitude compared with the cost of commercial systems. The system contains a high-precision valve system for fluid control and features automated data acquisition capability with high temporal resolution. Analyses with two well-established reaction kinetics yielded a dead time of approximately 8-9 ms.
Keywords: Stopped flow; Microfluidics; Low sample amounts; Short dead time

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols by Katja Dettmer; Nadine Nürnberger; Hannelore Kaspar; Michael A. Gruber; Martin F. Almstetter; Peter J. Oefner (1127-1139).
Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid–base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid–base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.
Keywords: Metabolomics; GC-MS; Adherent cells; Detachment; Extraction; Amino acids

Simultaneous analysis of cardiac glycosides in blood and urine by thermoresponsive LC-MS-MS by Sanae Kanno; Kanako Watanabe; Itaru Yamagishi; Seishiro Hirano; Kayoko Minakata; Kunio Gonmori; Osamu Suzuki (1141-1149).
A new thermoresponsive polymer separation column was applied to simultaneous analysis of four cardiac glycosides (CGs) being widely used for the treatment of arrhythmias and heart failure in human blood and urine. This column is composed of an N-isopropylacrylamide polymer, the surface of which undergoes a reversible alteration from hydrophilic to hydrophobic by changing temperature. The chromatographic separation and retention times can be easily be controlled by adjusting the column temperature. As the column temperature was changed from 50 to 10 °C over 8 min, five CGs, including deslanoside, digoxin, methyldigoxin, digitoxin, and digitoxigenin (internal standard) were better resolved. Using these LC conditions, we analyzed four CGs in human whole blood and urine simultaneously by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Validation data as functions of recovery rates, linearity, accuracy, and precision were carefully tested; all were generally satisfactory. The detection limits for the four CGs in both matrices were 0.2–0.3 ng/mL. The method was applied to analysis of methyldigoxin and its main metabolite digoxin in whole blood and urine samples obtained from a deceased person in actual autopsy case. To our knowledge, this is the first report describing an LC-MS-MS method using a thermoresponsive column for analysis of drug(s). The inclusion of the thermoresponsive column in an LC-MS-MS technique seems to extend the possibility for simultaneous analysis of compounds of different properties, such as hydrophobic precursors and their hydrophilic metabolites in biological samples within limited analysis times.
Keywords: Poly(N-isopropylacrylamide); Cardiac glycosides; Thermoresponsive; LC-MS-MS; Digoxin; Methyldigoxin

Tyrosinase (TYR: EC was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at −0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds. Figure Comparison of peak currents of tyrosinase/AO-modified CF and tyrosinase-modified CFs
Keywords: Tyrosinase; Acridine orange; Flow biosensor; Carbon felt; Phenolic compounds; Signal enhancement

Static secondary ion mass spectrometry for the surface characterisation of individual nanofibres of polycaprolactone functionalised with an antibacterial additive by Pieter Van Royen; Bart Boschmans; Ana dos Santos; Etienne Schacht; Peter Dubruel; Ria Cornelissen; Linda Beenaerts; Luc Van Vaeck (1163-1172).
Electrospinning (ES) of polymer solutions generates non-woven webs of nanofibres. The fibre diameter ranges between 10 nm and 1 μm depending on the operating conditions. Surface functionalisation can be performed by the use of suitable additives. Detailed characterisation of the molecular composition at the fibre surface is a key issue. Biodegradable nanowebs with potential antibacterial activity have been prepared by ES of solutions containing polycaprolactone (PCL) and a functionalising additive with PCL segments and hexyldimethylammonium groups (PCLhexaq). Static secondary ion mass spectrometry with Bi 3 + projectiles has been applied to individual nanofibres. The positive ion mass spectra contain several signals with high structural specificity allowing the presence of PCLhexaq to be traced back in spite of its low concentration (0.16–1.4% w/w relative to PCL) and its structural similarity to the PCL fibre matrix. Imaging of structural ions visualises the homogeneous distribution of PCLhexaq over the fibre surface. Quantifying the surface concentration of PCLhexaq relative to that of PCL reveals electric field-driven surface enrichment of the additive during ES. Finally, nanofibres subjected to leaching in water for up to 72 h have been analysed. The PCLhexaq surface concentration decreases almost linearly with time at a rate of 0.6% h−1. Figure Electrospinning is used to produce aligned nanofibres of polycaprolactone with functionalizing additives. Advanced S-SIMS is used for identification, quantification and imaging of the distribution of the additive and for the determination of the leaching kinetics
Keywords: Static secondary ion mass spectrometry; Nanofibres; Electrospinning; Polycaprolactone; Biomaterials; Leaching

In this study, a new type of localized surface plasmon resonance (LSPR) sensing substrate for phosphopeptides was explored. It has been known that LSPR response for target species is larger in the near-infrared region (NIR) than in the visible region of the electromagnetic spectrum. Several types of noble metal nanoparticles (NPs) with NIR absorption capacities have been previously demonstrated as effective LSPR-sensing nanoprobes. Herein, we demonstrate a straightforward approach with improved sensitivity by simply using layer-by-layer (LBL) spherical Au NPs self-assembled on glass slides as the LSPR-sensing substrates that are responsive in the NIR region of the electromagnetic spectrum. The modified glass slide acquired an LSPR absorption band in the NIR, which resulted from the dipole–dipole interactions between Au NPs. To enable the chip to sense phosphopeptides, the surface of the glass chip was spin-coated with thin titania film (TiO2-Glass@Au NPs). Absorption spectrophotometry was employed as a detection tool. Tryptic digest of α-casein was used as a model sample. The feasibility of using the new LSPR approach for detecting a potential risk factor leading to cancers (i.e., phosphorylated fibrinopeptide A) directly from human serum samples was demonstrated. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to confirm the results. Figure A label free LSPR sensing of phosphopeptides using the titania coated LBL spherical Au NPs coated glass slide (TiO2-Glass@Au NPs) as the sensing chip is demonstrated in this work
Keywords: Gold nanoparticles; LSPR; Phosphopeptides; Label-free sensing

Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection by Angelo Zinellu; Salvatore Sotgia; Valentina De Murtas; Paolo Cossu-Rocca; Maria Rosaria De Miglio; Maria Rosaria Muroni; Antonica Mura; Maria Gabriela Uras; Marcella Contini; Luca Deiana; Ciriaco Carru (1181-1186).
Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5–1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.
Keywords: Field-amplified sample injection; Capillary electrophoresis; 5-Methylcytosine; Cytosine; DNA methylation

The full elucidation of the enzymatic mechanisms leading to polyunsaturated ω-3 to ω-5 fatty acids (PUFAs) occurring in plants or microorganisms by analyzing their site-specific isotopic fractionation profiles is a challenging task. Isotropic SNIF-NMR® method is an historical, powerful tool for the determination of (2H/1H) ratios. However, the absence of accessible isotopic data on the enantiotopic hydrogen sites (CH2 groups) prevents the study of the enzymatic reaction stereoselectivity. Natural-abundance deuterium (NAD) 2D NMR experiment using chiral liquid crystals (CLC) as solvent is a new tool in this field, overcoming this limitation. In this work, we have explored various possibilities for optimizing the enantio-discrimination properties of CLC by changing the nature of the polypeptide and/or increasing the polarity of the organic co-solvents. We report also the first applications of TMU as co-solvent for preparing enantio-discriminating, homogenous polypeptide mesophases. The various experimental NAD NMR results recorded at an optimal sample temperature are discussed and compared in terms of number of discriminated 2H sites and magnitude of spectral separation for different PUFAs such as the linoleic and linolenic acids. The comparison of all NMR results shows that optimal results are obtained when CLC mixtures made of poly-γ-benzyl-l-glutamate (PBLG) and high polarity co-solvents are used. As new challenging examples of applications, we report the preliminary analytical results obtained from two ω-5 conjugated linolenic acids: the α-eleostearic acid (9Z, 11E, 13E) and the punicic acid (9Z, 11E, 13Z). NMR data are discussed in terms of molecular orientational ordering parameters and isotopic distribution. Figure The new challenges of the site-specific isotopic fractionation analysis of fatty acids using the natural-abundance deuterium NMR in polypeptide aligned media
Keywords: Polyunsaturated fatty acids; Site-specific isotopic fractionation; 2H NMR; Quadrupolar splittings; PBLG; Chirality; Ordering behavior

Thermal dependence of Raman descriptors of ceramides. Part II: effect of chains lengths and head group structures by Emmanuelle Guillard; Ali Tfayli; Michel Manfait; Arlette Baillet-Guffroy (1201-1213).
The outermost layer of the mammalian skin, the stratum corneum (SC), represents the main skin barrier. The SC lipids have a very exceptional composition, as the main lipid classes are ceramides (CER), long-chain fatty acids and cholesterol. Information on the function of each CER subclass and on the relation between CER lipid organisation and composition is of great importance to unravel the mechanism controlling the skin barrier function. Raman spectroscopy has been increasingly used for the study of intra- and inter-molecular structures of long-chain lipid compounds. In this study, we employed Raman spectroscopy to evaluate the effect of (1) the chain length and (2) the polar head architecture on the conformational order and organisational behaviour of CERs. The relation between the structure and the stability of the organisation was studied by monitoring the thermotropic response of each CER in the temperature range between 25 and 95 °C. This work enabled the determination of a correlation between the gauche/trans ratio in the νCC region and the state of the lateral packing. Moreover, it was shown that –OH groups in the α position of the fatty acids reduce the stability while long alkyl chains reinforces the intra- and inter-chains order.
Keywords: Ceramides; Stratum corneum; Raman spectroscopy; Conformational order; Lateral packing; Head group structures; Chain length

Sensitive and selective fluorescence detection of guanosine nucleotides by nanoparticles conjugated with a naphthyridine receptor by Piotr J. Cywinski; Artur J. Moro; Thomas Ritschel; Niko Hildebrandt; Hans-Gerd Löhmannsröben (1215-1222).
Novel fluorescent nanosensors, based on a naphthyridine receptor, have been developed for the detection of guanosine nucleotides, and both their sensitivity and selectivity to various nucleotides were evaluated. The nanosensors were constructed from polystyrene nanoparticles functionalized by (N-(7-((3-aminophenyl)ethynyl)-1,8-naphthyridin-2-yl)acetamide) via carbodiimide ester activation. We show that this naphthyridine nanosensor binds guanosine nucleotides preferentially over adenine, cytosine, and thymidine nucleotides. Upon interaction with nucleotides, the fluorescence of the nanosensor is gradually quenched yielding Stern–Volmer constants in the range of 2.1 to 35.9 mM−1. For all the studied quenchers, limits of detection (LOD) and tolerance levels for the nanosensors were also determined. The lowest (3σ) LOD was found for guanosine 3’,5’-cyclic monophosphate (cGMP) and it was as low as 150 ng/ml. In addition, we demonstrated that the spatial arrangement of bound analytes on the nanosensors’ surfaces is what is responsible for their selectivity to different guanosine nucleotides. We found a correlation between the changes of the fluorescence signal and the number of phosphate groups of a nucleotide. Results of molecular modeling and ζ-potential measurements confirm that the arrangement of analytes on the surface provides for the selectivity of the nanosensors. These fluorescent nanosensors have the potential to be applied in multi-analyte, array-based detection platforms, as well as in multiplexed microfluidic systems. Figure Naphthyridine_receptor
Keywords: Naphthyridine receptor; cGMP; Base pairing; Nucleotide nanosensor; Fluorescence spectroscopy

Ligand fishing with functionalized magnetic nanoparticles coupled with mass spectrometry for herbal medicine analysis by Lin-Sen Qing; Ying Xue; Wen-Long Deng; Xun Liao; Xue-Min Xu; Bo-Gang Li; Yi-Ming Liu (1223-1231).
The chemical composition of herbal medicines is very complex, and their therapeutic effects are determined by multi-components with sophisticated synergistic and/or suppressive actions. Therefore, quality control of herbal medicines has been a formidable challenge. In this work, we describe a fast analytical method that can be used for quality assessment of herbal medicines. The method is based on ligand fishing using human-serum-albumin-functionalized magnetic nanoparticles (HSA-MNPs) and mass spectrometry. To demonstrate the applicability of the proposed method, eight samples of Dioscorea panthaica were analyzed. The sampled plants were of both wild and cultivated origins. They grew at different geographical locations and were harvested at different times. The ligands bound to HSA-MNPs were isolated from the plant extracts and detected by using direct infusion electrospray ionization mass spectrometry (DI–ESI–MS). Chemical identity has been confirmed for five of the ligands isolated. From more than 15 peaks in the ESI–MS spectrum, 11 common peaks were selected for calculating the correlation coefficient and cosine ratio. The values of correlation coefficient and cosine ratio were >0.9824 and >0.9988, respectively, for all the samples tested. The results indicated a high level of similarity among the eight D. panthaica samples. Compared with chromatographic fingerprint analysis, the proposed HSA-MNP-based DI–ESI–MS/MS approach was not only fast and easy to carry out but also biological-activity-oriented, promising a more effective data interpretation and thus reliable assessment conclusions.
Keywords: Magnetic nanoparticles; Ligand fishing; ESI–MS/MS; Quality assessment of herbal medicines; Dioscorea panthaica

A chemo-electro-mechanical model is presented in this paper for transient simulation of the characteristics of the kinetic ionic-strength-sensitive hydrogel. It is termed the multi-effect-coupling ionic-strength-stimulus (MECis) model and it couples the chemical and electrical as well as mechanical effects together to predict responsive characteristics of the smart hydrogel to the ionic-strength stimulus of an environmental solution based on the laws of conservation of mass and momentum. The kinetic deforming characteristics simulated by the MECis model are compared with the experiments and achieve a good agreement. Then, the influence of the initial fixed charge density, as a material property of the hydrogel, on the kinetics of the ionic-strength-sensitive hydrogel is transiently analyzed, providing a deep view of the kinetics performance of the smart hydrogel. Figure Comparison of the kinetics swelling of the ionic-strength sensitive hydrogel between the MECis simulation results and experimental data
Keywords: Fixed charges; Ionic-strength-sensitive hydrogel; Transient behavior; Modeling

A European interlaboratory collaborative study was conducted to validate a method for the quantitative determination of lipophilic marine biotoxins based on high-performance liquid chromatography–tandem mass spectrometry. During this study, the diarrhetic shellfish poisoning toxins okadaic acid, dinophysis toxin1 and 2 including their esters, the azaspiracids 1-3, pectenotoxin2, and the yessotoxins were investigated at concentration levels near the limit of quantification and near the legal limit. Naturally contaminated blue mussels, both raw and cooked and spiked extracts of clams and oysters were studied and results were obtained for 16 test samples from 16 laboratories representing eight different countries. This article summarizes the study outcome concerning validation key parameters like specificity, linearity, limit of detection, accuracy/recovery, and precision. Further, influences of cooking of mussels before homogenization or hydrolysis on method robustness have been evaluated.
Keywords: Lipophilic marine biotoxins; Okadaic acid; Azaspiracid; Pectenotoxin; Yessotoxin; Mass spectrometry; Collaborative study; Validation study

Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species by Andrew D. Turner; Robert G. Hatfield; Monika Rapkova; Wendy Higman; Myriam Algoet; Benjamin A. Suarez-Isla; Marco Cordova; Catherine Caceres; Jeffrey van de Riet; Ryan Gibbs; Krista Thomas; Michael Quilliam; David N. Lees (1257-1270).
A refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. Figure Total saxitoxin equivalents in oysters (Pacific and native) quantified by ox-LC-FLD, LC-ox-FLD, HPLC-MS/MS and electrophysiological assay as compared with the MBA PSP toxicity reference method
Keywords: Paralytic shellfish poisoning; LC; AOAC 2005.06; Mouse bioassay; Shellfish; Saxitoxin; Oysters

Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes by Yuling Wang; Sandeep Ravindranath; Joseph Irudayaraj (1271-1278).
A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO2) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO2 were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO2, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 103 CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 103 CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices. Figure A rapid and sensitive method was developed for separation and detection of multiple pathogens in food matrix by magnetic-SERS nanoprobes. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO2 and pathogen specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices.
Keywords: Silica-coated magnetic nanoparticles; Surface-enhanced Raman scattering; Multiplex pathogen detection; Food matrix

A novel approach for the rapid analysis of ochratoxin A (OTA) in wine samples is presented. Mycotoxin was extracted and concentrated from matrix using dispersive liquid–liquid microextraction (DLLME). The final extract is analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry employing [2H5]-ochratoxin A as internal standard. Some important parameters, such as the nature and volume of extraction solvent and dispersive solvent, and salt effect were investigated and optimized to achieve the best extraction efficiency and higher enrichment factor. Under the optimum extraction condition, the method provided enrichment factor around 80 times and showed a high sensitivity with method detection and quantification limits of 0.005 and 0.015 ng mL−1, respectively. To test the accuracy of the analytical procedure, the optimized method was applied to the analysis of reference material T1755 (naturally contaminated white wine), with excellent results (accuracy of 103%) and showing a good precision with a CV (n = 6) of 5.8%. The proposed method, which is demonstrated to be quick, cheap, accurate and highly selective, was successfully applied to the analysis of Italian wines. Figure Analysis of ochratoxin A in wine by DLLME coupled to LC-MS/MS
Keywords: Ochratoxin A; Wine; Dispersive liquid–liquid microextraction; HPLC-MS/MS; Isotope dilution

Ionic liquids are a kind of environmentally friendly solvents which have drawn great attention in many fields. The potential of ionic liquid as dispersive liquid-phase microextraction (DLPME) solvent for the enrichment of typical persistent organic pollutants, dichlorodiphenyltrichloroethane (DDT), and its metabolites including 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethane and 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethylene has been investigated. Parameters that may influence the extraction efficiency, such as the type and volume of ionic liquid, the type and volume of disperser solvent, extraction time, and sample pH, were investigated and optimized in detail. The experimental results showed the excellent linear relationship between peak area and the concentration of DDT and its metabolites over the range of 1–50 μg L−1, and the precisions (RSDs) were 5.27–6.73% under the optimal conditions. The limits of detection could reach 0.33–0.63 μg L−1. Satisfied results were achieved when the proposed method was applied to determine the target compounds in real-world water samples with spiked recoveries over the range 94.4–115.3%. All these facts indicated that ionic liquid DLPME coupled to HPLC was an environmentally friendly alternative for the rapid analysis of DDT and its metabolites at trace level in environmental water samples. Figure Typical chromatograms of DDT and its metabolites in water samples by IL-DLPME
Keywords: Ionic liquid; Dispersive liquid-phase microextraction; High-performance liquid chromatography; DDT and its metabolites

A green, simple, non-toxic, and sensitive sample pretreatment procedure coupled with high-performance liquid chromatography (HPLC) was developed for the analysis of chloramphenicol (CAP) that exploits an aqueous two-phase system based on imidazolium ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate, [Bmim]BF4) and organic salt (Na3C6H5O7) using a liquid–liquid extraction technique. The influence factors on partition behaviors of CAP were studied, including the type and amount of salts, the pH value, the volume of [Bmim]BF4, and the extraction temperature. Extraction efficiency of the CAP was found to increase with increasing temperature and the volume of [Bmim]BF4. Thermodynamic studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the CAP. Under the optimal conditions, 90.1% of the CAP could be extracted into the ionic liquid-rich phase in a single-step extraction. This method was practical when applied to the analysis of CAP in feed water, milk, and honey samples with a linear range of 2~1,000 ng mL−1. The method yielded a limit of detection of 0.3 ng mL−1 and a limit of quantification of 1.0 ng mL−1. The recovery of CAP was 90.4–102.7% from aqueous samples of real feed water, milk, and honey samples by the proposed method. This novel process is much simpler and more environmentally friendly and is suggested to have important applications for the separation of antibiotics. Figure The schematic diagram of separation target molecule in ILATPS
Keywords: Ionic liquid extraction; Aqueous two-phase system; Chloramphenicol; Food; High-performance liquid chromatography

The selection of an appropriate calibration set is a critical step in multivariate method development. In this work, the effect of using different calibration sets, based on a previous classification of unknown samples, on the partial least squares (PLS) regression model performance has been discussed. As an example, attenuated total reflection (ATR) mid-infrared spectra of deep-fried vegetable oil samples from three botanical origins (olive, sunflower, and corn oil), with increasing polymerized triacylglyceride (PTG) content induced by a deep-frying process were employed. The use of a one-class-classifier partial least squares-discriminant analysis (PLS-DA) and a rooted binary directed acyclic graph tree provided accurate oil classification. Oil samples fried without foodstuff could be classified correctly, independent of their PTG content. However, class separation of oil samples fried with foodstuff, was less evident. The combined use of double-cross model validation with permutation testing was used to validate the obtained PLS-DA classification models, confirming the results. To discuss the usefulness of the selection of an appropriate PLS calibration set, the PTG content was determined by calculating a PLS model based on the previously selected classes. In comparison to a PLS model calculated using a pooled calibration set containing samples from all classes, the root mean square error of prediction could be improved significantly using PLS models based on the selected calibration sets using PLS-DA, ranging between 1.06 and 2.91% (w/w). Figure Sample classification employing PLS-DA, using statistical validation based on permutation testing, could be shown to improve the performance of PLS models applied to the quality control of deep frying oils of different botanic origins analyzed using ATR-FTIR spectroscopy.
Keywords: Polymerized triacylglycerides (PTG); Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR); Partial least squares-discriminant analysis (PLS-DA); Partial least squares (PLS) regression

Visible and near-infrared absorption spectroscopy by an integrating sphere and optical fibers for quantifying and discriminating the adulteration of extra virgin olive oil from Tuscany by Anna Grazia Mignani; Leonardo Ciaccheri; Heidi Ottevaere; Hugo Thienpont; Lanfranco Conte; Milena Marega; Angelo Cichelli; Cristina Attilio; Antonio Cimato (1315-1324).
Because of its high price, extra virgin olive oil is frequently targeted for adulteration with lower quality oils. This paper presents an innovative optical technique capable of quantifying and discriminating the adulteration of extra virgin olive oil caused by lower-grade olive oils. An original set-up for diffuse-light absorption spectroscopy in the wide 400–1,700 nm spectral range was experimented. It made use of an integrating sphere containing the oil sample and of optical fibers for illumination and detection; it provided intrinsically scattering-free absorption spectroscopy measurements. This set-up was used to collect spectroscopic fingerprints of authentic extra virgin olive oils from the Italian Tuscany region, adulterated by different concentrations of olive-pomace oil, refined olive oil, deodorized olive oil, and refined olive-pomace oil. Then, a straightforward multivariate processing of spectroscopic data based on principal component analysis and linear discriminant analysis was applied which was successfully capable of predicting the fraction of adulterant in the mixture, and of discriminating its type. The results achieved by means of optical spectroscopy were compared with the analysis of fatty acids, which was carried out by standard gas chromatography.
Keywords: Extra virgin olive oil; Absorption spectroscopy; Integrating cavity; Optical fibers; Adulteration; Chemometrics

Analytical method for assessing potential dermal exposure to pesticides of a non-agricultural occupationally exposed population by Olivier Delhomme; Caroline Raeppel; Olivier Briand; Maurice Millet (1325-1334).
To measure dermal exposure of a non-agricultural occupationally exposed population to pesticides, a new method has been developed for analysis of 13 pesticides from different classes (fungicides, herbicides, insecticides) on dermal patches. The method includes extraction of the patches and analysis of the pesticides by GC–MS and/or HPLC–fluorescence. Water-soluble pesticides (glyphosate and glufosinate) on patches were ultrasonically extracted twice with ultra-pure water for 10 min and analysed by HPLC–fluorescence after derivatisation with FMOC. Organic-soluble pesticides (bifenthrin, cyprodinil, difufenicanil, fludioxonil, oxadiazon, pyriproxyfen, clopyralid, 2,4-D, fluroxypyr, 2,4-MCPA, and triclopyr) were extracted ultrasonically twice for 10 min with 70:30 dichloromethane–acetonitrile and analysed by GC–MS directly or after derivatisation with N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide. Detection limits varied between 3 and 4 μg L−1 for water-soluble pesticides and between 1 and 10 μg L−1 for organic-soluble pesticides.
Keywords: Pesticides; Patches analysis; GC–MS; Derivatisation; LC–fluorescence

Study of the gas chromatographic behavior of selected alcohols and amines by Yasar Thewalim; Oscar Bruno; Anders Colmsjö (1335-1345).
The gas chromatographic behavior of selected linear and non-linear alcohols and amines was investigated using four capillary columns containing phenyl substitution levels of 0%, 5%, and 50% and 50% cyanopropyl substitution. In a previous study, the positions of specific compounds inside the capillary column were iteratively modeled using only two thermodynamic parameters (ΔH and ΔS). The present study addresses the validation of the two-parameter model for retention time prediction for selected alcohols and amines using thermodynamic data obtained from as few as two data points. The difference between predicted and observed retention times under different temperature conditions was generally less than 1% of the experimental value and the predicted order of elution was correct in the used model.
Keywords: Gas chromatography; Alcohols; Amines; Prediction; Retention time

The aim of this study was to test and develop techniques for the detection and identification of volatile compounds released as degradation products by Baltic amber. During a preliminary investigation, the off-gassing of acidic volatiles was detected through the corrosion of lead coupons. The corrosive compounds released by the material were then identified as formic acid and acetic acid by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry. During an advanced investigation, based on the use of artificial ageing to initiate degradation of model amber samples in different microclimates, the detected formic acid and acetic acid off-gassing appeared to be more intense in a dry environment with normal oxygen concentration. The release of formic and acetic acids by the amber was likely the result of radical reactions which should be investigated in further studies.
Keywords: Baltic amber; Headspace; Solid-phase microextraction; Gas chromatography–mass spectrometry

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [15N3]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots. Figure 15N3-labelled nicotianamine internal standard is synthesized by fission yeast cells growing on 15N-containing medium and expressing a nicotianamine synthase gene (NAS).
Keywords: UPLC; ESI-QTOF-MS; Heavy metals; Metal homeostasis; Iron; Zinc

A liquid chromatography–mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of alisol A and alisol A 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma using diazepam as an internal standard. A 200-μl plasma sample was extracted by methyl tert-butyl ether and the separation was performed on Kromasil C18 column (150 × 4.6 mm, 5 μm) with the mobile phase of acetonitrile (containing 0.1% of formic acid)–water (73:27, v/v) at a flow rate of 0.8 ml/min in a run time of 10 min. The two analytes were monitored with positive electrospray ionization by selected ion monitoring mode. The lower limit of quantitation for both alisol A and alisol A 24-acetate were 10 ng/ml. The calibration curves were linear in the measured range 10–1,000 ng/ml for alisol A and 10–500 ng/ml for alisol A 24-acetate. The mean extraction recoveries were above 74.7% for alisol A and above 72.4% for alisol A 24-acetate from biological matrixes. The intra- and inter-day precision for all concentrations of quality controls was lower than 14.1% (RSD %) for each analyte. The accuracy ranged from −12.3% to 9.8% (RE %) for alisol A, and −8.6% to 14.2% (RE %) for alisol A 24-acetate. The method was successfully applied to the study on the pharmacokinetics of alisol A and alisol A 24-acetate in rat plasma.
Keywords: Alisol A; Alisol A 24-acetate; Alisma orientale (Sam.) Juz.; LC-MS; Pharmacokinetics

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver by Yanhong Liu; Ximing Chen; Yanhong Ji; Jiangtao Chen; Guangxiang Wang; Youjun Shang; Xiangtao Liu; Jack Pan (1371-1380).
A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d6 as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d6 were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d6. The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d6 was to just compensate the variation of the injections. The detection limit was 5 ng g−1 swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.
Keywords: 2-Pyrrolidinone; LC-MS/MS; Matrix effect; Residue analysis

Miniaturized optical chemosensor for flow-based assays by Marta Pokrzywnicka; David J. Cocovi-Solberg; Manuel Miró; Víctor Cerdà; Robert Koncki; Łukasz Tymecki (1381-1387).
A cost-effective, highly compact, and versatile optoelectronic device constructed of two ordinary light emitting diodes compatible with optosensing films has been developed. This fibreless device containing chemoreceptor, semiconductor light source, and detector integrated in a miniaturized flow-through cell of low microliter internal volume works as a complete photometric chemical sensor suitable for detection in flow analysis. The operation of the developed device under nonstationary programmable-flow conditions offered by sequential injection analysis has been demonstrated using Prussian Blue film as a model optical chemoreceptor. The unique spectroelectrochemical properties of the sensing material enable its use for optical sensing of redox species, whereby ascorbic acid and hydrogen peroxide have been chosen as model analytes. The reported SI-sensor system features fast and reproducible determination of both analytes in the submillimolar range of concentrations. The construction concept demonstrated in this work can be easily applied to other kinds of optical sensors based on absorbance sensing films.
Keywords: Sensor; Sequential injection analysis; Optosensing; Light emitting diodes; Prussian blue

Synthesis of hydrochloric acid solution for total mercury determination in natural waters by Nathalie Patel-Sorrentino; Jean-Yves Benaim; Daniel Cossa; Yves Lucas (1389-1392).
Total mercury (HgT) determination requires the addition of concentrated hydrochloric acid solution (≥10 mol L−1 HCl) in relatively high amounts to preserve the samples and to prepare reagent solutions. A method for the preparation of concentrated HCl with HgT concentration of lower than 5 ng L−1 is described in this article. It is based on the well-known chemical reaction: 2 NH4Cl + H2SO4 → (NH4)2SO4 + 2 HCl. This method is validated thanks to the US Environmental Protection Agency method 1631 and standard reference materials BCR-579 (mercury in coastal seawater).
Keywords: Synthesis; Hydrochloric acid; Mercury determination; Green analytical chemistry

Erratum to: Simple fluorescence assay for quantification of OSCS in heparin by Susanne Lühn; Simone Schiemann; Susanne Alban (1393-1393).

Erratum to: Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples by Susanne Alban; Susanne Lühn; Simone Schiemann; Tanja Beyer; Jochen Norwig; Claudia Schilling; Oliver Rädler; Bernhard Wolf; Magnus Matz; Knut Baumann; Ulrike Holzgrabe (1395-1395).

Erratum: Measurement of zinc stable isotope ratios in biogeochemical matrices by double-spike MC-ICPMS and determination of the isotope ratio pool available for plants from soil by Tim Arnold; Maria Schönbächler; Mark Rehkämper; Schuofei Dong; Fang-Jie Zhao; Guy J. D. Kirk; Barry J. Coles; Dominik J. Weiss (1397-1397).