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Analytical and Bioanalytical Chemistry (v.398, #7-8)

Teaching in Hanoi – good mornings in Vietnam by Alexander Scheeline (pp. 2751-2753).

is Professor of Chemistry at the University of Illinois at Urbana-Champaign. His research interests are in instrument development. Current projects are focused on ultrasonically-levitated drops and diagnostics for reaction kinetics in such drops, and sensors for reactive oxygen species. The sensors are applied not only to studies in levitated drops but also to studies of noise-induced hearing loss, water purification, and photocatalysis. He is editor of the Journal of the Analytical Sciences Digital Library. He is a co-founder of Anchor Science LLC.

Expert texpert – analytical chemists as legal witnesses by John Fetzer (pp. 2755-2758).

is the author or co-author of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. Dr Fetzer worked for over 20 years as an analytical chemist for the Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Hercules, CA, USA. His book Career Management for Chemists—A Guide to Success in a Chemistry Career, was published by Springer.

Thanks to reviewers in 2010 (pp. 2759-2770).

Fritz Scholz (Ed.): Electroanalytical methods. Guide to experiments and applications, 2^nd ed. by Daniel Mandler (pp. 2771-2772).

Marvin C. McMaster: Buying and selling laboratory instruments: A practical consulting guide by Richard Turle (pp. 2773-2774).

Riccardo Flamini and Pietro Traldi: Mass spectrometry in grape and wine chemistry by Christelle Absalon (pp. 2775-2776).

Forty-third annual meeting of the German Society for Mass Spectrometry by Andrea Sinz; Jürgen Schmidt (pp. 2777-2778).

has been Full Professor (W3) of Pharmaceutical Chemistry at the Martin-Luther University Halle-Wittenberg (Germany) since 2007. She is the recipient of the 2004 Mattauch-Herzog award of the Deutsche Gesellschaft für Massenspektrometrie (DGMS). In 2006, she received the Innovation Prize in Medicinal/Pharmaceutical Chemistry of the Gesellschaft Deutscher Chemiker (GDCh) and the Deutsche Pharmazeutische Gesellschaft (DPhG). has been head of the mass spectrometry unit at the Department of Bioorganic Chemistry at the Leibniz Institute of Plant Biochemistry (IPB) in Halle/Saale (Germany) since 1992. His main fields of interest are mass spectrometry of organic compounds, especially natural products, metabolomics, and natural product chemistry. He has authored over 220 publications.

Computational mass spectrometry for metabolomics: Identification of metabolites and small molecules by Steffen Neumann; Sebastian Böcker (pp. 2779-2788).

The identification of compounds from mass spectrometry (MS) data is still seen as a major bottleneck in the interpretation of MS data. This is particularly the case for the identification of small compounds such as metabolites, where until recently little progress has been made. Here we review the available approaches to annotation and identification of chemical compounds based on electrospray ionization (ESI-MS) data. The methods are not limited to metabolomics applications, but are applicable to any small compounds amenable to MS analysis. Starting with the definition of identification, we focus on the analysis of tandem mass and MS n spectra, which can provide a wealth of structural information. Searching in libraries of reference spectra provides the most reliable source of identification, especially if measured on comparable instruments. We review several choices for the distance functions. The identification without reference spectra is even more challenging, because it requires approaches to interpret tandem mass spectra with regard to the molecular structure. Both commercial and free tools are capable of mining general-purpose compound libraries, and identifying candidate compounds. The holy grail of computational mass spectrometry is the de novo deduction of structure hypotheses for compounds, where method development has only started thus far. In a case study, we apply several of the available methods to the three compounds, kaempferol, reserpine, and verapamil, and investigate whether this results in reliable identifications.

Keywords: Mass spectrometry; Metabolomics; Compound identification; Spectral library; Structure elucidation

Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry by Vinzenz Handrick; Thomas Vogt; Andrej Frolov (pp. 2789-2801).

Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.

Keywords: Arabidopsis thaliana ; Pollen; Hydroxycinnamic acid amides (HCAAs); Tandem mass spectrometry; Relative quantification

Structure elucidation of the thermal degradation products of the nucleotide cofactors NADH and NADPH by nano-ESI-FTICR-MS and HPLC-MS by Diana Hofmann; Astrid Wirtz; Beatrix Santiago-Schübel; Ulrich Disko; Martina Pohl (pp. 2803-2811).

Redox cofactors like NADH and NADPH are essential for the catalytic activity of several oxidoreductases. Here, we describe a comparative study of the thermal degradation products of both cofactors in the dry and liquid states. The degradation products were first separated, detected, and quantified by high-performance liquid chromatography (HPLC). Subsequently, selected main fractions were investigated by nanoelectrospray ionization–Fourier transform ion cyclotron resonance mass spectrometry (MS). Additionally, HPLC-MS was used to elucidate the structure of all degradation products. From these data, degradation pathways for both the liquid and the solid states were elucidated. Thermal degradation in water is significantly faster compared to degradation in the solid state. Hydrolysis and oxidative ring opening of the reduced nicotinamide adenine dinucleotide (phosphate) were shown to be the main reaction paths. Surprisingly, no significant differences were observed between the degradation of both cofactors in solution and in the solid state. Our results demonstrate that the stability of both cofactors is not limiting at moderate temperatures if they are used in the dry state (e.g., solid/gas catalysis). Significant degradation of dry cofactors was only observed under conditions, which are usually not appropriate for biocatalysis (>95 °C). Besides, the situation is completely different in solution where degradation is already observed at moderate temperatures.

Keywords: FTICR-MS; HPLC-MS; NADH; NADPH; Chip-based nanoelectrospray; HILIC

Proton transfer reaction mass spectrometry for the sensitive and rapid real-time detection of solid high explosives in air and water by S. Jürschik; P. Sulzer; F. Petersson; C. A. Mayhew; A. Jordan; B. Agarwal; S. Haidacher; H. Seehauser; K. Becker; T. D. Märk (pp. 2813-2820).

Relying on recent developments in proton transfer reaction mass spectrometry (PTR-MS), we demonstrate here the capability of detecting solid explosives in air and in water in real time. Two different proton transfer reaction mass spectrometers have been used in this study. One is the PTR-TOF 8000, which has an enhanced mass resolution (m/Δm up to 8,000) and high sensitivity (~50 cps/ppbv). The second is the high-sensitivity PTR-MS, which has an improved limit of detection of about several hundreds of parts per quadrillion by volume and is coupled with a direct aqueous injection device. These instruments have been successfully used to identify and monitor the solid explosive 2,4,6-trinitrotoluene (TNT) by analysing on the one hand the headspace above small quantities of samples at room temperature and from trace quantities not visible to the naked eye placed on surfaces (also demonstrating the usefulness of a simple pre-concentration and thermal desorption technique) and by analysing on the other hand trace compounds in water down to a level of about 100 pptw. The ability to identify even minute amounts of threat compounds, such as explosives, particularly within a complex chemical environment, is vital to the fight against crime and terrorism and is of paramount importance for the appraisal of the fate and harmful effects of TNT at marine ammunition dumping sites and the detection of buried antipersonnel and antitank landmines. Figure Detecting TNT vapour with a high resolution PTR-TOF

Keywords: PTR-MS; Trace compound detection; Explosives; TNT; Direct aqueous injection

Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics by Iris Meisen; Sebastian Wisholzer; Jens Soltwisch; Klaus Dreisewerd; Michael Mormann; Johannes Müthing; Helge Karch; Alexander W. Friedrich (pp. 2821-2831).

Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C₁₈ reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 μg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs. Figure UV chromatograms recorded at l = 360 nm from unstained TCs separated on silica gel layers without (a) and with (b) EDTA plate pretreatment. The corresponding Fast Blue B stains are shown in the insets. For chromatographic separation 5 µg of individual TCs were applied. Lanes a OTC, lanes b CTC, lanes c TC, lanes d DC

Keywords: Tetracycline; Antibiotics; Mass spectrometry; IR-MALDI; Thin-layer chromatography; Reversed phase HPTLC

Thin-layer chromatography combined with MALDI-TOF-MS and³¹P-NMR to study possible selective bindings of phospholipids to silica gel by Kristin Teuber; Thomas Riemer; Jürgen Schiller (pp. 2833-2842).

High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and ³¹P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations. Figure Phospholipids separated by HPTLC were subsequently re-eluted from the plate and characterized by different methods. Spectra in blue represent the original mixture that was applied onto the HPTLC plate, while the red spectra represent the phospholipids subsequent to HPTLC. Although there were significant losses in the amount of each phospholipid, no changes in the relative and fatty acyl compositions occurred.

Keywords: High-performance thin-layer chromatography; MALDI-TOF MS; ³¹P NMR spectroscopy; Hen egg yolk; Phospholipids

Elucidation of the fragmentation pathways of different phosphatidylinositol phosphate species (PIPx) using IRMPD implemented on a FT-ICR MS by Nicole Zehethofer; Tom Scior; Buko Lindner (pp. 2843-2851).

This work reports on the fragmentation of phosphoinositides by tandem mass spectrometry (MS/MS) and MS³ experiments on a hybrid apex-Qe Fourier transform–ion cyclotron resonance mass spectrometer (FT-ICR MS) using internal infrared multiphoton dissociation (IRMPD). The fragmentation behavior of diacylphophatidylinositol triphosphate was intensively studied since an abundant loss of inositol biphosphate was observed. This loss was suggested to occur via phosphate migration along the inositol head group. Substantiation by MS³ experiments showed that this neutral loss is formed after the loss of water from the precursor ion, indicating phosphate migration along the inositol ring to the glycerol backbone. Further fragmentation of the ion formed by the loss of inositol biphosphate from diacylphophatidylinositol triphosphate resulted in the formation of a product ion with a molecular formula of C₃H₅O₇P₂, corresponding to a glycerol backbone linked to two phosphate groups. We suggested different structures for this ion and compared their stability using modeling experiments.

Keywords: Phosphoinositides; Phosphate migration; PIP; FT-ICR MS

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi by Markus Langsdorf; Alireza Ghassempour; Andreas Römpp; Bernhard Spengler (pp. 2853-2865).

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability. Figure So-far unknown natural frog skin peptides were identified by high-resolution CID and ECD MS/MS and by composition-based de novo sequencing. Sequences were confirmed by comparison of MS/MS spectra with synthesized analogs

Keywords: Frog peptides; Composition-based sequencing; Peptide fragmentation; Sequence rearrangements; Proline effect

Optimization of parameters for coverage of low molecular weight proteins by Stephan A. Müller; Tibor Kohajda; Sven Findeiß; Peter F. Stadler; Stefan Washietl; Manolis Kellis; Martin von Bergen; Stefan Kalkhof (pp. 2867-2881).

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.

Keywords: LTQ Orbitrap; Nano-HPLC; Nano-ESI-MS; MS; Proteomics; Low molecular weight proteome; Escherichia coli

Protein analysis of laser capture micro-dissected tissues revealed cell-type specific biological functions in developing barley grains by Stephanie Kaspar; Diana Weier; Winfriede Weschke; Hans-Peter Mock; Andrea Matros (pp. 2883-2893).

Both the nucellar projection (NP) and endosperm transfer cells (ETC) of the developing barley grain (harvested 8 days after flowering) were isolated by laser capture micro-dissection combined with pressure catapulting. Protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF mass spectrometry. The majority of the ∼160 proteins identified were involved in translation, protein synthesis, or protein destination. The NP proteome was enriched for stress defense molecules, while proteins involved in assimilate transport and the mobilization of nutrients were common to both the NP and the ETC. The combined qualitative and quantitative protein profiling allowed for the identification of several proteins showing tissue specificity in their expression, which underlines the distinct biological functions of these two tissues within the developing barley grain.

Keywords: Grain development; Barley; Micro-dissection; Nucellar projection; Endosperm transfer cells; Multiplexed LC-MS

Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer by Anna Laura Capriotti; Giulio Caracciolo; Giuseppe Caruso; Chiara Cavaliere; Daniela Pozzi; Roberto Samperi; Aldo Laganà (pp. 2895-2903).

Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the “protein corona” absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle–protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids. Scheme of protein adsorption onto nanoparticle surface

Keywords: Cationic liposomes; HPLC-Chip; Mass spectrometry; Nanoparticles; Plasma; Protein corona

A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues by Juan D. Chavez; William H. Bisson; Claudia S. Maier (pp. 2905-2914).

The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit. Figure Site-specific identification of protein semialdehydes

Keywords: Aldehyde-reactive probe; Protein carbonyls; Mass spectrometry; Metal-catalyzed oxidation; α-Aminoadipic semialdehyde; γ-Glutamic semialdehyde

Authenticity and provenance studies of copper-bearing andesines using Cu isotope ratios and element analysis by fs-LA-MC-ICPMS and ns-LA-ICPMS by Gisela H. Fontaine; Kathrin Hametner; Adolf Peretti; Detlef Günther (pp. 2915-2928).

Whereas colored andesine/labradorite had been thought unique to the North American continent, red andesine supposedly coming from the Democratic Republic of the Congo (DR Congo), Mongolia, and Tibet has been on the market for the last 10 years. After red Mongolian andesine was proven to be Cu-diffused by heat treatment from colorless andesine starting material, efforts were taken to distinguish minerals sold as Tibetan and Mongolian andesine. Using nanosecond laser ablation–inductively coupled plasma mass spectrometry (ICPMS), the main and trace element composition of andesines from different origins was determined. Mexican, Oregon, and Asian samples were clearly distinguishable by their main element content (CaO, SiO₂ Na₂O, and K₂O), whereas the composition of Mongolian, Tibetan, and DR Congo material was within the same range. Since the Li concentration was shown to be correlated with the Cu concentration, the formerly proposed differentiation by the Ba/Sr vs. Ba/Li ratio does not distinguish between samples from Tibet and Mongolia, but only between red and colorless material. Using femtosecond laser ablation multi-collector ICPMS in high-resolution mode, laboratory diffused samples showed variations up to 3‰ for ⁶⁵Cu/⁶³Cu within one mineral due to the diffusion process. Ar isotope ratio measurements proved that heat treatment will reduce the amount of radiogenic ⁴⁰Ar in the samples significantly. Only low levels of radiogenic Ar were found in samples collected on-site in both mine locations in Tibet. Together with a high intra-sample variability of the Cu isotope ratio, andesine samples labeled as coming from Tibet are most probably Cu-diffused, using initially colorless Mongolian andesines as starting material. Therefore, at the moment, the only reliable source of colored andesine/labradorite remains the state of Oregon. Figure Cu diffusion can be used to turn a plain, colorless andesine into a red gemstone. Cu and Ar isotope ratios in combination with main and trace elemental analysis can appoint andesine to thrie origin in Oregon, Mexico and Asia. In this study, red andesines sold as coming from Tibet and Mongolia are revealed to be most probably Cu-diffusion treated.

Keywords: Authenticity; Isotope ratios; Laser ablation; Gemstone analysis; Multi-collector ICPMS; Cu

Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study by M. Kosicek; S. Kirsch; R. Bene; Z. Trkanjec; M. Titlic; L. Bindila; J. Peter-Katalinic; S. Hecimovic (pp. 2929-2937).

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer’s disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC–MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole–time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

Keywords: Liquid chromatography; Mass spectrometry; Phospholipids; Sphingomyelin; Cerebrospinal fluid; Alzheimer’s disease

Constant activity of glutamine synthetase after morphine administration versus proteomic results by Anna Bodzon-Kulakowska; Piotr Suder; Anna Drabik; Jolanta Helena Kotlinska; Jerzy Silberring (pp. 2939-2942).

Glutamine synthetase is a key enzyme which has a regulatory role in the brain glutamate pool. According to previously published proteomic analysis, it was shown that the expression level of this enzyme is affected by morphine administration. In our study, we examined the activity of glutamine synthetase in various structures of rat brain (cortex, striatum, hippocampus and spinal cord) that are biochemically and functionally involved in drug addiction and antinociception caused by morphine. We were not able to observe any significant changes in the enzyme activity between morphine-treated and control samples despite previously reported changes in the expression levels of this enzyme. These findings stressed the fact that changes observed in the expression of particular proteins during proteomic studies may not be correlated with its activity.

Keywords: Addiction; Enzymatic activity; Glutamine synthetase; Morphine; Proteomics

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients by Roland J. W. Meesters; Robin Cornelissen; Rob J. van Klaveren; Robert de Jonge; Ethan den Boer; Jan Lindemans; Theo M. Luider (pp. 2943-2948).

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.

Keywords: Pemetrexed; NSCLC; MALDI-QqQ-MS/MS; Therapeutic drug monitoring; Alimta; Methotrexate

A one-step etching method to produce gold nanoparticle coated silicon microwells and microchannels by Teena James; Jeong Hyun Cho; Rohan Fernandes; Jatinder S. Randhawa; David H. Gracias (pp. 2949-2954).

Gold (Au) nanoparticles (NPs) have large surface areas and novel optical properties and can be readily functionalized using thiol-based chemistry; hence, they are useful in bioanalytical chemistry. Here, we describe a one-step, plasma-etching process that results in the spontaneous formation of Au NP coated recessed microstructures in silicon (Si). Mechanistically, the plasma etch rate of Si was enhanced in the vicinity of 10–100 nm thick Au patterns resulting in the formation of microwells or microchannels uniformly coated with 20–30 nm sized Au NPs. The methodology provides versatility in the types of microstructures that can be formed by varying the shape and dimensions of the Au patterns and the etch time. We also describe selective binding of antibodies to Au NP coated Si microwells using thiol-based surface modification.

Keywords: Microfluidics; Plasma; Nanofluidics; Nanotechnology

Enhancing the lateral-flow immunoassay for viral detection using an aqueous two-phase micellar system by Foad Mashayekhi; Ricky Y. T. Chiu; Alexander M. Le; Felix C. Chao; Benjamin M. Wu; Daniel T. Kamei (pp. 2955-2961).

Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5 × 10⁸ plaque forming units (pfu)/mL to 5 × 10⁷ pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA. Figure A schematic representation of concentrating viruses with an aqueous two-phase micellar system containing Triton X-114 surfactant prior to the detection of the virus through the lateral-flow immunoassay

Keywords: Lateral-flow immunoassay; Aqueous two-phase micellar systems; Point-of-care; Viral concentration and detection; Diagnostics

A novel electrochemical method to detect cell surface carbohydrates and target cells by Zhenyu Shao; Yun Li; Qianlu Yang; Jing Wang; Genxi Li (pp. 2963-2967).

Glycosylation of cell surfaces is a critical factor in many biological processes; however, the lack of effective analytical tools for the detection of cell surface carbohydrates has been the bottleneck for probing into the processes. In this paper, a novel electrochemical method is presented for the analysis of cell surface carbohydrates, which can be also used to detect the target cells. Firstly, 5-hydroxy-3-hexanedithiol-1,4-naphthoquinone (JUG_thio), the electrochemical reporter, and anti-selectin aptamer are successively modified onto the surface of a gold electrode. Different concentrations of intestinal human colon adenocarcinoma (LS180) cells are employed as the target cells for this study. Consequently, the specific carbohydrates on the surfaces of LS180 cells and anti-selectin aptamers will compete for combination with selectin in the system. As a result, the oxidation signal of JUG_thio is changed and the detection of the cell surface carbohydrates can be achieved easily and sensitively. Furthermore, the proposed method can be used to specifically detect LS180 cells in a wide concentration range, from 10³ to 10⁷ cells/mL, with a good linear relationship and low detection limit, which might be promising for the diagnosis of cancer and some other diseases in the future.

Keywords: Carbohydrates; L-selectin; Competitive recognition; Aptamer; Differential pulse voltammetry; Electroanalytical methods

Multivariate statistical differentiation of renal cell carcinomas based on lipidomic analysis by ambient ionization imaging mass spectrometry by Allison L. Dill; Livia S. Eberlin; Cheng Zheng; Anthony B. Costa; Demian R. Ifa; Liang Cheng; Timothy A. Masterson; Michael O. Koch; Olga Vitek; R. Graham Cooks (pp. 2969-2978).

Desorption electrospray ionization (DESI) mass spectrometry (MS) was used in an imaging mode to interrogate the lipid profiles of thin tissue sections of 11 sample pairs of human papillary renal cell carcinoma (RCC) and adjacent normal tissue and nine sample pairs of clear cell RCC and adjacent normal tissue. DESI-MS images showing the spatial distributions of particular glycerophospholipids (GPs) and free fatty acids in the negative ion mode were compared to serial tissue sections stained with hematoxylin and eosin (H&E). Increased absolute intensities as well as changes in relative abundance were seen for particular compounds in the tumor regions of the samples. Multivariate statistical analysis using orthogonal projection to latent structures treated partial least square discriminate analysis (PLS-DA) was used for visualization and classification of the tissue pairs using the full mass spectra as predictors. PLS-DA successfully distinguished tumor from normal tissue for both papillary and clear cell RCC with misclassification rates obtained from the validation set of 14.3% and 7.8%, respectively. It was also used to distinguish papillary and clear cell RCC from each other and from the combined normal tissues with a reasonable misclassification rate of 23%, as determined from the validation set. Overall DESI-MS imaging combined with multivariate statistical analysis shows promise as a molecular pathology technique for diagnosing cancerous and normal tissue on the basis of GP profiles. Figure Molecular disease diagnostics by DESI without sample preparation. a Good information is obtained by mapping the distribution of individual compounds in the tissue (e.g., PI(18:0/20:4). b Even better discrimination between tumor and healthy tissue is achieved using PLS-DA to consider all the data after having established through a training set of samples the features that correlate with disease as recognized by standard H&E stain pathological examination

Keywords: Ambient ionization; Kidney cancer; Lipidomics; Mass spectrometry; Molecular imaging; Phospholipids; Tissue analysis

Evaluating the effects of immunotoxicants using carbon fiber microelectrode amperometry by Bryce J. Marquis; Christy L. Haynes (pp. 2979-2985).

Carbon fiber microelectrode amperometry (CFMA) is explored as a technique for studying the effects of immunotoxicants on single-cell in vitro exocytosis function in a mouse peritoneal mast cell (MPMC)/fibroblast co-culture model. MPMCs were acutely exposed to between 10 and 100 μM of the immunotoxicants mono-2-ethylhexyl phthalate (MEHP) and bisphenol A (BPA), and release of serotonin was evaluated by CFMA. A significant decrease in the quantal content of serotonin was measured for all levels of exposure to both MEHP and BPA. The overall efficiency of the exocytotic function of MPMCs was found to be impaired by all exposure concentrations of BPA, but this efficiency was only impaired at the lowest exposure concentration of MEHP. This study illustrates the potential of CFMA as a technique for determining quantitative and biophysical chemical information in in vitro immunotoxicological studies. Figure Single-cell amperometry from a mast cell exposed to mono-2-ethylhexyl phthalate.

Keywords: Cell systems; Single-cell analysis; Electroanalytical methods; Pesticides; Endocrine disruptors; Bioanalytical methods; Exocytosis microelecrtodes

Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations by Danielle Cleveland; Stephen E. Long; Lane C. Sander; W. Clay Davis; Karen E. Murphy; Ryan J. Case; Catherine A. Rimmer; Lorena Francini; Anil K. Patri (pp. 2987-2995).

Speciation measurements of gadolinium in liposomal MRI contrast agents (CAs) are complicated by the presence of emulsifiers, surfactants, and therapeutic agents in the formulations. The present paper describes two robust, hyphenated chromatography methods for the separation and quantification of gadolinium in nanoemulsion-based CA formulations. Three potential species of gadolinium, free gadolinium ion, gadolinium chelated by diethylenetriamine pentaacetic acid, and gadolinium chelated by 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, were present in the CA formulations. The species were separated by reversed-phase chromatography (reversed phase high-performance liquid chromatography, RP-HPLC) or by high-pressure size-exclusion chromatography (HPSEC). For RP-HPLC, fluorescence detection and post-column online isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) were used to measure the amount of gadolinium in each species. Online ID-ICP-MS and species-specific isotope dilution (SID)-ICP-MS were used in combination with the HPSEC column. The results indicated that some inter-species conversions and degradation had occurred within the samples and that SID-ICP-MS should be used to provide the most reliable measurements of total and speciated gadolinium. However, fluorescence and online ID-ICP-MS might usefully be applied as qualitative, rapid screening procedures for the presence of free gadolinium ions. Figure Chromatographic methods, including size exclusion chromatography coupled to ICP-MS detection, were developed for the preclinical speciation and quantification of gadolinium in several liposome-based contrast agent formulations

Keywords: Gadolinium speciation; MRI contrast agent formulations; Species-specific isotope dilution analysis; ICP-MS; Fluorescence detection; High-pressure size-exclusion chromatography; Reversed-phase chromatography; Nanoemulsion; DTPA-DMPE; Liposome

Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell by Shunsuke Furutani; Hidenori Nagai; Yuzuru Takamura; Izumi Kubo (pp. 2997-3004).

For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells μL⁻¹ of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells μL⁻¹, chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Esherichia coli on the CD-shaped device.

Keywords: Isolation; Single cell; Compact disk; PCR; Salmonella

Rapid direct analysis in real time (DART) mass spectrometric detection of juvenile hormone III and its terpene precursors by Arti T. Navare; Jaime G. Mayoral; Marcela Nouzova; Fernando G. Noriega; Facundo M. Fernández (pp. 3005-3013).

Direct analysis in real time (DART) is a plasma-based ambient ionization technique that enables rapid ionization of small molecules with high sample throughput. In this work, DART was coupled to an orthogonal (oa) time-of-flight (TOF) mass spectrometer and the system was optimized for analyzing a vital hormonal regulator in insects, juvenile hormone (JH) III and its terpene precursors, namely, farnesol, farnesoic acid, and methyl farnesoate. Optimization experiments were planned using design of experiments (DOE) full factorial models to identify the most significant DART variables contributing to JH III analysis sensitivity by DART-TOF mass spectrometry (MS). The optimized DART-TOF MS method had femtomole to sub-picomole detection limits for terpene standards, along with mass accuracies below 5 ppm. Finally, the possibility of distinguishing between two farnesol isomers by in-source-collision-induced dissociation (CID) in the first differentially pumped region of the oaTOF mass spectrometer was investigated. DART-MS enabled high-throughput, sensitive analysis with acquisition times ranging from 30 s to a minute. To the best of our knowledge, this is the first report on the application of DART-MS to the detection and identification of volatile or semi-volatile insect terpenoids, and on the use of DOE approaches to optimize DART-MS analytical procedures.

Keywords: Mass spectrometry/ICP-MS; Biological samples; Bioanalytical methods

Biosilicated CdSe/ZnS quantum dots as photoluminescent transducers for acetylcholinesterase-based biosensors by Raluca Buiculescu; Maria Hatzimarinaki; Nikos A. Chaniotakis (pp. 3015-3021).

CdSe/ZnS core/shell quantum dots (QDs) are functionalized with mercaptoundecanoic acid (MUA) and subsequently covered with poly-l-lysine (PLL) as the template for the formation of the silica outer shell. This nanocomposite is used as a transduction and stabilization system for optical biosensor development. The covalent immobilization of the enzyme acetylcholinesterase from Drosophila melanogaster (AChE) during the formation of the biomimetically synthesized silica is used here as a model, relatively unstable enzyme, as a proof of principle. The enzyme is successfully immobilized onto the QDs and then stabilized by the PLL capping and the subsequent formation of the outer nanoporous silica thin shell, giving rise to the QD/AChE/PLL/silica biosensor. It is shown that the poly-l-lysine templated silica outer shell does not modify the optical properties of the quantum dots, while it protects the enzyme from unfolding and denaturation. The small pores of the silica shell allow for the free diffusion of the analyte to the active center of the enzyme, while it does not allow for the proteases to reach the enzyme. The response of the QD/AChE/PLL/silica nano-biosensor to its substrate, acetylcholine chloride, is evaluated by monitoring the changes in the QDs’ photoluminescence which are related to the changes in pH. These pH changes of the surrounding environment of the QDs are induced by the enzymatic reaction, and are associated with the analyte concentration in the solution. The biodetection system proposed is shown to be stable with a storage lifetime of more than 2 months. The data presented provides the grounds for the application of this nanostructured biosensor for the detection of AChE inhibitors.

Keywords: Quantum dots; Acetylcholinesterase (AChE); Biomimetically synthesized silica; Photoluminescence; Nano-biosensor

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays by Ferry Heus; Martin Giera; Gerdien E. de Kloe; Dick van Iperen; Joost Buijs; Tariq T. Nahar; August B. Smit; Henk Lingeman; Iwan J. P. de Esch; Wilfried M. A. Niessen; Hubertus Irth; Jeroen Kool (pp. 3023-3032).

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC₅₀ values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.

Keywords: Miniaturization; Acetylcholine binding protein; LED-based confocal fluorescence detection; Microfluidics; Bioassay

Simultaneous quantification of ten cytotoxic drugs by a validated LC–ESI–MS/MS method by Susanne Nussbaumer; Sandrine Fleury-Souverain; Paola Antinori; Farshid Sadeghipour; Denis F. Hochstrasser; Pascal Bonnabry; Jean-Luc Veuthey; Laurent Geiser (pp. 3033-3042).

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL⁻¹ for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces. Figure Preparation of cytotoxic formulations at the Pharmacy of Geneva University Hospitals

Keywords: Cytotoxic; Antineoplastic drugs; LC–MS/MS; Pharmaceutical formulation; SRM; Validation

Chemical characterization of exhaled breath to differentiate between patients with malignant plueral mesothelioma from subjects with similar professional asbestos exposure by G. de Gennaro; S. Dragonieri; F. Longobardi; M. Musti; G. Stallone; L. Trizio; M. Tutino (pp. 3043-3050).

Malignant pleural mesothelioma (MPM) is an aggressive tumour whose main aetiology is the long-term exposure to asbestos fibres. The diagnostic procedure of MPM is difficult and often requires invasive approaches; therefore, it is clinically important to find accurate markers for MPM by new noninvasive methods that may facilitate the diagnostic process and identify patients at an earlier stage. In the present study, the exhaled breath of 13 patients with histology-established diagnosis of MPM, 13 subjects with long-term certified professional exposure to asbestos (EXP) and 13 healthy subjects without exposure to asbestos (healthy controls, HC) were analysed. An analytical procedure to determine volatile organic compounds by sampling of air on a bed of solid sorbent and thermal desorption GC-MS analysis was developed in order to identify the compounds capable of discriminating among the three groups. The application of univariate (ANOVA) and multivariate statistical treatments (PCA, DFA and CP-ANN) showed that cyclopentane and cyclohexane were the dominant variables able to discriminate among the three groups. In particular, it was found that cyclohexane is the only compound able to differentiate the MPM group from the other two; therefore, it can be a possible marker of MPM. Cyclopentane is the dominant compound in the discrimination between EXP and the other groups (MPM and HC); then, it can be considered a good indicator for long-term asbestos exposure. This result suggests the need to perform frequent and thorough investigations on people exposed to asbestos in order to constantly monitor their state of health or possibly to study the evolution of disease over time.

Keywords: Biomarkers; Malignant pleural mesothelioma; Exhaled breath; Volatile organic compounds

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells by Dong-Hyun Kim; Roger M. Jarvis; J. William Allwood; Gavin Batman; Rowan E. Moore; Emma Marsden-Edwards; Lynne Hampson; Ian N. Hampson; Royston Goodacre (pp. 3051-3061).

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography–mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Keywords: HPV; Indinavir; Lopinavir; Raman chemical mapping; LC-MS

Effects of Lactobacillus rhamnosus on zebrafish oocyte maturation: an FTIR imaging and biochemical analysis by Elisabetta Giorgini; Carla Conti; Paolo Ferraris; Simona Sabbatini; Giorgio Tosi; Corrado Rubini; Lisa Vaccari; Giorgia Gioacchini; Oliana Carnevali (pp. 3063-3072).

The aim of this study was to verify the effects of probiotic Lactobacillus rhamnosus on zebrafish oocyte maturation using FPA (focal plane array) FTIR imaging together with specific biochemical assays (SDS–PAGE, real-time PCR and enzymatic assay). Oocyte growth is prevalently due to a vitellogenic process which consists of the hepatic synthesis of vitellogenin and its selective uptake during maturation. The administration of L. rhamnosus IMC 501 for 10 days induced chemical changes to oocyte composition, promoting the maturation process. Some interesting biochemical features, linked to protein secondary structure (amide I band) and to phospholipidic and glucidic patterns, were detailed by vibrational analysis. The spectroscopic results were supported by the early increase of the lysosomal enzyme involved in the final oocyte maturation, the cathepsin L. This enzyme increases during follicle maturation, with the highest levels in class IV oocytes. In treated females, class III oocytes showed higher cathepsin L gene expression and enzymatic activity, with levels comparable to class IV oocytes isolated from controls; this can be related to the proteolytic cleavage of the higher molecular mass yolk protein components, as evidenced by SDS–PAGE. Total absorbance cartogram of probiotic treated zebrafish oocyte (class III), reconstructed by integrating the area between 1,800-1,000 cm^-1 (a), together with the corresponding microphotograph (b).

Keywords: FPA FTIR imaging; Vibrational analysis; Fish oocytes; Probiotic; Ovary; Danio rerio

The design and synthesis of alanine-based indolicidin derivatives with identical physicochemical properties and their separation using capillary electrophoresis by Dustin S. A. Jones; Heidi E. K. Huttunen-Hennelly; Kingsley K. Donkor (pp. 3073-3079).

Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence. Fig. Electropherogram displaying the optimum separation of a mixture of indolicidin peptide derivatives having identical physicochemical properties. Conditions: detection, UV 214 nm; separation voltage, 10 kV; buffer, 5.5 M formic acid (pH 2.0)

Keywords: Capillary electrophoresis; Antimicrobial peptides; Peptide separation

A method for analysis of dimethyl selenide and dimethyl diselenide by LC-ICP-DRC-MS by Kristoffer Lunøe; Søren Skov; Charlotte Gabel-Jensen; Stefan Stürup; Bente Gammelgaard (pp. 3081-3086).

The aim of this work was to develop a simple and fast high performance liquid chromatography–inductively coupled argon plasma (ICP) mass spectrometry (MS) method capable of separating and detecting the two volatile selenium species dimethyl selenide (DMeSe) and dimethyl diselenide (DMeDSe) in biological samples. Dimethyl selenide and dimethyl diselenide were separated on a short reversed phase column using an eluent containing 40% methanol and detected by dynamic reaction cell ICP-MS monitoring the ⁸⁰Se isotope. The limit of detection was 8 nM for both species (corresponding to 0.6 and 1.3 μg Se/L for DMeDSe and DMeSe, respectively). Both compounds exhibited a linear signal–concentration relationship in the investigated concentration range of 0.1–1 μM with a precision on the determinations better than 3%. The method was applied for analysis of samples from cancer cell lines incubated with methylseleninic acid, selenomethionine, Se-methylselenocysteine, and sodium selenite. DMeDSe were detected in some samples. The method offers a simple and fast analysis of DMeDSe and DMeSe using standard liquid chromatography coupled with ICP-MS equipment and interfacing.

Keywords: DMeDSe; DMeSe; Selenium; HLPC-ICP-MS; Volatile

QSRR analysis of β-lactam antibiotics on a penicillin G targeted MIP stationary phase by Henrik Kempe; Maria Kempe (pp. 3087-3096).

The imprinting factors of the β-lactam antibiotics penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, and piperacillin on a poly(methacrylic acid-co-trimethylolpropane trimethacrylate) molecularly imprinted stationary phase targeted for penicillin G were correlated with molecular descriptors obtained by molecular computation. One-parameter linear regression and multivariate data analysis by principal component analysis and partial least square regression indicated that descriptors associated with molecular topology, shape, size, and volume were highly correlated with the imprinting factor and influential on the derived models.

Keywords: β-Lactam antibiotics; Molecular imprinting; Molecular computation; Multivariate data analysis; Principal component analysis; Partial least square regression

Biocatalytic nylon nanofibrous membranes by Alessandra Arecchi; Matteo Scampicchio; Oreste V. Brenna; Saverio Mannino (pp. 3097-3103).

Nylon-6 nanofibrous membranes (NFM) have been prepared, characterized and used to build-up electrochemical biosensing devices. The assembly and the functioning of biocatalytic NFM are described in connection with the physical and the covalent immobilization of glucose oxidase for the detection of glucose. Effects of the enzyme loading, the mediator, the pH, the surface acidity and the kinetic of the catalysis have been thoroughly investigated. The results show that NFM allow the binding of proteins without the need for the hydrolysis step, in contrast to the nylon film. Furthermore, the high surface-to-volume ratio of the NFM allow superior loading of the enzyme with respect to thin film technology. The immobilization step does not affect the permeability of the coating to the mediator used. These results give evidence that NFM are a promising and inexpensive coating for a novel electrochemical transducer. Figure Representation of the assembly of the biosensing unit and of the reactions occuring at the assembled biosensor

Keywords: Biosensors; Nanoparticles/nanotechnology; Electrochemical sensors; Bioanalytical methods

Immunosensor based on fluorescence quenching matrix of the conducting polymer polypyrrole by A. Ramanavicius; N. Ryskevic; Y. Oztekin; A. Kausaite-Minkstimiene; S. Jursenas; J. Baniukevic; J. Kirlyte; U. Bubniene; A. Ramanaviciene (pp. 3105-3113).

In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity. Figure

Keywords: Immunosensor; Fluorescence quenching; Conducting polymers; Polypyrrole; Nanotechnology

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 (pp. 3115-3125).

Analysis of naturally occurring isotopic variations is a promising tool for investigating Zn transport and cycling in geological and biological settings. Here, we present the recently installed double-spike (DS) technique at the MAGIC laboratories at Imperial College London. The procedure improves on previous published DS methods in terms of ease of measurement and precisions obtained. The analytical method involves addition of a ⁶⁴Zn–⁶⁷Zn double-spike to the samples prior to digestion, separation of Zn from the sample matrix by ion exchange chromatography, and isotopic analysis by multiple-collector inductively coupled plasma mass spectrometry. The accuracy and reproducibility of the method were validated by analyses of several in-house and international elemental reference materials. Multiple analyses of pure Zn standard solutions consistently yielded a reproducibility of about ±0.05‰ (2 SD) for δ⁶⁶Zn, and comparable precisions were obtained for analyses of geological and biological materials. Highly fractionated Zn standards analyzed by DS and standard sample bracketing yield slightly varying results, which probably originate from repetitive fractionation events during manufacture of the standards. However, the δ⁶⁶Zn values (all reported relative to JMC Lyon Zn) for two less fractionated in-house Zn standard solutions, Imperial Zn (0.10 ± 0.08‰: 2 SD) and London Zn (0.08 ± 0.04‰), are within uncertainties to data reported with different mass spectrometric techniques and instruments. Two standard reference materials, blend ore BCR 027 and ryegrass BCR 281, were also measured, and the δ⁶⁶Zn were found to be 0.25 ± 0.06‰ (2 SD) and 0.40 ± 0.09‰, respectively. Taken together, these standard measurements ascertain that the double-spike methodology is suitable for accurate and precise Zn isotope analyses of a wide range of natural samples. The newly installed technique was consequently applied to soil samples and soil leachates to investigate the isotopic signature of plant available Zn. We find that the isotopic composition is heavier than the residual, indicating the presence of loosely bound Zn deposited by atmospheric pollution, which is readily available to plants. Figure Zinc isotope ratio pools of bulk soil and the associated acid leach (estimated plant available pool) as measured by double-spike MC-ICPMS. δ^xZn_Lyon-JMC=(R_sample/R_JMC-Lyon -1)x10³, where R_sample and R_JMC-Lyon denote the ^xZn/⁶⁴Zn isotope ratio of the sample and standard (JMC-Lyon), respectively, and where x denotes either 66 or 68.

Keywords: Zinc isotopes; Stable isotope fractionation; Soil biogeochemistry; Double-spike; Mass bias correction; MC-ICPMS

Simultaneous determination of copper, mercury and zinc in water with a tailored fluorescent bipyridine ligand entrapped in silica sol-gel by Silvia C. Lopes Pinheiro; Ivo M. Raimundo Jr; María C. Moreno-Bondi; Guillermo Orellana (pp. 3127-3138).

A novel fluorescent ligand, (4-[(E)-2-(4′-methyl-2,2′-bipyridin-4-yl)vinyl]phenol) (abbreviated BSOH), has been designed and prepared for simultaneous determination of heavy metals in water. Its photophysical and photochemical properties in the absence and in the presence of Cd(II), Cu(II), Hg(II), Ni(II) and Zn(II) were determined, and the respective complexation constants (7.4 × 10³–2.8 × 10⁸ l mol⁻¹) and stoichiometries were extracted thereof. The Stern–Volmer emission intensity and lifetime plots indicate an efficient static quenching of the indicator dye with the heavy metals. The BSOH fluorescent reagent has been successfully immobilised in a silica sol-gel matrix for automation of the analytical method, and the sensing phase demonstrated a reversible response to Cu(II), Hg(II) and Zn(II) but not to Cd(II) and Ni(II). Characterisation of the sensor showed that its response to those heavy metals is linear in the 2.5 to 50 μmol l⁻¹ range, with a response time (t ₉₀) on the order of 100 min, providing detection limits of 9.0 × 10⁻⁷, 4.7 × 10⁻⁷ and 2.9 × 10⁻⁷ mol l ⁻¹ for Zn(II), Cu(II) and Hg(II), respectively. Due to the stability of the immobilised ligand, which presented no leaching from the sol-gel matrix, the simultaneous determination of the three cations in water was feasible by employing multivariate calibration techniques coupled to fluorescence quenching measurements. The sensor was validated with recovery tests by addition of Cu(II) and Hg(II) ions to spring waters, providing results with standard errors lower than 4.1 μmol l ⁻¹. Figure The green fluorescence of a novel 2,2'-bipyridine chelating ligand is suppressed by minute amounts of Cu(II). Other heavy metals also quench its emission so that some of them can be determined simultaneously after indicator immobilisation and multivariate calibration.

Keywords: Optical sensors; Fluorescence; Heavy metals; Multivariate calibration

Using one-dimensional (1D) and two-dimensional (2D) quantitative proton (¹H) nuclear magnetic resonance spectroscopy (q NMR) for the identification and quantification of taste compounds in raw onion (Allium cepa L.) bulbs and in aqueous solutions where onion tissues are soaked by Audrey Tardieu; Walter De Man; Hervé This (pp. 3139-3153).

Solutions obtained by soaking onion (Allium cepa L.) bulbs samples in water are frequently consumed, either directly or as part of dishes, both at home or in the food industry. However, little information is available regarding the extracted metabolites and the extraction mechanisms. In this article, the composition of raw onion extracts and of aqueous solutions where raw onion tissues were soaked was investigated directly by quantitative proton nuclear magnetic resonance spectroscopy (q ¹H NMR). The assignment of NMR signals was performed, with less than 3% (in area) of unidentified peaks. Analyses of one-dimensional ¹H NMR spectra with additional two-dimensional NMR studies showed 20 regions of interest where 3 saccharides, 17 amino acids, and 5 organic acids were detected and quantified. Resonance assignment with chemical shift was done for each saccharide, as well as for each amino acid and organic acid, with additional work on spin–spin coupling pattern and on observed and not observed correlations from correlation spectroscopy studies. Quantification of saccharides was performed and qualified by works on peak decomposition algorithms. Complementary studies by high-performance liquid chromatography, mass spectroscopy and tandem mass spectroscopy, and thin layer chromatography and preparative layer chromatography were carried out in order to validate the NMR results on identification.

Keywords: Taste; Quantitative ¹H NMR; Allium cepa L.; Saccharides; Amino acids; Organic acids

Determination of short-chain carbonyl compounds in drinking water matrices by bar adsorptive micro-extraction (BAμE) with in situ derivatization by N. R. Neng; J. M. F. Nogueira (pp. 3155-3163).

In this contribution, bar adsorptive micro-extraction using polystyrene–divinylbenzene sorbent phase and in situ derivatization with pentafluorophenyl hydrazine, followed by liquid desorption and high-performance liquid chromatography-diode array detection (BAμE(PS-DVB)_{PFPH in situ}-LD/HPLC-DAD), was developed for the determination of six short-chain carbonyl compounds (formaldehyde, acetaldehyde, propanal, acetone, butanone, and 2-hexenal) in drinking water matrices. PFPH presented very good specificity as an in situ derivatization agent for short-chain ketones and aldehydes in aqueous media, allowing the formation of adducts with remarkable sensitivity, selectivity and the absence of photodegradation. Assays performed on 30-mL water samples spiked at the 25.0 μg L⁻¹ levels, under optimized experimental conditions, yielded recoveries ranging from 47.4 ± 3.8% to 85.2 ± 3.8%, in which the PS-DVB proved to be a convenient sorbent phase. The analytical performance showed good accuracy, suitable precision (RSD < 13.0%), detection limits in between 47 and 132 ng L⁻¹ and remarkable linear dynamic ranges (r ² > 0.9907) from 1.0 to 80.0 μg L⁻¹. By using the standard addition methodology, the application of the present method to drinking water samples treated with different disinfectants, namely, chloride, ozone and both, allowed very good performances to monitor these priority compounds at the trace level. The proposed methodology proved to be a feasible alternative for polar compound analysis, showing to be easy to implement, reliable, sensitive and requiring a low sample volume to monitor short-chain aldehydes and ketones in drinking water matrices.

Keywords: Disinfection by-products; Short-chain carbonyl compounds; Bar adsorptive micro-extraction (BAμE); In situ derivatization; Pentafluorophenyl hydrazine (PFPH); Drinking water matrices

Simultaneous determination of NOGE-related and BADGE-related compounds in canned food by ultra-performance liquid chromatography–tandem mass spectrometry by Hong Zhang; Ming Xue; Yanyan Zou; Zhiyuan Dai; Kunhui Lin (pp. 3165-3174).

An improved analytical method enabling rapid and accurate determination and identification of bisphenol F diglycidyl ether (novolac glycidyl ether 2-ring), novolac glycidyl ether 3-ring, novolac glycidyl ether 4-ring, novolac glycidyl ether 5-ring, novolac glycidyl ether 6-ring, bisphenol A diglycidyl ether, bisphenol A (2,3-dihydroxypropyl) glycidyl ether, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A bis(3-chloro-2-hydroxypropyl) ether, and bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether in canned food and their contact packaging materials has been developed by using, for the first time, ultra-performance liquid chromatography coupled with tandem mass spectrometry. After comparison of electrospray ionization and atmospheric pressure chemical ionization in positive and negative-ion modes, tandem mass spectrometry with positive electrospray ionization was chosen to carry out selective multiple reaction monitoring analysis of novolac glycidyl ethers, bisphenol A diglycidyl ether, and its derivatives. The analysis time is only 5.5 min per run. Limits of detection varied from 0.01 to 0.20 ng g⁻¹ for the different target compounds on the basis of a signal-to-noise ratio (S/N) = 3; limits of quantitation were from 0.03 to 0.66 ng g⁻¹. The relative standard deviation for repeatability was <8.01%. Analytical recovery ranged from 87.60 to 108.93%. This method was successfully applied to twenty samples of canned food and their contact packaging materials for determination of migration of NOGE, BADGE, and their derivatives from can coatings into food.

Keywords: Mass spectrometry/ICP–MS; HPLC; Foods/beverages

Flow injection photoinduced chemiluminescence determination of imazalil in water samples by S. Meseguer-Lloret; S. Torres-Cartas; M. C. Gómez-Benito (pp. 3175-3182).

In this work, a fast, simple and economic method is proposed for the determination of imazalil in water samples by flow injection photoinduced chemiluminescence. In this method, imazalil degrades in basic media through the use of a photoreactor, and the resulting photofragments react with ferricyanide and generate the direct chemiluminescence signal. To the authors’ knowledge, this is the first time that a chemiluminescence method has been proposed for the determination of this fungicide. All physical and chemical parameters in the flow injection chemiluminescence system were optimized in the experimental setting. In the absence of preconcentration, the linear dynamic range for imazalil was 0.75–5 mg L⁻¹ and the detection limit was 0.171 mg L⁻¹. The application of solid-phase extraction with C18 cartridges allowed the elimination of interference ions, the reduction of the linear dynamic range to 15–100 μg L⁻¹, and a detection limit of 3.4 μg L⁻¹. This detection limit is below the maximum concentration level established by the Regulations of the Hydraulic Public Domain for pesticide dumping. The sample throughput after solid-phase extraction of the analyte was 12 samples h⁻¹. The intraday and interday coefficients of variation were below 9.9% in all cases. This method was applied to the analysis of environmental water samples, and recoveries of between 95.7 and 110% were obtained. Figure Flow injection chemiluminescence manifold for imazalil determination

Keywords: Imazalil; Chemiluminescence; Flow injection; Solid-phase extraction; Water

HPLC direct purity assay using ultra-purified materials as primary standards by Thierry Le Goff; Elodie Champarnaud; Fahmina Fardus (pp. 3183-3192).

Related substances using high-performance liquid chromatography, gas chromatography (GC), differential scanning calorimetryResidual solvents using headspace GC coupled to mass spectrometryInorganic content using ashing, acid digest ion couple plasma mass spectrometry or thermogravimetric analysisWater using oven coulometric Karl Fischer/direct addition coulometric Karl Fischer Related substances are not straightforward to quantify without an appropriate standard due to possible difference in response factor for the impurity relative to the main compound. In this article, existing LGC RMs certified for purity were purified further using semi-preparative HPLC. These ultra-purified organic substances were virtually free of related substances making their purity assessment faster and more straightforward, i.e., no need to identify impurities and subsequently quantify them. After characterization, these ultra-purified standards were used as calibrants to determine directly the mass fraction of the analyte in the original CRM using exact matching single-point HPLC calibration. This new approach opens the possibility of certifying the purity of low purity substances with a relative small uncertainty without the need of identifying the impurities present in the sample.

Keywords: Purity; Direct assay; HPLC; Organic compounds/trace organic compounds and reference materials

Ultrasensitive detection of malondialdehyde with surface-enhanced Raman spectroscopy by Dongmao Zhang; Rukshani Haputhanthri; Siyam M. Ansar; Karthikeshwar Vangala; Hondamuni I. De Silva; Andrzej Sygula; Svein Saebo; Charles U. Pittman Jr. (pp. 3193-3201).

Malondialdehyde (MDA) is a biomarker of lipid peroxidation that has been widely associated with food rancidity as well as many human diseases. Most current MDA detection methods involve MDA reaction with thiobarbituric acid (TBA), followed by UV–visible and/or fluorescence detection of high-performance liquid chromatography (HPLC)-separated TBA–MDA. Herein, we report the first proof-of-concept study of surface-enhanced Raman detection of a TBA–MDA adduct using silver nanoparticles as the SERS substrate and the 632.8 nm HeNe laser as a Raman excitation source. Current SERS detection limit of TBA–MDA is 0.45 nM, ~100 times higher than the 36 nM fluorescence sensitivity recently reported with the HPLC-purified TBA–MDA. Molecular specificity of the SERS technique was studied by comparing the SERS spectrum of TBA–MDA with those acquired with TBA adducts of other TBA-reactive compounds (TBARCs) that includes formaldehyde, acetaldehyde, butyraldehyde, trans-2-hexenal, and pyrimidine. Compared to TBA and TBA adducts with those TBARCs, the SERS activity of TBA–MDA adduct is significantly higher. The possibility of direct SERS detection of TBA–MDA in a reaction mixture (without HPLC separation) has also been investigated. Figure SERS Detection of Malondialdehyde

Keywords: Surface-enhanced Raman spectroscopy; Thiobarbituric acid; Malondialdehyde; Thiobarbituric acid reactive compounds

Analysis and aging of unsaturated polyester resins in contemporary art installations by NMR spectroscopy by Georgios Stamatakis; Ulla Knuutinen; Kai Laitinen; Apostolos Spyros (pp. 3203-3214).

Two original art installations constructed from unsaturated polyester resins (UPR) and four different reference UPR products (before and after UVB aging) were analyzed by high-resolution 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Breaking strain studies were also conducted for the four UPR model products before and after different aging procedures (moisture, UVB exposure, melt/freeze). NMR analysis of the chemical composition of the UPR resin extracts showed they contain several low MW organic compounds and oligomers rich in polar –OH groups that play a significant role in the degradation behavior of the composite UPR materials. Statistical analysis of the NMR compositional data showed that styrene and benzaldehyde contents can be used to differentiate between fresh and aged UPR samples. The phthalate and propylene glycol unit speciation (esterified, primary or secondary –OH) of the extracts provided evidence that UPR resin C was used in the construction of the two art installations, and direct comparison of ¹H and ¹³C NMR spectra verified this compositional similarity. UPR resin C was shown by both NMR and breaking strain studies to be the reference UPR most susceptible to degradation by different aging procedures, a characteristic attributed to the lower styrene content of resin C. Figure A1. NMaRt

Keywords: NMR spectroscopy; Unsaturated polyester resins (UPR); Art installations; Organics analysis; Polymer characterization; Aging

Determination of antioxidants in new and used lubricant oils by headspace-programmed temperature vaporization–gas chromatography–mass spectrometry by Miguel del Nogal Sánchez; Paul Glanzer; José Luis Pérez Pavón; Carmelo García Pinto; Bernardo Moreno Cordero (pp. 3215-3224).

A sensitive method is presented to determine antioxidants (2-, 3-, and 4-tert-butylphenol, 2,6-di-tert-butylphenol, 3-tert-butyl-4-hydroxyanisol, 2,6-di-tert-butyl-4-methylphenol, 1-naphthol, and diphenylamine) in new and used lubricant oil samples. Research was carried out on a GC device equipped with a headspace sampler, a programmed temperature vaporizer, and an MS detector unit. The proposed method does not require sample treatment prior to analyses, hence eliminating possible errors occurring in this step. Sample preparation is reduced when placing the oil sample (2.0 g) in the vial and adding propyl acetate (20 μL). Solvent vent injection mode permits a pre-concentration of the compounds of interest in the liner filled with Tenax-TA®, while venting other species present in the headspace. Thereby, both the life of the liner and the capillary column is prolonged, and unnecessary contamination of the detector unit is avoided. Calibration was performed by adding different concentrations of analytes to a new oil which did not contain any of the studied compounds. Limits of detection as low as 0.57 μg/L (2-tert-butylphenol) with a precision lower or equal to 5.3% were achieved. Prediction of the antioxidants in new oil samples of different viscosities (5W40, 10W40, and 15W40) was accomplished with the previous calibration, and the results were highly satisfactory. To determine antioxidants in used engine oils, standard addition method was used due to the matrix effect.

Keywords: Antioxidants; HS–PTV–GC–MS; Lubricant oil

Erratum to: Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry by Gianluca Maddalo; Mohammadreza Shariatgorji; Christopher M. Adams; Eva Fung; Ulrika Nilsson; Roman A. Zubarev; Jan Sedzik; Leopold L. Ilag (pp. 3225-3226).

Erratum to: Scientometric analysis of national university research performance in analytical chemistry on the basis of academic publications: Italy as case study by Anna Annibaldi; Cristina Truzzi; Silvia Illuminati; Giuseppe Scarponi (pp. 3227-3227).

2011 Bunsen-Kirchhoff Award for Analytical Spectroscopy by Detlef Günther (pp. 3229-3229).

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Analytical and Bioanalytical Chemistry (v.398, #7-8)