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Analytical and Bioanalytical Chemistry (v.395, #5)

Outcome and output-focused research: selling your research in difficult times by Robin Henderson (pp. 1181-1183).

started his professional life as a materials engineer, following his first degree with a PhD in modelling high-temperature ceramics. After working as a post-doctoral research fellow Robin was appointed as a lecturer at the University of Aberdeen where he led a number of research projects. During this period Robin became interested in supporting the learning of others and after a brief spell in staff development founded his own company ( www.myconsultants.net ) which works extensively with the higher education sector and in particular supports the development of early-stage career researchers.

Spectroscopy challenge 14 by Reinhard Meusinger (pp. 1185-1189).

Solution to quality assurance challenge 8 by Manfred Reichenbächer; Jürgen W. Einax (pp. 1191-1193).

Alexander P. Demchenko: Introduction to fluorescence sensing by Francesco Baldini (pp. 1195-1196).

Ljiljana Paŝa-Tolic and Mary S. Lipton (Eds.): Mass spectrometry of proteins and peptides. Methods and protocols, 2nd ed by Andreas Tholey (pp. 1197-1198).

Thomas J. Webster (Ed.): Safety of nanoparticles. From manufacturing to medical applications by Hugh J. Byrne (pp. 1199-1200).

Donna J. Dean: Getting the most out of your mentoring relationships. A handbook for women in STEM by Julie A. Stenken (pp. 1201-1202).

Mycotoxins by Rudolf Krska (pp. 1203-1204).

is head of the Center for Analytical Chemistry of the Department for Agrobiotechnology (IFA-Tulln) of the BOKU-University of Natural Resources and Applied Life Sciences, Vienna. In 2008 he was appointed Full Professor for Bio-Analytics and Organic Trace Analysis. Currently, he is on a temporary appointment as A/Chief of Food Research Division of the Bureau of Chemical Safety at Health Canada in Ottawa.

Recent advances in the development of novel materials for mycotoxin analysis by Chris M. Maragos (pp. 1205-1213).

For sensitive and specific toxin detection, mycotoxin immunoassays depend upon antibodies with high affinity and selectivity. While intact immunoglobulins remain the primary toxin-binding elements used in rapid assays, a number of alternatives have begun appearing in the literature. The alternatives can be broadly classified into those that are obtained by chemical synthesis and those that are obtained by altering biologically derived materials. Examples range from synthetically prepared polymers to recombinant fragments of antibodies, with a wide variety of synthetic and natural materials in-between. To date, obtaining the combination of selectivity and affinity needed for use in sensors has been more readily accomplished with biologically derived materials than with synthetic materials. Despite this, synthetic materials still offer certain potential advantages, such as high binding capacity and the ability to bind in environments that are too harsh for intact antibodies. This review focuses upon recent advances in the development of mycotoxin-binding materials and their potential for application in mycotoxin assays.

Keywords: Mycotoxin analysis; Antibody; Polymer; Aptamer; Cyclodextrins; Binding materials

Aflatoxin analysis at the beginning of the twenty-first century by Gordon S. Shephard (pp. 1215-1224).

Aflatoxin mycotoxins were first described in the early 1960s as important fungal toxins, which contaminate many different human foods and animal feeds. Accurate and sensitive determination of these carcinogenic compounds immediately became an important requirement to meet food safety concerns and new official legislated regulations. For these reasons, analytical methods for aflatoxins continued to develop over the decades, reflecting advances in analytical chemistry. Currently, a wide range of methods are available to analytical scientists, ranging from newly described multi-toxin liquid chromatography tandem mass spectrometry to rapid methods based on immunological principles. These latter methods can provide quantitative outputs or a simple rapid determination of contamination level above or below a pre-determined cutoff value. The newest official methods as validated by Association of Official Analytical Chemists International or Comité Européen de Normalisation rely on immunoaffinity column clean-up of conventional extracts, followed by high-performance liquid chromatography separation of the analogues with detection based on natural fluorescence or the fluorescence generated by various derivatisation methods. In selecting from this range of available methods, the analytical chemist must decide on the requirements of the analysis such that the method chosen is ‘fit for purpose’.

Keywords: Aflatoxin; HPLC; Mass spectrometry; Fluorescence; Mycotoxin

Review of secondary metabolites and mycotoxins from the Aspergillus niger group by Kristian Fog Nielsen; Jesper Mølgaard Mogensen; Maria Johansen; Thomas O. Larsen; Jens Christian Frisvad (pp. 1225-1242).

Filamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread food and feed contaminants known but they are also some of the most important workhorses used by the biotechnological industry. The Nigri section consists of six commonly found species (excluding A. aculeatus and its close relatives) from which currently 145 different secondary metabolites have been isolated and/or detected. From a human and animal safety point of view, the mycotoxins ochratoxin A (from A. carbonarius and less frequently A. niger) and fumonisin B₂ (from A. niger) are currently the most problematic compounds. Especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products where fumonisin-producing fusaria are not a problem, fumonisins pose a risk. Moreover, compounds such as malformins, naptho-γ-pyrones, and bicoumarins (kotanins) call for monitoring in food, feed, and biotechnology products as well as for a better toxicological evaluation, since they are often produced in large amounts by the black aspergilli. For chemical differentiation/identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested). Naptho-γ-pyrones are the compounds produced in the highest quantities and are produced by all six common species in the group (A. niger 134 of 140; A. tubingensis 169 of 177; A. acidus 44 of 47; A. carbonarius 40 of 40, A. brasiliensis 18 of 18; and A. ibericus three of three). Figure Image of Aspergillus niger growing on YES agar, and the resulting extract analysed by LCDAD-TOFMS

Keywords: Metabolomics; Fumonisin; Ochratoxin; Liquid chromatography–mass spectrometry; Polyketide synthase; Polyketide

Formation, determination and significance of masked and other conjugated mycotoxins by Franz Berthiller; Rainer Schuhmacher; Gerhard Adam; Rudolf Krska (pp. 1243-1252).

Mycotoxins are secondary metabolites of fungi poisonous for humans or animals which can be found on a great variety of food and feed commodities. Food is not necessarily safe just because the presence of well-known mycotoxins has been ruled out, as they might still be there in disguise. Mycotoxins may also occur in conjugated form, either soluble (masked mycotoxins) or incorporated into/associated with/attached to macromolecules (bound mycotoxins). These conjugated mycotoxins can emerge after metabolization by living plants, fungi and mammals or after food processing. Awareness of such altered forms of mycotoxins is increasing, but reliable analytical methods, measurement standards and occurrence and toxicity data are still lacking. In this paper currently known conjugated mycotoxins, their formation and determination are reviewed. For the latter, liquid chromatography-(tandem) mass spectrometry or ELISA methods are employed with or without conversion to the parent mycotoxins. Sample preparation to transform the bound forms into soluble forms can involve enzymatic or acidic/alkaline treatment. Especially mycotoxins which are in contact with living plants in the field are prone to be metabolized. This transformation process is not only important regarding food safety but also for the resistance of plants towards fungal-induced diseases, such as Fusarium head blight of wheat.

Keywords: Conjugated mycotoxins; Masked mycotoxins; Bound mycotoxins; Plant metabolism; Food processing; Mass spectrometry

Overview of analytical methods for beauvericin and fusaproliferin in food matrices by Antonello Santini; Rosalia Ferracane; Giuseppe Meca; Alberto Ritieni (pp. 1253-1260).

In recent years consumers and the scientific community have become increasingly interested in food safety, making it a major focus among the objectives of the international institutions responsible for food safety monitoring, e.g. the European Union or the EFSA. Aspects attracting much attention are the colonization of food by microscopic fungi which, under aerobic conditions, produce toxic secondary metabolites known as mycotoxins, and the accumulation of these toxins in the food chain. Numerous studies of surveillance, detoxification, prevention, and toxicological aspects reported in the literature mostly concentrate on major mycotoxins such as aflatoxins, ochratoxin A, trichothecenes, and fumonisins; studies on toxic secondary metabolites of mycotoxins are less common or are only just beginning. Among the molecules of interest, the family of beauvericin and fusaproliferin is certainly the most interesting. The objective of this review is to summarize reported data and the methods used to extract and quantify beauvericin and fusaproliferin in food matrices.

Keywords: Beauvericin; Fusaproliferin; Fusarium ; Solvent extraction; Liquid chromatography; Mass spectrometry

Analysis of selected phytotoxins and mycotoxins in environmental samples by Corinne C. Hoerger; Judith Schenzel; Bjarne W. Strobel; Thomas D. Bucheli (pp. 1261-1289).

Natural toxins such as phytotoxins and mycotoxins have been studied in food and feed for decades, but little attention has yet been paid to their occurrence in the environment. Because of increasing awareness of the presence and potential relevance of micropollutants in the environment, phytotoxins and mycotoxins should be considered and investigated as part of the chemical cocktail in natural samples. Here, we compile chemical analytical methods to determine important phytotoxins (i.e. phenolic acids, quinones, benzoxazinones, terpenoids, glycoalkaloids, glucosinolates, isothiocyanates, phytosterols, flavonoids, coumestans, lignans, and chalcones) and mycotoxins (i.e. resorcyclic acid lactones, trichothecenes, fumonisins, and aflatoxins) in environmentally relevant matrices such as surface water, waste water-treatment plant influent and effluent, soil, sediment, manure, and sewage sludge. The main problems encountered in many of the reviewed methods were the frequent unavailability of suitable internal standards (especially isotope-labelled analogues) and often absent or fragmentary method optimization and validation.

Keywords: Bioactive compounds; Natural toxins; Micropollutants; Phytoestrogens; Mycoestrogens; Allelopathy

Sampling and analytical variability associated with the determination of aflatoxins and ochratoxin A in bulk lots of powdered ginger marketed in 1-lb bags by Thomas B. Whitaker; Mary W. Trucksess; Carol M. Weaver; Andrew Slate (pp. 1291-1299).

Ginger has been used as a food, dietary supplement, and condiment for centuries. Mycotoxins such as the aflatoxins (AF) and ochratoxin A (OTA) have been reported in ginger roots in several studies. It is important to design effective sampling methods that will accurately and precisely predict the true mycotoxin level in a bulk lot. The objective of this study was to measure the sampling and analytical variability associated with the test procedure used to measure AF and OTA in a bulk lot of powdered ginger using a 5-g laboratory sample and HPLC analytical methods. Twelve 5-g laboratory samples were taken from each of two lots. Duplicate aliquots were removed from each 5-g laboratory sample/solvent blend, and each aliquot was simultaneously analyzed for AF and OTA by HPLC analytical methods. Using a balanced nested design, the total variance associated with the above AF and OTA test procedures was partitioned into sampling and analytical variance components for each lot. Averaged across both lots, the sampling and analytical variances accounted for 87% and 13% of the total variance, respectively, for AF and 97% and 3%, respectively, for OTA. The sampling and analytical coefficients of variation were 9.5% and 3.6%, respectively, for AF, and 16.6% and 2.9%, respectively, for OTA when using a single 5-g laboratory sample and HPLC analytical methods. Equations are derived to show the effect of increasing laboratory sample size and/or number of aliquots on reducing the variability of the test procedures used to estimate OTA and AF in powdered ginger.

Keywords: Aflatoxin; Ochratoxin A; Ginger; Sampling and analytical variability; Sampling; Foods/beverages; Quality assurance/control

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats by Natalia A. Burmistrova; Irina Yu. Goryacheva; Evgenia Yu. Basova; Ann-Sophie Franki; Dirk Elewaut; Katrien Van Beneden; Dieter Deforce; Carlos Van Peteghem; Sarah De Saeger (pp. 1301-1307).

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and α-zearalenol (α-ZOL) (69%) recognition, while cross-reactivities with α-zearalanol, zearalanone, β-zearalenol and β- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 μg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 µg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and α-ZOL). Figure Monoclonal antibodies against zearalenone were raised in mice and applied in three formats based on the competitive direct enzyme immunoassay principle: a microtiter plate enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay

Keywords: Zearalenone; Mycotoxin; Immunoassay; ELISA; Rapid test; Wheat

A rapid lateral flow test for the determination of total type B fumonisins in maize by Alexandra Molinelli; Karina Grossalber; Rudolf Krska (pp. 1309-1316).

A one-step lateral flow test was developed for the quantitative determination of total type B fumonisins in maize with a test range up to 4,000 μg/kg and a limit of detection of 199 μg/kg. The test presented gives a result within 4 min, including 1 min strip drying, and does not require any sample cleanup steps after a previous 3-min sample extraction. Quantitative readout with a compact photometric strip reader will also indicate the best suited measurement range when needed. The test is based on a competitive immunoassay format where a ready-to-use antibody–colloidal gold particle complex is mixed with 50 μL sample extract in a microwell and used as a signal reagent. The test strip is inserted into the well and the mixed content migrates onto the strip, which contains a test zone and a control zone. Mycotoxin–protein conjugate coated on the test zone captures free signal reagent, and colored particles concentrate, forming a visible line. The intensity of the test line is dependent on the total fumonisin concentration in the sample. Naturally contaminated quality-control maize material was used for matrix-matched calibration of photometric readout. The test presented is both quantitative and rapid, with no cross-reactivity to other mycotoxins. The applicability of the lateral flow test was shown by the screening of 23 naturally contaminated maize samples. Relative standard deviations ranged from 1.7 to 32.9%.

Keywords: Mycotoxins; Lateral flow device; Immunodiagnostic assay; Gold nanoparticles; Rapid test; Food and feed analysis

Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine by Francesco Zezza; Francesco Longobardi; Michelangelo Pascale; Sergei A. Eremin; Angelo Visconti (pp. 1317-1323).

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.

Keywords: Ochratoxin A; Fluorescence polarization immunoassay; Solid-phase extraction; Wine; Rapid methods

Enzymatic hydrolysis of T-2 toxin for the quantitative determination of total T-2 and HT-2 toxins in cereals by Veronica M. T. Lattanzio; Michele Solfrizzo; Angelo Visconti (pp. 1325-1334).

The selective enzymatic deacetylation of T-2 toxin to give HT-2 toxin has been investigated in aqueous crude extracts of different cereals and exploited to develop an analytical method for the determination of the sum of T-2 and HT-2 toxins. The method has been validated for the analysis of total T-2 and HT-2 toxins in maize, wheat, and oats, showing recoveries from 72 to 97% for maize, from 67 to 84% for wheat, and from 61% to 87% for oats, at spiking levels of 20–400 μg/kg, with relative standard deviation lower than 10%. Liquid chromatography-tandem mass spectrometry was used for quantitative toxin determination. The potential biological role of this enzymatic conversion and its perspectives for application in the development of antibody-based analytical techniques are discussed. Figure The selective deacetylation at the C-4 position of T-2 toxin to give HT-2 toxin catalyzed by cereal carboxylesterases

Keywords: T-2 toxin; HT-2 toxin; Carboxylesterase; Fusarium mycotoxins; Cereals; Mycotoxin analysis

Difficulties in fumonisin determination: the issue of hidden fumonisins by Chiara Dall’Asta; Mattia Mangia; Franz Berthiller; Alexandra Molinelli; Michael Sulyok; Rainer Schuhmacher; Rudolf Krska; Gianni Galaverna; Arnaldo Dossena; Rosangela Marchelli (pp. 1335-1345).

In this paper, the results obtained by five independent methods for the quantification of fumonisins B₁, B₂, and B₃ in raw maize are reported. Five naturally contaminated maize samples and a reference material were analyzed in three different laboratories. Although each method was validated and common calibrants were used, a poor agreement about fumonisin contamination levels was obtained. In order to investigate the interactions among analyte and matrix leading to this lack of consistency, the occurrence of fumonisin derivatives was checked. Significant amounts of hidden fumonisins were detected for all the considered samples. Furthermore, the application of an in vitro digestion protocol to raw maize allowed for a higher recovery of native fumonisins, suggesting that the interaction occurring among analytes and matrix macromolecules is associative rather than covalent. Depending on the analytical method as well as the maize sample, only 37–68% of the total fumonisin concentrations were found to be extractable from the samples. These results are particularly impressive and significant in the case of the certified reference material, underlying the actual difficulties in ascertaining the trueness of a method for fumonisin determination, opening thus an important issue for risk assessment.

Keywords: Fumonisins; Hidden fumonisins; Masked mycotoxins; Mass spectrometry; Maize

Simultaneous determination of deoxynivalenol, zearalenone, and their major masked metabolites in cereal-based food by LC–MS–MS by O. Vendl; F. Berthiller; C. Crews; R. Krska (pp. 1347-1354).

Cereals and cereal-based food have often been found to be contaminated with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON), after infection of the grain with the pathogenic fungus Fusarium. Both the pathogen and the infected plants can chemically modify DON and ZON, including acetylation, glucosidation, and sulfation. Analytical strategies for detection and quantification of DON and ZON are well known and established but often fail to recognize the respective metabolites, which are, therefore, also referred to as “masked” mycotoxins. However, several masked forms are also known to be harmful to mammals. Failure to detect these could lead to significant underestimation of the toxic potential of a particular sample. To monitor the levels of DON and ZON metabolites in cereals and cereal-based food, we have developed a LC–MS–MS method capable of simultaneous determination of DON, ZON, and eight of their masked metabolites, namely deoxynivalenol-3-glucoside (D3G), 3-acetyl-deoxynivalenol (3ADON), zearalenone-4-glucoside (Z4G), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalenol-4-glucoside (α-ZG), β-zearalenol-4-glucoside (β-ZG), and zearalenone-4-sulfate (Z4S). The suitability of several cleanup strategies including C₁₈-SPE, primary and secondary amines (PSA), MycoSep push-through columns, and immunoaffinity columns was evaluated. The final method used no sample cleanup and was successfully validated for four cereal-based food matrices, namely cornflour, porridge, beer, and pasta, showing good recoveries and precision for all analytes.

Keywords: Deoxynivalenol; Zearalenone; Masked mycotoxins; LC–MS–MS; Cereal-based food

Simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry by Vinay Vishwanath; Michael Sulyok; Roman Labuda; Wolfgang Bicker; Rudolf Krska (pp. 1355-1372).

This paper describes the application of a previously published multi-mycotoxin method for food and feed matrices based on liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) to the analysis of microbial metabolites in indoor matrices. The range of investigated analytes has been extended by 99 fungal and bacterial metabolites to cover now 186 compounds overall. The method is based on a single extraction step using an acidified acetonitrile/water mixture (which has been determined to be preferable to methanol and ethyl acetate) followed by analysis of the diluted crude extract. The analytical signal of one third of the investigated analytes was reduced by more than 50% due to matrix effects in a spiked extract of house dust, whereas the other investigated materials were less critical in that aspect. For determination of method performance characteristics, a spiked reference material for house dust was chosen as a model sample for an extremely complex matrix. With few exceptions, coefficients of variation of the whole procedure of <10% and limits of detection of <50 µg kg⁻¹ were obtained. The apparent recoveries were below 50% for half of the investigated analytes due to incomplete extraction and/or detection-related matrix effects. The application of the method to 14 samples from damp buildings revealed the presence of 20 different analytes at concentrations of up to 130 mg kg⁻¹. Most of these compounds have never been identified before in real-world samples, although they are known to be produced by indoor-relevant fungi. This underlines the great value of the described method for the on-site determination of microbial metabolites. Figure Mycotoxin concentrations determined in a damp room

Keywords: Liquid chromatography; Tandem mass spectrometry; Mycotoxins; Bacterial metabolites; Damp buildings; Indoor molds

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of Fusarium by MALDI linear TOF mass spectrometry by Hongjuan Dong; Jasmin Kemptner; Martina Marchetti-Deschmann; Christian Peter Kubicek; Günter Allmaier (pp. 1373-1383).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level. Figure Intact spore mass spectrometry - from fungal conidia spores to optimal MALDI TOF mass spectrum for species differentiation and identification.

Keywords: Fusarium ; Conidia spores; Sample preparation; ICMS; MALDI–TOF MS; Mass spectrometry

A reference-gene-based quantitative PCR method as a tool to determine Fusarium resistance in wheat by Kurt Brunner; Maria P. Kovalsky Paris; Guadalupe Paolino; Hermann Bürstmayr; Marc Lemmens; Franz Berthiller; Rainer Schuhmacher; Rudolf Krska; Robert L. Mach (pp. 1385-1394).

In recent years, plant breeders made great progress in breeding Fusarium-tolerant wheat lines. However, total resistance to this genus of plant pathogenic fungi has not yet been achieved as the resistance genes are located on several distinct genetic regions. Visual scoring of disease symptoms in combination with the analysis of mycotoxins is commonly applied to assess the tolerance of new lines. Both approaches are indirect methods and do not mandatorily determine the accumulated fungal biomass. Quantitative PCR is a useful tool to assess fungal biomass based on the abundance of organism-specific DNA. The aim of this study was the development of a quantitative PCR assay for trichothecene-producing Fusarium species and to adapt this method for resistance assessment of wheat lines artificially infected with Fusarium graminearum and Fusarium culmorum. Several DNA-extraction methods for wheat samples were evaluated and optimized for downstream real-time PCR analysis and furthermore, a new reference-gene-based approach for more accurate quantification of Fusarium biomass in cereals is presented. The co-determination of a plant gene was used to compensate for unequal DNA-extraction efficiencies.

Keywords: Fusarium ; Wheat; PCR; Resistance; Infection; Quantification

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision–mass spectrometry by Tatiana Pimenova; Lukas Meier; Bernd Roschitzki; Gabriela Paraschiv; Michael Przybylski; Renato Zenobi (pp. 1395-1401).

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP^C) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete suppression of nonspecific bPrP binding.

Keywords: Epitope excision; Polystyrene beads; Prion protein; Antibody–antigen interaction; Mass spectrometry

Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry by A. Vonaparti; E. Lyris; I. Panderi; M. Koupparis; C. Georgakopoulos (pp. 1403-1410).

In equine sport, salicylic acid is prohibited with a threshold level of 750 µg mL⁻¹ in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization in negative mode with full scan acquisition mode and product ion scan mode were chosen for the quantification and identification of salicylic acid, respectively. Run time was 2.0 min. The tested linear range was 2.5–50 µg mL⁻¹ (after 100-fold sample dilution). The relative standard deviations of intra- and inter-assay analysis of salicylic acid in horse urine were lower than 2.5% and 2.8%, respectively. Overall accuracy (relative percentage error) was less than 3.3%. Method was applied to two real samples found to be positive for salicylic acid, demonstrating simplicity, accuracy, and selectivity.

Keywords: Salicylic acid; Horse urine; Doping control; Quadrupole/time-of-flight mass spectrometry; Direct injection analysis; Matrix effects

Rapid, sensitive and simultaneous determination of fluorescence-labeled designated substances controlled by the Pharmaceutical Affairs Law in Japan by ultra-performance liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry by Jun Zhe Min; Suguru Hatanaka; Toshimasa Toyo’oka; Shinsuke Inagaki; Ruri Kikura-Hanajiri; Yukihiro Goda (pp. 1411-1422).

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 “designated substances” (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC™ BEH C₁₈ column (1.7 μm, 100 mm × 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile–methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H]⁺ obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection $$ left( {{ ext{S/N}} = 3} ight) $$ and the limits of quantification $$ left( {{ ext{S/N}} = 10} ight) $$ in 0.1 mL of human plasma and urine for the present method were 0.30–150 pmol and 1.0–500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products. Figure Structures of designated substances tested

Keywords: Designated substances (Shitei-Yakubutsu); Fluorescence labeling; Piperazines; Phenethylamines; 4-(N,N-Dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; Ultra-performance liquid chromatography; Time-of-flight mass spectrometry

Sulfonate group-modified FePtCu nanoparticles as a selective probe for LDI-MS analysis of oligopeptides from a peptide mixture and human serum proteins by Hideya Kawasaki; Tarui Akira; Takehiro Watanabe; Kazuyoshi Nozaki; Tetsu Yonezawa; Ryuichi Arakawa (pp. 1423-1431).

Bare FePtCu nanoparticles (NPs) are first prepared for laser desorption/ionization mass spectroscopy (LDI-MS) analysis as affinity probes to selectively trap oppositely charged analytes from a sample solution. Our present results demonstrate bare FePtCu NPs to be a potentially useful matrix for surface-assisted laser desorption/ionization mass spectroscopy (SALDI-MS), for the analysis of small proteins and peptides. The upper detectable mass range of peptides was approximately 5 kDa, and the detection limit for peptides approximately 5 fmol. Sulfonate group-modified FePtCu nanoparticles (FePtCu-SO ₃ ⁻ NPs), with ionization being independent of the solution pH, can interact with a positively charged analyte, and the analyte-bound NPs can be separated from the reaction supernatant by centrifugation or an external magnetic field. An oligopeptide, Gly-Gly-Tyr-Arg (GGYR) from an oligopeptide mixture containing Asp-Asp-Asp-Asp (DDDD), Gly-Gly-Gly-Gly (GGGG) and GGYR, was detected using SALDI-MS with FePtCu-SO₃(−) NPs employing electrostatic interaction. Furthermore, FePtCu-SO ₃ ⁻ NPs can detect lysozyme (Lyz) in human serum through the electrostatic attraction between positively charged Lyz and FePtCu-SO₃(−) NPs at pH 8, while detection of negatively charged albumin in human serum is not possible. Figure Sulfonate group-modified FePtCu nanoparticles as a selective probe for LDI-MS

Keywords: SALDI-MS; MALDI-MS; Magnetic particles; FePtCu; Affinity probe

Microwave-assisted sensing of tetracycline using europium-sensitized luminescence fibers as probes by Chi-Lap Kuong; Tsai-Jung Yu; Yu-Chie Chen (pp. 1433-1439).

In this paper, we describe a facile approach—using silicate fibers immobilized with Eu(III) ions [Eu(III) fibers] as affinity probes—to rapidly sense tetracycline (TC) in complex samples. The fabrication of the Eu(III) fibers is straightforward: Simply immerse a silicate fiber into a glass tube containing Eu(III) and irradiate with microwaves (power, 900 W) for 30 s. The Eu(III) fibers selectively trap TC from aqueous samples via chelation of the β-diketone functional group of TC with the Eu(III) center. Because the Eu(III)–TC complexes on the fibers are luminescent, as a result of intermolecular energy transfer from the TC moieties to the Eu(III) centers, they can be detected directly using a fluorophotometer. To accelerate the sensing process, we also used microwave irradiation (for only 15 s) to trap the TC molecules from the sample solutions onto the Eu(III) fibers. Furthermore, only a small volume (< 10 μL) of sample solution is required for these analyses. This approach allows TC to be detected in aqueous samples, with a detection limit of 50 nM.

Keywords: Europium; Tetracycline; Luminescence; Microwave heating

In vivo metabolism study of ginsenoside Re in rat using high-performance liquid chromatography coupled with tandem mass spectrometry by Liu Yang; Shunjun Xu; Chunjin Liu; Zhijun Su (pp. 1441-1451).

Ginsenoside Re is one of the major the bioactive triterpene saponins in ginseng root, a well-known adaptogen in traditional Chinese medicine. It is believed that the lead compound may be further developed into a promising new drug for preventing hypertension and cardiovascular disease. To better understand the pharmacological activities of the component, an investigation of its in vivo metabolism was necessary. In the present study, a high-performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-TOF-MS/MS) has been applied to discover and identify the metabolites of ginsenoside Re in rat urine following intravenous and oral administration of the component, respectively. The rat urine samples were collected and pretreated through C₁₈ solid-phase extraction cartridges prior to analysis. Negative electrospray ionization mass spectrometry was used to discern ginsenoside Re and its possible metabolites in urine samples. The metabolites were identified and tentatively characterized by means of comparing molecular mass, retention time, and fragmentation pattern of the analytes with those of the parent compound, ginsenoside Re. As a result, eleven and nine metabolites together with Re were detected and identified in rat urine collected after intravenous and oral administration, respectively. A possible metabolic pathway of ginsenoside Re was also investigated and proposed. Oxidation and deglycosylation were found to be the major metabolic processes of the constituent in rat. Figure Proposed metabolic pathways of 20(S)-ginsenoside Re in rats.

Keywords: High-performance liquid chromatography-mass spectrometry; Ginsenoside Re; Metabolite; In vivo metabolism

Myosin-catalyzed ATP hydrolysis elucidated by ³¹P NMR kinetic studies and ¹H PFG-diffusion measurements by Zhiyan Song; Kari J. Parker; Idorenyin Enoh; Hua Zhao; Olarongbe Olubajo (pp. 1453-1459).

We conducted ³¹P NMR kinetic studies and ¹H-diffusion measurements on myosin-catalyzed hydrolysis of adenosine triphosphate (ATP) under varied conditions. The data elucidate well the overall hydrolysis rate and various factors that significantly impact the reaction. We found that the enzymatic hydrolysis of ATP to adenosine diphosphate (ADP) was followed by ADP hydrolysis, and different nucleotides such as ADP and guanosine triphosphate acted as competitors of ATP. Increasing ATP or Mg²⁺ concentration resulted in decreased hydrolysis rate, and such effect can be related to the decrease of ATP diffusion constants. Below 50 °C, the hydrolysis was accelerated by increasing temperature following the Arrhenius’ law, but the hydrolysis rate was significantly lowered at higher temperature (~60 °C), due to the thermal–denaturation of myosin. The optimal pH range was around pH 6–8. These results are important for characterization of myosin-catalyzed ATP hydrolysis, and the method is also applicable to other enzymatic nucleotide reactions.

Keywords: ATP; Myosin; Hydrolysis; ³¹P NMR; ¹H diffusion

Quantitative determination of Phakellistatin 13, a new cyclic heptapeptide, in rat plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study by Hua Wei; Jun Wen; Rui Xie; Houwen Lin; Guorong Fan; Yutian Wu (pp. 1461-1469).

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation, has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 μL) in a 96-well plate by methanol (200 μL) containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical column (5 µm, 50 mm × 4.6 mm i.d.) using an eluent of methanol–water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity (r > 0.999) over the quantitation range of 0.1–5 ng/mL. The validation results demonstrated that this method was significantly specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50, and 100 µg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic studies on PK13.

Keywords: Phakellistatin 13; LC-MS/MS; Cyclic heptapeptide; Rat plasma; Pharmacokinetics

Evaluation of different calibration strategies for the analysis of pure copper and zinc samples using femtosecond laser ablation ICP-MS by Heike Traub; Markus Wälle; Joachim Koch; Ulrich Panne; Ralf Matschat; Heinrich Kipphardt; Detlef Günther (pp. 1471-1480).

Solution-doped metal powder pellets as well as aspirated liquids were used as calibration samples to analyze pure copper and zinc certified reference materials (CRMs) by femtosecond laser ablation ICP-MS. It was demonstrated that calibration by copper pellets resulted in relative deviations up to 20%, whereas fs-LA-ICP-MS among copper-based CRMs led to inaccuracies in the same range unless nominal mass fractions were chosen to be <3 mg/kg. Calibration by zinc pellets generally provided better accuracy. Depending on the analyte considered, deviations below 10% were obtained even for mass fractions close to the limit of quantification. Our data, therefore, indicate solution-doped metal powder pellets to be suitable as calibration samples for fs-LA-ICP-MS of metals. Furthermore, the utilization of liquid standards for calibration was found to result in stronger deviations of up to 50% for both copper and zinc samples which, in addition, turned out to be dependent on the plasma conditions. Figure Different calibration strategies for fs-LA-ICP-MS

Keywords: Laser ablation; ICP-MS; Calibration; Copper; Zinc; Metal

Bag-SPE—a convenient extraction method for screening of pharmaceutical residues in influent and effluent water from sewage treatment plants by J. A. Magnér; T. E. Alsberg; D. Broman (pp. 1481-1489).

Bag-SPE is a solid-phase extraction (SPE) technique here applied to sample pharmaceutical residues in wastewater. The device, consisting of 20 mg polystyrene-divinylbenzene (PS-DVB) enclosed in a woven polyester fabric was immersed into a 20-mL sample. Extraction of the analytes was performed under gentle rotation (25 rpm) until distribution equilibrium was achieved (4 h). The extraction efficiency for thirteen pharmaceuticals was evaluated for the bag-SPE sampler compared to a conventional SPE cartridge (Oasis HLB). All analyses were determined on an ultra-performance liquid chromatography (UPLC) coupled to a quadrupole time-of-flight (QToF) mass spectrometer. The detection limit of the bag-SPE technique for the analytes in wastewater ranged from 15–100 ng/L with recoveries between 20.7% and 58.2% and ion suppressions between 2.2% and 53.2%. Although the extraction efficiencies were lower with the bag-SPE sampler compared to the SPE technique, the two methods showed similar detection limits due to the lower ion suppression experienced with the bag-SPE. The results demonstrate that bag-SPE is an attractive alternative to the more, in terms of manual handling, demanding SPE technique.

Keywords: Bag-SPE; Solid-phase extraction; Pharmaceuticals; Wastewater; UPLC-QToF

Dispersive liquid–liquid microextraction using an in situ metathesis reaction to form an ionic liquid extraction phase for the preconcentration of aromatic compounds from water by Cong Yao; Jared L. Anderson (pp. 1491-1502).

A novel microextraction method is introduced based on dispersive liquid–liquid microextraction (DLLME) in which an in situ metathesis reaction forms a water-immiscible ionic liquid (IL) that preconcentrates aromatic compounds from water followed by separation using high-performance liquid chromatography. The simultaneous extraction and metathesis reaction forming the IL-based extraction phase greatly decreases the extraction time as well as provides higher enrichment factors compared to traditional IL DLLME and direct immersion single-drop microextraction methods. The effects of various experimental parameters including type of extraction solvent, extraction and centrifugation times, volume of the sample solution, extraction IL and exchanging reagent, and addition of organic solvent and salt were investigated and optimized for the extraction of 13 aromatic compounds. The limits of detection for seven polycyclic aromatic hydrocarbons varied from 0.02 to 0.3 µg L⁻¹. The method reproducibility produced relative standard deviation values ranging from 3.7% to 6.9%. Four real water samples including tap water, well water, creek water, and river water were analyzed and yielded recoveries ranging from 84% to 115%. Figure A method is introduced based on ionic liquid dispersive liquid–liquid microextraction (IL DLLME) in which an in-situ metathesis reaction forms a water immiscible ionic liquid that pre-concentrates aromatic compounds from water followed by separation using high performance liquid chromatography.

Keywords: Ionic liquid; Dispersive liquid–liquid microextraction; High-performance liquid chromatography; Metathesis reaction; Aromatic compounds; Water sampling; Polycyclic aromatic hydrocarbons

Chemometric tools for identification of volatile aroma-active compounds in oregano by Anne-Christin Bansleben; Ingo Schellenberg; Jürgen W. Einax; Kristin Schaefer; Detlef Ulrich; David Bansleben (pp. 1503-1512).

One of the purposes of chemical analysis is to find quick and efficient methods to answer complex analytical questions in the life sciences. New analytical methods, in particular, produce a flood of data which are often very badly arranged. An effective way to overcome this problem is to apply chemometric methods. As part of the following investigations, three brands of oregano were analysed to identify their volatile aroma-active compounds. Two techniques were applied—gas chromatograpy–olfactometry (GC–O) and human sensory evaluation. Aroma-impact compounds could be identified in the main brands of oregano with the aid of chemometric methods (principal-components analysis, hierarchical cluster analysis, linear discriminant analysis, partial least-squares regression). Therefore, it is possible to reduce the analysis of sensory and olfactometry to relevant attributes. This makes classifying new species easier, much faster, and less expensive and is the premise for quick and more economic identification of new potential genotypes for oregano plant breeding. A comprehensive list of oregano key odourants, determined by GC–O and human sensory evaluation using different methods of supervised and unsupervised pattern cognition, has not previously been published. Figure Oregano - a multi-faceted herb

Keywords: Chemometrics; Gas chromatography–olfactometry; Sensory evaluation; Oregano

Photostability and phytotoxicity of selected sunscreen agents and their degradation mixtures in water by Rosario Rodil; Monika Moeder; Rolf Altenburger; Mechthild Schmitt-Jansen (pp. 1513-1524).

The study on the photostability of six UV filters in aqueous solution was combined with investigations on the phytotoxicity of the produced degradation mixtures. During the exposure to artificial sunlight over 72 h, the degradation of three of the UV filters evaluated was observed with half-lives between 20 and 59 h. The structural changes of iso-amylmethoxy-cinnamate (IAMC), ethylhexyl-methoxy-cinnamate (EHMC), and 4-methylbenzyliden camphor (4-MBC) occurred during irradiation were consistent with isomerisation and polymerization (IAMC and EHMC) whereas 2-ethylhexyl-4-(dimethylamino)benzoate (OD-PABA) was degraded. The analysis of the UV filters and their degradation products was performed by stir bar sorptive extraction (SBSE) followed by thermodesorption–gas chromatography–mass spectrometry (TD-GC-MS) or liquid desorption–liquid chromatography–mass spectrometry (LD-LC-MS). The phytotoxicological potential of the UV filters was examined in vitro by evaluating reproduction inhibition of the chlorophyte microalgae Scenedesmus vacuolatus. Excess toxicity was calculated by comparing experimental derived median efficiencies after log-logistic modeling to predict effects assuming narcotic mode of action. Benzophenone-3 (BP-3) showed 43-fold higher toxicity than theoretically predicted and a more specific mode of action was assumed. The other UV filters tested indicated toxicity in the range of modeled narcosis. For IAMC, EHMC, and OD-PABA the phytotoxicity of their photodegradation mixtures was followed over time. Phytotoxicity decreased directly with the reduction of the parent substance from the solution. Five of the tested UV filters do not represent a risk at least for algae. Octocrylen and 4-MBC were found to be photostable but few toxic to algae. EHMC, IAMC, and OD-PABA were fast degraded during UV radiation and the phytotoxicity of the corresponding degradation mixtures was low and decreased onward during exposure. Thus, for the UV filters studied, it could be confirmed that sunlight can account noticeably for decontamination and detoxification of contaminated water. However, due to its potential accumulation in combination with a specific mode of action, BP-3 may imply probable environmental risks at least to algae. This study emphasizes the need of a combined chemical and toxicological evaluation for a reliable risk assessment concerning degradation processes exemplified here for UV-protecting agents.

Keywords: Excess toxicity; Photolysis; UV filter; GC-MS; HPLC-MS-MS; Stir bar sorptive extraction; Personal care products

The ability of a novel sorptive polymer to determine the freely dissolved fraction of polar organic compounds in the presence of fulvic acid or sediment by Jörgen A. Magnér; Tomas E. Alsberg; Dag Broman (pp. 1525-1532).

A novel plastic material, poly(ethylene-co-vinyl acetate-co-carbon monoxide) (PEVAC), was evaluated as an absorptive passive equilibrium sampler for determination of the freely dissolved fraction of seven polar organic contaminants (POCs) in the presence of fulvic acid and sediment. The seven compounds selected were imidacloprid, carbendazim, metoprolol, atrazin, carbamazepine, diazinon and chlorpyrifos, i.e. a mixture of pharmaceuticals and pesticides having logarithmic octanol/water partition coefficients (log K _OW) ranging from 0.2 to 4.77. The experiments demonstrated that the PEVAC sampler is well suited for determination of the freely dissolved fraction of chemicals in aquatic environments. Generally, the freely dissolved fraction of the POCs decreased with increasing hydrophobicity. However, strong interactions with functional groups of the organic matter seemed to dominate the partitioning for imidacloprid and carbendazim, having logarithmic dissociation partition coefficient log D < 1.47, and for metoprolol, which is positively charged at neutral pH.

Keywords: Partition coefficient; Polar organic compounds; Fulvic acid; Sediment; Poly(ethylene-co-vinyl acetate-co-carbon monoxide); Liquid chromatography–mass spectrometry

Determination of melamine in animal feed based on liquid chromatography tandem mass spectrometry analysis and dynamic microwave-assisted extraction coupled on-line with strong cation-exchange resin clean-up by Ligang Chen; Qinglei Zeng; Xiaobo Du; Xin Sun; Xiaopan Zhang; Yang Xu; Aimin Yu; Hanqi Zhang; Lan Ding (pp. 1533-1542).

In this work, a new method was developed for the determination of melamine (MEL) in animal feed. The method was based on the on-line coupling of dynamic microwave-assisted extraction (DMAE) to strong cation-exchange (SCX) resin clean-up. The MEL was first extracted by 90% acidified methanol aqueous solution (v/v, pH = 3) under the action of microwave energy, and then the extract was cooled and passed through the SCX resin. Thus, the protonated MEL was retained on the resin through ion exchange interaction and the sample matrixes were washed out. Some obvious benefits were achieved, such as acceleration of analytical process, together with reduction in manual handling, risk of contamination, loss of analyte, and sample consumption. Finally, the analyte was separated by a liquid chromatograph with a SCX analytical column, and then identified and quantitatived by a tandem mass spectrometry with positive ionization mode and multiple-reaction monitoring. The DMAE parameters were optimized by the Box–Behnken design. The linearity of quantification obtained by analyzing matrix-matched standards is in the range of 50–5,000 ng g⁻¹. The limit of detection and limit of quantification obtained are 12.3 and 41.0 ng g⁻¹, respectively. The mean intra- and inter-day precisions expressed as relative standard deviations with three fortified levels (50, 250, and 500 ng g⁻¹) are 5.1% and 7.3%, respectively, and the recoveries of MEL are in the range of 76.1–93.5%. The proposed method was successfully applied to determine MEL in different animal feeds obtained from the local market. MEL was detectable with the contents of 279, 136, and 742 ng g⁻¹ in three samples. Figure Schematic diagram of on-line coupling of dynamic microwave-assisted extraction with strong cation exchange resin clean-up

Keywords: Dynamic microwave-assisted extraction; On-line clean-up; Liquid chromatography tandem mass spectrometry; Melamine; Animal feed

Evaluation of the oxidative status of virgin olive oils with different phenolic content by direct infusion atmospheric pressure chemical ionization mass spectrometry by M. J. Lerma-García; J. M. Herrero-Martínez; E. F. Simó-Alfonso; G. Lercker; L. Cerretani (pp. 1543-1550).

Atmospheric pressure chemical ionization mass spectrometry was used to predict the oxidative status of virgin olive oils (VOO) during their storage. VOO samples, with and without phenolic compounds, were stored in the dark at 60 °C up to 7 weeks. The VOO samples were diluted in an alkaline propanol/methanol mixture and directly infused into an ion-trap mass spectrometer. The abundances of the [M−H]⁻ peaks of free fatty acids, oxidized fatty acids, tocopherols and phenolic compounds, jointly with their oxidized forms, were measured and used as predictors. Two linear discriminant analysis (LDA) models were constructed in order to classify samples according to their oxidative levels. The first model was constructed using both VOO samples (with and without phenols), considering as predictors only fatty acids and their oxidized products. The second LDA model was constructed with the VOO sample with phenolic compounds considering as predictors all the peaks measured. In both models, the samples divided in the eight different storage times were correctly classified (100%) by leave-one-out cross-validation with an excellent resolution among all the category pairs (for the first model Wilks’ lambda, λ _w = 0.229 and for the second λ _w = 0.928). This method is a very fast tool for on-line monitoring of VOO oxidation status.

Keywords: Direct infusion; Linear discriminant analysis; Lipid oxidation; Mass spectrometry; Oxidative status

Simultaneous extraction of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in agricultural soils by pressurized liquid extraction and determination by gas chromatography coupled to tandem mass spectrometry by José Luis Martínez Vidal; Antonia Garrido Frenich; María de las Nieves Barco Bonilla; Roberto Romero-González; Juan Antonio Padilla Sánchez (pp. 1551-1562).

A simple and rapid method based on pressurized liquid extraction has been validated for the simultaneous extraction of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) from agricultural soil samples. Effective extraction was carried out in less than 17 min for all the studied compounds, and good recoveries were obtained for PAHs and PCBs, ranging from 70% to 112%, when blank samples were spiked at 2.5 μg kg⁻¹, except for naphthalene with recoveries close to 40%. The separation and determination were performed by gas chromatography coupled to tandem mass spectrometry using a triple quadrupole mass analyzer. The target compounds were detected by electron impact with selected reaction monitoring, and mass spectrometric conditions were optimized in order to increase selectivity and sensitivity. The developed method was validated, and matrix-matched calibration was used for quantification purposes. Repeatability and interday precision ranged from 0.9% to 16.8% and from 1.6% to 22.3%, respectively. Limits of quantification ranged from 0.07 to 2.50 μg kg⁻¹. The proposed method was applied to the analysis of agricultural soil samples collected from Almeria (Spain), and PAHs and PCBs were detected in some samples at concentrations ranging from 0.1 to 210 μg kg⁻¹.

Keywords: Soils; PCBs; PAHs; Pressurized liquid extraction; GC-MS/MS

Fabrication of semiconductor nanowires by conjugation of quantum dots to actin filaments by Lan Yao; Gregory O. Andreev; Yana K. Reshetnyak; Oleg A. Andreev (pp. 1563-1566).

We demonstrate the feasibility of fabrication of semiconducting nanowires (quantum dots) using F-actin as a template. Three different approaches of assembling quantum dots into nanowires are described. The nanowires were characterized by fluorescence microscopy.

Keywords: Nanowires; Fabrication; Quantum dots; Fluorescence

2010 Bunsen-Kirchhoff Award for Analytical Spectroscopy (pp. 1567-1567).

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Analytical and Bioanalytical Chemistry (v.395, #5)

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Analytical and Bioanalytical Chemistry (v.395, #5)