Analytical and Bioanalytical Chemistry (v.398, #3)

Tandem MS for environmental and food analysis by Damià Barceló; Mira Petrovic (1143-1144).
is a full research professor at the Environmental Chemistry Department at IDAEA-CSIC in Barcelona, Spain, Director of the Catalan Institute for Water Studies (ICRA) in Girona, Spain, and Visiting Professor at the King Saud University, Saudi Arabia. He has been President of the Spanish Society of Mass Spectrometry (SEEM) since 2006. His research focuses on method development and the monitoring and fate of priority, new and emerging pollutants in water, using gas chromatography and liquid chromatography techniques coupled with advanced tandem and hybrid mass-spectrometric analysis combined with effect studies using bioassays and biosensors. He is the coordinator the project INNOVA-MED Innovative Processes and Practices for Wastewater Treatment and Reuse in the Mediterranean Region funded by the European Union and the project Assessing and Predicting Effects on Water Quantity and Quality in Iberian Rivers (SCARCE) funded by the CONSOLIDER-INGENIO 2010 programme of the Spanish Ministry of Science and innovation is an ICREA research professor at the Department of Environmental Chemistry, IDAE-CSIC, Barcelona, Spain. Her main expertise is in the field of analytical environmental chemistry, specifically analysis of organic micropollutants by advanced mass-spectrometric techniques and the study of their fate and behaviour in the aquatic environment and during wastewater and drinking water treatment.

Study of the performance of three LC-MS/MS platforms for analysis of perfluorinated compounds by Marta Llorca; Marinella Farré; Yolanda Picó; Damià Barceló (1145-1159).
The analytical suitabilities of three different liquid chromatography–tandem mass spectrometry (LC-MS/MS) systems, (1) triple quadrupole (QqQ), (2) conventional 3D ion trap (IT), and (3) quadrupole–linear IT (QqLIT), to determine trace levels of perfluorinated compounds (PFCs) in fish and shellfish were compared. Sample preparation was performed using alkaline extraction and solid-phase-extraction cleanup. This evaluation was focused on both quantitative (sensitivity, precision, and accuracy) and qualitative (identification capabilities) aspects. In the three instruments, the former facet was evaluated using selected reaction monitoring (SRM), which is the standard mode for quantitative LC-MS/MS analysis. Accuracy was similar in the three systems, with recoveries always over 70 %. Precision was better for the QqLIT and QqQ systems (7–15%) than for the IT system (10–17%). The QqLIT (working in SRM mode) and QqQ systems offered a linear dynamic range of at least 3 orders of magnitude, whereas that of the IT system was 2 orders of magnitude. The QqLIT system achieved at least 20-fold higher sensitivity than the QqQ system, and this was at least tenfold higher sensitivity than for the IT system. In the IT system, identification was based on sensitive full mass range acquisition and MS n fragmentation and in the QqLIT system, it was based on the use of an information-dependent-acquisition scan function, which allows the combination of an SRM or MS full scan acting as the survey scan and an enhanced product ion scan followed by MS3 as the dependent scan in the same analysis. Three instruments were applied to monitor the content in fish and shellfish (anchovies, swordfish, tuna, mussels, and oysters) obtained from Valencia and Barcelona markets (Spain). The eight target PFCs were detected at mean concentrations in the range from 10 ng  kg-1 (perfluoro-7-methyloctanoic acid and perfluoro-1-decanesulfonate) to 4,200 ng  kg-1 (perfluoropentanoic acid). Furthermore, perfluoroheptanoic and perfluoroundecanoic acids (not covered as target analytes) were identified in some samples. Figure PFCs in the environment
Keywords: Perfluorinated acids; Perfluorinated sulfonates; Liquid chromatography–tandem mass spectrometry; Triple quadrupole; Ion trap; Linear ion trap; Fish

Perfluorinated compounds (PFCs) have been recognised as emerging pollutants of global relevance. A fully automated method with inline solid-phase extraction coupled to electrospray ionisation liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) is presented and used for characterisation of soil adsorption and desorption for six PFCs: perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutane sulphonate (PFBS), and perfluorooctane sulphonate (PFOS). The method reduces sample turnaround time and solvent consumption and is suitable for low volume sampling. The only sample preparation necessary for water samples was sedimentation by centrifugation. The method has a total runtime of 21 min including inline sample cleanup (2 min for injection and SPE, 14 min for the chromatographic separation, 5 min for reconditioning). Negative AP-ESI with selective reaction monitoring (SRM) was used and the method was documented for quantification of the six environmentally important PFCs in subsoil matrix and related aqueous matrixes (groundwater and drainage water). Linearity was demonstrated in the range 5 to 2,500 ng/l and the LOD was between 2 and 8 ng/l in groundwater. Adsorption was characterised by linear Freundlich isotherms for all six compounds in two agricultural top soils (A horizon, sandy and clayey soil).Variability in sorption characteristics for soil types as well as compound properties were found, and correlation between the organic carbon normalised sorption coefficient (K OC) and PFC molecular weight was demonstrated. The K d values were in the range 0.1 to 33 (l/kg), and 0.3 to 65 (l/kg) for sorption and desorption respectively. Figure Hydrant sampling
Keywords: Fluorinated hydrocarbons; Inline SPE tandem mass spectrometry; Fluorochemical FC

Multiresidue method for the determination of 32 human and veterinary pharmaceuticals in soil and sediment by pressurized-liquid extraction and LC-MS/MS by Erkuden Pérez-Carrera; Martin Hansen; Víctor M. León; Erland Björklund; Kristine A. Krogh; Bent Halling-Sørensen; Eduardo González-Mazo (1173-1184).
An analytical chemical method has been developed for the simultaneous determination of 32 different pharmaceuticals in soils and sediments. The pharmaceuticals cover a varity of different compound groups. Soil samples were extracted with different solvents with the help of pressurized-liquid extraction (PLE) followed by clean-up using a solid-phase extraction (SPE) procedure. The purified extracts were analyzed by LC-MS/MS. The extraction method was evaluated by testing the following variables: extraction solvents, solvent pH, and temperature. Applying 20 g of soil/sediment and extracting with a mixture of methanol with aqueous ammonia solution (0.1 mol L−1) at 80 °C for 5 min in five cycles provided satisfactory recoveries between 66 and 114% with SD of between 1 and 14%. For preconcentration and purification tandem MAX-HLB cartridges were used. The volume and composition was optimized and the highest recoveries were obtained with a combination of methanol—aqueous ammonia solution. The limits of quantification (LOQs) were between 0.2 and 2 ng g−1 and linearity higher than 0.98 for the majority of the selected pharmaceuticals. The method was successfully applied to soil samples collected from the Jerez de la Frontera agricultural region, irrigated with treated wastewater, and to sediment samples from the River Guadalete. The detection of nine pharmaceuticals including stimulants, antirheumatics, analgesics, anti-inflammatories, tranquilizers, and veterinary medicines at ng g−1 concentration levels was achieved.
Keywords: Pharmaceuticals; Soil; Sediment; Pressurized-liquid extraction; Solid-phase extraction; PLE-SPE-LC-MS/MS

Determination of multi-class pharmaceuticals in wastewater by liquid chromatography–tandem mass spectrometry (LC–MS–MS) by Sandra Babić; Dragana Mutavdžić Pavlović; Danijela Ašperger; Martina Periša; Mirta Zrnčić; Alka J. M. Horvat; Marija Kaštelan-Macan (1185-1194).
An analytical method for multi-class pharmaceuticals determination in wastewater has been developed and validated. Target compounds were: sulfonamides (sulfadiazine, sulfaguanidine, sulfamethazine, sulfamethoxazole), fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin), diaminopyrimidine (trimethoprim), anaesthetic (procaine), anthelmintic (praziquantel and febantel), and macrolide (roxithromycin). The method involves pre-concentration and clean-up by solid-phase extraction (SPE) using Strata-X extraction cartridges at pH 4.0. Target analytes were identified and quantitatively determined by liquid chromatography–tandem mass spectrometry using multiple reaction monitoring (MRM). Recoveries were higher than 50% with relative standard deviation (RSD) below 18.3% for three concentrations. Only for sulfaguanidine was low recovery obtained. Matrix effect was evaluated using matrix-matched standards. The method detection limit (MDL) was between 0.5 and 5 ng L−1 in spiked water samples. The precision of the method, calculated as relative standard deviation, ranged from 0.5 to 2.0% and from 1.4 to 8.3 for intra-day and inter-day analysis, respectively. The described analytical method was used for determination of pharmaceuticals in effluent wastewaters from the pharmaceutical industry.
Keywords: Pharmaceuticals; Wastewater; Liquid chromatography–tandem mass spectrometry; Solid phase extraction

A fully automated method has been developed for analysis of eighteen antibacterial compounds, including penicillins, cephalosporins and sulfonamides, in animal feed with limits of quantification in the range 0.25–5.79 μg kg−1. The method is based on pressurized liquid extraction of 3 g homogenized feed with water and online clean-up of 500 μL of the extract with C18HD cartridges. The purified sample was directly analysed by liquid chromatography–electrospray tandem mass spectrometry (SPE–LC–ESI-MS–MS). Chromatographic separation was achieved within 10 min by use of a C12 Phenomenex Hydro-RP reversed-phase analytical column and a mobile phase gradient (water + 0.1% formic acid–methanol + 0.1% formic acid). The method was validated, revealing capability for detection of concentrations as low as 0.09 μg kg−1, decision limits (CCα) and detection capabilities (CCβ) in the range 10–174 μg kg−1 and 22–182 μg kg−1, respectively, and inter-day precision ranging from 0.7 to 8.3%. Recovery, with internal standard correction, was in the range 93–134% for all analytes. The method was then applied to analysis of fifteen feed samples, nine of which contained at least one antimicrobial at concentrations between 0.006 and 1.526 mg kg−1. The performance data and results from the method were compared with those from a previous method developed by our group, using offline SPE, by analyzing the same set of samples by both methods. The online SPE approach resulted in slightly improved sensitivity, with LODs of 0.09–2.12 μg kg−1 compared with 0.12–3.94 μg kg−1 by the offline approach. In general, better recovery was achieved by use of online purification (for 72% of the analytes) and the correlation between the two methods was good. The main advantages of the new online method are rapid and automated sample pre-treatment, and reduction of sample manipulation, enabling high-throughput analysis and highly accurate results. Because of all these characteristics, the proposed method is applicable and could be deemed necessary within the field of food control and safety.
Keywords: β-lactams; Penicillins; Cephalosporins; Sulfonamides; Online SPE; LC–ESI-MS–MS; Animal feed

Municipal wastewater has been examined for steroids, β2-agonists, stimulants, diuretics, and phosphodiesterase type V inhibitors (PDE type V inhibitors), which are “dual-use-drugs” applied either as anabolic, doping, and lifestyle drugs or for treatment of diverse diseases. To identify their origin, fitness centre discharges under suspicion of being point sources and sewage-treatment plant feed and effluents were sampled and concentrations determined. Sensitive and selective methods for determination and quantification based on solid-phase extraction (SPE) followed by high-performance liquid chromatography–high resolution mass and tandem mass spectrometry (HPLC–(HR)MS and HPLC–MS–MS) were developed and established for analysis of these compounds in wastewater and to assess their effect on the environment. The methods developed enabled quantification at trace concentrations (limit of quantification (LOQ): 5 ng L−1). Of the steroids and stimulants under investigation, testosterone, methyltestosterone, and boldenone or ephedrine, amphetamine, and MDMA (3,4-methylendioxy-N-methylamphetamine) were observed at up to 5 μg L−1 (ephedrine). Of the β2-agonists salbutamol only, and of the diuretics furosemide and hydrochlorothiazide were confirmed in the extracts. Quite high concentrations of the PDE type V inhibitors sildenafil, tadalafil, and vardenafil and their metabolites were confirmed in fitness centre discharges (sildenafil: 1,945 ng L−1) whereas their concentrations in municipal wastewater did not exceed 35 ng L−1. This study identified anabolic and doping drugs in wastewater for the first time. Results obtained from wastewater treatment plant effluents proved that these “dual-use-drugs”, with the exception of hydrochlorothiazide, were mostly eliminated. Figure Anabolic, doping and lifestyle drugs on their ways into the environment
Keywords: Anabolic; Doping; Lifestyle drugs; Wastewater; LC–MS; LC–MS n ; Elimination; Degradation

LC-MS analyses of microcystins in fish tissues overestimate toxin levels—critical comparison with LC-MS/MS by J. Kohoutek; O. Adamovský; M. Oravec; Z. Šimek; M. Palíková; R. Kopp; L. Bláha (1231-1237).
Microcystins are cyclic peptide toxins with hepatotoxic and tumour-promoting properties which are produced in high quantities in freshwater cyanobacterial water blooms, and several studies have reported microcystin accumulation in fish with possible food transfer to humans. In this study, we provide the first comparison of liquid chromatography with single mass-spectrometric and with tandem mass-spectrometric detection for analyses of microcystins in complex fish tissue samples. Use of traditional single mass spectrometry (i.e. monitoring of ions with m/z 519.5 for microcystin-RR and m/z 995.5 for microcystin-LR) was found to provide false-positive responses, thus overestimating the concentrations of microcystins in the tissue samples. More selective tandem mass spectrometry seems to provide more reliable results. The concentrations of microcystins detected by tandem mass spectrometry in fish from controlled-exposure experiments were more than 50% lower in comparison with concentrations obtained by single mass spectrometry. Extensive analyses of edible fish parts—muscles (148 fish specimens from eight different species from five natural reservoirs with dense cyanobacterial water blooms)—showed negligible microcystin concentrations (all analyses below the limit of detection; limit of detection of 1.2–5.4 ng/g fresh weight for microcystin-RR, microcystin-YR and microcystin-LR in multiple reaction monitoring mode). Our findings have practical consequences for critical re-evaluation of the health risks of microcystins accumulated in fish.
Keywords: Microcystin; Fish tissue; Liquid chromatography–tandem mass spectrometry

The present work describes the development and validation of an analytical method based on liquid chromatography (LC), coupled with tandem mass spectrometry (MS/MS) that allows the determination and confirmation of several endocrine-disrupting chemicals (EDCs) in honey. The EDCs studied were nine phenols of different nature: chlorophenols (2,4-dichlorophenol, 2,4,5-trichlorophenol, and pentachlorophenol), alkylphenols (4-tert-butylphenol, 4-tert-octylphenol, and 4-n-octylphenol) bisphenols (bisphenol-A and bisphenol-F), and 4-tert-butylbenzoic acid. The method incorporates a restricted-access material (RAM), coupled on-line to the LC-MS/MS system, which allows direct injection of the matrix into the RAM-LC-MS/MS system. The optimized method developed, RAM-LC-MS/MS, was applied to fortified honey samples, affording detection limits in the 0.6–7.2 ng g−1 range, calculated for a signal-to-noise ratio of 3. In addition, the method was validated as a quantitative confirmatory method according to European Union Decision 2002/657/EC. The validation criteria evaluated were linearity, repeatability, reproducibility, recovery, decision limits, detection capabilities, specificity, and ruggedness. Repeatability and within-laboratory reproducibility were evaluated at two concentration levels, being ±11% or below at 20 ng g−1. The decision limits (CCα) and detection capabilities (CCβ) were in the 1.7–12.6 and 2.8–21.6 ng g−1 range, respectively.
Keywords: Endocrine-disrupting chemicals; Phenolic compounds; Honey; Liquid chromatography–tandem mass spectrometry; Validation according to 2002/657/EC; Restricted-access materials

A simple method of surface functionalisation for immuno-specific immobilisation of proteins by R. P. Kengne-Momo; Y. L. Jeyachandran; A. Assaf; C. Esnault; P. Daniel; J. F. Pilard; M. J. Durand; F. Lagarde; E. Dongo; G. Thouand (1249-1255).
We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.
Keywords: Surface functionalisation; Protein immobilisation; Raman spectroscopy; QCM

A novel FRET approach for in situ investigation of cellulase–cellulose interaction by Liqun Wang; Yiqing Wang; Arthur J. Ragauskas (1257-1262).
A novel real-time in situ detection method for the investigation of cellulase–cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)–cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase–cellulose study using cellulosic fiber.
Keywords: FRET; Cellulase; Cellulose; Carboxymethyl cellulose (CMC)

Quantum dots for positional registration in live cell-based arrays by Maureen A. Walling; Shengchun Wang; Hua Shi; Jason R. E. Shepard (1263-1271).
For a better understanding of complex biological processes, it is desirable to simultaneously follow the dynamics of multiple components in living cells or organisms in real time. An encoding scheme was developed that enables the observation of multiple cell populations with single-cell resolution. Specifically, different yeast cell types were labeled with quantum dots and added to an array of microwells, where they randomly self-assemble into the complementary-sized cavities. Quantum dots conjugated to cells externally, internally, or in combination generated unique optical patterns to differentiate various cell types in the array. For the model system described herein, cells were monitored for their lacZ expression levels through the processing of a fluorescent precursor by ß-galactosidase. The encoding schemes employed were independent of the reporter emission and had no affect on the cellular activity. The live cell array platform allowed analysis of hundreds of individual cells simultaneously and continuously in real time. By coupling this platform with quantum dot cell labeling, the utility of this array format is extended to mixed cellular populations. Figure Three images of individual cells localized on a microarray: (left) white light image. (center) false color image of the quantum dot-encoded cells, and (right) an overlay image of the white light and encoding images.
Keywords: Quantum dot; Live cell array; Encoding; Yeast

Investigating the relationship between cell cycle stage and diosgenin-induced megakaryocytic differentiation of HEL cells using sedimentation field-flow fractionation by Clementine Cailleteau; Ludovic Micallef; Clemence Lepage; Philippe Jean-Paul Cardot; Jean-Louis Beneytout; Bertrand Liagre; Serge Battu (1273-1283).
Differentiation therapy could be one strategy for stopping cancer cell proliferation. A plant steroid, diosgenin, is known to induce megakaryocytic differentiation in human erythroleukemia (HEL) cells. In recent studies, the use of sedimentation field-flow fractionation (SdFFF) allowed the preparation of subpopulations that may differ in regard to sensitivity to differentiation induction. The specific goal of this study was to determine the relationship between cell cycle stage and sensitivity to megakaryocytic differentiation induction of HEL cells. After first confirming the capacity of diosgenin to specifically select targets, hyperlayer SdFFF cell sorting was used to prepare fractions according to cell cycle position from crude HEL cells. The sensitivities of these fractions to diosgenin-induced differentiation were then tested. The coupling of SdFFF cell separation to imaging flow cytometry showed that G1-phase cells were more sensitive to differentiation induction than S/G2M-phase cells, confirming the relationship between cell status at the start of induction, the extent of the biological event, and the potential of SdFFF in cancer research.
Keywords: Sedimentation field-flow fractionation; Cell separation; Diosgenin; Human erythroleukemia cells; Megakaryocyte; Cell cycle; Differentiation

Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using two-dimensional gas chromatography time-of-flight mass spectrometry by Kishore Kumar Pasikanti; Juwita Norasmara; Shirong Cai; Ratha Mahendran; Kesavan Esuvaranathan; Paul C. Ho; Eric Chun Yong Chan (1285-1293).
In this study, gas chromatography mass spectrometry (GC-MS) and two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) were employed for the metabolic footprinting of a pair of immortalized human uroepithelial cells namely HUC-1 (nontumorigenic) and HUC T-2 (tumorigenic). Both HUC-1 and HUC T-2 cell lines were cultivated in 1 mL of Ham’s F-12 media. Subsequent to 48 h of incubation, 200 μL of cell culture supernatant was protein-precipitated using 1.7 mL of methanol and an aliquot of 1.5 mL of the mixture was separated, dried, trimethylsilyl-derivatized, and analyzed using GC-MS and GC×GC-TOFMS. Metabolic profiles were analyzed using multivariate data analysis techniques to evaluate the changes of the metabolomes. Both GC-MS and GC×GC-TOFMS analyses showed distinct differences in metabolic phenotypes of the normal and tumorigenic human bladder cells (partial least squares-discriminant analysis (PLS-DA) of GC×GC-TOFMS data; two latent variables, R 2 X = 0.418, R 2 Y = 0.977 and Q 2 (cumulative) = 0.852). Twenty metabolites were identified as being statistically different between the two cell types. These metabolites revealed that several key metabolic pathways were perturbed in tumorigenic urothelial cells as compared to the normal cells. Application of GC×GC-TOFMS offered several advantages compared to classical one-dimensional GC-MS which include enhanced chromatographic resolution (without increase in analytical run time), increase in sensitivity, improved identification of metabolites, and also separation of reagent artifacts from the metabolite peaks. Our results reinforced the advantages of GC×GC-TOFMS and the role of metabolomics in characterizing bladder cancer biology using in vitro cell culture models. Figure Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using GCxGCTOFMS
Keywords: Metabolomics; Metabolic footprinting; Metabolic profiling; Two-dimensional gas chromatography time-of-flight mass spectrometry; Bladder cancer; Metabonomics

Novel solid-phase refolding method for preparation of scFv-immobilized polystyrene plates with high-antigen-binding activity by Yoichi Kumada; Yuki Shiritani; Kyoko Hamasaki; Aya Nakagawa; Eiju Sasaki; Michimasa Kishimoto (1295-1303).
In the present study, we demonstrated site-specific immobilization and solid-phase refolding of single-chain Fv antibodies on hydrophilic polystyrene (phi-PS) plates that was mediated by novel polystyrene binding peptides (PS-tags: RIIIRRIRR), which were originally isolated and optimized in previous studies. Three PS-tag-fused scFvs, namely scFv-PS, scFv-(PS), and scFv-PSII, which were over-expressed in the insoluble fraction of Escherichia coli cells were denatured and site-specifically immobilized onto hydrophilic PS plates in the presence of 0.5~4 M urea and 0.1% Tween 20. The antigen-binding activity of the scFvs was efficiently recovered by washing the surface of the plate with PBS that contained 0.1% Tween 20 (PBST). The solid-phase refolding mediated by PS-tag was successfully applied to several scFvs such as mouse anti-CRP antibodies and an anti-RNase antibody, although further investigation of the versatility of scFv-PSII is needed. The maximal density of PS-tag-fused scFvs was increased more than 15-fold compared with a whole monoclonal antibody (mAb) immobilized on Maxisorp™ and, consequently, the sensitivity of PS-tag-fused scFvs for CRP in a sandwich ELISA was increased 25-fold. Thus, the novel, solid-phase, refolding method mediated by a PS-tag will be very useful for preparation of solid supports coated with recombinant antibody fragments, which can be used in immunoassays and immuno-separation.
Keywords: Site-specific immobilization; Solid-phase refolding; Recombinant antibody; ScFv; PS-tag; ELISA

Mass spectrometric detection of siRNA in plasma samples for doping control purposes by Maxie Kohler; Andreas Thomas; Katja Walpurgis; Wilhelm Schänzer; Mario Thevis (1305-1312).
Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL−1) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies. Online abstract figure A method for the mass spectrometric detection of siRNA molecules in doping control is described. siRNA, which blocks the translation of genes, could be used by athletes for illicit performance enhancement by e.g. down-regulating the myostatin gene for enhanced muscle growth.
Keywords: Doping; Sport; Exactive mass spectrometry; Oligonucleotide; Duplex

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin by Maria Tsivou; Helen A. Dimopoulou; Dimitris G. Georgakopoulos; Michael Α. Koupparis; Julia Atta-Politou; Costas G. Georgakopoulos (1313-1318).
The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes’ urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 °C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes’ urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation. Figure Changes in hCG concentration (mIU/ml) due to protease addition (200 μg/ml) with and without the chemical stabilization mixture following a 4-day incubation period at 37 °C
Keywords: Stabilization; Urine; Human chorionic gonadotropin; Proteases; Microorganisms; Doping control analysis

A method was developed for the simultaneous determination of six toxic alkaloids (aconitine, hypaconitine, gelsemine, raceanisodamine, strychnine, brucine) in blood and urine by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray tandem mass spectrometry. Ephedrine was selected as the internal standard. Samples were extracted and cleaned up by solid-phase extraction (SPE) using Oasis MCX cartridges. Separation parameters such as organic modifier, buffer pH, and concentration of buffer salt were investigated. Gradient separation and analysis were achieved for six alkaloids on a 3-μm Atlantis HILIC column using a mobile phase consisting of 30 mM ammonium formate and acetonitrile at pH 3. Two multiple reaction monitoring (MRM) transitions for each substance were monitored to provide sufficient identification of alkaloid. The retention mechanisms were explored in the method development. Validation included assessment of linearity, limit of quantification, accuracy, and precision. Bias was less than 15.1% and precision was better than 8.3% for both blood and urine samples. A total of 54 clinical samples were examined by this validated method.
Keywords: Hydrophilic interaction chromatography; LC–MS/MS; Alkaloids; SPE; Simultaneous determination

Bioactive molecules in Kalanchoe pinnata leaves: extraction, purification, and identification by Saïda El Abdellaoui; Emilie Destandau; Alix Toribio; Claire Elfakir; Michel Lafosse; Isabelle Renimel; Patrice André; Perrine Cancellieri; Ludovic Landemarre (1329-1338).
Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinnatum; family Crassulaceae) is a popular plant used in traditional medicine in many temperate regions of the world and particularly in South America. In Guyana, the leaves are traditionally used as an anti-inflammatory and antiseptic to treat coughs, ulcers, and sores. The purpose of this study was to implement a method for targeting and identifying molecules with antimicrobial activity, which could replace chemical preservatives in cosmetic applications. The leaves were extracted by a method based on pressurized liquid extraction (PLE), using different solvents. A study of antimicrobial activity and cytotoxicity tests were performed to select the most interesting extract. To isolate one or more active molecules, the selected crude extract was fractionated by centrifugal partition chromatography (CPC) and then antimicrobial activity and cytotoxicity of each fraction were tested under the same procedure. The last step consisted of identifying the main compounds in the most active fraction by LC-MS/MS.
Keywords: Kalanchoe pinnata ; Centrifugal partition chromatography; LC/MS/MS; Antimicrobial activity; Cytotoxicity

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 μL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0–10.0 × 103 ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible.
Keywords: Dried plasma spots; Butanol/HCl; Gabapentin; Spot homogeneity; Mass spectrometry

The design of boronic acid sensors for photometric detection of carbohydrates has relied on exploiting differences in the thermodynamic stability of complex formation for molecular recognition. Herein, we introduce a direct method for analysis of sugar alcohols using 3-nitrophenylboronic acid (NPBA) as an electrokinetic probe in capillary electrophoresis (CE). Dynamic complexation of neutral polyols by NPBA during electromigration allows for their simultaneous resolution and UV detection based on formation of an anionic ternary boronate ester complex in phosphate buffer. Unlike conventional boronic acid sensors, thermodynamic and electrokinetic processes in CE allow for improved selectivity for the resolution of sugar alcohol stereoisomers having different vicinal polyol chain lengths even in cases when binding affinity is similar due to differences in their complex mobility. Three complementary approaches were investigated to compare the thermodynamics of polyol chelation with NPBA, namely direct binding assays by CE, UV absorbance spectroscopy and an indirect pK a depression method. Overall, CE offers a convenient platform for characterization of reversible arylboronic acid interactions in free solution while allowing for direct analysis of complex mixtures of neutral/UV-transparent polyols without complicated sample handling. Figure 3-nitrophenylboronic acid (NPBA) functions as an electrokinetic probe in capillary electrophoresis for the separation and detection of polyols based on dynamic ternary boronate ester complexation in free solution.
Keywords: Capillary electrophoresis; Electrokinetic probe; Nitrophenylboronic acid; Polyols; Thermodynamics; UV absorbance

The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture. Figure AFM images and fluorescence anistrotropy profile of fluorescent probe inserted in different lipids used as mimetic systems for E. coli. The techniques confirm that cardiolipin plays an important role in mimicking the E. coli membrane.
Keywords: Lipid membranes; Cardiolipin; Dynamic light scattering; Fluorescence anisotropy; Atomic force microscopy

Quantitative estimation of riluzole in human plasma by LC-ESI-MS/MS and its application to a bioequivalence study by Babu Rao Chandu; Sreekanth Nama; Kanchanamala Kanala; Balasekhara Reddy Challa; Rihana Parveen Shaik; Mukkanti Khagga (1367-1374).
A novel simple, sensitive, selective, and rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantification of riluzole in human plasma. The chromatography was performed by using a Zorbax-SB-C18 (4.6 × 75 mm, 3.5 μm) column , isocratic mobile phase 0.1% formic acid/acetonitrile (10:90 v/v), and an isotope-labeled internal standard (IS), [13C,15N2]riluzole. The extraction of drug and internal standard was performed by liquid–liquid extraction and analyzed by MS in the multiple reaction monitoring (MRM) mode using the respective [M+H]+ ions, m/z 235.0/165.9 for riluzole and m/z 238.1/169.0 for the IS. The calibration curve was linear over the concentration range 0.5–500.0 ng/ml for riluzole in human plasma. The limit of quantification (LOQ) was demonstrated at 0.5 ng/ml. The within-batch and between-batch precision were 0.6–2.3% and 1.4–5.7%, and accuracy was 97.1–101.1% and 98.8–101.2% for riluzole respectively. Drug and IS were eluted within 3.0 min. The validated method was successfully applied in a bioequivalence study of riluzole in human plasma.
Keywords: Liquid chromatography; Mass spectrometry; Liquid–liquid extraction; Bioequivalence study; Pharmacokinetic study; Riluzole

Tracking bacterial infection of macrophages using a novel red-emission pH sensor by Yuguang Jin; Yanqing Tian; Weiwen Zhang; Sei-Hum Jang; Alex K.-Y. Jen; Deirdre R. Meldrum (1375-1384).
The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria. Figure Time-lapse confocal image analysis of bacterial infection into murine macrophage RAW 264.7. A) 0 sec, before infection; B) 25 sec; C) 7 min; D) 15 min; E) 20 min after bacterial infection; and F) the spectral analysis of the sensors in macrophages infected by E. coil shown in D. Spot 3 represents fluorescence from background. Spot 1, 2, 4, and 5 represent spectra from four selected areas in image D. Size-bars of 10 μm are included in the figures
Keywords: Red emitter; pH sensor; Bacterial infection; Mouse macrophage

Disposable sensors for rapid screening of mutated genes by T. García; M. G. Fernández-Barrena; M. Revenga-Parra; A. Núñez; E. Casero; F. Pariente; J. Prieto; E. Lorenzo (1385-1393).
A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH3)5 L]2+, where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at –0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/μL PCR product can be detected.
Keywords: DNA biosensor; Gene mutation; Single-nucleotide polymorphism; Mutation screening

Troponin T (TnT) is a useful biomarker for studying drug-induced toxicity effects on cardiac cells. We describe how a surface plasmon resonance (SPR) biosensor was applied to monitor the release of TnT from active HL-1 cardiomyocytes in vitro when exposed to cardiotoxic substances. Two monoclonal human TnT antibodies were compared in the SPR immunosensor to analyse the TnT release. The detection limit of TnT was determined to be 30 ng/ml in a direct assay set-up and to be 10 ng/ml in a sandwich assay format. Exposure of the cardiomyocytes to doxorubicin, troglitazone, quinidine and cobalt chloride for periods of 6 and 24 h gave significant SPR responses, whereas substances with low toxicity showed insignificant effects (ascorbic acid, methotrexate). The SPR results were verified with a validated immunochemiluminescence method which showed a correlation of r 2 = 0.790.
Keywords: Surface plasmon resonance; Troponin T; Cardiotoxicity; In vitro toxicity; HL-1 cardiomyocytes; Cytotoxicity screening

Electro-catalysis by immobilised human flavin-containing monooxygenase isoform 3 (hFMO3) by Silvia Castrignanò; Sheila J. Sadeghi; Gianfranco Gilardi (1403-1409).
Human flavin-containing monooxygenases are the second most important class of drug-metabolizing enzymes after cytochromes P450. Here we report a simple but functional and stable enzyme-electrode system based on a glassy carbon (GC) electrode with human flavin-containing monooxygenase isoform 3 (hFMO3) entrapped in a gel cross-linked with bovine serum albumin (BSA) by glutaraldehyde. The enzymatic electrochemical responsiveness is characterised by using well-known substrates: trimethylamine (TMA), ammonia (NH3), triethylamine (TEA), and benzydamine (BZD). The apparent Michaelis–Menten constant (KM) and apparent maximum current (Imax) are calculated by fitting the current signal to the Michaelis–Menten equation for each substrate. The enzyme-electrode has good characteristics: the calculated sensitivity was 40.9 ± 0.5 mA mol−1 L cm−2 for TMA, 43.3 ± 0.1 mA mol−1 L cm−2 for NH3, 45.2 ± 2.2 mA mol−1 L cm−2 for TEA, and 39.3 ± 0.6 mA mol−1 L cm−2 for BZD. The stability was constant for 3 days and the inter-electrode reproducibility was 12.5%. This is a novel electrochemical tool that can be used to investigate new potential drugs against the catalytic activity of hFMO3.
Keywords: Flavin-containing monooxygenase; Glassy carbon electrode; Glutaraldehyde gel; Bovine serum albumin

A glassy carbon electrode modified with palladium nanoparticles decorated multiwalled carbon nanotubes (GCE/nanoPd-MWCNTs) was fabricated. Incorporation of palladium nanoparticles onto the carbon nantube surface by thermal decomposition of palladium acetate led to the fabrication of a sensor with a significant decrease in hydrazine electrooxidation potential. The sensor exhibited low detection limits, high sensitivity and selectivity, rapid response, and good stability toward hydrazine detection. Figure SEM images and cyclic voltammograms for a bare GCE, GCE/MWCNTs, and GCE/nanoPd-MWCNTs in the absence (dashed line) and presence (solid line) of 1 mM N2H4. Conditions: supporting electrolyte, phosphate buffer solution (0.1 M and pH 7); scan rate, 50 mVs−1.
Keywords: Palladium nanoparticles; Multiwalled carbon nanotubes; Hydrazine; Electrocatalysis; Amperometric sensor

Studies of climate change increasingly recognize the diverse influences exerted by terpenes in the atmosphere, including roles in particulates, ozone formation, and their oxidizing potential. Measurements of key terpenes suggest atmospheric concentrations ranging from low pmol/mol (parts per trillion) to nmol/mol (parts per billion), depending on location and compound. To accurately establish concentration trends, assess the role of terpenes in atmospheric chemistry, and relate measurement records from many laboratories and researchers, it is essential to have good calibration standards. The feasibility of preparing well-characterized, stable gas cylinder standards for terpenes at the nmol/mol level is not yet well established. Several of the world’s National Metrology Institutes (NMIs) are researching the feasibility of developing primary and secondary reference gas standards at the nmol/mol level for terpenes. The US NMI, the National Institute of Standards and Technology, has prepared several nmol/mol mixtures, in treated aluminum gas cylinders, containing terpenes in dry nitrogen at nominal 5 nmol/mol for stability studies. Overall, 11 terpenes were studied for stability. An initial gas mixture containing nine terpenes, one oxygenate, and six aromatic compounds, including benzene as an internal standard, was prepared. Results for four of the nine terpenes in this initial mixture indicate stability in these treated aluminum gas cylinders for over 6 months and project long term (years) stability. Interesting results were seen for β-pinene, which when using a linear equation rate decline predicts that it will reach a zero concentration level at day 416. At the same time, increases in α-pinene, d-limonene (R-(+)-limonene), and p-cymene were observed, including camphene, a terpene not prepared in the gas mixture, indicating a chemical transformation of β-pinene to these species. Additional mixtures containing combination of either α-pinene, camphor, α-terpinene, and benzene indicate a second-order quadratic rate decline for the α-pinene and α-terpinene, a linear rate decline for camphor, and a second-order quadratic rate increase of camphene.
Keywords: Terpenes; Internal standard; Growth rate; Isomerization; Degradation; Gas mixtures in aluminum cylinders; nmol/mol; pmol/mol

In this paper, the direct coupling between stir membrane extraction and infrared spectroscopy working under transmission mode is presented for the sensitive and selective determination of the total hydrocarbon index in waters. For this purpose, a new extraction unit was built using stainless steel in order to maximize the adsorption of the target analytes in the 40-μm-thick polytetrafluoroethylene membrane. The method allows the determination of hydrocarbons in the presence of grease, using hexadecane and stearic acid as model compounds, respectively. The proposal is optimized in depth, taking into account the main experimental variables such as membrane thickness, extraction time, and stirring and sample volume. Later on, the method was characterized on the basis of its linearity, precision, and limits of detection. The combination allows the determination of the hydrocarbon index with a limit of detection of 18 μg L−1, the precision being (expressed as relative standard deviation) better than 4.3%. The analytical method provides a high sample throughput since some extractions can be performed in parallel, the relative standard deviation between devices being better than 8%. The proposed analytical method is finally compared in terms of analytical figures with counterpart ASTM method, recently presented. Stir membrane extraction using infrared-transparent materials is a useful tool for the determination of the hydrocarbon index in water samples. The hydrocarbons are isolated and preconcentrated in a polymeric membrane which is later on monitored by infrared spectroscopy. This approach allows the determination of the hydrocarbon index with a limit of detection of 18 μgL-1 and precision better than 4.3%.
Keywords: Stir membrane extraction; Infrared spectroscopy; Hydrocarbon index; Waters

Comprehensive multidimensional separation methods by hyphenation of single-photon ionization time-of-flight mass spectrometry (SPI-TOF-MS) with GC and GC×GC by Markus S. Eschner; Werner Welthagen; Thomas M. Gröger; Marc Gonin; Katrin Fuhrer; Ralf Zimmermann (1435-1445).
One- and comprehensive two-dimensional gas chromatography were hyphenated with soft photoionization mass spectrometry. The characteristics of these two- and three-dimensional comprehensive separation techniques are discussed in detail. Using the innovative electron beam pumped excimer light source (EBEL) for single-photon ionization (SPI), organic molecules with ionization energies (E i ) of below 9.8 eV can be detected by a time-of-flight mass spectrometer (TOF-MS). SPI with 126 nm vacuum ultraviolet (VUV) photons enables the universal and soft ionization of organic molecules. SPI-TOF-MS hyphenated to one-dimensional gas chromatography results in a comprehensive two-dimensional separation method (GC×MS). To demonstrate this, diesel fuel was analyzed, and the resulting GC×MS chromatograms are discussed in depth. A three-dimensional separation method was also realized by combining comprehensive two-dimensional gas chromatography (GC×GC) with SPI-MS. In the resulting separation space, constituents originating from mineral oil diesel blended with biodiesel were dispersed along the two GC separation axes, while the molecular mass axis served as a third separation dimension. Figure Sketch of the innovative VUV light source EBEL and three-dimensional representation of a GC×MS analysis of diesel fuel
Keywords: Multidimensional separation; Single-photon ionization; Time-of-flight mass spectrometry; Excimer light source; Diesel fuel

Screening of perfluorinated chemicals (PFCs) in various aquatic organisms by María Fernández-Sanjuan; Johan Meyer; Joana Damásio; Melissa Faria; Carlos Barata; Silvia Lacorte (1447-1456).
The aim of this study was to evaluate the occurrence of five perfluorinated chemicals (perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonic acid (PFHxS), and perfluorobutane sulfonic acid) in aquatic organisms dwelling in either freshwater or marine ecosystems. Organisms selected were insect larvae, oysters, zebra mussels, sardines, and crabs, which are widespread in the environment and may represent potential bioindicators of exposure to PFCs. The study comprises the optimization of a solid–liquid extraction method and determination by high-performance liquid chromatography coupled to tandem mass spectrometry. Using spiked zebra mussels at 10 and 100 ng/g level, the method developed provided recoveries of 96% and 122%, and 82% to 116%, respectively, and a limit of detection between 0.07 and 0.22 ng/g ww. The method was highly sensitivity and robust to determine PFC compounds in a wide array of biological matrices, and no matrix interferents nor blank contamination was observed. Among organisms studied, none of the bivalves accumulated PFCs, and contrarily, insect larvae, followed by fish and crabs contained levels ranging from 0.23 to 144 ng/g ww of PFOS, from 0.14 to 4.3 ng/g ww of PFOA, and traces of PFNA and PFHxS. Assessment of the potential use of aquatic organisms for biomonitoring studies is further discussed.
Keywords: Perfluorinated compounds; LC-MS/MS; Aquatic organisms; Biomonitoring

Chloramphenicol (CAP), an effective antibiotic against many microorganisms, is meanwhile banned in the EU for treatment of food-producing animals due to adverse health effects. The Institute for Reference Materials and Measurements (IRMM) is currently developing a certified reference material (CRM) for CAP in pork, intended for validation and method performance verifications of analytical methods. The material will be certified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methods and has a target CAP level around the minimum required performance limit (MRPL) of 0.3 μg/kg. To prove that the material can be applied as a quality control tool for screening methods, a commutability study was conducted, involving five commercially available enzyme-linked immunosorbent assay kits and one biosensor assay (BiaCore kit). Meat homogenates (cryo-milled wet tissue) with CAP concentrations around the MRPL and the candidate CRM (lyophilised powder) were measured by LC–MS/MS and GC–MS as well as the six screening methods. Pairwise method comparisons of results obtained for the two sample types showed that the CRM can successfully be applied as quality control (QC) sample to all six screening methods. The study suggests that ERM-BB130 is sufficiently commutable with the investigated assays and that laboratories applying one of the investigated kits therefore benefit from using ERM-BB130 to demonstrate the correctness of their results. However, differences among the assays were observed, either in the abundance of bias between screening and confirmatory LC and GC methods, the repeatability of test results, or goodness of fit between the methods.
Keywords: Commutability; Certified reference material (CRM); ELISA; Biosensor; Chloramphenicol

Hydrodistillation–liquid-phase microextraction for infrared analysis of food by Ana Gonzálvez; Salvador Garrigues; Sergio Armenta; Miguel de la Guardia (1467-1476).
A combination of hydrodistillation (HD) and liquid-phase microextraction (LPME) has been successfully developed to improve sensitivity and selectivity in attenuated total reflection (ATR) infrared determination of semivolatile organic compounds from high water content plant and food matrices contributing to solve extraction efficiency drawbacks. The HD sampling facilitates the extraction of the semivolatile analytes from the sample matrix compared to headspace sampling, while the liquid-phase microextraction using a water immiscible solvent allows analyte preconcentration prior to ATR analysis. Experimental conditions regarding temperature and time of extraction, water effect and number of consecutive extractions have been deeply studied. The qualitative and quantitative capability of the developed methodology has been evaluated through the identification of the main semivolatile substances in plant and food matrices like spices and citrus peels and the effect of different drying treatments on the volatile composition of rosemary samples was studied through the quantification of camphor and eucalyptol.
Keywords: Attenuated total reflectance; Infrared; Hydrodistillation; Liquid-phase microextraction; Semivolatile organic compounds; Plant materials; Spices

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods by Isa Ruppen Canás; Keith O’Callaghan; Cian Moroney; Brett Hamilton; Kevin J. James; Ambrose Furey (1477-1491).
The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg−1 are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.
Keywords: Marine toxins; Azaspiracids; Azaspiracid shellfish poisoning; AZP; Mass spectrometry; Quadrupole ion-trap MS; LC–MS–MS; QTOF MS

Follow-up of stable isotope analysis of organic versus conventional milk by Joachim Molkentin; Anette Giesemann (1493-1500).
Analysis of the stable isotope ratio of carbon (δ 13C) and α-linolenic acid (C18:3ω3) content in milk fat is a useful indicator of organic milk production. Referring to corresponding measurements, further analyses of stable isotope ratios were performed in 120 samples of conventionally and organically produced whole milk collected from German retailers during a period of 18 months. Conventional milk predominantly exhibited higher δ 15N values than organic milk, the latter of which never exceeded a maximum δ 15N threshold value of 5.50‰. Measurements of δ 34S did not differ significantly between organic and conventional milk. Because δ 13C, in general, is related to maize consumption, δ 13C in milk protein and δ 13C in milk fat were equally suited for authentication of organic milk. Thus, a high correlation (r = 0.99) was established between δ 13C in milk protein and lipids. Although occurring on different levels in organic and conventional milk, the relatively constant fractionation of carbon isotopes between protein and fat will allow for the advanced detection of adulteration in processed milk products, such as fraudulent combinations of organic milk fat and conventional skim milk. In addition to the strong correlation between C18:3ω3 and δ 13Cprotein (r = −0.91), a mutual dependence was identified between both δ 13Cprotein and δ 15N (r = 0.66) and C18:3ω3 and δ 15N (r = −0.61). Thus, multi-variable analyses are useful to increase robustness and reduce the number of exceptions in organic milk authentication. Future work involving multivariate statistical analysis can possibly further improve milk authentication in various respects including differentiating between brands of retail milk. Figure Correlation (r = 0.66) between δ 13Cprotein and δ 15N in conventional (CRM) and organic (ORM) retail milk
Keywords: Nitrogen; Sulfur; Carbon; Stable isotopes; Organic milk; Authentication

A method involving simultaneous extraction and sample clean-up procedure: hollow fiber sorptive microextraction, coupled with gas chromatography–mass spectrometric detection for quantification of seven organochlorine pesticides in Radix et Rhizoma Rhei is described. SiO2 hollow fiber with porous structure was synthesized for the first time. The internal diameter of SiO2 hollow fiber is 380 μm and average wall thickness is 100 μm. Aggregated SiO2 particles deposited on the surface of the hollow fiber in a regular array lead to porous structure. SiO2 hollow fiber was applied to the determination of organochlorine pesticides in Radix et Rhizoma Rhei to avoid sample clean-up and minimize the matrix effects. Extraction solvent, extraction temperature and equilibration time were optimized. Fiber to fiber repeatability over the concentration ranges were less than 10%. Recoveries were satisfactory (between 63% and 115%) for most of organochlorine pesticides at spiking levels. Furthermore, the proposed method was also applied to determine seven organochlorine pesticides in 43 commercial Radix et Rhizoma Rhei samples, in which the selected pesticides were found in eight samples. The results have been further confirmed by solvent extraction methods according to China Pharmacopoeia (2005). Figure GC-MS-SIM chromatograms of organochlorine pesticides in Radix et Rhizoma Rhei extracted with SiO2 hollow fibers (inset SEM of SiO2 hollow fiber).
Keywords: Organochlorine pesticides; SiO2 hollow fiber; Hollow fiber sorptive microextraction; Radix et Rhizoma Rhei; Gas chromatography–mass spectrometry

In this work, a simple and low-cost method based on matrix solid-phase dispersion (MSPD) and gas chromatography to determine eight multi-class pesticides such as vinclozolin, dichlofluanid, penconazol, captan, quinoxyfen, fluquinconazol, boscalid, and pyraclostrobin in grapes is described. Fungicide residues were identified and quantified using gas chromatography–mass spectrometry in selected ion monitoring mode (GC-MS, SIM). The experimental variables that affect the MSPD method, such as the amount of solid phase, solvent nature and elution volume were optimized using an experimental design. The best results were obtained using 0.5 g of grapes, 1.0 g of silica as clean-up sorbent, 1.50 g of C18 as bonded phase and 10 mL of dichloromethane/ethyl acetate (1:1, v/v) as eluting solvent. Significant matrix effects observed for most of the pesticides tested were eliminated using matrix-matched standards. The pesticide recoveries in grapes samples were better than 80% except for captan. Intra-laboratory precision in terms of Horwitz ratio of the pesticides evaluated was below 0.5, suggesting ruggedness of the method. The quantification limits of the pesticides were in the range of 3.4–8.7 μg kg−1, which were lower than the maximum residue limits (MRLs) of the pesticides in grapes samples established by the European legislation. Decision limits (CCα) and detection capability (CCβ) have been calculated. The expanded uncertainties at two levels of concentration were <20% for all analytes.
Keywords: Matrix solid-phase dispersion; Experimental design; Uncertainty; Grapes; Gas chromatography

A compact miniaturized continuous flow system for the determination of urea content in milk by Willian Toito Suarez; Osmundo Dantas Pessoa-Neto; Vagner Bezerra dos Santos; Ana Rita de Araujo Nogueira; Ronaldo Censi Faria; Orlando Fatibello-Filho; Mar Puyol; Julián Alonso (1525-1533).
A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the urea concentration range from 1.0 × 10−4 to 5.0 × 10−3 mol L−1 with a limit of detection of 8.0 × 10−6 mol L−1. The relative standard deviation (RSD) for a 2.0 × 10−3 mol L−1 urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h−1. To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the data are in close agreement, at a 95% confidence level.
Keywords: Urea; Milk; Low temperature co-fired ceramics; Berthelot’s reaction; Multicommutation; Mini-photometer

Simultaneous determination of ochratoxin A, mycophenolic acid and fumonisin B2 in meat products by Louise Marie Sørensen; Jesper Mogensen; Kristian Fog Nielsen (1535-1542).
Here we present a method for simultaneous determination of the fungal metabolites mycophenolic acid, ochratoxin A (OTA) and fumonisin B2 (FB2) in meat products. Extraction was performed with water–acetonitrile, followed by acetone-induced precipitation of salts and proteins. Purification and identification of analytes was performed by mixed-mode reversed-phase anion-exchange chromatography in direct ion-exchange mode, followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection. Quantification was based on isotope dilution with fully 13C-labelled FB2 and OTA, and matrix-spiked calibration curves. Fermented sausages inoculated with an OTA- and FB2-producing strain of Aspergillus niger were analysed, but no analytes were detected. Analysis of 22 retail products showed one Parma meat with a very high level of OTA contamination (56–158 μg/kg) that clearly exceeded the Italian regulatory limit of 1 μg/kg. This sample and uninfected control samples were subsequently reanalysed, and the high OTA content was verified by two other techniques: (i) LC–time-of-flight MS confirmed the accurate mass as well as chlorine isotope pattern; and (ii) sample methylation in methanol–BF3 and subsequent LC-MS/MS provided indirect confirmation by detection of the OTA methyl ester. In the contaminated Parma ham, the high OTA level most likely originated from growth of Penicillium nordicum on the meat.
Keywords: Filamentous fungi; Meat products; Mycotoxins; Isotope dilution; Mixed-mode reversed-phase anion exchange; LC-MS/MS