Analytical and Bioanalytical Chemistry (v.403, #10)

Recent advances in food analysis by Jana Hajslova; Rudolf Krska; Michel W. F. Nielen (2795-2796).
is a Professor at the Department of Food Chemistry and Analysis at the Institute of Chemical Technology in Prague (CZ). Her research interests focus on the fate of contaminants and toxicants in food chains and issues related to the implementation of novel strategies for their analysis. She is a board member of the EU-backed Advisory Group on Food Quality and Safety and a member of the European Union’s Scientific Committee on Emerging and Newly Identified Health Risks. Professor Hajšlová has participated in many international research projects and has established close collaboration with several institutions, for example the UN’s WHO and FAO, and the European Commission’s Joint Research Centre. She has published widely on organic contaminants and food safety. is Full Professor of Bioanalysis and Organic Trace Analysis and is head of the Department of Agrobiotechnology, IFA-Tulln, at the University of Natural Resources and Life Sciences, Vienna (BOKU). He obtained his degree in chemistry at the Vienna University of Technology and is an expert in food and feed analysis by chromatographic, mass spectrometric, and immunoanalytical techniques. As member of JECFA (Joint Expert Committee for Food Additives) of the FAO/WHO he has evaluated the effect of trichothecene mycotoxins on humans. He has received 6 scientific awards and is (co-)author of more than 258 scientific publications. In 2009/2010 he worked for a year as A/Chief of Health Canada´s Food Research Division in Ottawa. is senior scientist in residue analysis at RIKILT and scientific director of TI-COAST, the Dutch public–private partnership on analytical science and technology. He holds a chair in detection of food contaminants at Wageningen University. He obtained his degree in analytical chemistry at the Free University of Amsterdam. He is co-founder and co-chairman of the symposium series on recent advances in food analysis. He is (co-)author of more than 150 peer-reviewed publications.

Dioxin food crises and new POPs: challenges in analysis by Jean-François Focant (2797-2800).
is Associate Professor in the Chemistry Department of the University of Liège, Belgium. He has worked on the development and implementation of emerging analytical strategies for food control and human biomonitoring since the very beginning of his career. Known as a dioxin expert, he is also active in other areas of separation science, for example characterization of complex mixtures of volatile organic compounds (VOCs) for medical and forensic applications. Working on the hyphenation of state-of-the-art analytical techniques to solve practical analytical issues is what he enjoys doing most

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS3 quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS3 (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg−1 (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90–108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg−1. A quantitation limit of 2–6 μg kg−1 was achieved by applying an MRM transition quantitation strategy. Under MS3 mode, a quantitation limit of 4–10 μg kg−1 was achieved. Relative standard deviations of 2–10% and 2–11% were reported for MRM transition and MS3 quantitation, respectively. The successful utilization of MS3 enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition. Figure The power of QTRAP® technology
Keywords: LC-MS/MS; Grains; MRM transition; MS/MS/MS (MS3); Quantitation; Tricothecenes

Rapid screening of pesticides present on the surfaces of fruits and vegetables has been facilitated by using a Direct Analysis in Real Time (DART®) open air surface desorption ionization source coupled to an Exactive® high-resolution accurate mass benchtop orbitrap mass spectrometer. The use of cotton and polyester cleaning swabs to collect and retain pesticides for subsequent open air desorption ionization is demonstrated by sampling the surface of various produce to which solutions of pesticides have been applied at levels 10 and 100 times below the tolerance levels established by the United States Environmental Protection Agency (US EPA). Samples analyzed include cherry tomatoes, oranges, peaches and carrots each chosen for their surface characteristics which include: smooth, pitted, fuzzy, and rough respectively. Results from the direct analysis of fungicides on store-bought oranges are also described. In all cases, the swabs were introduced directly into the heated ionizing gas of the DART source resulting in production of protonated pesticide molecules within seconds of sampling. Operation of the orbitrap mass spectrometer at 25,000 full-width half maximum resolution was sufficient to generate high-quality accurate mass data. Stable external mass calibration eliminated the need for addition of standards typically required for mass calibration, thus allowing multiple analyses to be completed without instrument recalibration.
Keywords: Direct analysis in real time (DART); Pesticide screening; High-resolution accurate mass (HRAM); Benchtop orbitrap; Surface analysis

In the presented study, comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC–TOFMS) was shown to be a powerful tool for the simultaneous determination of various groups of contaminants including 18 polychlorinated biphenyls (PCBs), seven polybrominated diphenyl ethers (PBDEs), and 16 polycyclic aromatic hydrocarbons (PAHs). Since different groups of analytes (traditionally analyzed separately) were included into one instrumental method, significant time savings were achieved. Following the development of an integrated sample preparation procedure for an effective and rapid isolation of several groups of contaminants from fish tissue, the GC × GC–TOFMS instrumental method was optimized to obtain the best chromatographic resolution and low quantification limits (LOQs) of all target analytes in a complex mixture. Using large-volume programmable temperature vaporization, the following LOQs were achieved—PCBs, 0.01–0.25 μg/kg; PBDEs, 0.025–5 μg/kg; PAHs 0.025–0.5 μg/kg. Furthermore, several capillary column combinations (BPX5, BPX50, and Rxi-17Sil-ms in the first dimension and BPX5, BPX50, Rt-LC35, and HT8 in the second dimension) were tested during the experiments, and the optimal separation of all target analytes even of critical groups of PAHs (group (a): benz[a]anthracene, cyclopenta[cd]pyrene and chrysene; group (b): benzo[b]fluoranthene, benzo[j]fluoranthene and benzo[k]fluoranthene; group (c): dibenz[ah]anthracene, indeno[1,2,3–cd]pyrene and benzo[ghi]perylene) was observed on BPX5 × BPX50 column setup. Moreover, since the determination of target analytes was performed using TOFMS detector, further identification of other non-target compounds in real life samples was also feasible.
Keywords: Fish; GC × GC–TOFMS; PAH; PBDE; PCB; PTV

Development and validation of an SPE HG-AAS method for determination of inorganic arsenic in samples of marine origin by Rie R. Rasmussen; Rikke V. Hedegaard; Erik H. Larsen; Jens J. Sloth (2825-2834).
The present paper describes a novel method for the quantitative determination of inorganic arsenic (iAs) in food and feed of marine origin. The samples were subjected to microwave-assisted extraction using diluted hydrochloric acid and hydrogen peroxide, which solubilised the analytes and oxidised arsenite (AsIII) to arsenate (AsV). Subsequently, a pH buffering of the sample extract at pH 6 enabled selective elution of AsV from a strong anion exchange solid-phase extraction (SPE) cartridge. Hydride generation atomic absorption spectrometry (HG-AAS) was applied to quantify the concentration of iAs (sum of AsIII and AsV) as the total arsenic (As) in the SPE eluate. The results of the in-house validation showed that mean recoveries of 101–104% were achieved for samples spiked with iAs at 0.5, 1.0 and 1.5 mg·kg−1, respectively. The limit of detection was 0.08 mg kg−1, and the repeatability (RSDr) and intra-laboratory reproducibility (RSDIR) were less than 8% and 13%, respectively, for samples containing 0.2 to 1.5 mg kg−1 iAs. The trueness of the SPE HG-AAS method was verified by confirming results obtained by parallel analysis using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. It was demonstrated that the two sets of results were not significantly different (P < 0.05). The SPE HG-AAS method was applied to 20 marine food and feed samples, and concentrations of up to 0.14 mg kg−1 of iAs were detected.
Keywords: Inorganic arsenic; Marine samples; SPE HG-AAS; Microwave-assisted extraction; Food and feed control; Speciation

Quantification of nanoparticles in aqueous food matrices using Particle-Induced X-ray Emission by Omar Lozano; Jorge Mejia; Tijani Tabarrant; Bernard Masereel; Jean-Michel Dogné; Olivier Toussaint; Stéphane Lucas (2835-2841).
Nanoparticles (NPs) of SiO2 (15 nm) or Ag (20 – 40 nm) were dispersed in water, coffee and milk at several aqueous dilutions. The NPs dispersions concentrations were quantified with an ion beam technique: Particle-Induced X-ray Emission. Additional measurements in relation to the state of the NPs dispersions were done: particle size distribution by centrifuge liquid sedimentation and the extreme surface composition by X-ray photoelectron spectroscopy. The particle size distribution of SiO2 and Ag NPs dispersions in water and Ag NPs in coffee remained mostly as primary particles with hydrodynamic diameters close to the reported pristine NPs diameter. SiO2 NPs agglomerated in coffee. In milk, both NPs presented an adsorption with milk lipids. Extreme surface composition corroborated adsorption in milk and showed that SiO2 agglomerates adsorb some coffee components. A linear tendency in the measurement of the concentration dilutions of all dispersions was measured, and a lack of media influence in the slope of each curve was found. Limits of detection with the current setup were estimated at 0.5 and 0.3 mg/ml for SiO2 and Ag NPs, respectively.
Keywords: PIXE; Nanoparticle; Liquids; Food; Particle size distribution

Development of surface plasmon resonance-based sensor for detection of silver nanoparticles in food and the environment by Sabina Rebe Raz; Maria Leontaridou; Maria G. E. G. Bremer; Ruud Peters; Stefan Weigel (2843-2850).
Silver nanoparticles are recognized as effective antimicrobial agents and have been implemented in various consumer products including washing machines, refrigerators, clothing, medical devices, and food packaging. Alongside the silver nanoparticles benefits, their novel properties have raised concerns about possible adverse effects on biological systems. To protect consumer’s health and the environment, efficient monitoring of silver nanoparticles needs to be established. Here, we present the development of human metallothionein (MT) based surface plasmon resonance (SPR) sensor for rapid detection of nanosilver. Incorporation of human metallothionein 1A to the sensor surface enables screening for potentially biologically active silver nanoparticles at parts per billion sensitivity. Other protein ligands were also tested for binding capacity of the nanosilver and were found to be inferior to the metallothionein. The biosensor has been characterized in terms of selectivity and sensitivity towards different types of silver nanoparticles and applied in measurements of real-life samples—such as fresh vegetables and river water. Our findings suggest that human MT1-based SPR sensor has the potential to be utilized as a routine screening method for silver nanoparticles, that can provide rapid and automated analysis dedicated to environmental and food safety monitoring. Figure Surface plasmon resonance biosensor for rapid detection of silver nanoparticles was developed. Human metallothionein 1A (hMT1A) was immobilized on the sensor chip surface and showed dose dependent binding to the silver nanoparticles with part per billion sensitivity.
Keywords: Silver nanoparticles; Metallothionein; Food; Environment; Surface plasmon resonance

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit by Vincent Dehalu; Stefan Weigel; Sabina Rebe; Ringo Grombe; Raimar Löbenberg; Philippe Delahaut (2851-2857).
Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL−1 using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined—0.42, 0.27, 0.26, and 0.24 μg mL−1 for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.
Keywords: Gelatin nanoparticles; Nano-sensor; Antibodies; ELISA screening

DART-Orbitrap MS: a novel mass spectrometric approach for the identification of phenolic compounds in propolis by Elena S. Chernetsova; Maciej Bromirski; Olaf Scheibner; Gertrud E. Morlock (2859-2867).
This is the first direct analysis in real-time mass spectrometry (DART-MS) study of propolis and a first study on the analysis of bee products using high-resolution DART-MS (DART-HRMS). Identification of flavonoids and other phenolic compounds in propolis using direct analysis in real-time coupling with Orbitrap mass spectrometry (DART-Orbitrap MS) was performed in the negative ion mode for minimizing the matrix effects, while the positive ion mode was used for the confirmation of selected compounds. Possible elemental formulae were suggested for marker components. The duration of one sample analysis by DART-MS analysis lasted ca. 30 s, and all benefits of high-resolution mass spectrometry were used upon data processing using the coupling of DART with the Orbitrap mass spectrometer. The possibility for scanning analysis of dried propolis extract spots on a planar porous surface was investigated in the heated gas flow of the DART ion source with adjustable angle. As an independent method, the approach of scanning analysis is of high interest and of future potential for confirmation of the results obtained from liquid sample analysis. Scanning analysis is highly promising for further development in the bioanalytical field due to the convenience of the storage and transportation of dried sample spots.
Keywords: Direct analysis in real-time; DART-Orbitrap MS; Negative ion; Phenolic compounds; Propolis; Dried extract spots

Adhesives used in food packaging to glue different materials can provide several substances as potential migrants, and the identification of potential migrants and migration tests are required to assess safety in the use of adhesives. Solid-phase microextraction in headspace mode and gas chromatography coupled to mass spectrometry (HS-SPME-GC-MS) and ChemSpider and SciFinder databases were used as powerful tools to identify the potential migrants in the polyurethane (PU) adhesives and also in the individual plastic films (polyethylene terephthalate, polyamide, polypropylene, polyethylene, and polyethylene/ethyl vinyl alcohol). Migration tests were carried out by using Tenax® and isooctane as food simulants, and the migrants were analyzed by gas chromatography coupled to mass spectrometry. More than 63 volatile and semivolatile compounds considered as potential migrants were detected either in the adhesives or in the films. Migration tests showed two non-intentionally added substances (NIAS) coming from PU adhesives that migrated through the laminates into Tenax® and into isooctane. Identification of these NIAS was achieved through their mass spectra, and 1,6-dioxacyclododecane-7,12-dione and 1,4,7-trioxacyclotridecane-8,13-dione were confirmed. Caprolactam migrated into isooctane, and its origin was the external plastic film in the multilayer, demonstrating real diffusion through the multilayer structure. Comparison of the migration values between the simulants and conditions will be shown and discussed. Figure Cross-section of multilayer packaging material (polyethylene terephthalate/adhesive /oriented polyamide/adhesive/cast polypropylene) viewed by optical microscopy
Keywords: Food safety; Multilayer food packaging; Polyurethane adhesives; Migration; NIAS

Rapid analysis of caffeine in various coffee samples employing direct analysis in real-time ionization–high-resolution mass spectrometry by Hana Danhelova; Jaromir Hradecky; Sarka Prinosilova; Tomas Cajka; Katerina Riddellova; Lukas Vaclavik; Jana Hajslova (2883-2889).
The development and use of a fast method employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) for the quantitative analysis of caffeine in various coffee samples has been demonstrated in this study. A simple sample extraction procedure employing hot water was followed by direct, high-throughput (<1 min per run) examination of the extracts spread on a glass rod under optimized conditions of ambient mass spectrometry, without any prior chromatographic separation. For quantification of caffeine using DART-TOFMS, an external calibration was used. Isotopically labeled caffeine was used to compensate for the variations of the ion intensities of caffeine signal. Recoveries of the DART-TOFMS method were 97% for instant coffee at the spiking levels of 20 and 60 mg/g, respectively, while for roasted ground coffee, the obtained values were 106% and 107% at the spiking levels of 10 and 30 mg/g, respectively. The repeatability of the whole analytical procedure (expressed as relative standard deviation, RSD, %) was <5% for all tested spiking levels and matrices. Since the linearity range of the method was relatively narrow (two orders of magnitude), an optimization of sample dilution prior the DART-TOFMS measurement to avoid saturation of the detector was needed.
Keywords: Ambient mass spectrometry; Direct analysis in real time; Coffee; Caffeine

The analytical capabilities of liquid chromatography with single-stage high-resolution mass spectrometry have been investigated with emphasis on qualitative aspects related to selective detection during screening and to identification. The study involved 21 different vegetable and fruit commodities, a screening database of 556 pesticides for evaluation of false positives, and a test set of 130 pesticides spiked to the commodities at 0.01, 0.05, and 0.20 mg/kg for evaluation of false negatives. The final method involved a QuEChERS-based sample preparation (without dSPE clean up) and full scan acquisition using alternating scan events without/with fragmentation, at a resolving power of 50,000. Analyte detection was based on extraction of the exact mass (±5 ppm) of the major adduct ion at the database retention time ±30 s and the presence of a second diagnostic ion. Various options for the additional ion were investigated and compared (other adduct ions, M + 1 or M + 2 isotopes, fragments). The two-ion approach for selective detection of the pesticides in the full scan data was compared with two alternative approaches based on response thresholds. Using the two-ion approach, the number of false positives out of 11,676 pesticide/commodity combinations targeted was 36 (0.3 %). The percentage of false negatives, assessed for 2,730 pesticide/commodity combinations, was 13 %, 3 %, and 1 % at the 0.01-, 0.05-, and 0.20-mg/kg level, respectively (slightly higher with fully automated detection). Following the SANCO/12495/2011 protocol for validation of screening methods, the screening detection limit was determined for 130 pesticides and found to be 0.01, 0.05, and ≥0.20 mg/kg for 86, 30, and 14 pesticides, respectively. For the detected pesticides in the spiked samples, the ability for unambiguous identification according to EU criteria was evaluated. A proposal for adaption of the criteria was made. Figure Extracted ion chromatograms showing availability of various diagnostic ions after analysis by LC with full scan high resolution MS, enabling selective detection and identification of pesticides. Example shown: propoxur at 0.01 mg/kg in apple.
Keywords: Screening; LC–high-resolution MS; Validation; Identification; Pesticides; Vegetables and fruits

Common wheat determination in durum wheat samples through LC/MS analysis of gluten peptides by Barbara Prandi; Mariangela Bencivenni; Tullia Tedeschi; Rosangela Marchelli; Arnaldo Dossena; Gianni Galaverna; Stefano Sforza (2909-2914).
A method to detect the presence of common wheat in durum wheat flour samples was developed and tested. Flour samples, or ground wheat samples, were digested by pepsin and chymotrypsin, and the peptide mixture obtained was analyzed by LC/ESI-MS and LC/ESI-MS/MS, which led to the identification of two marker peptides. One peptide was coded only in the DD genome, and thus present only in common wheat; the second was present in all wheat samples (both common and durum), so it was used as marker of the total wheat content. The ratio of the chromatographic areas of these two peptides, as determined by LC/ESI-MS, was related to the proportion of common wheat in the sample using a calibration curve that was constructed with standards of known composition. The proportions of common wheat in samples obtained by mixing different common and durum wheat varieties were accurately determined by this method. Finally, the method was applied in a survey of several durum wheat flour brands present on the Italian market. The results of the survey revealed that contamination of durum wheat flour with common wheat is commonplace.
Keywords: Common wheat; Durum wheat; Food authenticity; Gluten peptides; LC/MS

This study describes a rapid and sensitive analytical method for the determination of amino acids in foods and drinks. The method entailed dilution or extraction of amino acids from foods using the mixture of acetonitrile and 0.1% aqueous formic acid (50:50, v/v). Chromatographic separation of underivatized amino acids was performed using a hydrophilic interaction liquid chromatography within a runtime of 6 min. Both hydrophobicity and charge of the side chain played important roles on the elution order of amino acids under the chromatographic conditions. High-resolution mass spectrometry allowed qualitative and quantitative detection of amino acids in complex food matrices. Its response was found linear over a concentration range of 0.25–10 μg/ml. The method could be successfully applied to various foods and drinks to profile individual amino acids. Mean percentage recoveries of amino acids from different matrices were 88.5% or higher with residual standard deviation of less than 5.0%.
Keywords: Amino acids; Food; Hydrophilic interaction liquid chromatography; High-resolution mass spectrometry

Identification and quantification of six major α-dicarbonyl process contaminants in high-fructose corn syrup by Sabrina Gensberger; Stefan Mittelmaier; Marcus A. Glomb; Monika Pischetsrieder (2923-2931).
High-fructose corn syrup (HFCS) is a widely used liquid sweetener produced from corn starch by hydrolysis and partial isomerization of glucose to fructose. During these processing steps, sugars can be considerably degraded, leading, for example, to the formation of reactive α-dicarbonyl compounds (α-DCs). The present study performed targeted screening to identify the major α-DCs in HFCS. For this purpose, α-DCs were selectively converted with o-phenylendiamine to the corresponding quinoxaline derivatives, which were analyzed by liquid chromatography with hyphenated diode array–tandem mass spectrometry (LC-DAD-MS/MS) detection. 3-Deoxy-d-erythro-hexos-2-ulose (3-deoxyglucosone), d-lyxo-hexos-2-ulose (glucosone), 3-deoxy-d-threo-hexos-2-ulose (3-deoxygalactosone), 1-deoxy-d-erythro-hexos-2,3-diulose (1-deoxyglucosone), 3,4-dideoxyglucosone-3-ene, methylglyoxal, and glyoxal were identified by enhanced mass spectra as well as MS/MS product ion spectra using the synthesized standards as reference. Addition of diethylene triamine pentaacetic acid and adjustment of the derivatization conditions ensured complete derivatization without de novo formation for all identified α-DCs in HFCS matrix except for glyoxal. Subsequently, a ultra-high performance LC-DAD-MS/MS method was established to quantify 3-deoxyglucosone, glucosone, 3-deoxygalactosone, 1-deoxyglucosone, 3,4-dideoxyglucosone-3-ene, and methylglyoxal in HFCS. Depending on the α-DC compound and concentration, the recovery ranged between 89.2% and 105.8% with a relative standard deviation between 1.9% and 6.5%. Subsequently, the α-DC profiles of 14 commercial HFCS samples were recorded. 3-Deoxyglucosone was identified as the major α-DC with concentrations up to 730 μg/mL HFCS. The total α-DC content ranged from 293 μg/mL to 1,130 μg/mL HFCS. Significantly different α-DC levels were not detected between different HFCS specifications, but between samples of various manufacturers indicating that the α-DC load is influenced by the production procedures.
Keywords: High-fructose corn syrup (HFCS); Sugar degradation products; α-Dicarbonyl compound (α-DC); o-Phenylenediamine (OPD); Quinoxaline derivatives; Ultra high-performance liquid chromatography (UHPLC)

Glycidyl fatty acid esters in food by LC-MS/MS: method development by A. Becalski; S. Y. Feng; B. P.-Y. Lau; T. Zhao (2933-2942).
An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70–150 μg/kg for each analyte using 10 mg sample and 1–3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5–10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to the survey of glycidyl fatty acid esters in food products on the Canadian market.
Keywords: Glycidyl fatty acid esters; Liquid chromatography tandem mass spectrometry (LC-MS/MS); Food analysis; Oils

The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5–50 μg kg−1. The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters—decision limit CCα (range, 7.5–12.9 μg kg−1), detection capability CCβ (range, 9.4–19.9 μg kg−1), within-laboratory reproducibility RSDwR (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSDr (<20% except for tylvalosin with 21.1%), and recovery (range, 92–106%)—were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.
Keywords: Honey; Veterinary drug residues; Antibiotics; Multi-group method; LC-MS/MS; Validation

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography–tandem mass spectrometry by Piotr Jedziniak; Teresa Szprengier-Juszkiewicz; Konrad Pietruk; Edyta Śledzińska; Jan Żmudzki (2955-2963).
Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers’ health. For this reason an LC–MS–MS method was developed for detection of wide range of NSAIDs, including both “acidic” NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and “basic” NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC–MS–MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7–28%, and accuracy was in the range 71–116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 μg kg−1). An additional advantage is the possibility of simultaneous determination of “acidic” NSAIDs and metamizole metabolites.
Keywords: Drugs in milk; Non-steroidal anti-inflammatory drugs; Diclofenac; Metamizole

LC-MS/MS fast analysis of androgenic steroids in urine by Barbara Wozniak; Iwona Matraszek-Zuchowska; Jan Zmudzki (2965-2972).
The liquid chromatography–tandem mass spectrometry method (LC-MS/MS) was developed and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone, methylboldenone, testosterone, methyltestosterone, 17β-1-testosterone and their metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on C18 SPE column and purification of the extract using liquid–liquid extraction with n-pentane and SPE NH2 column. For the chromatographic separation of steroids, the Poroshell 120-EC C18 column (150 × 2.1 mm, 2.7 μm) was used. Mass spectrometric measurement was achieved using the API 4000 triple quadrupole (QqQ) instrument with a TurboIon-Spray source operating in positive electrospray ionization mode. The procedure was validated according to the Decision 2002/657/EC. Recovery ranged from 76.5% to 118.9% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the 25%. The linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limit (CCα) ranged from 0.10 to 0.17 μg L−1 for all analytes, whereas the detection capability (CCβ) ranged from 0.17 to 0.29 μg L−1. The application of an innovative Poroshell column allowed for very good chromatographic separation of steroids with a much shorter time of analysis. Figure MRM chromatogram of 30 ng of androgenic steroids
Keywords: Androgenic steroids; Validation; Liquid chromatography; Mass spectrometry

Thyreostatic drugs, stability in bovine and porcine urine by J. Vanden Bussche; S. S. Sterk; H. F. De Brabander; M. H. Blokland; Y. Deceuninck; B. Le Bizec; L. Vanhaecke (2973-2982).
Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981. For monitoring their illegal use, sensitive and specific analytical methods are required. In this context, the knowledge of the stability in a matrix is of primary importance. This study aimed at evaluating the effects of preservation, number of freeze–thaw cycles, and matrix-related variables on the stability of thyreostatic drugs in the urine of livestock. Finally, the developed conservation approach was applied on incurred urine samples, which displayed traces of the thyreostat thiouracil below the recommended concentration of 10 μg L−1. The stability study confirmed the negative influence of preservation (8 h) at room temperature and at −70 °C, decreases in concentration of more than 78.0% were observed for all thyreostats, except for 1-methyl-2-mercaptoimidazole and 2-mercaptobenzimidazole. Additionally, investigation of matrix-related variables indicated significant impacts of the presence of copper (p = 0.001) and the pH (p = 0.002). Next, an optimised pre-treatment (pH 1 and 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate) significantly differing from the original conservation approach (p < 0.05) was developed, which proved capable of delaying the decrease in concentration and improved the detection in time for both spiked as well as incurred urine samples. In the future, it seems highly advisable to apply the developed pre-treatment on incurred urines upon sampling, before thyreostat analysis. Additionally, it is recommendable to limit preservation of urine samples at room temperature, but also in the freezer prior to thyreostat analysis. Figure Schematic representation of experimental design
Keywords: Thyreostatic drugs; Stability; Urine; UPLC; Mass spectrometry

Fungal contamination of food causes health and economic concerns. Several species of lactic acid bacteria (LAB) have antifungal activity which may inhibit food spoilage fungi. LAB have GRAS (generally recognised as safe) status, allowing them to be safely integrated into food systems as natural food preservatives. A method is described herein that enables rapid screening of LAB cultures for 25 known antifungal compounds associated with LAB. This is the first chromatographic method developed which enables the rapid identification of a wide range of antifungal compounds by a single method with a short analysis time (23 min). Chromatographic separation was achieved on a Phenomenex Gemini C18 100A column (150 mm × 2.0 mm; 5 μm) by use of a mobile-phase gradient prepared from (A) water containing acetic acid (0.1%) and (B) acetonitrile containing acetic acid (0.1%), at a flow rate of 0.3 µL min−1. The gradient involved a progressive ramp from 10–95% acetonitrile over 13 min. The LC was coupled to a hybrid LTQ Orbitrap XL fourier-transform mass spectrometer (FTMS) operated in negative ionisation mode. High mass accuracy data (<3 ppm) obtained by use of high resolution (30,000 K) enabled unequivocal identification of the target compounds. This method allows comprehensive profiling and comparison of different LAB strains and is also capable of the identification of additional compounds produced by these bacteria.
Keywords: LAB; Antifungal compounds; LTQ Orbitrap XL; High mass accuracy data; LC–FTMS

Accurate mass screening of pharmaceuticals and fungicides in water by U-HPLC–Exactive Orbitrap MS by Carmen Lidia Chitescu; Efraim Oosterink; Jacob de Jong; Alida Adriana Maria (Linda) Stolker (2997-3011).
The use of pharmaceuticals in livestock production is a potential source of surface water, groundwater and soil contamination. Possible impacts of antibiotics on the environment include toxicity and the emergence of antibiotic resistance. Monitoring programs are required to record the presence of these substances in the environment. A rapid, versatile and selective multi-method was developed and validated for screening 43 pharmaceutical and fungicides compounds, in surface and groundwater, in one single full-scan MS method, using benchtop U-HPLC–Exactive Orbitrap MS at 50,000 (FWHM) resolution. Detection was based on calculated exact masses and on retention time. Sample volume, pH conditions and solid-phase extraction (SPE) sample clean-up conditions were optimized. In the final method, 74 % of the compounds had recoveries higher than 80 %, 15 % of the compounds had recoveries between 60 % and 80 %, and 7 % of the compounds had recoveries between 40 % and 50 %. One of the compounds (itraconazole) had a recovery lower than 10 % and nystatin was not detected. The level of detection was 10 ng L−1 for 61 % of the compounds, 50 ng L−1 for 32 % and 100 ng L−1 for 5%. In-house validation, based on EU guidelines, proves that the detection capability CCβ is lower than 10 ng L−1 (for β error 5 %) for 37 % of the compounds, lower than 50 ng L−1 for 35 % of the compounds and lower than 100 ng L−1 for 14 % of compounds. This study demonstrates that the ultra-high resolution and reliable mass accuracy of Exactive Orbitrap MS permits the detection of pharmaceutical residues in a concentration range of 10–100 ng L−1, applying a post target screening approach, in the multi-method conditions.
Keywords: Antibiotics; Antifungal compounds; Water contamination; Mass accuracy; Post-target approach; Multi-residue analysis

Quantum dot based rapid tests for zearalenone detection by N. V. Beloglazova; E. S. Speranskaya; S. De Saeger; Z. Hens; S. Abé; I. Yu. Goryacheva (3013-3024).
Three different kinds of immunosorbent assays with luminescence detection were developed for the determination of zearalenone (ZEN), a secondary toxic metabolite of Fusarium fungi. CdSe/ZnS core/shell quantum dots (QDs) were used as a label in quantitative micro-well plate immunoassays (fluorescent-labeled immunosorbent assay, FLISA) and in qualitative column test methods. As carriers for QD-based column tests, sepharose gel (for covalent binding of antibody) and polyethylene frits (for physical absorption of antibody) were used and compared. The application of QDs as a label resulted in a fourfold decrease in the IC50 value with FLISA (0.1 ng mL−1) with a detection limit of 0.03 ng mL−1 when compared with the traditional immunosorbent assay which makes use of horseradish peroxidase as the enzyme label. The cutoff levels for both qualitative column test methods were selected based on the maximum level for ZEN in unprocessed cereals established by the European Commission (100 μg kg−1) as 5 ng mL−1 taking into account extraction and dilution. The different developed immumoassays were tested for ZEN determination in raw wheat samples. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using FLISA and both qualitative column test methods for the analysis of analytes with very low established maximum limits, even in very complicated food matrices, owing to the high dilution of the sample extract.
Keywords: Quantum dots; Immunoassay; On-site method; Enzyme-linked immunoassay; Fluorescent-labeled immunoassay; Zearalenone

Development of a molecularly imprinted polymer-matrix solid-phase dispersion method for selective determination of β-estradiol as anabolic growth promoter in goat milk by J. Gañán; A. Gallego-Picó; R. M. Garcinuño; P. Fernández-Hernando; S. Morante; I. Sierra; J. S. Durand (3025-3029).
A simple, fast, and sensitive method for determination of 17 β-estradiol (E2) in goat milk samples has been developed by combining selective molecularly imprinted matrix solid-phase dispersion (MIP–MSPD) and liquid chromatography with diode-array detection (DAD). The molecularly imprinted polymer was synthesized by use of 17β-estradiol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker monomer, azobisisobutyronitrile as initiator, and acetonitrile as porogen, and was used as selective solid support for matrix solid-phase dispersion. The selected dispersant had high affinity for E2 in the goat milk matrix and the extract obtained was sufficiently clean for direct injection for HPLC analysis without any interferences from the matrix. The proposed MIP–MSPD method was validated for linearity, precision, accuracy, decision limit (CCα) and detection capability (CCβ), in accordance with European Commission Decision 2002/657/EC criteria. Linearity ranged from 0.3–10 μg g−1 (correlation coefficient r 2 > 0.999). Mean recovery of E2 from goat milk samples at different spiked levels was between 89.5 and 92.2%, with RSD values within 1.3–2%. CCα and CCβ values were 0.36 and 0.39 μg g−1, respectively. The developed MIP–MSPD method was successfully applied to direct determination of E2 in goat milk samples. Figure Determination of 17β-Estradiol by using a MIP-MSPD method in goat milk sample
Keywords: β-estradiol; Growth promoters; Milk; MSPD; HPLC

Quantification of cow’s milk percentage in dairy products – a myth? by Helmut K. Mayer; J. Bürger; N. Kaar (3031-3040).
The substitution of ewe’s and goat’s milk for cheaper cow’s milk is still a fraudulent practice in the dairy industry. Moreover, soy-based products (e.g., soy milk, yoghurt) have to be checked for cow’s milk as they are an alternative for people suffering from an allergy against bovine milk proteins. This work reports the evaluation of different protein-based electrophoretic methods and DNA-based techniques for the qualitative detection as well as the quantitative determination of cow’s milk percentage in dairy and soy milk products. Isoelectric focusing (IEF) of γ-caseins using an optimized pH gradient was appropriate not only for the detection of cow’s milk, but also for an estimation of cow’s milk percentage in mixed-milk cheese varieties. Urea-polyacrylamide gel electrophoresis (PAGE) proved the method of choice to detect cow’s milk in soy milk products, whereas IEF and SDS-PAGE of proteins were not applicable due to false-positive results. Polymerase chain reaction (PCR) analysis was used to confirm the results of protein-based electrophoretic methods. Problems inherent in quantitative analysis of cow’s milk percentage using protein-based techniques and even more using DNA-based methods were emphasized. Applicability of quantitative real-time PCR for the determination of cow’s milk percentage in mixed-milk cheese was shown to be hampered by several factors (e.g., somatic cell count of milk; technological parameters influencing the final DNA concentration in ripened commercial cheese samples). The implementation of certified reference standards (of major relevant cheese groups) containing 50% cow’s milk was urgently recommended to enable at least a yes/no decision in commercial mixed-milk cheese samples. Figure Austrian cheese variety manufactured from cow’s, sheep’s and goat’s milk
Keywords: Milk species identification; Cow’s milk percentage; Mixed-milk cheese; Electrophoresis; PCR; Quantitative real-time PCR

The development of DNA-based methods for the identification and quantification of fish in food and feed samples is frequently focused on a specific fish species and/or on the detection of mitochondrial DNA of fish origin. However, a quantitative method for the most common fish species used by the food and feed industry is needed for official control purposes, and such a method should rely on the use of a single-copy nuclear DNA target owing to its more stable copy number in different tissues. In this article, we report on the development of a real-time PCR method based on the use of a nuclear gene as a target for the simultaneous detection of fish DNA from different species and on the evaluation of its quantification potential. The method was tested in 22 different fish species, including those most commonly used by the food and feed industry, and in negative control samples, which included 15 animal species and nine feed ingredients. The results show that the method reported here complies with the requirements concerning specificity and with the criteria required for real-time PCR methods with high sensitivity.
Keywords: Real-time PCR; Nuclear DNA; Fish DNA; Quantification; Rhodopsin gene; Degenerate primers/probe

Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs by J. Porter; N. O’Loan; B. Bell; J. Mahoney; M. McGarrity; R. I. McConnell; S. P. Fitzgerald (3051-3056).
Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71–135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output.
Keywords: Anthelmintic drugs; Biochip array; Multiplex screening; Simultaneous immunoassays

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCα) and detection capabilities (CCβ) were established for both the estrogen and androgen RGAs. All samples were compliant with CCα and CCβ in both bioassays. Recovery rates were 96 % for 17β-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at −20 °C. Specificity, good repeatability (coefficients of variation (CV), 12–25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.
Keywords: Validation; Sport supplements; Androgen; Estrogen; Reporter gene assay; Bioassay

Normalisation of data from allergens proficiency tests by Mark Sykes; Dominic Anderson; Bhavna Parmar (3069-3076).
The problem of allergen analysis using ELISA kits from different commercial products giving significantly different results is widely acknowledged. The effect on proficiency testing results is that different assigned values have to be generated for the different kits used. Some experimental Food Analysis Performance Assessment Scheme (FAPAS) proficiency tests aimed to establish whether the use of a standardised calibrant could be used to normalise the complete data set without recourse to differentiation. Three recent FAPAS proficiency tests (2776 peanut, 2778 soya and 2781 gluten) sent out a second spiked sample, in addition to the usual spiked and unspiked samples. Further analysis of the data was undertaken after the completion of the tests. The ratio of the submitted results for the two spiked samples yielded complete data sets which could be tested for normality of the distribution. Where the raw data for each individual test sample was clearly non-normal and multi-modal, the ratio data yielded a much more normal and symmetrical distribution. The use of one of the test samples as a single-point calibrant has some limitations but the principle of applying a standardisation clearly works. The development of internationally recognised sets of certified reference calibration standards for use by allergens testing laboratories would greatly benefit the analysis.
Keywords: Proficiency testing; Allergens; Calibration standard