New efficient methodology for screening of selected organic micropollutants in raw- and drinking water from 90 Swedish water treatment plants.

A new, efficient method for analysis of organic micropollutants (OMPs) in water samples was developed, validated and applied in a nationwide survey. The goal with the survey was to identify common compounds with relatively high concentrations to be used as markers e.g. for routine monitoring of removal efficiencies. The method comprises sample concentration by evaporation, and large volume injection on a standard UHPLC-MS/MS system. The OMPs selected for this approach were mainly semi-polar and non-volatile, with molecular weights below 300 Da. Except one outlier, the limit of detection (LoD) ranged from 0.01 to 1 ng/L which is sufficient for most surface waters, and also useful for many ground waters. The method requires low manual labor and comparably small sample volumes, enabling a cost-efficient nationwide screening. Raw- and drinking water from 90 Swedish water treatment plants (WTPs) were investigated for occurrence of the selected OMPs. Carbamazepine and tramadol were the most widespread compounds, detected in around 50% of the surface waters. Ground water from rock aquifers were least contaminated, while soil aquifers were more similar to surface waters. Due to the frequent use of ground water in Sweden, many samples did not contain any of the investigated compounds (i.e. below LoD). In the positive samples, the median estimated concentrations of individual OMPs were generally <1 ng/L in raw water and <0.5 ng/L in drinking water. Swedish waters were in general less contaminated than those investigated in similar Brazilian, Chinese, pan-European and US studies. Altogether, the presented methodology gave a cost-efficient overview on the occurrence and estimated concentrations of OMPs in raw- and drinking water on a national level in Sweden. From the data, WTPs can find suitable OMPs to monitor at their site, for example for measuring removal efficiencies on a routine basis.

Non-targeted analysis of unexpected food contaminants using LC-HRMS.

A non-target analysis method for unexpected contaminants in food is described. Many current methods referred to as "non-target" are capable of detecting hundreds or even thousands of contaminants. However, they will typically still miss all other possible contaminants. Instead, a metabolomics approach might be used to obtain "true non-target" analysis. In the present work, such a method was optimized for improved detection capability at low concentrations. The method was evaluated using 19 chemically diverse model compounds spiked into milk samples to mimic unknown contamination. Other milk samples were used as reference samples. All samples were analyzed with UHPLC-TOF-MS (ultra-high-performance liquid chromatography time-of-flight mass spectrometry), using reversed-phase chromatography and electrospray ionization in positive mode. Data evaluation was performed by the software TracMass 2. No target lists of specific compounds were used to search for the contaminants. Instead, the software was used to sort out all features only occurring in the spiked sample data, i.e., the workflow resembled a metabolomics approach. Procedures for chemical identification of peaks were outside the scope of the study. Method, study design, and settings in the software were optimized to minimize manual evaluation and faulty or irrelevant hits and to maximize hit rate of the spiked compounds. A practical detection limit was established at 25 µg/kg. At this concentration, most compounds (17 out of 19) were detected as intact precursor ions, as fragments or as adducts. Only 2 irrelevant hits, probably natural compounds, were obtained. Limitations and possible practical use of the approach are discussed.

Are additive effects of dietary surfactants on intestinal tight junction integrity an overlooked human health risk? - A mixture study on Caco-2 monolayers.

Surfactants may cause dysfunction of intestinal tight junctions (TJs), which is a common feature of intestinal autoimmune diseases. Effects of dietary surfactants on TJ integrity, measured as trans-epithelial resistance (TEER), were studied in Caco-2 cell monolayers. Cytotoxicity was assessed as apical LDH leakage. Monolayers were apically exposed for 60 min to the dietary surfactants solanine and chaconine (SC, potato glycoalkaloids, 0-0.25 mM), perfluorooctane sulfonic acid (PFOS, industrial contaminant, 0-0.8 mM), and sucrose monolaurate (SML, food emulsifier E 473, 0-2.0 mM) separately and as a mixture. Dose-response modelling of TEER EC50 showed that SC were 2.7- and 12-fold more potent than PFOS and SML, respectively. The mixture was composed of 1 molar unit SC, 2.7 units PFOS and 12 units SML ("SC TEER equivalent" proportions 1:1:1). Mixture exposure (0-0.05 mM SC equivalents) dose-response modelling suggested additive action on TJ integrity. Increasing SC and SML concentrations caused increased LDH leakage, but PFOS decreased LDH leakage at intermediate exposure concentrations. In the mixture PFOS appeared to protect from extensive SC- and SML-induced LDH leakage. Complex mixtures of surfactants in food may act additively on intestinal TJ integrity, which should be considered in risk assessment of emulsifier authorisation for use in food production.

A new method for analysis of underivatized free ß-methylamino-alanine: Validation and method comparison.

A new method was developed for analysis of free ß-Methylamino-alanine (BMAA) in biological matrices. The method is based on direct analysis of the underivatized molecule, using an amide column for separation by Hydrophilic Interaction Liquid Chromatography (HILIC) and detection by tandem mass spectrometry (MS/MS) using a deuterium labeled internal standard. The use of Ultra-High Performance Liquid Chromatography (UHPLC) combined with MS/MS detection allowed for high chromatographic resolution and a low limit of detection (0.025 µg/g wet weight (ww) in mussels). The method was validated by analyzing spiked blank mussels from the Baltic Sea (0.15-4.4 µg/g (ww), trueness 99%-105%, RSD 2%-8%). An inter-laboratory comparative analysis of extracts of mussel was performed. The mussels were extracted according to an established protocol for analysis of free BMAA, and the extracts were then analyzed in parallel by the new method and a validated procedure based on detection of BMAA derivatized with dansyl chloride. Both methods detected BMAA in similar concentrations. Thus, derivatization with dansyl chloride did not influence the results compared to direct detection. The new method presents an alternative to the commonly applied derivatization step, and is proved through validation and method comparison to reliably identify and quantify free BMAA at low concentration levels.

BMAA detected as neither free nor protein bound amino acid in blue mussels.

The results of this study imply that ß-methylamino-alanine (BMAA) obtained from extracts of blue mussels from the Swedish west coast is neither free nor protein bound. The results were obtained by separation (precipitation and ultrafiltration) of low and high molecular weight compounds from neutral extracts of blue mussels, and treatment of these extracts with low and high concentrations of acids, varying time and temperature. The main portion of BMAA was obtained from the low molecular weight fraction, released or formed at 95 °C in dilute acids. The measured amount of BMAA did not increase by strong acid treatment. Lysine was used as reference and was only released at significant amounts when treating the high molecular weight fraction with concentrated acid. The results also indicated that breakage of peptide bonds was not involved in the formation/release of BMAA in these extracts unless any BMAA peptide bond would be significantly more susceptible to dilute acid than e.g. the monitored lysine peptide bond. BMAA was measured using isotope dilution and detection of the underivatized compound by HILIC-UHPLC-MS/MS (Hydrophilic Interaction Liquid Chromatography, Ultra-High Performance Liquid Chromatography, tandem Mass Spectrometry). The findings might add to the understanding of conflicting data in the literature regarding the occurrence of BMAA, and have implications for studies on possible biomagnification of BMAA in the food chain and bioavailability from food.

Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

Quantitative analysis of cereulide toxin from Bacillus cereus in rice and pasta using synthetic cereulide standard and 13C6-cereulide standard - a short validation study.

A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with 13C6-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1-500 ng/g (R2 = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from -2% to +6% relative bias at different concentration levels in cooked rice and pasta.

Serum levels of unconjugated bisphenol A are below 0.2ng/ml in Swedish nursing women when contamination is minimized.

In this study serum levels of bisphenol A (BPA) were investigated in primiparous women from Uppsala County, Sweden, sampled 3weeks after delivery 1996-2011, in both yearly pools of serum (n=39, temporal trend study) and in 208 individual samples also present in the pools. Possible contamination risks of BPA from blood sampling equipment and sample tubes, as well as from handling of the samples were evaluated. The unconjugated form of BPA was analyzed using a UPLC-MS/MS method with a limit of quantification (LOQ) of 0.2ng/ml. The results show that the levels of unconjugated BPA generally were <0.2ng/ml. The sampling equipment used when taking blood samples from the women and the tubes used for storage and processing of samples did not show any detectable BPA leakage. In the first analysis of the serum samples, unconjugated BPA levels ≥0.2ng/ml were found in 12% of the individual samples and in 21% of the trend samples. However, in reanalyses of individual serum samples from the same aliquot or from new aliquots, samples with BPA levels ≥0.2ng/ml in the first analysis did not have quantifiable BPA levels. Moreover, 11% of BPA spiked calibration samples (over 200) had higher levels than could be explained by the random error of the method. Thus BPA contamination of the biobanked samples probably occurred randomly during sample handling, pooling and processing. Equipment used for sampling of children and repeated blood sampling were leaking BPA. The results show the difficulties in analyzing compounds where samples are easily contaminated from exogenous sources. It also points out that it is questionable to use biobanked samples unless absence of BPA contamination from the sampling and storage materials, and during handling of the samples, can be proven.

A concept study on non-targeted screening for chemical contaminants in food using liquid chromatography-mass spectrometry in combination with a metabolomics approach.

A generic method to screen for new or unexpected contaminants at ppm levels in food has been developed. The method comprises an acidic acetonitrile extraction, detection with ultra-high-pressure liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry and statistical evaluation using a metabolomics approach comparing suspected contaminated food with uncontaminated foods. The method was tested for 26 model contaminants from 100 µg/g down to 0.4 µg/g in three brands of fresh orange juice. Blinded statistical evaluation revealed signals from all added contaminants detectable by liquid chromatography-electrospray ionisation using positive ionisation mode, while only two false-positive signals were reported. The method is primarily intended to be used for investigation of food samples suspected to be contaminated with unknown substances. Additionally it could be used to continuously monitor for appearance of new food contaminants as a complement to the specific targeted analysis that is today's foundation of food safety analysis.

Analysis of the mushroom nephrotoxin orellanine and its glucosides.

Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C18 HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum.

Evaluation of a generic multi-analyte method for detection of >100 representative compounds correlated to emergency events in 19 food types by ultrahigh-pressure liquid chromatography-tandem mass spectrometry.

A generic extraction procedure combined with triple quadrupole mass spectrometric detection was evaluated for multi-residue analysis in 19 different foods. Measurable peaks could be obtained at relevant concentrations for 108 out of a total of 127 targeted compounds representing a wide range of physicochemical properties and compound classes related to emergency situations. Recoveries were determined for all 19 foods spiked with the 108 compounds. Seventy-five percent of the compounds had extraction recoveries of 70% or higher, with no compound below 46%. Suppression or enhancement effects on the MS response of the compounds dissolved in the extracts were low, as more than 80% of them had matrix effects between -35% and +20% and no compound was below -44% compared to matrix-free standard. In a validation, all compounds could be quantified at 200 µg/kg and 400 µg/kg food sample and 81% of the compounds at 40 µg/kg. It is concluded that the method is useful for the detection of various types of organic chemical toxicants at levels generally well below concentration thresholds for severe acute intoxication.

Validation of a food frequency questionnaire measurement of dietary acrylamide intake using hemoglobin adducts of acrylamide and glycidamide.

OBJECTIVE: Acrylamide, a probable human carcinogen, is formed during high-heat cooking of many common foods. The validity of food frequency questionnaire (FFQ) measures of acrylamide intake has not been established. We assessed the validity of acrylamide intake calculated from an FFQ using a biomarker of acrylamide exposure. METHODS: We calculated acrylamide intake from an FFQ in the Nurses' Health Study II. We measured hemoglobin adducts of acrylamide and its metabolite, glycidamide, in a random sample of 342 women. Correlation and regression analyses were used to assess the relationship between acrylamide intakes and adducts. RESULTS: The correlation between acrylamide intake and the sum of acrylamide and glycidamide adducts was 0.31 (95% CI: 0.20-0.41), adjusted for laboratory batch, energy intake, and age. Further adjustment for BMI, alcohol intake, and correction for random within-person measurement error in adducts gave a correlation of 0.34 (CI: 0.23-0.45). The intraclass correlation coefficient for the sum of adducts was 0.77 in blood samples collected 1-3 years apart in a subset of 45 women. Intake of several foods significantly predicted adducts in multiple regression. CONCLUSIONS: Acrylamide intake and hemoglobin adducts of acrylamide and glycidamide were moderately correlated. Within-person consistency in adducts was high over time.

Determination of the neurotoxin BMAA (beta-N-methylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry).

A highly specific method for the analysis of beta-N-methylamino-L-alanine (BMAA) by LC-MS/MS (liquid chromatography tandem mass spectrometry) has been developed and applied for cycad seeds and cyanobacteria. BMAA was analysed as a free fraction or as total BMAA after acidic hydrolysis to release any protein-bound BMAA. Deuterium labelled BMAA was synthesised and used as internal standard. The method comprises HILIC (hydrophilic interaction chromatography) and positive electrospray ionisation of the native compound, i.e. no derivatisation was used. For safe identification five specific product ions (m/z 102, 88, 76, 73 and 44), all derived from a precursor ion of m/z 119 and originating from different parts of the molecule, were detected (typical relative abundance 100%, 16%, 14%, 12% and 22% respectively). Cyanobacteria or muscle tissue was spiked with BMAA (10 to 1000 microg g(-1)) to validate the method (accuracy 95% to 109%, relative standard deviation 1% to 6%). The detection limit for free and total BMAA in tissue was <1 microg g(-1) and <4 microg g(-1) respectively. BMAA was successfully identified and quantified in cycad seeds, whereas previously reported findings of BMAA in samples of cyanobacteria could not be confirmed. Instead, the presence of alpha-,gamma-diamino butyric acid (DAB), an isomer of BMAA, was confirmed in one sample. The possible implications of this finding are discussed.

Moisture enhances acrylamide reduction during storage in model studies of rye crispbread.

The effect of storage conditions on the residual acrylamide content of unfermented rye crispbread was studied in a model system. When milled samples were stored at -80 to 6 degreesC for up to 224 days in double sealed plastic bags, no change in acrylamide content was observed. However, when the milled samples were stored under warmer conditions (20 and 40 degreesC), a notable reduction in acrylamide was noted (22% and 29%, respectively). When stored at 40 degreesC for 70 days in glass tubes, acrylamide content in the samples decreased by 37% in the capped samples, while the decrease in the uncapped samples was in the order of 15%. Finally, a notable reduction of 80% was found when samples were stored at increased moisture level at 40 degreesC for 70 days in capped glass containers. These results highlight that moisture content seems to be of importance for reduction of acrylamide content during storage of food and analytical samples.

Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food.

The aim of this study was to investigate if consumption of ordinary carbohydrate-rich food prepared in different ways has an impact on chromosome stability, i.e., on the formation of micronucleated young erythrocytes in humans. Twenty-four persons, divided into two groups, participated during 4 days in a semi-controlled food-consumption study. One group (low-heated-food-group, LowHF-group) consumed only food boiled in water (max 100 degrees C) and the other group (high-heated-food-group, HighHF-group) consumed preferentially strongly heated (fried) food. From each of the subjects, blood samples were drawn, before and after 4 days. The frequency (f) of micronucleated (MN) very young erythrocytes (transferrin-positive reticulocytes, Trf-Ret), fMNTrf-Ret, was determined, and the difference in the frequency, before and after the eating period, was calculated. The obtained mean differences for the two groups were compared. As an indicator of highly heated food the acrylamide (AA) content in part of the consumed foodstuffs was analysed by use of LC/MS-MS and the AA intake estimated. In the blood samples the hemoglobin-adduct levels from AA were analysed as a measure of the internal AA dose. The differences between the mean fMNTrf-Ret, before and after the eating period, were -0.15 per thousand for the LowHF-group and +0.17 per thousand for the HighHF-group, p<0.005 (t-test, one-tailed). The mean total AA intake in the HighHF-group during 4 days was estimated to about 3000+/-450microg per person. For the LowHF-group, the mean AA intake was low, 20+/-10microg per person. The lowest dose of AA that caused a significant increase of micronucleated erythrocytes in mice is more than a hundred times higher than the AA level in this study. Thus, it is unlikely that the exposure to AA is the major cause behind the observed difference. The answer is probably to be found in other compounds produced at the same time during heating of the food.

Retention studies of acrylamide for the design of a robust liquid chromatography-tandem mass spectrometry method for food analysis.

A wide range of solid phases for SPE (solid-phase extraction) (n=14) and HPLC (n=9) were compared regarding the chromatographic retention of acrylamide. For SPE, a hydroxylated polystyrene-divinylbenzene copolymer phase (ENV+) gave the strongest retention. Twenty millilitre of water per gram solid phase could be passed with less than 5% loss of acrylamide from the column, thus enabling significant enrichment of food extracts. Other polymer phases gave varying degrees of retention, while silica bonded phases gave low retention. For HPLC, columns were evaluated both in reversed-phase and aqueous normal-phase (hydrophilic interaction chromatography) modes. The best retention was obtained with a phase comprising porous graphitic carbon (Hypercarb), giving a k-value of 4 with water as the mobile phase. Based on these investigations, a method for analysis of acrylamide in food using liquid chromatography-tandem mass spectrometry was designed to meet the demands of a collaborative validation trial. A comparative investigation of solid phases has not been published earlier. Thus, the paper should provide a base for new method developments regarding clean-up, enrichment and chromatography of acrylamide. In addition, the detailed standard operating procedure (SOP) method, as used in a collaborative validation trial, is provided as an electronic supplement (www.elsevier.com).

Food allergen detection with biosensor immunoassays.

An optical biosensor was used to develop both direct and sandwich immunoassays for the detection of proteins from milk, egg, hazelnut, peanut, shellfish, and sesame in food samples. Affinity-purified polyclonal antibodies raised against the proteins were immobilized on the biosensor chip. Food samples were injected and the proteins that bound to the antibodies on the surface were detected by a shift in the resonance angle. By adding a second antibody in a sandwich assay, matrix effects could be overcome and the sensitivity and selectivity enhanced. Detection of allergen levels down to 1-12.5 microg/g in food samples was demonstrated for the various assays. Good agreement of results was also obtained from parallel analysis with alternative immunoassays, including rocket immunoelectrophoresis, enzyme immunoassay, and immunoblotting. The present study demonstrates that the sensitivity of the described biosensor technique is comparable to the most sensitive enzymed-linked immunosorbent assays.

The HEATOX Project.

HEATOX is the acronym for a European Union-funded project entitled Heat-Generated Food Toxicants: Identification, Characterization, and Risk Minimization. Acrylamide will be the main experimental focus, but identification of unknown toxicants in heated carbohydrate-rich foods will also be attempted. The project includes research on formation chemistry, food technology, analytical methods, hazard characterization, and exposure assessment. The results will finally be used in risk assessment and risk management advice.

Matrix effects in immunobiosensor determination of clenbuterol in urine and serum.

The construction of immunochemical inhibition assays for beta-agonist and hormone residues have previously been described. In the present work the beta-agonist assay was further optimised for application to biological samples, using urine as the main model matrix. Matrix interferences with the antigen-antibody interaction and non-specific binding (NSB) of matrix components to the sensor surface were systematically studied. A full factorial design experiment was employed for evaluating the effects of assay buffer composition. In addition, the influence of antibody concentration and sample dilution on the matrix background was investigated. NSB from urine was highly affected by buffer pH and salt concentration, while buffer composition had little effect on matrix interferences with the antigen-antibody interaction. Ultra-filtration efficiently prevented NSB from urine and serum samples. Increased antibody dilution reduced the matrix background while sample dilution had an opposite effect.

Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry.

A method using liquid chromatography tandem mass spectrometry (LC-MS-MS) with electrospray for the analysis of acrylamide in foods is reported. The method comprises the addition of deuterium-labelled acrylamide-d3, extraction with water, mixed mode solid phase extraction, ultrafiltration and a graphitised carbon column for chromatography. The transitions m/z 72 > 55, 72 > 54, 72 > 44, 72 > 27, 72 > 72 and 75 > 58 were recorded in multiple reaction monitoring mode for identification and quantification. In-house validation data for products from potatoes and cereals (30 to 10,000 microg kg(-1)) are presented (accuracy 91 to 102%, relative standard deviation 3 to 21%). Interlaboratory validation data (comparison with gas chromatography mass spectrometry, 25 to 2000 microg kg(-1)) showed excellent results (r2 = 0.998).