Bruno Le Bizec

École Nationale Vétérinaire, Agroalimentaire et de l'Alimentation Nantes-Atlantique | Oniris · LABERCA - Laboratory of Food Contaminants and Residue Analysis

Background: Persistent organic pollutants (POPs) are known to accumulate in adipose tissues (AT). This storage may be beneficial by diverting POPs from other sensitive tissues or detrimental because of chronic release of pollutants as indirectly suggested during weight loss. The aim is to study the biological and/or toxic effects that chronic POP release from previously contaminated grafted AT could exert in a naïve mouse. Methods: C57BL/6J male mice were exposed intraperitoneally to 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD); their epididymal fat pads were collected and grafted on the back skin of uncontaminated recipient mice whose brain, liver, and epididymal ATs were analyzed (TCDD concentration, relevant gene expression). Kinetics of release and redistribution were modeled using Physiologically Based PharmacoKinetics (PBPK). Results: The grafts released TCDD over a period of 10 weeks with different kinetics of distribution in the three organs studied. A PBPK model was used to simulate the AT releasing process and the incorporation of TCDD into the major organs. At three weeks post-graft, we observed significant changes in gene expression in the liver and the host AT with signatures reminiscent of inflammation, gluconeogenesis and fibrosis as compared to the control. Conclusions: This study confirms that AT-stored TCDD can be released and distributed to the organs of the recipient hence leading to distinct changes in gene expression. This original model provides direct evidence of the potential toxic-relevant effects when endogenous sources of contamination are present.

In the context of human and veterinary drugs identification, ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) may provide a relevant complementary piece of information to mass-to-charge ratio (m/z), the so-called collision-cross-section (CCS). Up to now, however, the application of CCS as identification parameter has not been fully investigated due to the reduced number of these drugs that have being characterized in terms of CCS. This work proposes a CCS database for 92 human and veterinary drugs, including eighteen benzimidazoles, eleven 5-nitroimidazoles, eleven aminoglycosides, nineteen quinolones, eighteen β-lactams, ten sulfonamides and five tetracyclines. Among them, 37 drugs have been characterized in terms of CCS for the first time. The CCS values of the other 55 compounds have been compared with those from a recently published database in order to evaluate inter-laboratory reproducibility, which is crucial for the implementation of the CCS as identification parameter. CCS values were measured by traveling wave ion mobility spectrometry (TWIMS) under positive ionization conditions. Nitrogen was used as drift gas in the ion mobility cell. The proposed database covers 173 ions including [M+H]⁺ and [M+Na]⁺ species. High correlation between m/z and CCS has been observed for [M+H]⁺ (R² = 0.9518, n = 91) and [M+Na]⁺ (R² = 0.9135, n = 82) ions. As expected, CCS values for sodium adducts are generally greater than for protonated molecules because they exhibit higher molecular weight. However, sodium adducts of aminoglycosides, β-lactams, and of several quinolones and benzimidazoles, were characterized as more compact ions than their related protonated molecule. In addition, this work describes the fragmentation pattern observed for the studied molecules. For the first time, the main fragment ions for most of the compounds have also been characterized in terms of CCS, involving a total of 238 ions. As proof of concept, for the application of this database to biological matrices, eleven veterinary drugs in bovine urine samples were characterized in terms of CCS, showing that this parameter was not influenced by the matrix.

Within the particular context of controlling chemical residues in food, an alternative to targeted approaches has emerged; it consists in the characterization of physiological perturbations induced upon exposure of animals to a given chemical substance/class of substances to highlight suitable biomarkers addressing safety and/or regulatory issues. Metabolomics in particular has been investigated in the hope of identifying such biomarkers, and a range of studies have demonstrated the efficiency of the strategy. Until very recently, steps remained to be taken toward official or commercial implementation of corresponding tools. In particular, the lack of guidelines and criteria to validate such methods that do not target specific chemical species per se, constituted a bottleneck. In the present work, a metabolomics model dedicated to the detection of β-agonist administration in bovines has been developed and fully validated; criteria (selectivity, robustness, stability, suspicion threshold definition, false positive and false negative rates) have been proposed in agreement with EU expectations (Dec 2002/657), enabling demonstration that performances comply with screening requirements. Although some of the biomarkers involved in the prediction model remain un-elucidated, the corresponding LC-HRMS method has recently been ISO17025 accredited, allowing for the very first official implementation of a metabolomics based strategy within French National Monitoring Plans.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production but little is known about the synthesis of backdoor androgens despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human male fetuses using multi-dimensional and high-resolution mass-spectrometry. Results show that androsterone is the principal backdoor androgen in the fetal circulation and that DHT is undetectable (<1ng/ml). Backdoor pathway intermediates are found primarily in the placenta and fetal liver with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are mostly undetectable in the fetal testes. This is consistent with transcript levels of enzymes involved in the backdoor pathway (SRD5A1, AKR1C2/4, CYP17A1), as measured by qPCR. These data identify androsterone as the predominant backdoor androgen in the human fetus and show that it is formed primarily in non-gonadal tissue with placental progesterone the likely substrate. Masculinization of the human fetus depends, therefore, on androgen synthesis by both the fetal testes and non-gonadal tissues leading to DHT formation at the genital tubercle. Our findings provide, for the first time, a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans

A steroidomics workflow has been developed in the objective of monitoring a wide range (n >150) of steroids in urine. The proposed workflow relies on the optimization of an adequate SPE extraction step followed by an UHPLC-HRMS/MS simultaneous analysis of both free and conjugated forms of C18, C19 and C21 steroid hormones. On the basis of 44 selected steroids, representative of main classes of steroids constituting the steroidome, the performances of the developed workflow were evaluated in terms of selectivity, repeatability (< 13%) and linearity (R2> 0.985 on in the concentration range [0.01 – 10 ng/mL]). As metabolites identification and characterization constitute the bottleneck of such profiling approaches, a homemade database was created encompassing a large number of characterized free and conjugated steroids (n> 150) for putative steroid-like biomarkers identification purposes. The efficiency of the workflow in highlighting fine modifications within the urinary steroidome was assessed in the frame of an anabolic treatment involving an intra-muscular administration of boldenone undecylenate (2 mg/kg) to veals (n=6) and the investigation of potential steroid biomarkers. Besides monitoring known phase II metabolites of boldenone in the bovine specie, namely, boldenone glucuronide and sulfate, the applied strategy also permitted to observe, upon boldenone administration, a modified profile of epiboldenone glucuronide. Furthermore, 31 signals corresponding to non-identified steroid species could also be highlighted as impacted upon the exogenous steroid treatment. This study is the first to simultaneously investigate both free and conjugated C18, C19 and C21 steroid hormones in their native form using UHPLC-HRMS/MS and allowing their comprehensive profiling. This strategy was probed in-vivo.

It is generally accepted that androgens produced by fetal Leydig cells (FLC) control proper masculinization of the male external genitalia. Here, we hypothesized that the human genital tubercle (GT) has potential to synthesize androgens independently of FLC at early pregnancy. We observed that human GT of both genders have capacity to synthesize steroids of the Δ4, Δ5 and alternative pathway of DHT synthesis including the androgen itself. The presence of steroids in the GT was associated with the expression of corresponding steroidogenic enzymes. Levels of steroids and the expression of steroidogenic enzymes were similar in the GT from male and female fetuses. In contrast to the GT, the human fetal testis synthesized DHT from testosterone but not via the alternative pathway. Our findings strongly suggest that the human GT at early pregnancy can synthesize DHT via the alternative pathway, which may play an important role in organogenesis of the urethra.

Introduction Although it is still at a very early stage compared to its mass spectrometry (MS) counterpart, proton nuclear magnetic resonance (NMR) lipidomics is worth being investigated as an original and complementary solution for lipidomics. Dedicated sample preparation protocols and adapted data acquisition methods have to be developed to set up an NMR lipidomics workflow; in particular, the considerable overlap observed for lipid signals on 1D spectra may hamper its applicability. Objectives The study describes the development of a complete proton NMR lipidomics workflow for application to serum fingerprinting. It includes the assessment of fast 2D NMR strategies, which, besides reducing signal overlap by spreading the signals along a second dimension, offer compatibility with the high-throughput requirements of food quality characterization. Method The robustness of the developed sample preparation protocol is assessed in terms of repeatability and ability to provide informative fingerprints; further, different NMR acquisition schemes—including classical 1D, fast 2D based on non-uniform sampling or ultrafast schemes—are evaluated and compared. Finally, as a proof of concept, the developed workflow is applied to characterize lipid profiles disruption in serum from β-agonists diet fed pigs. Results Our results show the ability of the workflow to discriminate efficiently sample groups based on their lipidic profile, while using fast 2D NMR methods in an automated acquisition framework. Conclusion This work demonstrates the potential of fast multidimensional ¹H NMR—suited with an appropriate sample preparation—for lipidomics fingerprinting as well as its applicability to address chemical food safety issues.

Dechlorane related compounds (DRCs), including Dechlorane Plus (syn-DP and anti-DP), Dechlorane-601, -602, -603 and Chlordene Plus (CP), constitute a group of polychlorinated flame retardants (FRs) that are still of industrial use. In particular, DRCs have been detected in various environmental matrices and in different aquatic and terrestrial biota, thus exhibiting bioaccumulation and biomagnification potentials. The present study aimed at producing first occurrence data of a range of DRCs in Silurus spp. samples from different rivers located in France. Determination was carried out by gas chromatography high-resolution mass spectrometry after a sample clean-up based on a multilayer silica column and gel permeation chromatography. The concentration of monitored ΣDRCs ranged from 1.58 to 408 pg g-1 wet weight (54-11100 pg g-1 lipid weight). The fractional abundance of syn- and anti-DP stereoisomers was similar to that reported by other studies with an average equal to 0.60. Dec-601 was not detected in any sample. Detection frequencies ranged between 34 and 100% for other DRCs. Investigated correlations between DRCs and polychlorobiphenyls (PCBs) suggest a link with lipid content but independent contamination sources.

Determination of the occurrence levels of legacy and novel BFRs is today required to better understand the trends of BFRs contamination in food consecutive to the EU PBDEs restrictions and to proceed to a recent human food exposure in parallel. Therefore, concentrations of a large set of brominated flame retardants (BFRs) (n = 27) including PBDEs, HBCDDs, TBBPA and novel flame retardants (nBFRs) have been determined in more than 600 food and feed samples collected between 2014 and 2016 in the context of French monitoring plans. Although legacy BFRs had already been studied in France, such a survey constituted the very first determination of nBFRs occurrence in foodstuffs at the national level. The concentration levels measured in fish and fish products were in general higher than in the other food categories. PBDEs were detected in 70% of the samples and were observed as the most abundant congeners (representing 80% of the sum of the monitored BFRs), while α-HBCDD could also be considered as a predominant congener (up to 26% of the sum of the monitored BFRs in fishes). nBFRs concentration levels were most of the time below the LOQ, except PBT, PBBz and HBBz which were more frequently detected at low levels. Also investigated in the study, BRPs exhibited high concentration levels in crustaceous (maximum value > 2700 pg/g ww).

The majority of L-cysteine is obtained industrially by hydrolysis of animal materials, such as poultry feathers. Despite widespread belief, there is little evidence that human hair is used as a source material and its use is explicitly banned in the European Union (2000/63/EC decision). We developed an isotope ratio mass spectrometric (EA-IRMS) method to determine carbon and nitrogen isotopic ratio in cysteine preparations and related compounds, e.g. cystine and carbocysteine. A threshold relying on the ¹⁵N/¹⁴N was established to differentiate between hair and feathers; a value below 6.6‰ indicates a poultry feathers origin. Global uncertainty of measurement was found to be 0.1‰ for δ¹⁵N (sample size of 0.5–1.8 mg).

β-agonistic drugs have been forbidden as growth promoters in rearing animals in Europe since the late 1980s (Dir 96/22/EC). Specific and sensitive analytical methods based on UHPLC-MS/MS allow to monitor a large set of these substances. However, optimal performances are not observed for all the target analytes, especially for those exhibiting the highest polarities. We developed an SFC-MS/MS approach to cover the huge elution window of β-agonists, from the most polar which are usually eluted in the void volume when using reversed phase chromatography in conventional HPLC to the most apolar ones. The objective was to reach performances in accordance with the European Union recommended level in feeding stuff, i.e. 50 μg kg-1. LC/MS and SFC/MS performances were thoroughly compared in terms of analytical validation data (linearity, selectivity, recovery rates, reproducibility, compounds identification, trueness, decision limit (CCα) and detection capability (CCβ)) for 6 β-agonistic drugs, namely bromobuterol, clenbuterol, isoxsuprine, ractopamine, salbutamol and zilpaterol. As a result, the SFC approach appeared complementary to the LC one because the elution order of compounds was totally different from the one obtained with a classical C18 stationary phase. Moreover, the UPLC-MS/MS approach gave a better response linearity and more accurate values, whereas SFC-MS/MS provided greater data for identification purposes, reproducibility and sensitivity. Both analytical approaches enabled the detection of targeted β-agonists at a lower concentration than the recommended one (50 μg kg-1).

To assess the presence of prohibited anabolic substances used to promote growth in livestock, calf urine is the most relevant matrix. However, the sample preparation methods (required to remove unwanted matrix components and fractionate isobaric species that may be unresolved by gas chromatography- mass spectrometry GC/MS) are long and complex. In this context, semi-preparative supercritical fluid chromatography (SFC) was considered to possibly simplify the sample preparation in reducing the number of procedures. Fifteen stationary phases were screened with SFC combined with UV and evaporative light-scattering detection (ELSD), among which two columns (Cosmosil π-NAP and Princeton DIOL) were retained for their ability to isolate steroid hormones from other matrix components and, for the second column, for the additional possibility to fractionate steroid hormones into different families (estrogens, mono-hydroxylated and di-hydroxylated androgens). The fractions were further analysed with GC/MS showing the benefit of class fractionation. The final method allows for significant time, solvent and money savings compared to the previously widely used method (solid-phase extraction combined with semi-preparative high-performance liquid chromatography).

Brominated Flame Retardants (BFRs) are still widely used for industrial purposes. These contaminants may enter the food chain where they mainly occur in food of animal origin. The aim of our work was to provide a unique method able to quantify the widest range of BFRs in feed and food items. After freeze-drying and grinding, a pressurized liquid extraction was carried out. The extract was purified on acidified silica, Florisil® and carbon columns, the four separated fractions were analyzed by gas and liquid chromatography coupled to high resolution and tandem mass spectrometry. Isotopic dilution was preferentially used when commercial labelled compounds were available. Analytical sensitivity was in accordance with the expectations of Recommendation 2014/118/EU for PBDEs, HBCDDs, TBBPA, TBBPA-bME, EHTBB, BEHTEBP and TBBPA-bME. Additional BFRs were included in this analytical method with the same level of performances (LOQs below 0.01 ng g-1 ww). These are PBBs, pTBX, TBCT, PBBz, PBT, PBEB, HBBz, BTBPE, OBIND and T23BPIC. However, some of the BFRs listed in Recommendation 2014/118/EU are not yet covered by our analytical method, i.e. TBBPA-bOHEE, TBBPA-bAE, TBBPA-bGE, TBBPA-bDiBPrE, TBBPS, TBBPS-bME, TDBPP, EBTEBPI, HBCYD and DBNPG. The uncertainty measurement was fully calculated for 21 of the 31 analytes monitored in the method. Reproducibility uncertainty was below 23% in isotopic dilution. Certified reference materials are now required to better characterize the trueness of this method, which was applied in the French National Control Plans.

Ion mobility spectrometry enhances the performance characteristics of liquid chromatography-mass spectrometry workflows intended to steroid profiling by providing a new separation dimension and a novel characterization parameter, the so-called collision cross section (CCS). This work proposes the first CCS database for 300 steroids (i.e. endogenous, including phase I and phase II metabolites, and exogenous synthetic compounds), which involves 1080 ions and covers the CCS of 127 androgens, 84 estrogens, 50 corticosteroids and 39 progestagens. This large database provides information related to all the ionized species identified for each steroid in positive electrospray ionization mode as well as for estrogens in negative ionization mode. CCS values have been measured using nitrogen as drift gas in the ion mobility cell. Generally, direct correlation exists between mass-to-charge ratio (m/z) and CCS because both are related parameters. However, several steroids mainly steroid glucuronides and steroid esters have been characterized as more compact or elongated molecules than expected. In such cases, CCS results in additional relevant information to retention time and mass spectral data for the identification of steroids. Moreover, several isomeric steroid pairs (e.g. 5β-androstane-3,17-dione and 5α-androstane-3,17-dione) have been separated based on their CCS differences. These results indicate that adding the CCS to databases workflows increases selectivity, thus improving the confidence in steroids analysis. Consequences in terms of identification and quantification are discussed. Quality criteria and a construction of an inter-laboratory reproducibility approach are also reported for the obtained CCS values. The CCS database described here is made publicly available.

Tap water is used in France to reconstitute powder infant formula, although it is not sterile and possibly contaminated by microbiological and chemical hazards. The present study aims to quantify risks of using tap water in France for the preparation of infant formula, during the first six months of life. Cryptosporidium and arsenic were selected as hazards of greatest concern in microbiology and chemistry, respectively. A probabilistic model was developed using French (when available) and European (alternatively) data. Second order Monte Carlo simulation was used to separate uncertainty and variability of inputs. Outputs were expressed at the individual level as probability of illness and at the population level, using a common metric, the DALY (Disability Adjusted Life Year). Two scenarios of milk preparation were considered: with un-boiled or boiled tap water. Consuming infant formula rehydrated with un-boiled tap water during the first six months of life led to a total of 2250 DALYs per 100,000 infants (90% uncertainty interval [960; 7650]) for Cryptosporidium due to diarrhea, and 1 DALY [0.4; 2] for arsenic due to expected lifetime risk of lung and bladder cancer as a result of early exposure in life. For the entire population, boiling water would suppress the risk from Cryptosporidium. In contrast, the incremental cancer risk was low at the population level but elevated for 5% of the population exposed to high levels of arsenic. A stringent monitoring of tap water supply points should be continued. This multi-risk assessment model could help public health authorities and managers in evaluating both microbiological and chemical safety issues associated with using infant formula prepared with tap water.

An untargeted strategy aiming at identifying non-intentionally added substances (NIAS) migrating from coatings was developed. This innovative approach was applied to two polyester-polyurethane lacquers, for which suppliers previously provided the identity of the monomers involved. Lacquers were extracted with acetonitrile and analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Data, acquired in the full scan mode, were processed using an open-source R-environment (xcms and CAMERA packages) to list the detected features and deconvolute them in groups related to individual compounds. The most intense groups, accounting for more than 85% of cumulated feature intensities, were then investigated. A homemade database, populated with predicted polyester oligomer combinations from a relevant selection of diols and diacids, enabled highlighting the presence of 14 and 17 cyclic predicted polyester oligomers in the two lacquers, including three mutual combinations explained by common known monomers. Combination hypotheses were strengthened by chromatographic considerations and by the investigation of fragmentation patterns. Regarding unpredicted migrating substances, four monomers were hypothesised to explain several polyester or caprolactam oligomer series. Finally, considering both predicted and tentatively elucidated unpredicted oligomers, it was possible to assign hypotheses to features representing up to 82% and 90% of the cumulated intensities in the two lacquers, plus 9% and 3% (respectively) originating from the procedural blank. Graphical abstractElucidation of non-intentionally added substances Elucidation of non-intentionally added substances

The gold-standard matrix for measuring the internal levels of persistent organic pollutants (POPs) is the adipose tissue, however in epidemiological studies the use of serum is preferred due to the low cost and higher accessibility. The interpretation of serum biomarkers is tightly related to the understanding of the underlying causal structure relating the POPs, serum lipids and the disease. Considering the extended benefits of using serum biomarkers we aimed to further examine if through statistical modelling we would be able to improve the use and interpretation of serum biomarkers in the study of endometriosis. Hence, we have conducted a systematic comparison of statistical approaches commonly used to lipid-adjust the circulating biomarkers of POPs based on existing methods, using data from a pilot case-control study focused on severe deep infiltrating endometriosis. The odds ratios (ORs) obtained from unconditional regression for those models with serum biomarkers were further compared to those obtained from adipose tissue. The results of this exploratory study did not support the use of blood biomarkers as proxy estimates of POPs in adipose tissue to implement in risk models for endometriosis with the available statistical approaches to correct for lipids. The current statistical approaches commonly used to lipid-adjust circulating POPs, do not fully represent the underlying biological complexity between POPs, lipids and disease (especially those directly or indirectly affecting or affected by lipid metabolism). Hence, further investigations are warranted to improve the use and interpretation of blood biomarkers under complex scenarios of lipid dynamics.

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

Ce numéro comprend les articles correspondant aux présentations du Colloque Casdar 2018

In a proof of concept perspective, Rapid Evaporative Ionization Mass Spectrometry (REIMS) was explored for the direct analysis of meat samples from β-agonist treated livestock. In this context, the combination of REIMS with untargeted metabolomics was investigated to identify carcasses from treated animals on the basis of a modification of indirect metabolites profile. The REIMS analysis generated specific lipid profiles which enabled the differentiation of meat samples collected from pigs treated with ractopamine via their feeding regime. Furthermore, the strategy was found successful when tested on different muscle types (loin, shoulder and thigh), which further expands its applicability. Classification performances were greater than 95% accurate which fully answers requirements of a screening strategy. This research indicates that REIMS implemented in an untargeted-metabolomics workflow can be considered as a high-throughput and accurate strategy for real-time meat classification in relation to ractopamine (and wider β-agonists) treatment in pig production. This approach may subsequently be implemented as a rapid screening test, at the slaughterhouse or at boarder inspection points, to detect such practice.

Shells fired during World War I exhibited different explosive compounds and some of these weapons also contained a wide variety of chemical warfare agents. At the end of the war, for safety purposes, the large quantity of weapons remaining on the former front needed to be dismantled and destroyed. A large amount of the remaining shells was destroyed in specific sites which led to the contamination of the surroundings in Belgium and France. In the 1920s, 1.5 million chemical shells and 30,000 explosive shells were destroyed in a place close to the city of Verdun, in the East of France. In this paper, the risk for human health related to the consumption of foodstuffs produced on this site was assessed. To this end, food products of plant and animal origin were sampled in 2015-2016 and contaminant analyses were conducted. Human exposure was assessed using a specifically built methodology. The contaminants considered in this study were trace elements (TEs - primarily Zn, As, Pb and Cd), nitroaromatic explosives (trinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitroluene and 4-amino-2,6-dinitrotoluene), phenylarsenic compounds including diphenylarsinic acid and triphenylarsine, perchlorate, tetrabromoethane and vinyl bromide. Depending on the compound, different approaches were used to assess the risk for both adults and children. Exposure to these contaminants through the consumption of foodstuffs produced locally on the considered site was unlikely to be a health concern. However, as for inorganic arsenic, given the presence of highly contaminated zones, it was suggested that cereals should not be grown on certain plots.

Body mass index is a known breast cancer risk factor due to, among other mechanisms, adipose-derived hormones. We developed a method for steroid hormone profiling in adipose tissue to evaluate healthy tissue around the tumor and define new biomarkers for cancer development. A semi-automated sample preparation method based on gel permeation chromatography and subsequent derivatization with trimethylsilyl (TMS) is presented. Progestagens and androgens were determined by GC-EI-MS/MS (LOQ 0.5 to 10 ng/g lipids). For estrogen measurement, a highly sensitive GC-APCI-MS/MS method was developed to reach the required lower limits of detection (0.05 to 0.1 ng/g lipids in matrix, 100–200 fg on column for pure standards). The combination of the two methods allows the screening of 27 androgens and progestagens and 4 estrogens from a single sample. Good accuracies and repeatabilities were achieved for each compound class at their respective limit of detection. The method was applied to determine steroid hormone profiles in adipose tissue of 51 patients, collected both at proximity and distant to the tumor. Out of the 31 tested steroid hormones, 14 compounds were detected in human samples. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), and androstendione accounted together for 80% of the observed steroid hormone profiles, whereas the estrogens accounted for only 1%. These profiles did not differ based on sampling location, except for ß-estradiol; steroid hormone conversions from androgens to estrogens that potentially take place in adipose or tumoral tissue might not be detectable due a factor 100 difference in concentration of for example DHEA and ß-estradiol.

During a national monitoring plan, a pork fat sample was declared non-compliant for the sum of dioxins and PCB-DL (EU regulation). The National Reference Laboratory together with competent authorities conducted extended investigations to determine rapidly the contamination source at the farm level. A range of samples (n = 129), representative of potential contamination sources, was selected for further characterization (fat, feed, materials, dust, soil) and was analyzed for PCDD/Fs and DL-PCBs by GC-HRMS. A hot spot was localized in the farm, which corresponded to a pre-feed storage tank, the paints of which presented huge DL-PCB concentrations (>1 × 106 pg g-1), responsible for the contamination. The present case report describes a new source of PCB contamination, previously undescribed.

The present study addresses the hypothesis that the concentration of tetrahydroxylated Polycylic Aromatic Hydrocarbons (tetra-OH-PAHs) in hair might be a useful biomarker of human exposure to PAHs, providing quantitative assessment of the internal dose, as well as information on the associated toxicity in relation to individual's specific metabolism. By means of animal models, this work aimed at identifying new tetra-OH-PAHs which can be released from the hydrolysis of DNA-adducts and can also be directly detected in biological matrices usually used in the field of biomonitoring such as hair and urine. Results obtained from a targeted gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) approach, demonstrated the presence of 8 tetrahydroxylated metabolites in DNA and 23 in hairs of rats exposed to mixtures of PAHs, which had never been analyzed before. Ten tetra-OH-PAHs were clearly characterized by using their analytical standards, corresponding to 4 parent PAHs (phenanthrene, chrysene, benz[. a]anthracene and benzo[. a]pyrene) whereas 13 tetra-OH-PAHs from 3 other parents (anthracene, fluoranthene and benz[k]fluoranthene) were detected but not yet characterized. No tetrahydroxylated metabolite has been clearly identified for naphthalene, fluorene, benzo[. b]fluoranthene, benzo[g,h,i]perylene, or dibenzo[a,h]anthracene, which can all potentially form adducts.The relevance of tetra-OH-PAH analysis in hair as biomarkers of PAH exposure was evaluated in a dose-response study conducted on 64 rats (Long Evans females/n[U+202F]=[U+202F]8 per groups) under repeated exposure (3 times per week) to a mixture of 16 PAHs at low doses (0.01-0.8[U+202F]mg/kg) for 90 days. Most of the tetra-OH-PAHs targeted in the method were detected in the hairs of the rats, regardless of the dose of exposure. Significant linear relationships (R² ranging from 0.558 to 0.964, p[U+202F]<[U+202F]0.001) were observed between the administered dose and the tetra-OH-PAH concentrations in the hairs for 20 out of the 23 metabolites. By widening the range of PAH metabolites used as biomarkers of exposure so as to include the analysis of PAH tetrahydroxylated forms (especially those exhibiting more than 5 aromatic rings), the present methodology will enable multi-exposure assessments which are more accurately representative of actual situations of exposure to PAHs.

Bisphenol A (BPA) is used in a wide variety of products and objects for consumers use (digital media such as CD's and DVD's, sport equipment, food and beverage containers, medical equipment). For humans, the main route of exposure to BPA is food. Based on previous estimates, almost 20% of the dietary exposure to BPA in the French population would be from food of animal origin. However, due to the use of composite samples, the source of the contamination had not been identified. Therefore, 322 individual samples of non-canned foods of animal origin were collected with the objectives of first updating the estimation of the exposure of the French population and second identifying the source of contamination of these foodstuffs using a specific analytical method. Compared to previous estimates in France, a decline in the contamination of the samples was observed, in particular with regard to meat. The estimated mean dietary exposures ranged from 0.048 to 0.050 μg (kg bw)(-1) d(-1) for 3-17 year children and adolescents, from 0.034 to 0.035 μg (kg bw)(-1) d(-1) for adults and from 0.047 to 0.049 μg (kg bw)(-1) d(-1) for pregnant women. The contribution of meat to total dietary exposure of pregnant women, adults and children was up to three times lower than the previous estimates. Despite this downward trend in contamination, the toxicological values were observed to have been exceeded for the population of pregnant women. With the aim of acquiring more knowledge about the origin the potential source(s) of contamination of non-canned foods of animal origin, a specific analytical method was developed to directly identify and quantify the presence of conjugated BPA (BPA-monoglucuronide, BPA-diglucuronide and sulphate forms) in 50 samples. No conjugated forms of BPAs were detected in the analysed samples, indicating clearly that BPA content in animal food was not due to metabolism but arise post mortem in food. This contamination may occur during food production. However, despite extensive sampling performed in several different shops (butcheries, supermarkets …. ) and in different conditions (fresh, prepared, frozen …), the source(s) of the contamination could not be specifically identified.

Endometriosis is a gynaecological disease characterized by the presence of ectopic endometrial tissue. Histologically, it appears as different sub-types, being peritoneal endometriosis, ovarian endometrioma (OvE) and deep infiltrating endometriosis (DIE), which are of major relevance due to their varying clinical presentations. A number of persistent organic pollutants (POPs) have been associated with the onset of endometriosis, yet the overall set of existing studies remains fairly divergent. In this preliminary case-control study we aimed to assess the potential associations between the internal exposure to POPs and the presence of DIE with or without concurrent OvE. Adipose tissue and serum samples were collected from surgically confirmed cases (n=55) and controls (n=44) enrolled during 2013 and 2015 in Pays de la Loire, France. Targeted pollutants (76 historical or more emerging POPs including dioxins, polychlorobiphenyls (PCB), polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), hexabromocyclododecanes (HBCDs) and organochlorine pesticides (OCPs) were quantified by chromatography coupled to mass spectrometry. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated from unconditional logistic regression adjusted for known confounding variables. The results showed significant associations between DIE and adipose tissue levels of 1.2.3.7.8 - PeCDD, OCDF, PCB 105, 114, 118 and 123, PBDE 183, PBB 153, and several OCPs including trans‑nonachlor, cis‑heptachlor epoxide, dieldrin, β-hexachlorocyclohexane and hexachlorobenzene. The largest associations were observed for OCDF followed by cis‑heptachlor epoxide, exhibiting adjusted ORs (95% CI) of 5.42 (2.73-12.85) and 5.36 (2.44-14.84) per 1-SD increase, respectively. The stratified analysis comparing both disease sub-types suggested that adipose tissue exposure markers may be more associated with DIE concurrent with OvE, however these results need to be confirmed in a larger population.

The aim of the current study was to describe the fate of ingested α-hexabromocyclododecane (α-HBCDD) in fast-growing (FG) and slow-growing (SG) broilers, through an exposure to a dietary concentration of 50 ng α-HBCDD g−1 feed during 42 and 84 days, respectively. Depuration parameters were assessed in SG broilers successively exposed during 42 days and depurated during 42 days. At market age, SG broilers had ingested 42% more feed than FG broilers, while their body weight gain per g of feed ingested was 34% lower. No isomerization of α- to β- or γ-HBCDD forms occurred, while OH-HBCDD was identified as a product of α-HBCDD metabolism. Irrespective of the strain, abdominal fat displayed the highest α-HBCDD concentration on a lipid weight basis, followed leg muscles and then breast muscle, liver and plasma. The accumulation ratios of α-HBCDD were slightly higher in SG (6.7, 2.1, 2.6 and 9.9 in leg muscles, breast muscle, liver and abdominal fat, respectively) than in FG broilers (5.2, 2.2, 1.1 and 8.4, respectively). The elimination half-lives in SG broilers were 20, 12 and 19 d in leg muscles, breast muscle and abdominal fat, respectively, to which dilution through growth contributed for around 50%. The overall assimilation efficiency of α-HBCDD was estimated at 58 and 50% in FG and SG broilers, respectively, while 22 and 17% of α-HBCDD ingested were estimated to be eliminated in excreta as metabolites.

Ion mobility spectrometry (IMS) has recently caught the attention of researchers from different fields including food safety. In general, IMS has been considered as analytical detection tool for the analysis of residues and contaminants in foodstuffs due to its high sensitivity, quick response and portability. However, IMS also provides an extra separation dimension when it is coupled to traditional liquid chromatography or gas chromatography-mass spectrometry methods. Due to the enhancement of the resolving power, target analytes can be easier isolated from chemical background as well as isobaric and isomeric compounds are separated. In addition, collision cross section databases for residues and contaminants have been recently reported. It supposes the first attempt for considering this IMS-related parameter as an additional dimension for chemical structure elucidation in food safety control. This review presents an overview of the current state of IMS in the field and discusses its main perspectives in the area.

The overall concentration of hexabromocyclododecane (HBCDD) in eggs is low although abnormally high concentrations exceeding 3000 ng g⁻¹ lw have been reported. In order to test whether these contaminations may originate from the ingestion of insulating materials in rearing buildings, a group of 55 hens raised in a collective cage was provided with a 64-g piece of extruded polystyrene (XPS, 2.59% HBCDD of which 75, 15 and 10% as α-, β- and γ-HBCDD, respectively). Hens entirely consumed the piece within 3 days, leading to a mean daily exposure of 4.7 mg HBCDD per kg body weight. Whole egg HBCDD concentration reached a maximum of 1037 ng HBCDD g⁻¹ fresh weight (fw), recorded 2 days after the piece had disappeared, and decreased down to 86 ng g⁻¹ fw within the 19 following days. In all these samples, HBCDD was made of 98.7 ± 0.7 and 1.3 ± 0.6% α- and β-HBCDD, respectively, and 0.1% γ-HBCDD when quantified; it was enriched in (−)α- and (+)β-HBCDD with enantiomeric fractions of 0.438 ± 0.009 and 0.579 ± 0.030, respectively. HBCDD was quantified in all the individual eggs collected the last day of experiment at concentrations ranging between 0.47 and 1361 ng g⁻¹ fw, according to a lognormal distribution. The ingestion of XPS in degraded rearing buildings is thus a plausible cause of on-farm egg contamination by HBCDD which should be strictly avoided.

Introduction: Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites. Objectives: In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting. Methods: The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces. Results: A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed. Conclusion: The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

Perfluorooctane sulfonamidoethanol based phosphate diester (SAmPAP) is a potential PFOS-precursor. To examine whether SAmPAP exposure would result in fish contamination by perfluoroalkyl and polyfluoroalkyl substances (PFASs), juvenile Eurasian perch were dietary exposed to this compound (dosed group) or exposed to the same tank water but fed control feed (control group). SAmPAP and metabolites were monitored in the muscle, liver and serum during the 45-day exposure phase and 35-day depuration phase. SAmPAP was only detected in the dosed group and absorption efficiency (0.04-2.25%) was very low possibly related to low bioavailability in the gastrointestinal tract, steric constraints in crossing biological membranes and clearing by entero-hepatic circulation. Although SAmPAP was biotransformed and eliminated at a slow rate (t1/2>18 days), its biomagnification factor was low. The observed metabolites in fish were N-ethyl perfluorooctane sulfonamidoacetic acid (NEtFOSAA), perfluorooctane sulfonamidoacetic acid (FOSAA), perfluorooctane sulfonamide (FOSA) and PFOS. Considering SAmPAP was the only source of PFASs in the tanks, the occurrence of metabolites indicates that SAmPAP could be biotransformed in fish and contribute to PFOS bioaccumulation. However, levels of metabolites were not significantly different in the dosed and control group indicating that metabolite excretion followed by re exposure to these metabolites from water was the main uptake route.

IntroductionThe surveillance of illegal anabolic practices in bovine meat production is necessary to guarantee consumers’ health. Screening strategies based on the recognition of indirect biological effects are considered by the community as promising tools to overcome some limitations of classical analytical methods and might therefore concur to ensure safer food for the consumer. Objectives The present work aims at characterizing the metabolic profile induced in liver by administration of anabolic steroids, and at identifying potential disturbances in the hepatic metabolism. MethodsA total of 32 liver samples, 16 from untreated bulls and 16 from bulls treated with an ear implant (Revalor-XS®) containing trenbolone acetate (200 mg) and β-estradiol (40 mg), were analyzed following a LC–MS-based metabolomic analysis combining RP and HILIC chromatographic separations. Different multivariate statistical tools were applied to the datasets to select common metabolites that may be considered as potential markers based on their significant changes in concentrations after administration of sexual steroids. ResultsEight candidate markers were identified. Moreover, a subset of four markers was also validated by a different laboratory that performed the same analysis using an independent instrumental and elaboration platform, confirming the robustness of the results achieved. Conclusion This study was performed mimicking experimental conditions that may be used during a potential misuse practice. It is promising in the objective of setting up an analytical strategy to highlight sexual steroids abuse in livestock animals.

IntroductionRactopamine, a β-agonist used as growth promoter in livestock, is with great controversy, and it has been forbidden in most countries worldwide. However, due to economic benefits, the possibility of widespread abuse of ractopamine still exists. “Omics” strategies, based on the observation of physiological perturbations, are promising approaches to tackle drug misuse in breeding animals. ObjectivesA study was performed to determine if serum-metabolomics could be used to establish a predictive tool for identifying ractopamine misuse in pigs. Methods Our aim was to set up a high performance liquid chromatography—high resolution mass spectrometry based metabolomics workflow for screening pig serum for ractopamine administration. Therefore, an untargeted metabolomics approach was developed to characterize and compare serum metabolic profiles from control and treated pigs. Two different extraction strategies were investigated, and the results showed that the combination of methanol extraction and methanol–water extraction protocols significantly improve the metabolites coverage. A two-level data analysis using univariate and multivariate statistical analyses was carried out to establish descriptive and predictive models. ResultsThe discrimination of treated animals from control animals could be achieved. A number of candidate biomarkers that contributed the most in the observed discrimination could be listed. Conclusion This research indicates that metabolomics approach can be considered as a powerful strategy to highlight biomarkers related to ractopamine treatment in pig which may subsequently be implemented as screening strategy to predict for such illicit practices.

The chemical contamination levels of both conventional and organic meats were assessed. The objective was to provide occurrence data in a context of chronic exposure. Environmental contaminants (17 PCDD/F, 18 PCBs, 3 HBCD isomers, 6 mycotoxins, 6 inorganic compounds) together with chemical residues arising from production inputs (75 antimicrobials, 10 coccidiostats and 121 pesticides) have been selected as relevant compounds. A dedicated sampling strategy, representative of the French production allowed quantification of a large sample set (n=266) including both conventional (n=139) and organic (n=127) raw meat from three animal species (bovine, porcine, poultry). While contamination levels below regulatory limits were measured in all the samples, significant differences were observed between both species and types of farming. Several environmental contaminants (Dioxins, PCBs, HBCD, Zn, Cu, Cd, Pb, As) were measured at significantly higher levels in organic samples.

A LC-ESI(−)-HRMS method dedicated to the analysis of 6 HBCDD enantiomers at trace levels in animal matrices was developed, using a cellulose based stationary phase with a particle size of 2.5 μm. This method was applied to a sample set derived from a kinetic study of α-HBCDD previously conducted in fast- and slow-growing chickens (Gallus gallus domesticus, n = 49, plus controls), in order to study the enantiomer specific accumulation and depuration of α-HBCDD in various tissues. Regarding abdominal adipose tissue, muscle and liver, the average enantiomeric fractions of α-HBCDD (EFα) for continuously exposed groups ranged between 0.434 and 0.467, with standard deviations below 0.014, showing a significant enrichment in (−)α enantiomer even accentuated for slow growing individuals during depuration with EFα reduced by about 0.020. Similar trends were observed for pooled plasma. Then, EFα of circulating plasma α-HBCDD appeared to closely reflect EFα in storage tissues and liver, suggesting some equilibrium. The racemic elimination of α enantiomer in excreta during the contamination phase indicated that no preferential gastrointestinal absorption took place. By contrast, preferential excretion of (−)α-HBCDD from the circulating compartment to the intestinal lumen occurred during the depuration. Finally, the method was applied to samples collected in three chicken farms, selected for total HBCDD levels in muscle in the ng/g range, as a tool to determine whether the contamination occurred ante- or post-mortem, according to the chiral signature. Ante-mortem contamination was hypothesised for 2 farms, with feed being excluded as contamination source.

A probabilistic and interdisciplinary risk–benefit assessment (RBA) model integrating microbiological, nutritional, and chemical components was developed for infant milk, with the objective of predicting the health impact of different scenarios of consumption. Infant feeding is a particular concern of interest in RBA as breast milk and powder infant formula have both been associated with risks and benefits related to chemicals, bacteria, and nutrients, hence the model considers these three facets. Cronobacter sakazakii, dioxin-like polychlorinated biphenyls (dl-PCB), and docosahexaenoic acid (DHA) were three risk/benefit factors selected as key issues in microbiology, chemistry, and nutrition, respectively. The present model was probabilistic with variability and uncertainty separated using a second-order Monte Carlo simulation process. In this study, advantages and limitations of undertaking probabilistic and interdisciplinary RBA are discussed. In particular, the probabilistic technique was found to be powerful in dealing with missing data and to translate assumptions into quantitative inputs while taking uncertainty into account. In addition, separation of variability and uncertainty strengthened the interpretation of the model outputs by enabling better consideration and distinction of natural heterogeneity from lack of knowledge. Interdisciplinary RBA is necessary to give more structured conclusions and avoid contradictory messages to policymakers and also to consumers, leading to more decisive food recommendations. This assessment provides a conceptual development of the RBA methodology and is a robust basis on which to build upon.

Among pregnant women ibuprofen is one of the most frequently used pharmaceutical compounds with up to 28% reporting use. Regardless of this, it remains unknown whether ibuprofen could act as an endocrine disruptor as reported for fellow analgesics paracetamol and aspirin. To investigate this, we exposed human fetal testes (7–17 gestational weeks (GW)) to ibuprofen using ex vivo culture and xenograft systems. Ibuprofen suppressed testosterone and Leydig cell hormone INSL3 during culture of 8–9 GW fetal testes with concomitant reduction in expression of the steroidogenic enzymes CYP11A1, CYP17A1 and HSD17B3, and of INSL3. Testosterone was not suppressed in testes from fetuses younger than 8 GW, older than 10–12 GW, or in second trimester xenografted testes (14–17 GW). Ex vivo, ibuprofen also affected Sertoli cell by suppressing AMH production and mRNA expression of AMH, SOX9, DHH, and COL2A1. While PGE2 production was suppressed by ibuprofen, PGD2 production was not. Germ cell transcripts POU5F1, TFAP2C, LIN28A, ALPP and KIT were also reduced by ibuprofen. We conclude that, at concentrations relevant to human exposure and within a particular narrow ‘early window’ of sensitivity within first trimester, ibuprofen causes direct endocrine disturbances in the human fetal testis and alteration of the germ cell biology.

Starting from a critical analysis of a first “proof of concept” study on the utility of the liver volatolome for detecting livestock exposure to environmental micropollutants (Berge et al., 2011), the primary aim of this paper is to improve extraction conditions so as to obtain more representative extracts by using an extraction temperature closer to livestock physiological conditions while minimizing analytical variability and maximizing Volatile Organic Compound (VOC) abundancies. Levers related to extraction conditions and sample preparation were assessed in the light of both abundance and coefficient of variation of 22 candidate VOC markers identified in earlier volatolomic studies. Starting with a CAR/PDMS fiber and a 30 minutes extraction, the reduction of SPME temperature to 40 °C resulted in a significant decrease in the area of 14 candidate VOC markers (p < 0.05), mainly carbonyls and alcohols but also a reduction in the coefficient of variation for 17 of them. In order to restore VOC abundances and to minimize variability, two approaches dealing with sample preparation were investigated. By increasing sample defrosting time at 4 °C from 0 to 24 h yielded higher abundances and lower variabilities for 15 and 13 compounds, respectively. Lastly, by using additives favouring the release of VOCs (1.2 g of NaCl) the sensitivity of the analysis was improved with a significant increase in VOC abundances of more than 50% for 13 out of the 22 candidate markers. The modified SPME parameters significantly enhanced the abundances while decreasing the analytical variability for most candidate VOC markers. The second step was to validate the ability of the revised SPME protocol to discriminate intentionally contaminated broiler chickens from controls, under case/control animal testing conditions. After verification of the contamination levels of the animals by national reference laboratories, data analysis by a multivariate chemometric method (Common Components and Specific Weights Analysis − ComDim) showed that the liver volatolome could reveal dietary exposure of broilers to a group of environmental pollutants (PCBs), a veterinary treatment (monensin), and a pesticide (deltamethrin), thus confirming the usefulness of this analytical set-up.

Conducting an MS based metabolomics study : critical review, experiences and Outlook SUMMARY Metabolomics is a methodological approach emerged in the late 2 000s in line with other «- omics» : genomics, transcriptomics and proteomics. Like these other approaches, metabolomics is used in various fields and aims at measuring simultaneously all small endogenous metabolites (< 1 000 Da) impacted by a disturbance affecting a given biological system. Ultimately, these measures give insights in differences, often tenuous, between different states of a biological system. To achieve with confidence such a degree of sensitivity, it is necessary to master all the different steps of the approach. Metabolomics involves several disciplines (biology, chemistry, bioinformatics, and statistics) and such diversity multiplies the possibilities of confounding factors to arise and thus increases the sources of results misinterpretation. The expertise acquired over the years by researchers from the different areas involved in metabolomics can be briefly summarized in a generic workflow that from the experimental design to the biological interpretation of the data, intends at minimizing all sources of undesirable variability. All this is done in order to respond to this «simple» question : are the differences observed at the end of the process really and only linked to a biological reality ? Here, we discuss the first steps of a metabolomics study: experimental design, sample preparation, data acquisition and pre-processing, with the primary objective to provide clues for a critical assessment of the results and to provide the foundation for the successful implementation of a metabolomics study, in other words, a study meeting the quality criteria for an unbiased interpretation. Examples of illustration mainly refer to studies of mass spectrometric based untargeted metabolomics in the field of chemical food safety.

L’utilisation des promoteurs de croissance est interdite en élevage au sein de l’Union européenne depuis 1988. Afin de garantir au consommateur des denrées exemptes de résidus de ce type de substances, un dispositif européen de surveillance et de contrôle accompagne cette mesure, qui en France est organisé depuis 1988 dans le cadre des plans de surveillance et de contrôle mis en place par la direction générale de l’Alimentation. Le présent article décrit le cadre réglementaire, les modalités de mise en oeuvre en termes de composés d’intérêt, d’espèces animales concernées, de matrices biologiques pertinentes et de stratégies analytiques adaptées. Les données issues des plans 2014 illustrent l’ensemble du dispositif.

Urine stability during storage is essential in metabolomics to avoid misleading conclusions or erroneous interpretations. Facing the lack of comprehensive studies on urine metabolome stability, the present work performed a follow-up of potential modifications in urinary chemical profile using LC-HRMS on the basis of two parameters: the storage temperature (+4°C, -20°C, -80°C and freeze-dried stored at -80°C) and the storage duration (5 to 144 days). Both HILIC and RP chromatographies have been implemented in order to globally monitor the urinary metabolome. Using an original data processing associated to univariate and multivariate data analysis, our study confirms that chemical profiles of urine samples stored at +4°C are very rapidly modified, as observed for instance for compounds such as :N-acetyl Glycine, Adenosine, 4-Amino benzoic acid, N-Amino diglycine, creatine, glucuronic acid, 3-hydroxy-benzoic acid, pyridoxal, L-pyroglutamic acid, shikimic acid, succinic acid, thymidine, trigonelline and valeryl-carnitine, while it also demonstrates that urine samples stored at -20°C exhibit a global stability over a long period with no major modifications compared to -80°C condition. This study is the first to investigate long term stability of urine samples and report potential modifications in the urinary metabolome, using both targeted approach monitoring individually a large number (n>200) of urinary metabolites and an untargeted strategy enabling assessing for global impact of storage conditions.

Boar taint is an offensive odor that can occur while cooking pork or pork products and is identified in some uncastrated male pigs that have reached puberty. It is widely held that boar taint is the result of the accumulation in back-fat of two malodorous compounds: androstenone and skatole. The purpose of the present study was to assess a mass spectrometry-based metabolomics strategy to investigate the metabolic profile of urine samples from pig carcasses presenting low (untainted) and high (tainted) levels of androstenone and skatole in back fat. Urine samples were analyzed by LC-ESI(+)-HRMS. Discrimination between tainted and untainted animals was observed by application of multivariate statistical analysis, which allowed to highlight candidate urinary biomarkers. These urinary metabolites were positively correlated to androstenone and skatole levels in back fat. Therefore, the present study suggested that the measurement of these urinary metabolites might provide information with regard to androstenone and skatole levels in live pigs

Organophosphate esters (OPEs) are chemical compounds incorporated into materials as flame‐proof and/or plasticizing agents. In this work, 13 non‐halogenated and 5 halogenated OPEs were studied. Their mass spectra were interpreted and compared in terms of fragmentation patterns and dominant ions via various ionization techniques [electron ionization (EI) and chemical ionization (CI) under vacuum and corona discharge atmospheric pressure chemical ionization (APCI)] on gas chromatography coupled to mass spectrometry (GC‐MS). The novelty of this paper relies on the investigation of APCI technique for the analysis of OPEs via favored protonation mechanism, where the mass spectra were mostly dominated by the quasi‐molecular ion [M + H]⁺. The EI mass spectra were dominated by ions such as [H4PO4]⁺, [M–R]⁺, [M–Cl]⁺, and [M–Br]⁺, and for some non‐halogenated aryl OPEs, [M]+● was also observed. The CI mass spectra in positive mode were dominated by [M + H]⁺ and sometimes by [M–R]⁺, while in negative mode, [M–R]⁻ and more particularly [X]‐ and [X2]‐● were mainly observed for the halogenated OPEs. Both EI and APCI techniques showed promising results for further development of instrumental method operating in selective reaction monitoring mode. Instrumental detection limits by using APCI mode were 2.5 to 25 times lower than using EI mode for the non‐brominated OPEs, while they were determined at 50‐100 times lower by the APCI mode than by the EI mode, for the two brominated OPEs. The method was applied to fish samples, and monitored transitions by using APCI mode showed higher specificity but lower stability compared with EI mode. The sensitivity in terms of signal‐to‐noise ratio varying from one compound to another. Copyright © 2016 John Wiley & Sons, Ltd.

Selective androgen receptor modulators (SARMs) are a novel class of androgen receptor ligands. They are intended to exhibit the same kind of effects as androgenic drugs, like anabolic steroids, but be much more selective in their action, targeting particular tissues without any undesirable effects on others. While the main applications of these synthetic substances are for therapeutic purposes, they also have a high potential for misuse in veterinary practice and the sporting world. In order to guarantee the consumers with food from animal origin free from any residues of such compounds, analytical strategies are required to ensure safe food and also enable fair trade between producers. In this context an animal experiment involving bovines administered with enobosarm has been conducted to provide the study with biological matrices. Different animal matrices (urine and faeces) have been investigated to select most appropriate matrix to be used for control purposes, in terms of metabolite relevance and detection time window. Based on ultra-high pressure liquid chromatography (UHPLC) hyphenated to tandem mass spectrometry (LC-MS/MS) this work highlighted the presence of sulfonated and glucuronated-conjugated forms of the molecule in the urine of treated animals. Enobosarm could be detected in urine up to 9 days after the administration when samples undergo phase II hydrolysis. Faeces was demonstrated to be the main matrix of excretion of enobosarm since values up to 500 times higher compared to urine could be detected, during 21 days. There was no difference between the kinetic profiles when a deconjugation step was or was not was applied.

Besides their development for therapeutic purposes, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth associated pathways as ligands of androgenic receptors (AR). They present thus a potential for abuse in sports and food producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non-steroidal SARMs used in human treatment of non-metastatic prostate cancer because of its antiandrogenic activity exhibiting no anti-anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and feces) have been investigated and SARMs metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and fecal eliminations of bicalutamide in a calf, and investigate Phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in feces (ppm) than urine (ppb); although both matrices were assessed as suitable for residues control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions).

Food consumption data of the populations of four Sub-Saharan Africa countries were derived from food expenditure data of 72.979 households. A food classification system was set up for the purpose of the study. The approach enables to take into consideration the local specificity of food consumption patterns in the completion of dietary exposure assessment.

Marine ecosystems are constantly being threatened by contaminants produced by human activities. There is an urge to better understand their impacts on marine organisms and develop reliable tools for biomonitoring studies, while also assessing their potential impacts on human health. Given their position on top of food webs, sharks are particularly susceptible to bioaccumulation, making them potential sentinel species of marine contamination. The main objective of this study was to find suitable biomarkers for future marine pollution biomonitoring studies by correlating biochemical responses with tissue contaminant body burden in blue sharks (Prionace glauca), a species heavily caught and consumed by humans, while also addressing their general health. The chemical contaminants analysed comprised different persistent organic pollutants (POPs) families from polychlorinated compounds to brominated flame retardants (BFRs) and perfluorinated compounds (PFCs) and different trace and heavy metals. Concentrations of some contaminants in sharks' tissues were found to be above the legally allowed limits for human consumption. A canonical correspondence analysis (CCA) was performed and some strong associations were found between biochemical responses and contaminants' accumulation levels. DNA damage and lipid peroxidation levels, as well as the inhibition of the antioxidant enzyme glutathione peroxidase, were the main effects and consequences of contamination. The impact of contamination on these vital macromolecules underlines the suboptimal conditions of the sampled P. glauca, which can ultimately lead to the degradation of core ecological aspects, such as swimming, feeding, and reproduction. It can be concluded that P. glauca demonstrates great potential to be used as environmental sentinel and suitable biomarker candidates were identified in this work. Moreover, this study also highlights the risks that the consumption of blue shark derived products can pose to human health, which is of upmost interest as the sampled organisms were still juveniles and already presented values above regulatory limits.

Several studies have assessed the potential role of environmental chemicals in the onset, growth, and/or physiopathology of endometriosis. However, their contour in terms of considered exposure markers remains limited. The present study aimed to characterize the internal exposure levels of 78 persistent organic pollutants (POPs, including dioxins, polychlorobiphenyls, brominated flame retardants and organochlorine pesticides) in a set of 113 adult French women (45 controls, 68 cases), and to characterize the distribution of these POPs within three biological compartments (omental adipose tissue, parietal adipose tissue, and serum). For all targeted substances, the correlation between the concentrations measured in omental versus parietal adipose tissue was found strongly significant (p<0.0001). An equivalence of the measures performed in parietal and omental adipose tissue was moreover observed with median levels of 6.4 vs. 7.4pg/gl.w. for WHO-TEQ2005 PCDD/F, 4.5 vs. 4.7pg/gl.w. for WHO-TEQ2005 dl-PCB, 137.1 vs. 147.9ng/gl.w. for sum of 6 ndl-PCB, and 2.1 vs. 2.0ng/gl.w. for sum of 7 i-PBDE, respectively. The same observation was made for individual targeted OCs compounds. Significant correlations were also observed between these concentrations determined in adipose tissue and those measured in serum for dioxins (WHO-TEQ2005 PCDD/F=6.1pg/gl.w), PCBs (WHO-TEQ2005 dl-PCB=3.6pg/gl.w., sum of 6 ndl-PCB=81.1ng/gl.w.), and brominated flame-retardants (sum of 7 i-PBDE=0.9ng/gl.w.). The circulating versus stored ratio of some exposure markers (Sum PCDDs, 1,2,3,6,7,8-HxCDF, slightly versus highly chlorinated PCBs ratio, PBDE 99 and PBB 153) was found statistically different for control and case individuals. These extended exposure data from deep infiltrating endometriosis patients are the first ones available for France and give a new insight about the equilibrium of chemicals between storage and circulating compartments that should be further considered as new marker of exposure in the context of exposure-health relationship studies.

The present study compares concentrations and chemical profiles of an extended range of persistent organic pollutants (dioxins, polychlorobiphenyls, brominated flame retardants and organochlorine pesticides) in breast milk samples from French (n = 96), Danish (n = 438) and Finnish (n = 22) women. Median exposure levels observed in French women (WHO-TEQ2005 PCDD/F = 6.1 pg/g l.w., WHO-TEQ2005 dl-PCB = 4.3 pg/g l.w., sum of 6 ndl-PCB = 85.2 ng/g l.w., sum of 7 i-PBDE = 1.5 ng/g l.w.) appeared overall lower than in Danish and Finnish women for all examined POPs, except for α-HBCD (2-fold higher level at 0.6 ng/g l.w.). Furthermore, the observed exposure levels of dioxins and PCBs were higher in Danish women (WHO-TEQ2005 PCDD/F = 13.2 pg/g l.w., WHO-TEQ2005 dl-PCB = 6.6 pg/g l.w., sum of 6 ndl-PCB = 162.8 ng/g l.w.) compared to Finnish women (WHO-TEQ2005 PCDD/F = 9.0 pg/g l.w., WHO-TEQ2005 dl-PCB = 4.6 pg/g l.w., sum of 6 ndl-PCB = 104.0 ng/g l.w.), whereas the concentrations of PBDEs were similar for Danish and Finnish women (sum of 7 i-PBDE = 4.9 and 5.2 ng/g l.w. respectively). The organochlorine (OC) pesticide contamination profile, determined in a subset of French samples, was dominated by p,p’-DDE (56.6%), followed by β-HCH (14.2%), HCB (9.7%) and dieldrin (5.2%), while other compounds were only minor contributors (<5%). The three countries appeared to be discriminated by the observed contamination patterns of the PCDD/F versus PCB, and the 1,2,3,6,7,8-HxCDD versus 1,2,3,4,7,8-HxCDD ratios, in addition to the relative contributions of specific congeners to the contamination profile (PCBs #118 and #156, PBDEs #28, #47, #99 and #153). In conclusion, unique chemical signatures were observed for each country on the basis of some POP congeners. Future biomonitoring studies will need to consider the high variability of individual exposure profiles in relation to multiple exposure sources but also physiological and metabolic differences.

ScopeResveratrol has a diverse array of healthful effects on metabolic parameters in different experimental paradigms but has also potential to inhibit steroidogenesis in rodent adrenals. The aim of the present study was to characterize the effects of resveratrol on human fetal adrenal steroidogenesis at gestational weeks (GW) 9-12. Methods and resultsAdrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/mL) with or without resveratrol (10 M) for 24 h. The production of steroids by HFAC was analyzed by gas and liquid chromatography coupled to tandem/mass spectrometry. The expression of steroidogenic enzymes at GW 9-12 was quantified by automated Western blotting. We observed that resveratrol significantly suppressed synthesis of dehydroepiandrosterone (DHEA), androstenedione and 11-deoxicortisol by ACTH-activated and unstimulated HFAC, which was associated with inhibition of the activities and expression of cytochromes 17-hydroxylase/17,20 lyase (CYP17) and 21-hydroxylase (CYP21) in these fetal adrenocortical cells. Conclusion Our in vitro findings on the sensitivity of human fetal adrenal steroidogenesis to resveratrol at GW9-12 suggest that intake of this polyphenol at high doses by women who are at early stages of pregnancy is undesirable.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants present in dietary fats. Most studies evaluating PCB effects have been conducted with a single compound or a mixture of PCBs given as a single acute dose. The purpose of this study was to evaluate in vivo PCB toxicity in a realistic model of exposure: a low daily dose of PCBs (twice the tolerable daily intake (TDI)), chronically administered (8 weeks) to rats in contaminated goat milk. Liver and brain PCB toxicities were investigated by evaluating oxidative stress status and mitochondrial function. PCB toxicity in the liver was also estimated by transaminase enzymatic activity. This study shows that even at low doses, chronic PCB exposure resulted in a statistically significant reduction of mitochondrial function in liver and brain. In the liver, oxygen consumption in the condition of adenosine triphosphate (ATP) production (state 3) decreased by 22-29% (p < 0.01), according to the respiratory substrates. In the brain, respiratory chain complexes II and III were reduced by 24% and 39%, respectively (p < 0.005). The exposed rats presented higher lipid peroxidation status (+20%, p < 0.05) and transaminase activity (+30%, p < 0.05) in the blood. Thus, our study showed that exposure of rats to a daily realistic dose of PCBs (twice the TDI in a food complex mixture of environmental origin) resulted in multiple disruptions in the liver and brain.

In the present work, we addressed the question of global seeking/screening organohalogenated compounds in a large panel of complex biological matrices, with a particular focus on unknown chemicals that may be considered as potential emerging hazards. A fishing strategy was developed based on untargeted profiling among full scan acquisition datasets provided by high resolution mass spectrometry. Since large datasets arise from such profiling, filtering useful information stands as a central question. In this way, we took advantage of the exact mass differences between Cl and Br isotopes. Indeed, our workflow involved an innovative Visual Basic for Applications script aiming at pairing features according to this mass difference, in order to point out potential organohalogenated clusters, preceded by an automated peak picking step based on the centWave function (xcms package of open access R programming environment). Then, H/Cl-scale mass defect plots were used to visualize the datasets before and after filtering. The filtering script was successfully applied to a dataset generated upon liquid chromatography coupled to ESI(-)-HRMS measurement from one eel muscle extract, allowing for realistic manual investigations of filtered clusters. Starting from 9,789 initial obtained features, 1,994 features were paired in 589 clusters. Hexabromocyclododecane, chlorinated paraffin series and various other compounds have been identified or tentatively identified, allowing thus broad screening of organohalogenated compounds in this extract. Although realistic, manual review of paired clusters remains time consuming and much effort should be devoted to automation. https://advanceseng.com/chemical-engineering/screening-halogenated-environmental-contaminants-biota-based-isotopic-pattern-mass-defect-provided-high-resolution-mass-spectrometry-profiling/

A gas chromatography tandem mass spectrometry method using atmospheric pressure chemical ionisation was developed for the monitoring of 16 brominated flame retardants (7 usually monitored polybromodiphenylethers (PBDEs) and BDE #209 and 8 additional emerging and novel BFRs) in food and feed of animal origin. The developed analytical method has decreased the run time by three compared to conventional strategies, using a 2.5 m column length (5% phenyl stationary phase, 0.1 mm i.d., 0.1 μm f.t.), a pulsed split injection (1:5) with carrier gas helium flow rate at 0.48 mL min−1 in one run of 20 min. For most BFRs, analytical data were compared with the current analytical strategy relying on GC/EI/HRMS (double sector, R = 10000 at 10% valley). Performances in terms of sensitivity were found to meet the Commission recommendation (118/2014/EC) for nBFRs. GC/APCI/MS/MS represents a promising alternative for multi-BFRs analysis in complex matrices, in that it allows the monitoring of a wider list of contaminants in a single injection and a shorter run time.

b-agonist drugs are synthetic molecules used for therapeutic purposes in human as well as in veterinary medicine for their anti-asthmatic, bronchodilator, tocolytic and cardiotonic properties. However, and despite their ban for growth promoting purposes [Dir 1996/22/EC], their illegal use in food producing animals is regularly suspected in Europe and in edible tissues imported from third countries. The presence of residues and their associated harmful effects on humans makes the control of such veterinary drug residues a key component in ensuring consumer protection [Dir 1996/23/EC]. The control of b-agonists misuse received extra attention after outbreaks of food poisoning in 1990 in Spain caused by consumption of bovine liver. This was the first time that pharmacological residues present in slaughtered cattle were found to have caused acute intoxication in consumers. Although clenbuterol is still probably the most popular known b-agonist illegally used in farm, a wide range of other b-agonists exists which constitutes a challenge in detecting their use. Besides classical protocols of administration, some practices consisting in the use of “cocktails” composed of mixtures of low amounts of several substances that exert a synergistic effect and exhibiting similar growth promotion properties have also been reported as additional challenge to be overcome by control laboratories. Emerging screening strategies based on biomarkers monitoring have proven their efficiency in highlighting in a generic way a range of b-agonists based practices, while confirmatory strategies targeting b-agonists residues in various relevant biological matrices (urine, serum, tissues, retina, hair) enable long term detection of forbidden practices. Although histology is not implemented as screening strategy to detect b-agonists misuse, its application should be encouraged to investigate microscopic changes in lungs of treated animals.

Plurality and individuality of hepatocellular carcinoma: PPPM perspectives Krishna Chander Sridhar, Olga Golubnitschaja Department of Radiology, Univeristy of Bonn, Bonn, Germany Correspondence: Olga Golubnitschaja (olga.golubnitschaja@ukb.uni-bonn.de) – Department of Radiology, Univeristy of Bonn, Bonn, Germany The EPMA Journal 2016, 7(Suppl 1):A72 The EPMA Journal 2016, Volume 7 Suppl 1 Page 35 of 42 Keywords: Predictive preventive personalized medicine, Innovative screening methods, Patient-specific therapy, Multilevel diagnostics, Molecular markers, CTCs and CNAPS, Stratification Hepatocellular Carcinoma (HCC) may arise due to various risk fac- tors such as genetic predisposition, chronic hepatic infections, al- coholabuse,hemochromatosis,cirrhosis,obesity,fatty liver disease, etc. [1, 2]. Low mean 5-year survival rate (15 %) and high treatment costs imply the need for more effective treatments tai- lored to the person [3]. The predictive, preventive and personal- ized medicine (PPPM) aims at paradigm change in management of HCC from delayed to advanced approaches creating innovative screening programs, early/predictive diagnostics and targeted therapy [1]. For that, better patient stratification is needed utilizing multilevel diagnostics. Molecular mechanisms which underlie tumor manifestation, progression and aggressiveness may vary substantially inHCC dependingonanumber of factorssuchas localization of the primary tumor, tumor’scharacteristics / sub- type, family history, life-style and co-morbidities. Consequently, a creation of individualized patient profiles is of particular import- ance in patient stratification, predictive/early diagnostics and tar- geted treatments of HCC (see Fig. 4). Further, highly sensitive and specific biomarker-panels play a crucial role in sub-categorization of HCC, e.g. to distinguish between local and distanced metastatic activities, where the latter are functionally linked to circulating en- tities such as circulating tumour cells (CTC) and circulating nucleic acids in plasma and serum (CNAPS) [4]. The integrative PPPM-strategies advancing HCC management (both curative and palliative) may have great impacts on improved screen- ing of individuals at risk, life-quality of targeted patient cohorts, bet- ter healthcare economy and benefits to diagnostic and pharmaceutical industry. References 1. Berliner L, Lemke HU (eds.). An Information Technology Framework for Predictive, Preventive and Personalized Medicine: A Use-Case with Hepato- cellular Carcinoma. In: Advances in Predictive, Preventive and Personalised Medicine, Vol 8in Cham: Springer International Publishing; 2015 2. Fox RK. Surveillance for Hepatocellular Carcinoma. Hepatitis C Online, http:// www.hepatitisc.uw.edu/go/evaluation-staging-monitoring/surveillance- hepatocellular-carcinoma/core-concept/all (2013) Accessed 15 Aug 2015. 3. Thein HH, Isaranuwatchai W, Campitelli MA, Feld JJ, Yoshida E, Sherman M, et al. Healthcare costs associated with hepatocellular carcinoma: a population-based study. Hepatology 2013;58(4):1375-84. 4. Zhao YJ, Ju Q, Li GC. Tumor markers for hepatocellular carcinoma. Mol Clin Oncol. 2013;1(4):593-598.

Currently, no analytical method is available to demonstrate progesterone administration in biological samples collected in rearing animals, and therefore, tracking the abuse of this popular growth promoter is arduous. In this study, a method is presented to reveal progesterone (PG) treatment on the basis of carbon isotope measurement of 5β-pregnane-3α, 20α-diol (BAA-PD), a major PG metabolite excreted in bovine urine, by gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry (GC-MS/C/IRMS). 5-Androstene-3β,17α-diol (AEdiol) is used as endogenous reference compound. Intermediate precisions (n=11) of 0.56‰ and 0.68‰ have been determined for AEdiol and BAA-PD, respectively. The analytical method was used for the very first time to successfully differentiate urine samples collected in treated and untreated animals.

The aim of the current study was to describe the fate of ingested α-hexabromocyclododecane (α-HBCDD) in laying hens. Individuals were exposed to two dietary concentrations of α-HBCDD (50 and 5 ng g-1 feed) for 18 or 11 weeks followed by a 7-week decontamination period. The results show that no isomerization of α- to β- or γ-HBCDD forms occurred, while OH-HBCDD was identified as a product of α-HBCDD metabolism. Irrespective of the level of feed contamination, estimates of steady state accumulation ratios were 5.2, 3.6 and 9.2 and half-lives were estimated at 17.4, 22.8 and 35.3 days, in egg yolk, liver tissue and abdominal fat, respectively. The steady state carry over rate to eggs was 22.9%. Thus, α-HBCDD ingested by laying hens is readily transferred to eggs and significantly accumulates in adipose tissue.

The safety and quality of infant milk, whether it is breast milk (BM) or infant formula (IF), are a major concern for parents and public health authorities. BM is recommended as the gold standard at WHO level. However, nowadays IF appears as an essential alternative in Western countries, challenging producers to optimise nutritional quality and safety of IF. The aim of the present paper is to give an overview on the assessment and comparison of risks and benefits associated with BM and IF consumption. To date, this intensively debated subject has been mainly investigated. It has been shown that both diets could be sources of beneficial health effects in terms of nutrition and also risks in terms of chemical safety. Moreover, microbiologists have demonstrated that IF consumption can cause illness due to product contamination or inappropriate milk preparation. The paper concludes on the bottlenecks and gaps which should be investigated to further progress the quantification of the impact of early diet on infant health. Performing a multi-disciplinary risk-benefit assessment with DALY as endpoint, might be a future option to help prioritise management options.

STUDY QUESTION Do sex and maternal smoking effects on human fetal anogenital distance (AGD) persist in a larger study and how do these data integrate with the wider literature on perinatal human AGD, especially with respect to sex differences?

Currently under development for therapeutic purposes in human medicine, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth associated pathways. As such, they present a potential for abuse in sports and food-producing animals as interesting alternative anabolic substances. Forbidden since 2008 by the World Anti-Doping Agency (WADA) these compounds are however easily available and could be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detection of these compounds in biological matrices. Using an innovative analytical platform constituted of supercritical fluid chromatography coupled to ion mobility-mass spectrometry, the present study enabled efficient separation and identification in urine of 4 of these drugs (andarine, bicalutamide, hydroxyflutamide, and enobosarm) in accordance with European Union criteria (Commission Decision 2002/657/EC). Besides providing information about compounds structure and behaviour in gas phase, such a coupling enabled reaching low limits of detection (LOD < 0.05 ng.mL(-1) for andarine and limits of detection < 0.005 ng.mL(-1) for the three others) in urine with good repeatability (CV < 21 %). The workflow has been applied to quantitative determination of enobosarm elimination in urine of treated bovine (200 mg, oral). Copyright © 2016 John Wiley & Sons, Ltd.

Invasive breast carcinoma is the most common cancer in women as in non-ovariectomised pet dogs, which are already identified as a valuable spontaneous preclinical model for that disease. Geographical and time trends suggest that environmental factors may play an important role in the etiology and pathogenesis of breast cancer. Persistent organic pollutants (POPs) fit perfectly with these trends and are known to interact with hormonal receptors implicated in breast cancer subtyping. The aim of this innovating study was to evaluate the interest of the companion dog model in assessing chemical exposure and breast cancer associations, in order to identify common etiological features with the human disease in a context of comparative oncology. We monitored a hundred of molecules belonging to a large panel of POPs (dioxins, dioxin-like and non dioxin-like polychlorobisphenyls, organochlorine pesticides, brominated flame retardants, perfluorinated alkylated substances) in companion dogs diagnosed for mammary adenocarcinoma (n = 54) and non cancer controls (n = 47). All targeted chemical families were able to be detected in canine samples. We identified pollutants associated with mammary cancer belonging to the dioxin like-PCB family (notably PCB-118, -156, -105, -114) that were already pointed out in human epidemiological studies on breast cancer, and that fit with the fundamental role of the Aryl Hydrocarbon Receptor in the promotion of breast cancer. Similarities observed in the spontaneous dog model are very helpful to progress in interpretation of human breast cancer-environment relationships. This study provides a new insight focusing on this discrete but recurrent signature.

Humans are significantly exposed to phthalates via food packaging, cosmetics and medical devices such as tubings and catheters. Testicular Leydig cells (LCs) are suggested to be among the main targets of phthalate toxicity in the body. However, their sensitivity to phthalates is species-dependent. This paper describes the response of the LCs from different species (mouse, rat and human) to phthalate exposure in different experimental paradigms (in vivo, ex vivo and in vitro), with particular focus on mechanisms of phthalate action on LC steroidogenesis. A comprehensive analysis of the impact of phthalate diesters and phthalate monoesters on LCs in different stages of their development is presented and possible mechanisms of phthalates action are discussed. Finally novel, not yet fully elucidated sites of action of phthalate monoesters on the backdoor pathway of 5α-dihydrotestosterone biosynthesis in immature mouse LCs and their effects on steroidogenesis and redox state in adult mouse LCs are reported.

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and the number of colonies, for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, could influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain, with two different spatial distributions, either big or small colonies in order to monitor the production of the major ripening metabolites such as sugars, organic acids, peptides, free amino acids and volatile metabolites, over one month of ripening. The monitored metabolites were qualitatively the same for both cheeses but many of them were more abundant in the small colony cheeses than in the big colony cheeses over one month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis led to modulate the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work deepens the exploration of the immobilization of bacteria as colonies within cheese and highlights its consequences on cheese ripening.

Multi-residue methods permitting the high-throughput and affordable simultaneous determination of an extended range of endocrine disrupting chemicals (EDCs) with reduced time and cost of analysis is of prime interest in order to characterize a whole set of bioactive compounds. Such a method based on UHPLC-MS/MS measurement and dedicated to 13 estrogenic EDCs was developed and applied to biological matrices. Two molecular recognition-based strategies, either molecular imprinted polymer (MIP) with phenolic template or estrogen receptors (ERα) immobilized on a sorbent, were assessed in terms of recovery and purification efficiency. Both approaches demonstrated their suitability to measure ultra-trace levels of estrogenic EDCs in aqueous samples. Applicability of the MIP procedure to urine and serum samples has also been demonstrated.

An analytical strategy dedicated to 4 major phthalate diesters (DiBP, DnBP, BBzP and DEHP) monitoring in food items has been developed and validated according to normalized guidelines. The method has been applied to a wide range of foodstuffs (n = 54) to generate first-ever occurrence data at the French level. This method involves separation and detection using gas chromatography coupled to tandem mass spectrometry, in electron ionisation with highly specific selected reaction monitoring, quantification being performed according to the isotope dilution principle. A particular attention has been paid to background contamination management at any stage of the analytical process, from the sampling to the expression of the results. Limits of reporting, defined as statistically different from background contamination, were found to be 2.7, 0.53, 0.18 and 3.4 lg kg�1, and relative combined uncertainties were finally found to be 7.6%, 12.2%, 12.0% and 14.1%, for DiBP, DnBP, BBzP and DEHP, respectively.

Glycerophospholipids have been highlighted as the major lipids class affected by trenbolone acetate/estradiol implant administration in bovines. Non-targeted hydrophilic interaction chromatography (HILIC) and reverse phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry have been successfully applied for characterizing lipid profile disruption in serum of implanted bovines. HILIC data pointed towards significant decrease of C22 fatty acids in implanted animals, i.e., C22:3 (p < 0.05), C22:4 (p < 0.05) and C22:6 (p < 0.01), whilst RPLC data confirmed depletion of glycerophospholipids (phosphatidylglycerols, phosphatidylcholine, phosphatidic acid and phosphatidylethanolamine) with C22 fatty acid chains. Using these two complementary methods, complex lipids with the same alkyl chain have been putatively characterized and could further serve to understand the biological mechanism of illicit administration of trenbolone acetate/estradiol implant. These metabolites underpin glycerophospholipid metabolism as potential pathway, which was identified using metabolomic pathway analysis and MetExplore platforms. We hypothesized that implantation of exogenous androgenic and estrogenic steroids induces a depletion of glycerophospholipids with C22 FA chains, by decreasing high-density lipoproteins, the main transporters of glycerophospholipids in plasma.

While the coupling of travelling wave ion mobility spectrometry (TWIMS) and mass spectrometry is mainly reported for structural purposes, we studied its potential in enhancing compounds analysis such as growth promoters used in livestock animals at trace concentrations. β-adrenergic agonists have been selected as model compounds since they exhibit a range of close physico-chemical properties leading to analytical issues using classical approaches. In this paper, the potential of Synapt G2-S (Q-TWIM-TOF MS) has been investigated for sensitive and specific detection of a range of these synthetic phenethanolamines in various complex biological matrices (retina, meat and urine) from bovine considered as relevant in the context of detecting -adrenergic agonists use in animals. In particular, the specificity of the additional information provided by the TWIMS (i.e. collision cross section) together with the interest of the extra dimension of separation is discussed.

The administration of synthetic homologues of naturally occurring steroids can be demonstrated by measuring (13)C/(12)C isotopic ratios of their urinary metabolites. Gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry (GC-MS/C/IRMS) was used in this study to appraise in a global approach isotopic deviations of two 17β-testosterone metabolites (17α-testosterone and etiocholanolone) and one 17β-estradiol metabolite (17α-estradiol) together with those of 5-androstene-3β,17α-diol as endogenous reference compound (ERC). Intermediate precisions of 0.35‰, 1.05‰, 0.35‰ and 0.21‰, respectively, were observed (n=8). To assess the performance of the analytical method, a bull and a heifer were treated with 17β-testosterone propionate and 17β-estradiol-3-benzoate. The sensitivity of the method permitted the demonstration of 17β-estradiol treatment up to 24 days. For 17β-testosterone treatment, the detection windows were 3 days and 24 days for the bull and the heifer, respectively. The capability of GC-MS/C/IRMS to demonstrate natural steroid abuse for urinary steroids was eventually compared to those of mass spectrometry (LC-MS/MS) when measuring intact steroid esters in blood and hair.

One major concern regarding perfluoroalkyl acids (PFAAs) is their potential role in onset of health troubles consecutive to early exposure during the perinatal period. In the present work, the internal exposure levels of 18 targeted PFAAs were determined in ca. 100 mother-newborn pairs recruited in France between 2010 and 2013. In serum, the cumulated concentrations of the 7 most frequently detected compounds were 5.70ng/mL and 2.83ng/mL (median values) in maternal and cord serum, respectively. Perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexylesulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) contributed to around 90% of the total PFAAs contamination, with concentration levels and contamination profiles in accordance with other published work in Europe. Levels measured in breast milk were far lower (20 to 150 fold) than those determined in serum. Associations between the different monitored substances as well as between levels determined in the different investigated biological matrices mostly do not appear statistically significant. The estimated materno-foetal transfer would be thus substance-dependant, mainly driven by the physico-chemical properties of the different PFAAs (nature of polar group and length of alkylated side chain). We conclude that trans-placental passage and breastfeeding are both significant routes of human exposure to PFAAs. Copyright © 2015. Published by Elsevier Ltd.

Chlordecone is an organochlorine pesticide (OCP) considered as a Persistent Organic Pollutant (POP) as it persists in the environment, bio-accumulates through the food web, causes adverse effects to human health and the environment and transports across international boundaries far from its sources. The atypical physico-chemical properties of chlordecone make its inclusion in classical analytical approaches non applicable. The aim of our work was to include chlordecone in a multi organochlorine residue method preventing any degradation during the analytical process and thus allowing quantification at ppt (ngkg(-1) or ngL(-1)) levels for a wide range of OCPs in breast milk, human serum and adipose tissue. After GC-HRMS vs. MS/MS and EI vs. APCI comparisons, the major improvement in terms of sensitivity was found in decreasing the length and film thickness of the gas chromatography column. Thanks to a linear correlation between relative response and quantity of chlordecone injected, LC-(ESI-)-MS/MS was finally preferred. An acetonitrile based gradient optimized on a C30 coreshell HPLC column has led to reaching limits of quantification as low as 8ngL(-1), 25pgmL(-1) and 0.2ngg(-1) fat for breast milk, serum and adipose tissue, respectively, allowing multiresidue OCP quantification at concentration levels compatible with biomonitoring purposes and pre-requisites. Copyright © 2015 Elsevier B.V. All rights reserved.

Voir le texte complet en pdf (en anglais)

Résumé Certaines substances β-adrénergiques peuvent être utilisées chez l’animal d’élevage à des fins zootechniques. Ces β-agonistes peuvent être incorporés à des doses très faibles dans les aliments destinés aux animaux. Ces molécules modifient certains processus biologiques liés à la croissance des tissus en augmentant la lipolyse et la synthèse protéique, en diminuant la lipogenèse et la dégradation des protéines. Ces substances sont considérées comme des « agents de répartition ». Les méthodes classiques dites ciblées permettent de manière routinière de contrôler une quarantaine de b-agonistes dont l’usage est interdit chez les animaux de production. La simplicité relative des procédés de synthèse pour ce type de molécules permet d’envisager une possible circulation sur le marché noir en Europe de substances dont la structure chimique n’a pas encore été officiellement décrite. Cette hypothèse de travail a conduit le LNR français compétent en la matière à développer dès 2007 des stratégies de criblage, non plus ciblées uniquement sur les molécules natives ou leurs métabolites, mais sur des marqueurs moléculaires d’effet, consécutifs à l’administration de la dite substance connue ou pas. Ces approches dites non ciblées ou métabolomiques sont utilisées officiellement depuis 2013 en France pour le contrôle officiel. Il s’agit d’une première mondiale en la matière.

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL(-1) and 0.5 ng mL(-1) for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R (2) > 0.995, RSD < 20 %, and bias < 30 %) through the use of suitable labeled internal standards. Comparison with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol.

Recombinant bovine somatotrophin (rbST) is widely used in some countries to increase milk production. Since 1994, both marketing and use of this substance have been prohibited within the European Union. In this context, the targeted plasma biochemical and hormonal profiling was assessed as a potential screening strategy to highlight rbST (ab)use in cattle. Twenty-one routinely measured clinical blood parameters, representative of main biological profiles (energetic, proteic, etc.), were measured in the plasma of six lactating cows before and after rbST treatment throughout a 23-day study period. Appropriate multivariate statistical analyses [principal component analysis (PCA) and orthogonal partial least square (OPLS)] enabled discriminating animal samples before and after treatment (days 0 vs. 2 to 9, P = 2.10(-9)) and highlighted the five most relevant blood parameters in this discrimination. Based on each five-analyte contribution, a simple mathematically weighted equation was suggested to predict the status of samples. A suspicious threshold was proposed, and the model was further tested with the status prediction of the supplementary samples from untreated (n = 20) and treated cows (n = 22). The calculated false-positive (10 %) and false-negative (4.5 %) rates were in accordance with the EU requirements for screening methods. Although the model needs to be further validated with additional samples, such targeted plasma biochemical and hormonal profiling already appears as a potential promising screening strategy to highlight rbST (ab)use in cattle.

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10-8M and 10-5M for 72h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48h in both strains. BPA at 10-7-10-5M for 72h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10-8M, 10-7M, and 10-5M for 72h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.

The chemical contamination of the Loire estuary by three classes of persistent organic pollutants (POPs): the polychlorinated biphenyls (PCBs), the polybrominated diphenyl ethers (PBDEs) and the perfluorinated and polyfluorinated alkyl substances (PFAS), and three families of organic contaminants, the alkylphenols (APs), the polycyclic aromatic hydrocarbon metabolites (OH-PAHs) and the bisphenol A (BPA) were investigated in the muscles and bile of European eel (Anguilla anguilla). Yellow eels (n = 30) were caught in three different points along the estuary to highlight variations between sites and sources of contaminations. Silver eels (n = 15) were also studied to compare contaminant impregnation between different life stages of the species. Average concentrations in the muscles of the eel ranged between: 857 and 4358 ng/g LW for the PCBs, 26 and 46 ng/g LW for the PBDEs, 130 and 1293 ng/g LW for the PFAS; and in bile: 31 and 286 μg/g protein for the APs, 9 and 26 μg/g protein for the OH-PAHs and ND-1213 μg/g protein for the BPA. Among PCBs, PCB 153 (40% contribution to the sum of PCBs) was predominant in all eel muscles. PBDE 47 (60%) was the most predominant PBDE congeners, while perfluorooctanesulfonic acid (85%) was the most widely detected PFAS. For APs, 4p-nonylphenol (91%) was the most abundant and for the OH-PAHs, it was 1OH-Pyrene (63%). All the eels exceeded the environmental quality standards (EQS) for biota for the PBDEs and about 75% were higher than the EQS specific to PFOS. Finally, 20% of the analyzed eels presented TEQ concentrations above the maximum limits for lipid-rich species. These results supplied new data on the occurrence, levels, and patterns of 53 organic chemicals in the eels from the Loire estuary and they highlighted the need of further investigations focused notably on the potential effects of these chemicals on this species and their analysis in the water and sediments of the estuary.

Bisphenol A (BPA) is an industrial chemical widely used in the production of polycarbonate and epoxy resins. Identified as an endocrine-disrupting chemical (EDC), BPA is a matter of existing or ongoing restrictive regulations and then is increasingly being replaced by other analogues used as BPA’s substitutes. Human biomonitoring studies focusing on both BPA and emerging related analogues consequently appear as a requirement either for documenting the efficiency of regulatory actions toward BPA and for fuelling incoming risk assessment studies toward BPA’s substitutes. In particular, the increasing concern about the late effects consecutive to early exposures naturally identify human breast milk as a target biological matrix of interest for priority exposure assessment focused on critical sub-populations such as pregnant women, fetuses, and/or newborns. In this context, an accurate and sensitive analytical method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed for the quantification of 18 “BPA-like” compounds in breast milk samples at trace levels (<0.05 μg kg−1). The method includes a preliminary protein precipitation step followed by two successive solid-phase extraction (SPE) stages. Quantification of the targeted compounds was achieved according to the isotopic dilution method using 13C12-BPA as internal standard. The method was validated according to current EU guidelines and criteria. Linearity (R 2) was better than 0.99 for each molecule within the concentration range 0–5 μg kg−1. The detection and quantification limits ranged from 0.001 to 0.030 μg kg−1 and from 0.002 to 0.050 μg kg−1, respectively. The analytical method was successfully applied to the first set of human breast milk samples (n = 30) originating from French women in the Region Pays-de-la-Loire. The measured levels of BPA were found in the <LOQ–1.16 μg kg−1 range. BPS was detected in only one sample at 0.23 μg kg−1, while the other targeted molecules were not detected. The proposed methodology then appeared suitable for the further monitoring of a potential decrease of BPA levels and an increase of other BPA analogue levels as reflective of the expected incoming trend in terms of human exposure.

Thiouracil, 1 (Figure 1) is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a ?recommend concentration? (RC) of 10 ?g/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC, led to demands for international surveys defining an epidemiologic threshold. Therefore, six European Member States (France, Poland, the Netherlands, United Kingdom, Norway and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines and small livestock with 95 and 99 % percentiles of 8.1 and 18.2 ?g/L for bovines (n = 3894); 7.4 and 13.5 ?g/L for porcines (n = 654) and 7.4 ?g/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (non-adults significantly higher levels for bovines) and higher levels were observed in male bovines compared to females.

The use of anabolic agents in livestock production is a subject of much concern. Although prohibited for more than 20 years within the EU, growth promoting practices are still widely suspected. To meet the current challenges for detecting illicit practices, ‘omics’ strategies have recently been demonstrated as important new investigative tools. These investigations, based on the observation of physiological disturbances, mainly in urine, demonstrated the possibility to monitor biomarkers enabling high throughput determination of animal status in terms of hormonal treatment. In this context, serum was investigated for the first time as an alternative and potential complementary sample type. A metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry, was exploited in order to, highlight metabolic perturbations in serum of Revalor-XS® (trenbolone acetate/estradiol) implanted bovines. Univariate and multivariate statistical analyses were carried out to establish descriptive and predictive models. These models enabled the discrimination of anabolised from control animals, and highlighted a number of metabolites which contributed the most in the observed discrimination. Further, a screening model combining a set of selected markers intensities was generated and it successfuly classified animals according to their status, up to 4 weeks post Revalor-XS® implant. This research indicates, for the first time, that serum metabolomics has an important role in screening to detect for anabolic misuse in bovines.

Background: In the food safety field, risk assessment, including microbial and chemical components, has been applied for many years. However, a whole and integrated public health assessment also depends on the nutritional composition of food. While the fact that foods and diets can be a source of both risks and benefits now appears undisputed, carrying out a risk-benefit assessment (RBA) is still an emerging and challenging scientific subject. Aims: The purpose of the present review was to synthesize RBA studies associated with food consumption and to summarize the current methodological options and/or tendencies carried out in this field. Methods: The different data sources explored included around 20 accessible databases using the main terms “risk”, “benefit” and “food” as keyword enquiries in article title and full-text. The initial research process led to 3293 screened papers, 160 of which were examined in detail. Results: There were 126 articles dealing with RBA studies and 34 with the RBA methodological framework. Most of the available papers dealt with the comparison of nutritional beneficial effects

New approaches, mainly based on mass spectrometry techniques, are being developed and appear as a must in the modern food science and microbiology research to investigate food quality and safety. To date, the investigation of cheese ripening mechanisms has mostly used targeted approaches. The aims of the present project were to assess the use of untargeted metabolomics as an approach to investigate the influence of altering one ripening parameter to generate fine differences in the microbial metabolism within cheese. Two cheeses were made which varied with respect to the spatial distribution of bacterial colonies, leading to cheeses with only big or only small colonies. Liquid chromatography high resolution mass spectrometry metabolic fingerprints were acquired on cheese extracts collected after 2, 13 and 27 days of ripening using two different extraction methods (water or acetonitrile) and analyzed using two different simultaneous ionization modes (positive and negative electrospray). Data processing involving XCMS and multivariate statistical analysis highlighted significant discriminant profiles of the cheese metabolomes according to the two different spatial distributions compared. The different fractions investigated (water and acetonitrile extractions in two ionization modes) were complementary and resulted in a view as global as possible of the cheese metabolome which had been modulated by the spatial distribution of bacterial colonies. Some of the metabolites were then identified using an in-house database. These results show the relevance of cheese LC–HRMS fingerprinting to understand the influence of a ripening parameter generating fine differences on microbial metabolism within cheese.

This review aims to describe the most significant applications of mass spectrometry-based metabolomics in the field of chemical food safety. A particular discussion of all the different analytical steps involved in the metabolomics workflow (sample preparation, mass spectrometry analytical platform and data processing) will be addressed.

Metabolomics approaches, particularly those based on mass spectrometry (MS) techniques,are being developed and appear as a must in the modern food science and microbiologyresearch to investigate food quality and safety. Cheese ripening mechanisms has always beeninvestigated with targeted approaches. Our objective was to assess the relevance ofuntargeted metabolomics to explore cheese ripening. MS metabolic fingerprints were foundto differ significantly depending on the incubation time, pointing out the capacity of thisapproach to study the evolution of bacterial metabolism within cheese. Forty-five metaboliteswere identified on the basis of an internal data bank. Variations over time of a large diversityof well-known cheese metabolites such as 12 amino acids and 25 volatile compounds, butalso less studied ones such as 4 vitamins and L-carnitine were highlighted. From now on,untargeted metabolomics offer new perspectives for the understanding of cheese ripeningmechanisms.

The emerging field of metabolomics, aiming to characterize small molecule metabolites present in biological systems, promises immense potential for different areas such as medicine, environmental sciences, agronomy… The purpose of this paper is to guide the reader through the history of the field, then through the main steps of the metabolomics workflow, from study design to structure elucidation, and help the reader to understand the key phases of a metabolomics investigation and the rationale underlying the protocols and techniques used. This article is not intended to give standard operating procedures as several papers related to this topic were already provided, but is designed as a tutorial aiming to help beginners understanding the concept and challenges of Mass Spectrometry-based metabolomics. A real case example is taken from the literature to illustrate the application of the metabolomics approach in the field of doping analysis. Challenges and limitations of the approach are then discussed along with future directions in research to cope with these limitations. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP18).This article is protected by copyright. All rights reserved

Polychlorobiphenyls (PCBs) are persistent pollutants that are widespread in the environment and in foodstuffs, particularly in freshwater fish, which frequently exceed the maximum levels set by European regulations. First, we describe the consumption of freshwater fish and serum PCB levels in French anglers, a population expected to have the highest level of dietary PCB exposure. Second, we investigated whether there is a statistical relationship between serum PCB levels and the angler consumption of freshwater fish with high PCB bioaccumulation potential (PCB-BP(+) freshwater fish) in order to make recommendations with regard to safe consumption of freshwater fish. We conducted a survey of anglers from six sites with contrasting PCB contamination levels. The survey included a food consumption frequency questionnaire and blood samples were taken to assess serum PCB levels. We used a regression model to determine the main factors contributing to serum PCB levels. Consumption of PCB-BP(+) freshwater fish was relatively infrequent. Serum PCB levels of the study population and of women of childbearing age were in the same range as those observed in the French population and in neighbouring European countries, but higher than in the North American population. The two factors with the highest positive association with serum PCB levels were age (R(2)=61%) and the consumption of PCB-BP(+) freshwater fish (R(2)=2%). Using the regression model, we calculated, for several scenarios depending on the age and gender of the population, the maximum annual frequencies for PCB-BP(+) freshwater fish consumption that do not exceed the critical body burden threshold. Following the results of this study, the French agency for food, environmental and occupational health and safety (ANSES) issued an opinion and recommended some specific maximum freshwater fish consumption frequencies to protect the French general population. Copyright © 2014 Elsevier B.V. All rights reserved.

High throughput screening is essential for doping, forensic, and food safety laboratories. While hyphenated chromatography-mass spectrometry (MS) remains the approach of choice, recent ambient MS techniques, such as direct analysis in real time (DART), offer more rapid and more versatile strategies and thus gain in popularity. In this study, the potential of DART hyphenated with Orbitrap-MS for fast identification and quantification of 21 anabolic steroid esters has been evaluated. Direct analysis in high resolution scan mode allowed steroid esters screening by accurate mass measurement (Resolution = 60 000 and mass error < 3 ppm). Steroid esters identification was further supported by collision-induced dissociation (CID) experiments through the generation of two additional ions. Moreover, the use of labelled internal standards allowed quantitative data to be recovered based on isotopic dilution approach. Linearity (R2 > 0.99), dynamic range (from 1 to 1000 ng mL-1), bias (<10%), sensitivity (1 ng mL-1), repeatability and reproducibility (RSD < 20%) were evaluated as similar to those obtained with hyphenated chromatography-mass spectrometry techniques. This innovative high throughput approach was successfully applied for the characterization of oily commercial preparations, and thus fits the needs of the competent authorities in the fight against forbidden or counterfeited substances. Copyright © 2014 John Wiley & Sons, Ltd.