Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

International audienceA Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia

Bisphenol A determination in baby bottles by chemiluminescent enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4¿-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL¿1, respectively. The LOD of LFIA was 0.1 ¿g mL¿1. A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL¿1, with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL¿1 and 17.4 ng mL¿1, respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 ¿g mL¿1) and by the Directive 2004/19/EC (0.6 ¿g mL¿1), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assaysThis work was supported by a grant from the University of Bologna (RFO-Focused Fundamental Research Projects 2000-2011) grants.Maiolini, E.; Ferri, E.; Pitasi, AL.; Montoya Baides, Á.; Di Giovanni, M.; Errani, E.; Girotti, S. (2014). Bisphenol A determination in baby bottles by chemiluminescent enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry. Analyst. 139(1):318-324. doi:10.1039/c3an00552fS3183241391The Bisphenol-A, http://www.bisphenol-a.org, accessed 04 July 2013Diamanti-Kandarakis, E., Bourguignon, J.-P., Giudice, L. C., Hauser, R., Prins, G. S., Soto, A. M., … Gore, A. C. (2009). Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement. 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Baldi and A.Mantovani, Baby bottle: Struggling for safety, Food and Veterinary Toxicology Unit – ISS, http://www.iss.it/prvn/focu/cont.php?id=281&lang=2&tipo=42, accessed 04 July 2013K. Aschberger , P.Castello, E.Hoekstra, S.Karakitsios, S.Munn, S.Pakalin and D.Sarigiannis, European Commission Joint Research Centre Institute for Health and Consumer Protection, http://publications.jrc.ec.europa.eu/repository/bitstream/111111111/14221/1/eur%2024389_bpa%20%20baby%20bottles_chall%20%20persp%20%282%29.pdf, accessed 04 July 2013Ballesteros-Gómez, A., Rubio, S., & Pérez-Bendito, D. (2009). Analytical methods for the determination of bisphenol A in food. Journal of Chromatography A, 1216(3), 449-469. doi:10.1016/j.chroma.2008.06.037Feng, Y., Ning, B., Su, P., Wang, H., Wang, C., Chen, F., & Gao, Z. (2009). An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates. Talanta, 80(2), 803-808. doi:10.1016/j.talanta.2009.07.070Mudiam, M. K. R., Jain, R., Dua, V. K., Singh, A. K., Sharma, V. P., & Murthy, R. C. (2011). Application of ethyl chloroformate derivatization for solid-phase microextraction–gas chromatography–mass spectrometric determination of bisphenol-A in water and milk samples. Analytical and Bioanalytical Chemistry, 401(5), 1695-1701. doi:10.1007/s00216-011-5226-6Negre, M., Monterumici, C. M., Vindrola, D., & Piccone, G. (2011). Changes in chemical and biological parameters during co-composting of anaerobically digested sewage sludges with lignocellulosic material. Journal of Environmental Science and Health, Part A, 46(5), 509-517. doi:10.1080/10934529.2011.551737Noonan, G. O., Ackerman, L. K., & Begley, T. H. (2011). Concentration of Bisphenol A in Highly Consumed Canned Foods on the U.S. Market. Journal of Agricultural and Food Chemistry, 59(13), 7178-7185. doi:10.1021/jf201076fSchecter, A., Malik, N., Haffner, D., Smith, S., Harris, T. R., Paepke, O., & Birnbaum, L. (2010). Bisphenol A (BPA) in U.S. Food. Environmental Science & Technology, 44(24), 9425-9430. doi:10.1021/es102785dViganò, L., Benfenati, E., Cauwenberge, A. van, Eidem, J. K., Erratico, C., Goksøyr, A., … Urbatzka, R. (2008). Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays. Chemosphere, 73(7), 1078-1089. doi:10.1016/j.chemosphere.2008.07.057Fukata, H., Miyagawa, H., Yamazaki, N., & Mori, C. (2006). Comparison of Elisa- and LC-MS-Based Methodologies for the Exposure Assessment of Bisphenol A. Toxicology Mechanisms and Methods, 16(8), 427-430. doi:10.1080/15376520600697404Rezaee, M., Yamini, Y., Shariati, S., Esrafili, A., & Shamsipur, M. (2009). Dispersive liquid–liquid microextraction combined with high-performance liquid chromatography-UV detection as a very simple, rapid and sensitive method for the determination of bisphenol A in water samples. 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BIOCHRONOLOGY OF SELECTED MAMMALS, MOLLUSCS AND OSTRACODS FROM THE MIDDLE PLIOCENE TO THE LATE PLEISTOCENE IN ITALY. THE STATE OF THE ART

The Authors have elaborated four range charts of mammalian (large and micro), molluscs and fresh-water and brackish ostracodes faunas, for the selected Plio-Pleistocene fossiliferous localities of the Italy. A new Mammal Age (Aurelian) correlatable to late Middle and Late Pleistocene has been defined. Inside this age two Faunal Units (Torre in Pietra and Vitinia) have been defined as characteristic for Early and Middle Aurelian, while no gisements have been chosen for the late Aurelian. Biochronological units are calibrated on magnetostratigraphic and isotopic scales and by radiometric datings.   &nbsp

BIOCHRONOLOGY OF SELECTED MAMMALS, MOLLUSCS AND OSTRACODS FROM THE MIDDLE PLIOCENE TO THE LATE PLEISTOCENE IN ITALY. THE STATE OF THE ART

The Authors have elaborated four range charts of mammalian (large and micro), molluscs and fresh-water and brackish ostracodes faunas, for the selected Plio-Pleistocene fossiliferous localities of the Italy. A new Mammal Age (Aurelian) correlatable to late Middle and Late Pleistocene has been defined. Inside this age two Faunal Units (Torre in Pietra and Vitinia) have been defined as characteristic for Early and Middle Aurelian, while no gisements have been chosen for the late Aurelian. Biochronological units are calibrated on magnetostratigraphic and isotopic scales and by radiometric datings.   &nbsp

Measurement of the anomalous precession frequency of the muon in the Fermilab Muon g-2 Experiment

The Muon g-2 Experiment at Fermi National Accelerator Laboratory (FNAL) has measured the muon anomalous precession frequency $\omega_a$ to an uncertainty of 434 parts per billion (ppb), statistical, and 56 ppb, systematic, with data collected in four storage ring configurations during its first physics run in 2018. When combined with a precision measurement of the magnetic field of the experiment's muon storage ring, the precession frequency measurement determines a muon magnetic anomaly of $a_{\mu}({\rm FNAL}) = 116\,592\,040(54) \times 10^{-11}$ (0.46 ppm). This article describes the multiple techniques employed in the reconstruction, analysis and fitting of the data to measure the precession frequency. It also presents the averaging of the results from the eleven separate determinations of \omega_a, and the systematic uncertainties on the result.Comment: 29 pages, 19 figures. Published in Physical Review

Magnetic Field Measurement and Analysis for the Muon g-2 Experiment at Fermilab

The Fermi National Accelerator Laboratory has measured the anomalous precession frequency $a^{}_\mu = (g^{}_\mu-2)/2$ of the muon to a combined precision of 0.46 parts per million with data collected during its first physics run in 2018. This paper documents the measurement of the magnetic field in the muon storage ring. The magnetic field is monitored by nuclear magnetic resonance systems and calibrated in terms of the equivalent proton spin precession frequency in a spherical water sample at 34.7$^\circ$C. The measured field is weighted by the muon distribution resulting in $\tilde{\omega}'^{}_p$, the denominator in the ratio $\omega^{}_a$/$\tilde{\omega}'^{}_p$ that together with known fundamental constants yields $a^{}_\mu$. The reported uncertainty on $\tilde{\omega}'^{}_p$ for the Run-1 data set is 114 ppb consisting of uncertainty contributions from frequency extraction, calibration, mapping, tracking, and averaging of 56 ppb, and contributions from fast transient fields of 99 ppb

Beam dynamics corrections to the Run-1 measurement of the muon anomalous magnetic moment at Fermilab

This paper presents the beam dynamics systematic corrections and their uncertainties for the Run-1 data set of the Fermilab Muon g-2 Experiment. Two corrections to the measured muon precession frequency $\omega_a^m$ are associated with well-known effects owing to the use of electrostatic quadrupole (ESQ) vertical focusing in the storage ring. An average vertically oriented motional magnetic field is felt by relativistic muons passing transversely through the radial electric field components created by the ESQ system. The correction depends on the stored momentum distribution and the tunes of the ring, which has relatively weak vertical focusing. Vertical betatron motions imply that the muons do not orbit the ring in a plane exactly orthogonal to the vertical magnetic field direction. A correction is necessary to account for an average pitch angle associated with their trajectories. A third small correction is necessary because muons that escape the ring during the storage time are slightly biased in initial spin phase compared to the parent distribution. Finally, because two high-voltage resistors in the ESQ network had longer than designed RC time constants, the vertical and horizontal centroids and envelopes of the stored muon beam drifted slightly, but coherently, during each storage ring fill. This led to the discovery of an important phase-acceptance relationship that requires a correction. The sum of the corrections to $\omega_a^m$ is 0.50 $\pm$ 0.09 ppm; the uncertainty is small compared to the 0.43 ppm statistical precision of $\omega_a^m$