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Bisphenol-A Detection in Human Urine

Bisphenol-A Detection in Human Urine. Megan Babcock Fall 2008. Industrial BPA. BPA is a synthetic estrogen and monomer component of plastics and resins that are used in production of food and beverage containers. This environmental toxin has become a serious health concern at low doses.

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Bisphenol-A Detection in Human Urine

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  1. Bisphenol-A Detection in Human Urine Megan Babcock Fall 2008

  2. Industrial BPA • BPA is a synthetic estrogen and monomer component of plastics and resins that are used in production of food and beverage containers. • This environmental toxin has become a serious health concern at low doses.

  3. Problem • BPA leaches into food and drink at concentrations shown to have biological consequences • Cell differentiation and proliferation • Rate of body growth • Sexual maturation • Reproductive function • Development and brain structure • Objective: investigate the environmental exposure levels of BPA in humans

  4. Samples (1) • BPA analyses in human urine • 57-98% is metabolized to glucuronides • 1-12% is unmetobolized • Urine samples will be collected from individuals • Buffered with sodium acetate & hydrolyzed, some w/ β-glucuronidase • This deconjugates the glucuronides • HCl added and extracted w/ ethyl acetate • Centrifuged, residue dissolved in 60% acetonitrile in water • Spiked mixtures (w/ known concentrations) will be prepared in same manor • Standards (99%, Aldrich) prepared in water

  5. BPA Analytical Methods • GS/MS • Gas chromatogram: detects retention time • Mass spectrum: fragmentation of analyte • UV absorption • Absorbing electromagnetic radiation • UV spectra’s do not have enough fine structure to permit an analyte to be identified • Electrochemistry • Electrical properties of an analyte • Provides information about activities not concentration • All methods are acceptable, but GS/MS is better for selectivity & structural information

  6. GC/MS • GC-components of vaporized sample are separated as it is partitioned between a mobile gaseous phase and a liquid/solid stationary phase. • MS separates/identifies ionized atoms/molecules based on mass-to-charge (m/z) • Obtain structural info. about compound from fragmentation patterns that arise from electron impact ionization (EI) • Quantitative analysis is possible by measuring the relative intensities of the mass spectra and comparing to calibration standards.

  7. Characteristics of Analytical Method (3) • LOD determined at 20 ng/L • Limit of quantification determined at 100ng/L representing the S/N ratio 10:1 • Retention time 26.4 mins (2) • Recovery 88.2-94.2%

  8. GC/MS model • Hewlett Packard 6890 GC (fitted with HP 7673 Automatic Liquid Sampler), Hewlett Packard 5972A MS (with an EI of 70 eV). (3) • LOD identified at a signal/noise ratio 3:1 • Calibration curve with a good linear trend • Quantitation repeatability • High sensitivity www.gmi-inc.com/AnyLab/HP%205973%20Mass%20Sperometer.htm

  9. Operating conditions of GC/MS for BPA analysis (3) • Parameters for GC • Carrier gas Helium (purity > 99.99%) • Column DB-5 30m • Stationary phase 5% diphenyl-95% dimethyl polysiloxane • Total flow rate 100 mL minˉ¹ • Carrier gas flow rate 1.8 mL min ˉ¹ • Pressure 100 kPa • Inlet temperature 250 ºC • Injected volume 2 μL • Injection mode Splitless • Oven program Initial temperature 50 ºC, hold 1 min, 25 ºC/min to 150 ºC, hold 1 min, 10 ºC/min to 270 ºC, hold 2 min • Parameters for MS • GC-MS interface temperature 280 ºC • Electron multiplier voltage 2400–2600 V • Selected ions (m/z) in BPA 119, 213, 228

  10. Results GC chromatograms (2) MS Spectrum (3) 213 Bisphenol-A 228 [M+] 119 a) BPA standard in water (0.53 ng/ml b) Unspiked human urine extract c) Unspiked human urine extract--deconjugated

  11. Conclusion (2) • Estimated daily intake for adult <0.0015mg/kg/bw, this is above our LOD, allowing detection of analyte • Linear correlation determined upon analysis of spiked samples (0.25-10 ng BPA/mL) • Methods will be validated by analysis of duplicate samples • Sensitivity can be improved using different volumes of spiked urine samples and analyzing. Standard addition and recovery in human urine.

  12. References • 1) Matsumoto, A., Kunugita, N., Kitagawa, K., Isse, T., Oyama, T., Foureman, G., Morita, M., Kawamoto, T.: Bisphenol A Levels in Human Urine. Environ. Health Perspectives, V 111, 1, Jan. 2003. • 2) Brock, J., Yoshimura, Y., Barr, J., Maggio, V., Graiser, S., Nakazawa, H., Needham, L.: Measurement of Bisphenol A levels in humans urine. J. of Exposure to Analysis and Environment Epidemiology. V 11, 323-328, 2001. • 3) Hu, J., Yuan, T., Ong, S., Song, L., Ng. W., Identification and quantification of bisphenol A by gas chromatography and mass spectrometry in lab-scale dual membrane system. J. Environ. Monit., 2003, 5, 141-144 • 4) Skoog, D., Holler, E.J., Crouch, S.: Principles of Instrumental Analysis. Thompson/Brooks/Cole. 6th ed. 2007

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