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. Properties of BPA MW BP 200 ºC at 4 mm Hg

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 %

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

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 228 [M+] 213 GC chromatograms (2)MS Spectrum (3) 119 Bisphenol-A 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 ( 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 ) 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, , ) 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, ) Skoog, D., Holler, E.J., Crouch, S.: Principles of Instrumental Analysis. Thompson/Brooks/Cole. 6 th ed. 2007