BisphenolA Detection in Human Urine

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Bisphenol-A Detection in Human Urine

• Megan Babcock
• Fall 2008

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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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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

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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/ ß-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

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BPA Analytical Methods

• GS/MS
• Gas chromatogram detects retention time
• Mass spectrum fragmentation of analyte
• UV absorption
• Absorbing electromagnetic radiation
• UV spectras 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

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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.

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Characteristics of Analytical Method (3)

• LOD determined at 20 ng/L
• Limit of quantification determined at 100ng/L
  representing the S/N ratio 101
• Retention time 26.4 mins (2)
• Recovery 88.2-94.2

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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 31
• Calibration curve with a good linear trend
• Quantitation repeatability
• High sensitivity

www.gmi-inc.com/AnyLab/HP20597320Mass20Speromet
er.htm
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Operating conditions of GC/MS for BPA analysis (3)

• Parameters for GC
• Carrier gas Helium (purity gt
  99.99)
• Column
  DB-5 30m
• Stationary phase
  5 diphenyl-95 dimethyl polysiloxane
  
• Total flow rate
  100 mL min¹
• Carrier gas flow rate
  1.8 mL min ¹
• 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
  24002600 V
• Selected ions (m/z) in BPA
  119, 213, 228

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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
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Conclusion (2)

• Estimated daily intake for adult lt0.0015mg/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.
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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