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Perfluorooctanoic acid levels in human blood (Andrew Szeliga)

Perfluorooctanoic acid (PFOA) is a perfluoronated water and oil repellant used to treat carpeting and tile. In addition, it is used in the synthesis of polytetrafluoroethylene, also known as Teflon, as well fluoroelastomers. It has been found in elevated levels in both animals and humans, and is a known carcinogen. One of the major sources of contamination is through the waste water of factories that use PFOA.

The central hypothesis of this study is that people living or work in close proximity to sources of PFOA dumping will have dangerously elevated levels of PFOA in their bloodstream. This hypothesis is best tested by taking blood samples in potential risk areas and comparing them against a control of blood samples taken in blood banks across the nation. The analyte, PFOA, will have to be detected in the matrix, blood, quickly and efficiently. The instruments chosen to measure PFOA levels will have to be able to either filter out interfering factors.

UV-Vis Absorption Spectrometry

UV-Vis spectra for perfluorooctanoic acid is not freely available. Only IR spectra for perfluorooctanoic acid were posted to spectra databases. The carbon-fluorine bonds
are not visible in UV-Vis spectra, but the carboxyl group in isolation would have a maximum wavelength of absorption of 204 nm in ethanol. Carbonxyl's molar absorptivity is 41 L/mol*cm. Since perfluorooctanoic acid is soluble in blood, a water based matrix, it is reasonable to assume that it will be soluble in ethanol. A preliminary experiment would have to be done in order to determine if the adjacent carbon-fluorine bonds alter the absorption spectra from the prediction. Comparing two similar simple compounds, one an organic molecular and the other the perfluorinated version, would show if there is a significant effect. One possible pair is acetic acid and trifluoroacetic acid.

Separation Through Chromatography

PFOA is suitable to be extracted by several methods of chromatography. It is large enough to be seperated with size exclusion chromatography, it can be deprotonated in basic conditions and extracted with ion exchange chromatography, it can be derivatized with diazomethane and extracted with gas chromatography, or it can be extracted with reverse phase chromatography. HILIC can be used but it is not as effective as reverse phase. Affinity and chiral chromatography can not be used as the molecule has no notable stereoisomers or notable ligands.

Reverse phase chromatography is the most efficient choice of the available options. It is effective and widely used. Extraction has previously been demonstrated with a Genesis C8 column [7]. A Genesis C8 column has a length of 50 mm, an inner diameter of 2.1 mm. It is filled with silica with a particle size of 4 μm that remains stable between pH 1 to 10 [8]. The column can be bought from Crawford Scientific, catalog number 5109766. The mobile phase is a gradient elution of 2mM ammonium acetate in water and methanol.

The mobile phase can be directly injected into a tandem MS/MS with electrospray ionization. When measured against a standard solution of 1 μg/mL PFOA the exact levels of PFOA in the bloodstream can be quickly and accurately measured.

Capillary Electrophoresis

CZE is the most effective type of capillary electrophoresis for separating my analyte. MEKC has no benefit as my analyte contains a polar acid group and a nonpolar fluorinated hydrocarbon tail. Its preference of phase is not easily predictable. As my analyte has a pKa of approximately 0 [10], and is neutral normally, cIEF has no benefit either. CGE is not needed as the analyte is not a macromolecule and it is not helpful to sort by size.

The appropriate buffer for perfluorooctanoic acid is a 50 mM disodium hydrogen phosphate buffer at 9.5 pH diluted with 40% isopropanol. The capillaries should be fused silicon (75 mm ID, 30 cm total length) for optimal resolution and separation time [9].

While UV spectroscopy has been used as a detection method in the past, mass spectrometry is the best way to measure the concentration of the analyte. Mass spec gives a lower limit of detection and quantification than UV spec.

Suppressed Conductivity Detection
http://blog.lib.umn.edu/cgi-bin/mt.cgi?__mode=view&_type=entry&id=309094&blog_id=8764
PFOA can be successfully detected in concentrations as low as 0.5 micrograms per liter by a combination of reversed-phase HPLC and suppressed conductivity detection [11]. The PFOA is separated on a Acclaim PA2 (3 mm 2.1 x 150 mm) column using a combination of 70:30 (v:v) acetonitrile to water, 100mM H3BO3 and 9mM KOH at a pH of 8, and deionized water. The eluent was suppressed by a Dionex ASRS ULTRA II 2 mm suppressor using 25 meq/L H2SO4 at approx 0.5 mL/min as the regenerant. Once extracted, the PFOA concentration can be measured with an electrical conductivity meter and compared to a standard sample.

Standard Supplier

The perfluorooctanoic acid used to make the standards for the calibration curve will be purchased from Sigma-Aldrich. It is listed under catalog number 171468 and costs $34 for 5 grams.

Perfluorooctanoic Acid Structure

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Derivatized Perfluorooctanoic Acid Mass Spectrum

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Similar Analytical Problems

This problem is most similar to four other analytical problems, three which have the same matrix and one that shares an analyte. Rajvi Mehta's study of DEHP leeching from PVC, Osman Jamshed's study of prion detection, Matt Marah's study of cadmium levels in blood and my study are all conducted in a blood matrix. The other studies concentrate on different analytes however, DEHP, prions, and Cd respectively. Megan Hartmann's study of PFOA and Teflon shares the PFOA analyte with my study, however she is studying PFOA released from cookware so she has an air matrix. Matt Marah has the closest hypothesis to mine, he is testing if Cd levels are higher in the blood of people who live near plants that use Cd. My hypothesis also theorizes that analyte levels increase in people who live near a plant that uses the analyte. Rajvi Mehta's hypothesis that DEHP builds up in the human body and causes adverse effects, is based on constant exposure but also concentrates on the specific level of analyte. Osman Jamshed's hypothesis that prions are present in unregulated meat also requires the measurement of the level of an analyte but seeks to find an analyte that is naturally generated. Megan Hartmann's hypothesis that Teflon pans release PFOA differs from my hypothesis in that I seek quantitative levels and she is looking for qualitative conformation of the release of PFOA.

1. Substance flow analysis for Switzerland: Perfluorinated surfactants perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA); Federal Office for the Environment (FOEN) : Bern 2009

2. Olsen, G. Decline in Perfluorooctanesulfonate and Other Polyfluoroalkyl Chemicals in American Red Cross Adult Blood Donors, 2000−2006. Environ. Sci. Technol., 2008, 42 (13), pp 4989-4995

3. National Institute of Standards and Technology. Chemistry WebBook: Perfluorooctanoic acid http://webbook.nist.gov/cgi/cbook.cgi?ID=B6009805&Mask=80 (Accessed 9/29/11)

4. Skoog , D, Holler, J, Crouch, S. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007.

5. Sigma-Aldrich Catalog, Perfluorooctanoic Acid. http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=171468|ALDRICH&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC

6. Liu, Wen-Lin, and W. Liu. "Headspace solid phase microextraction in-situ supercritical fluid extraction coupled to gas chromatography-tandem mass spectrometry for simultaneous determination of perfluorocarboxylic acids in sediments." Journal of chromatography 1218.43 (2011):7857.

7. Flaherty, John M "Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry" Journal of Chromatography B 819.2 (2005): 329-338

8. Jones Catalog, Genesis C8 Column, http://www.discoverysciences.com/uploadedFiles/Home/HPLCCols_OthrColsJonesCols_p64to66.pdf

9. Wójcik, L., Szostek, B., "Separation of perfluorocarboxylic acids using
capillary electrophoresis with UV detection" Electrophoresis 2005, 26, 1080-1088

10. Goss, K.-U. "The pKa Values of PFOA and Other Highly Fluorinated Carboxylic Acids (Addition/Correction)", Environ. Sci. Technol., 2008, 42 (13), pp 5032-5032

11. de Borba B., Rohrer J. "Analysis of PFOA and PFOS in Water Using Reversed-Phase HPLC with Suppressed Conductivity Detection." LC-GC Europe. March 2007;20:10

Comments

Blog 13? - 1 pt.

Blogs 9 and 11. Good answers.
Blog 8? (-1 pt).

Blog 8? (-1 pt)

Good answers for Blogs 6 and 7.

My analyte is not suitable for atomic spectrometry. I need to use mass spectrometry instead. My analyte is perfluorooctanoic acid, with a molecular mass of 414.07 Daltons. If I can isolate my analyte ahead of time, Electrospray-Quadrupole mass spectrometry is the best way to analyze my analyte. It has lower resolution than time of flight mass spec but can detect lower concentrations. If I have to measure my analyte in a raw matrix I have to derivatize my analyte into a butyl ether to allow seperation by gas chromotography. The analyte can then be ionized by negative chemical ionization and measured by time of flight.

Answers to Blogs 4 and 5? (-1 pt each).

BLOG 2 - Posting in other people's entries? (-0.15 pt).

BLOG 3 - Parts (b) and (c)? (-0.5 pt).

Our problems are similar because we are both looking for PFOA.

Great post. Informative and succinct!