1. Detection of Perfluorooctanoic acid concentration in human plasma Andrew Szeliga

2. Perfluorooctanoic acid (PFOA) PFOA is a perfluorinated carboxylic acid 1 The primary use of PFOA is as a processing aid in the production of fluoropolymers pKa: 2.5 Melting point: 55-56°C 2 Boiling Point: 189°C Exact mass: 413.973700 Daltons Not fluorescently active

3. Hypothesis People who work or live in close proximity to places that use PFOA will have elevated and potentially harmful levels of PFOA in their bloodstream if PFOA is leaking into their water supply The level of PFOA in the bloodstream of this at- risk population should be compared against the national average If the level of PFOA is significantly elevated, then there is evidence of improper disposal

4. How PFOA Enters the Human Body PFOA is an industrial pollutant. Improper disposal leads to elevated levels in the water supply which causes it to accumulate in the bloodstream PFOA can also leech out of fluoropolymers present in many household goods 1 Due to this exposure, PFOA exists at a 3.4 ng/mL concentration in the blood of the average American 3

5. Methods of Separation MethodAdvantagesDisadvantages Reverse-Phase HPLC 4 Fast (14 min trial) High Resolution Widespread use in medical research High use of solvent Gas Chromatography 5 High Resolution Prepares sample for Mass Spec. Requires derivatization with n-butanol Slow (26 min trial) Capillary Zone Electrophoresis 6 Fast (14 min trial) Low resolution Requires several organic additives

6. Methods of Detection MethodAdvantagesDisadvantages Suppressed Conductivity Detection 4 Can measure directly Requires no derivatization Simple Low equipment cost Must be modified to exclude teflon and other fluoropolymers from its construction High limit of detection Mass Spectrometry 5 Extremely selective Extremely sensitive Requires GC Expensive equipment required Fluorescence Detection 6 Extremely selective PFOA must be measured indirectly High limit of detection

7. Reverse-phase HPLC HPLC System: Dionex ICS-3000, replacing the sample loop with a concentrator and ASI-100 autosampler 4. Column: Acdaim PA2 (3 mm 2.1 x 150 mm) Concentrator: Acclaim PA2 (5 mm 4.3 x 10 mm guard cartridge and holder Selected for lack of fluoropolymers in internal construction http://pioneer.netserv.chula.ac.th/~skitipat/hplc/hplcman.html Mobile Phase A 17% 100 mM H 3 BO 3 and 9 mM KOH, pH 8 83% DI water Mobile Phase B 30% 70:30 (v:v) acetonitrile:water 30% 100 mM H 3 BO 3 and 9 mM KOH, pH 8 40% DI water

8. Suppressed Conductivity Detection Conductivity Detector: ED50A Electrochemical Detector with conductivity cell and DS3 Detector Stabilizer 4 Suppressor: Dionex ASRS ULTRA II 2 mm, with external regenerant Regenerant: 25 meq/L H2SO4 at approx 0.5 mL/min Limit of Detection: 0.5 μg/mL http://www.chromatography-online.org/HPLC/Electrical-Conductivity/rs29.html

9. Procedure The PFOA in the original plasma sample is purified by precipitating the proteins in acetonitrile. The solvent extracts are then saved for analysis The solvent extracts are spiked with a standard solution of PFOA The solvent extracts are concentrated on a column before being injected into the separation column The fractions move through a suppression filter before entering the conductivity detector

10. The future of PFOA detection While no robust procedure has been developed, better electrochemical methods for the detection of PFOA are possible Ion selective electrodes have been used to quantify the concentration of perfluorooctanoate in solution 7 In a basic environment, PFOA may be quantifiable with an ion-selective electrode with minimal sample preparation

11. Conclusion HPLC/Suppressed Conductivity Detection is the most cost effective method for large scale analysis of PFOA concentration in blood samples The results can be compared to the national average to look for evidence of localized PFOA contamination Future methods may be more portable or more cost effective

12. References 1.Substance flow analysis for Switzerland: Perfluorinated surfactants perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA); Federal Office for the Environment (FOEN) : Bern 2009 2.Sigma-Aldrich Catalog, Perfluorooctanoic Acid. http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=171468|A LDRICH&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC 3.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 4.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 5.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. 6.Wójcik, L., Szostek, B., "Separation of perfluorocarboxylic acids using capillary electrophoresis with UV detection" Electrophoresis 2005, 26, 1080-1088 7.The Influence of Sodium Perfluorooctanoate on the Conformational Transitions of Human Immunoglobulin Paula V. Messina,†,#Gerardo Prieto,†Francisco Salgado,§, Carla Varela,§, Montserrat Nogueira,§, Verónica Dodero,‡Juan M. Ruso,† and, and Félix Sarmiento*,† The Journal of Physical Chemistry B 2007 111 (28), 8045-8052