Document dQK2YZD8yDNdMJLg48RKQg146

AR226-0191 OES `Compound-Specific, Quantitative Characterizationof Organic Fluorochemicals in Biological Matrices Kcisen) Hae;LisA.Clemens; Mark . Elton,Harold O Johnson (+Conspoandhicnilg: Kansacnom; phone 65177-6018 x 651-7617) HFM Ereminyent Laboratory Soom sss Abstract Since the early 1980s, there has been a steady increase in the useof nonvolatile fluorinated organic compounds for a varietyofindustrial applications. The industrial use ofthese relatively stable compounds has initiated debate over the fate of fluorochemicals in the environment and, ultimately, the bioavailability of these compounds (1,2). Until recently, levels of organic fluorochemicals in biological matrices have been determined by non-chemical specific analytical methods such as total fluoride analysis (2-6). In this manuscript, we present a compound-specific method for the extractionofextremely low levelsofseveral commercial organic fluorochemicals from sera and liver with quantitative detection by negative ion electrospray tandem mass spectrometry. This technique represents a robust, previously undescribed approach to quantifying specific organic fluorochemicals in biological matrices. This method should prove useful in future studies designed to determine the levels of organic fluorochemicals in humans and the environment. Results from a study of 65 human sera samples purchased from biological supply companies and the detailsof the analytical method for the quantitative analysis of specific organic fluorine containing compounds are described. 04567 \ Introduction `The unique chemical/physical propertiesoffluorine make fluorinated organic compounds useful for many commercial applications, and industrial productionofthese compounds has increased significantly since the early 1980s. Fluorinated organics are used as refrigerants, surfactants, and polymers and as componentsofpharmaceuticals, fire retardants, lubricants, and insecticides (1). Fluorochemical compounds that are not perfluorinated may be susceptible to partial chemical breakdown at functional group bonds (7). However, given the energyofthe carbon-fluorine bond, it is expected that many organic fluorochemical compounds will be resistant to hydrolysis, photolysis, biodegradation, or metabolism (8). For example, even in the high-energy environment of the stratosphere, the carbon-fluorine bonds in chlorofluorocarbons are exceptionally stable (9). In 1974, Guy et al. reported results for the determinationoforganic fluorine levels in plasma from 106 individuals from five cities in the United States (2). These researchers demonstrated that although levelsof inorganic fluorine in human plasma could be correlated to fluoride levels in drinking water, organic fluorine levels showed no such correlation. Guy et al. showed that the organic fluorine levels measured from samples collected within a particular city were, basically, log normally distributed with few outliers. The average organic fluorine level in human plasma samples included in the study was reported to be 1.35 0.85 micromolar R-F (approximately 26 ppb organic fluorine). By concentrating the organic fluorine from 20 litersofplasma and performing nuclear magnetic resonance (NMR) analysis, Guy et al. postulated that the 04568 2 perfluorooctanoate anion (PFOA) or a structurally related compound may be the source of the organic fluorine. Further, they suggested that there may be three or more different `components of organic fluorine in plasma samples collected from the general population. Although the source ofthe organic fluorine in general population blood has been debated and never definitively determined, some have postulated that contaminationof the environment with industrial fluorochemicals is the source of the organic fluorine compounds (4). Others suggest that the organic fluorine is likely to have a natural source (10). Despite the routeof exposure, researchers agree that such low levelsoforganic. fluorochemicals are unlikely to cause toxic effects (2,11,12). A large number of studies in both humans and animals have been conducted to study the toxicity associated with PFOA. In these studies, when determinationsofthe PFOA levels in tissues were necessary, a total organic fluorine method was employed or a study using radiolabeled material was designed, because easy, sensitive, compound-specific methods have not been available (11-17). Historically, low-level detection of fluorochemicals such as PFOA and perfluorooctanesulfonate (PFOS) has been limited to relatively insensitive or non-mass-specific detection methods, such as gas chromatography-flame ionization detection, gas chromatography-electron capture detection and high performance liquid chromatography (HPLC)-ultraviolet detection (18-20). In the work presented here, a new methodfor the analysisofseveral low-level fluorinated organic compounds in sera and liver tissue is described. Aer initial extractionof the tissue with an ion-pairing reagent, extracts are analyzed with HPLC-negative ion electrospray tandem mass spectrometry (HPLC-ESMSMS). The ability to select a unique 04569 3 product ion upon fragmentationofthe molecular ion provides a very selective analysis that is not as likely to be affected by biological interferences. Detection limits and the linear rangeofthe method were determined for four fluorinated organic compounds [PFOA, PFOS, perfluorooctanesulfonylamide (PFOSA), and perfluorhexanesulfonate (PFHS)] in both liver and sera by spiking cach matrix with standard material and quantitatively recovering the compounds. Although detection limits can be improved by concentrating sample extracts, extraction of non-concentrated sera produced detection limits for all target analytes of 1-3 ppb. The method presented here has been used to quantitatively analyze four organic fluorochemicals in 65 human sera samples collected from several biological supply companies in the United States. High-resolution time-of-flight mass spectrometry was used to confirm the identity of PFOS, PFOA, PFHS, and PFOSA extracted from a single representative sera sample. Experimental Materials andMethods Rabbit and rat sera were purchased from Sigma (St. Louis, MO). HPLC-grade methyltert-butyl-cther (MTBE) and methanol were purchased from EM. Science (Gibbstown, NJ); the tetra-butyl ammonium (TBA) hydrogen sulfate was purchased from Kodak (Rochester, NY); the pHofthe TBA solution was adjusted with sodium hydroxide (.T. Baker; Phillipsburg, NJ). Before use, water was purified with a Milli-Q" system (Millipore; Bedford, MA). Human sera samples were purchased from the following biological supply companies: Sigma (St. Louis, MO); Golden West Biologicals 004570 4 (Temecila, CA); Biological Specialty Corporation (Colmar, PA); and Lampire Biological Laboratories (Pipersville, PA) New Zealand White [Hra:(NZW)SPF] rabbit liver was obtained from Covance Laboratories, Inc, in Madison, WL; Sprague Dawley rats were purchased from Harlan (Indianapolis, IN), and rat liver samples were harvested by 3M Toxicology personnel (St. Paul, MN) The PFOS and PFOA used as standards and as matrix spikes were purchased from Fluka (Milwaukee, WI); standards of PFHS and PFOSA were made available from 3M Company (St. Paul, MN). The internal standard, 14, 1H,2H,2H perfluorooctane sulfonate (THPFOS), was purchased from ICN (Costa Mesa, CA). Extraction Procedure: OnehalfmL of sera, uLofinternal standard, 1 mL of 0.5 M TBA solution (adjusted to pH 10), and 2 mL of 0.25 M sodium carbonate buffer were added 10a 15-mL polypropylene tube for extraction. After thorough mixing, mL of MTBE was added to the solution, and the mixture was shaken for 20 minutes. The organic and aqueous layers were separated by centrifugation, and an exact volume of MTBE (4.0 mL) was removed from the solution. The aqueous mixture was rinsed with MTBE and separated twice more; all rinses were combined in a second polypropylene tube. The solvent was allowed to evaporate under nitrogen before being reconstituted in 0.5 mL of methanol. The sample was vortex mixed for 30 seconds and passed through a 0.2 um nylon mesh filter into an autovial. Depending upon the speciesoftest animal, the serum extract was typically either colorless or light yellow. 04571 5 For the extraction ofliver samples, a liver homogenate of 1 gram of liver to 5 mL of Milli-Q water was prepared. One mLofthe homogenate was added to a polypropylene tube, and the sample was extracted according to the procedure for sera (described above), Teflon or glass containers were avoided in this procedure; the former may cause. analytical interferences, and the latter may bind the surfactants in an aqueous solution. Disposable polypropylene or plastic lab wear was used to minimize the possibility of sample contamination that can occur when glassware is reused. Any glassware used in the preparationofthe reagents was thoroughly rinsed with methanol prior to use. To ensure that target analytes were not introduced to the matrix prior to extraction, blood collection supplies were extracted and analyzed. Blood bags were purchased from Baxter (Deerfield, IL), and five different typesofVacutainersTM (two labeled "gel and clot activator," two labeled "KsEDTA," and one labeled "no activator") were purchased from Becton Dickinson (Franklin Lakes, NJ). Additional blood collection materials tested consistedof3-cc and 10-cc syringes, 19G1 1/2 Precision Guide sterile needles, multiple sample Vacutainer sterile needles, and Terumo winged infusion sets, all of which were obtained from Becton Dickinson. `The inside surfacesofall blood collection supplies were exposed to methanol (from 0.5 mL to 80 mL, depending on the particular supply) for 1 hour. The extraction solvent was dried and reconstituted to exactly 1 mL of methanol. A second set of samples was 4572 6 spiked with analyte and extracted in exactly the same way as the firstsetto ensure that analyte could be recovered. Extraction blanks were prepared using Milli-Q water, and matrix blanks were prepared from rabbit or rat tissue spiked with THPFOS. Analyte separation was performed using a Hewlett-Packard HP1100 liquid chromatograph modified with low dead-volume internal tubing. Prior to the autosampler, a1 em Hypercarb cartridge from Keystone (Bellefonte, PA) was added. Ten pLs of extract were injected onto a 50 x 2mm (5 um) Keystone Betasil Cis column with a 2 mM ammonium acetate/methanol mobile phase starting at 45% methanol. At a flow rate of 300 uL/minute, the gradient increased to 90% methanol before reverting to original conditions at 9 minutes. Column temperature was maintained at 25 C. For quantitative determination, the HPLC system was interfaced to a Micromass (Beverly, MA) Quattro II atmospheric pressure ionization tandem mass spectrometer operated in the clectrospray negative mode. Instrumental parameters were optimized to transmit the [M-H] ion for all analytes. When possible, multiple daughter ions were monitored, but quantitation was based on a single product ion. Refer to Table 2 for a summaryoftransitions monitored. In all cases, the capillary was held between 1.6-3.2 kV. For PFOA determination, the quantitation ion (m/z=169) corresponds toCsFs' the product ion m/z=99 corresponds to 04573 7 FSOy for quantitative determination of PFOS. QuantitationofPFOSA occurs at m/z=78, corresponding to SO;N; quantitation of PHS occurs at m/z=80 (SO), In the ESMSMS system, the 499 Da. 80 Da. transition can provide a stronger signal than the 499 Da. 99 Da. transitionofthe PFOS analysis. However, in the analysis of tissue samples collected from some speciesof animals, an unidentified interferent was present in the 499 Da.> 80 Da. transition. Although this interferent was rarely observed, to ensure complete selectivity, quantitation was based on the 499 Da. => 99 Da. transition. Exact mass determination was achieved by interfacing the chromatographic system to eithear Micromass" LCT; product ion spectra were collected with a Micromass" Q-TOF. Both the LCT and Q-TOF are high-resolution time-of-flight mass spectrometers. An 800 ng/mL solutionofraffinose in 50/50 ACN/water was infused into the source at 20 uL/hr as a lock mass (503.1612 Da). PFOS, PFOSA, and PFHS were measured at a cone voltageof 70 V; PFOA was measured at a 10-V cone voltage. For analysis ofall analytes, the capillary was maintained at 3200 V. Results and Discussion Characterization of the Method A seriesofexperiments, described in more detail below, was designed to characterize the analytical method. In general, all curves, extracted or unextracted, were plotted using linear regression, weighted 1/X. Tables 3 and 4 show the extraction efficiency, limit of detection, and linear range for the target analytes. 04574 5 With the exceptionofPFOA, the extraction efficiency was determined by extracting and analyzing six replicate rat or rabbit sera samples spiked at approximately the following levels: 10 ng/mL, 50 ng/mL, 100 ng/mL, and 500 ng/mL. For PFOA, only the three higher levels were used for extraction efficiency calculations. Extraction efficiency in liver was determined by extracting samples spiked at 50 ng/g, 100 ng/g, and 500 ng/g For both sera and liver, the extracted samples were evaluated versus the average curve produced by two unextracted solvent curves analyzed before and after the extracts. The extraction efficiency for PFHS and PFOSA from liver was determined to be significantly Tower than those determined for the PFOS and POAA. However, because sample analysis is conducted using extracted curves, the relatively low recoveries should not affect the results. Table 3 shows the compiled average for all spike levels along with the standard deviation. For both sera and liver analyses, the internal standard was used for quantitative determination of PFOS and PFOA, only. The limitofdetection was determined as per EPA Regulation 40 CFR part 136, Appendix B. For each analyte, seven low-level spikes were prepared and analyzed. Based on the standard deviation associated with the replicate analysis, a limitofdetection was calculated. This calculated limit of detection was verified by analyzing a sample that was spiked at that level and extracted. 04575 9 `The linear range was determined by analyzing duplicate curves extracted from each `matrix over awide range. Starting with the highest standard, points were removed from the curve until the correlation coefficient for the 1x weighted fit was greater than 0.99. For the sera curves, all points except for the lowest standard level were evaluated to be `within 20%ofthe expected value. For the standard curves extracted from liver, all points except the lowest point were within + 30. Characterization of Blanks Method blanks were prepared from Milli-Q water. Because analyte-free (less than 1 ng/mL) human sera matrix could not be located, surrogate matrix blanks were prepared from rabbit sera. None of the analytes were detected in either set of blanks. Instrument blanks, consistingofHPLC-grade methanol, were analyzed after high-level-standardcurve points and after periodic calibration checks, to monitor potential carry-over. No. carry-over was observed Methanol extractsofblood collection supplies were analyzed; noneofthe target analytes were detected in these extracts. In addition, the Teflon cap liners ofglass jars used for reagent storage were extracted with methanol. Low-levelsofPFOS and PFOA were detected in someofthe extractsofthe Teflon liners. These materials were removed from the extraction procedure. Figure | compares the resultsofthe multiple response monitoring (MRM) analysis for PFOS in an extraction blank, in unspiked rabbit sera, and in unspiked, commercially available human sera. 04576 10 Identification of Target Analytes The retention timesofthe analytes extracted from human sera were matched to within 2%of the retention timeof standard material spiked into and extracted from rabbit sera MRM analysis was used for verificationofanalyte identity. For each analyte except PFOSA, at least two characteristic product ions were monitored, although quantitation was based on the response ofa single product ion. PFOSA was detected at such low levels, only a single product ion could be monitored, even for qualitative purposes. In the human sera samples, for all analytes except PFOSA, the relative abundancesoftwo or more product ions collected by MRM were confirmed to within 20% of standards as criteria for analyte verification (21). To further verify the identityof the detected analytes, a 30-fold concentrated extraction of one sera sample was prepared. This concentrated extract was used for exact mass determinationofall four analytes using high-resolution time-of-flight mass spectrometry. `The concentrationofthe detected analytes were confirmed to within S ppm for all target analytes. Figure 2 shows the resultsofthe PFOS and PFOSA high-resolution analysis. `Additionally, using high-resolution time-of-flight mass spectrometry, full product ion spectra were collected for each analyte in the concentrated extract. The product ion spectra for the PFOS identified in human sera is shown in Figure 3 Quantitation of Target Analytes in Human Sera Quantitationof the analytes was based on comparison ofa single product ion peak area to the response of two standard curves, weighted 1/X, bracketing each sample set. Mid-level calibration checks were analyzed every five to ten samples. Based on the precision (30)4ves u determined from repeat injectionsof the standard curves, results were considered quantitative to + 30%. Quantitative results, presented as compound-specific average concentrations in sera are presented in Table 5. In addition to the average analyte concentration, the concentrationoforganic fluorine represented by each compound is presented. For example, by weight, PFOS is 65% fluorine; for samples reported here, the. average PFOS concentration was determined to be 33 ng/mL of PFOS. This corresponds 10 about 22 ng/mLoforganic fluorine Added together, the four specific fluorochemicals measured in this small set of samples `account for approximately 31 ng of organic fluorine per milliliter of sera. Within experimental error associated with each technique, this value compares closely to the value obtained by Guy et al. (approx. 26 ng/mL) more than 20 years ago (2). Also in accordance with Guy et al, PFOA has specifically been identified in the sera samples, although not necessarily as the major component. For the 65 samples reported here, PFOS was present at the highest concentration. Each analyte measured was detected in every sample, with the following significant exceptions: PFOSA was not measured above the detection limit in 60of the 65 samples; PFHS was not detected in one sample. A combinationofextraction and analytical methods that do no require chemical derivitization, use smal volumes of samples, and are highly sensitive and mass specific were developed for the low-level analysisofseveral fluorinated organic compounds in sera and liver. Using these methods, samplesof human sera collected from biological supply companies were analyzed for four separate fluorochemicals, PFOA, PFOS, PFHS, . S04578 and PFOSA. Taken together, these fluorochemicals account for 31 ng/mLoforganic fluorine in an examination of 65 human sera samples from biological supply companies, consistent with historical reportsoftotal organic fluorine studies. Although this study comprises a relatively small sample set, it does suggest the possibility ofa more complete characterizationof the organic fluorine compounds present in human sera. Additionally, these compound-specific analyses should be paired with a total organic fluorine analysis to determine what fractionof the total organic fluorine present is due to the four fluorochemicals quantified in this study. 04579 1 Acknowledgements The authors are grateful to Dr. Andrew Seacat and Deanna Luebker of 3M Toxicology for supplying rat liver for method development, and to Dr. George Moore for providing standard materials. Dr. Robert Voyksner is acknowledged for his thorough and timely review of this work. 04580 1 References 1 Key, B.D, Howell, RD., and Criddle, Environ. Sci. Technol. 1997, 31, 2445. C.S. Fluorinated Organics in the Biosphere. 2 Guy, WS, Taves, DR, and Brey, W.S,, Jr. Organic Fluorocompounds in Human Plasma: Prevalence and Characterization. Biochemistry Involving Carbon-Fluorine Bonds, Edition number; Publisher; Place of Publication; Year; Volume number; pp. 117-XXX 3 Taves, D. Evidence that thereare Two Forms of Fluoride in Human Serum, Nature 1968, 217, 1051 4 Taves, D.R. Comparisonof `Organic' Fluoride in Human and Nonhuman Serum. Nature 1971, 50,783 5 CBeolnitselen,t1o,faWnhdolHaegBelno,odD,.F.SeMruemt/hPoldasfioar,thaenDdeOttehremrinBaitoiloongoifcalthSeaTmoptlalesFlAunaolr.ine Biochem. 1978, 87, 545, 6 YFoarmmasmooftoF,luGo,rinYeosihnitWahkoel,eK.B,loSaotdoo,fT.H,uKmiamunraM,alT,e Aanna.d BAinodoc,heT.m.Di1s9t8r9i,bu1t8i2o,n a3n7d1 7 7Hagen, DE. Belisle, J. Johnson, 1.D.; Venkateswarlu, V. Characterization of Fluorinated Metabolites by a Gas Chromatgraphic Helium Microwave Plasma. Detecto-r The Biotransformation of 1H,1H,2H,2H-Perfluorodecanol to Perfluorooctanoate. Anal. Biochem. 1981, 118, 336. 8 Organoftuorine Chemistry Principles and Commercial Applications, Smart, BE; Tatlow, J.C., Eds; Pelnum Press: New York, 1994. Banks, R.E.; 9 Atmospheric Chemistry, Sons: New York, 1986, Finlayson-Pitts, B.J, Pitts, JN. Jr, Eds; John Wiley & 10 Belisle, J. Organic Fluorine in Human Serum: Science 1981, 212, 1509. Natural Versus Industrial Sources. 11 GEinlzlyilmaensd,, FL.iDp.o,praontdeiMnasn,daenld, CJh.5o.leSsteerruolm: PeArfSlutourdoyoocftaOncociucpAactiiodnaanldlyHEepxaptoisced Men Am. J. Ind. Med. 1996, 29, 560. 12 Griffith, F.D., and Long, Perfluorooctanoate. Am. J. E. Ind. HAygn.imAaslsoTco.xiJ.ci1t9y8S0t,u4d1i,es57w6i.th Ammonium 04581 15 13 Kennedy, JR, Toxicol. Appl. and Gerald, Pharmacol. L ; Dermal Toxicity 1985, 81, 348. of Ammonium Perfluorooctanoate 14 Kennedy, G.L., Jr, Hall, GT, Britelli, MR., and Chen, of Ammonium Perfluorooctanoate. Food Chem. Toxicol. H.C. Inhalation 1986, 24, 1325. Toxicity 15 Hanhijarvi, H., Ophaug, RH. and Singer, L. The Sex-related Difference in Perfluorooctanoate Excretion in the Rat. Proceedings of the Societyfor Experimental Biology and Medicine 1982, 171, 50. 16 Venden Heuvel, J.P, Kuslikis, B.1, Van Rafelghem, M.J., and Peterson, R.E. TMiaslseueanDdisFtreimbaultieonR,atMs.etJa.bBoiloicshme,m.andToExliciomli.na1t9i9o1n,o6f, Perfluorooctanoic 83. Acid in 17. Venden Heuvel, JP, Davis, J.W., I, Sommers, R., and Peterson, R.E.; Renal Excretion of Perfluorooctanoic Acid in Male Rats: Inhibitory Effect of Testosterone. J. Biochem. Toxicol. 1992, 7,31 18 Ohya, T, Kudo, N., Suzuki, E., and Kawashima, Y., Determination of Perfluorinated Carboxylic Acids in Biological Samples by High-performance Liquid Chromatography. J. Chromatogr. 1998, 720, 1. 19 BAecliidslei,n BJ,loaonddaHnadgeOnt,heDr.FB.ioAlogMiectahloSdamfpolretsh.e ADneatle.rmBiinoacthieomn.of1P9e8r0f,lu1o0r1,oo3c6t9.anoic 20 Ylinen, M., Hanhijarvi, Determination of PFOA H., as Peura, P., the Benzyl Ramo, Ester O. Quantitative Gas in Plasma and Urine. Chromatographic Arch. Environ. Contam. Toxicol. 1985, 14,713 21 Lily Y.T, Campbell, D.A,, Bennett, P.K., and Henion, J. Acceptance Criteria for Ultratrace HPLC-Tandem Mass Spectrometry: Quantitative and Qualitative DeterminationofSulfonylurea Herbicides in Soil, Anal. Chem. 1996, 68, 3397. 04582 16 Conrousp PFOA PFOS PFHS PFOSA_ PRIMARY ION [oN PRODUCTIONS [DN 413 119, 169%, 219 499 | 80,99% 130 399 | 80,99 130 08 78 Er COME VOLTAGE (V) 25 0 60 | 0 Fr a ENERGY (EV) 20 s| 45 [5 *Product ions were used for quantitation. Table 2. Summary of Primary lons, Product Tons, and ESMSMS Conditions 04583 1" ANALYTE PFOA PFOS PFOSA PFHS EXTRACTION [ree eve DEVIATION 1019% 939% | 95%6% 8547% [ DETECTION [re RANGE, EXTRACTED 1.0 ppb 5-100 ppb 1.7 ppb 5-1000 ppb 15ppb | 5-1000 ppb 2.0 ppb 5-1000 ppb CORRELATION COEFFICIENT 0.998 0995 | 0.998 0.998 Table 3. Method Characteristics for the AnalysisofSpecific Organic Fluorochemicals in Sera. (All concentrations are expressed as ng/g.) 04584 1s I PFOA PFOS PFOSA PFHS EXTRACTION I 87:12% 100=13% S6%11% 723% Lint oF 85 ppb 35ppb 2.0 ppb [re RANGE, CORRELATION Rr 10-1000 0.989 51000 0.991 | 5-100 ppb 5-1000 ppb 0995 | 0.99 Table 4. MinetLihvoerd. C(hAalrlacctoenrciesnttircastfioornsthaereAneaxlpyrseissseodf Saspencgi/gf.i)c Organic Fluorochemicals C04585 19 PFOA 6.63% 1-13 46 PFHS 6.45% 1-13 37 1 PFOSA 1.803% <1-2 a * Several samples were determined to contain the target analyte below the limit of quantitation, therefore, average concentration is estimated. Table 5. Concentrations (ng/mL)of Various Organic Fluorochemicals in Human Sera 04586 o Figure 2. High Resolution Analysis of PFOS and PFOSA 04587 21 Figure 3. Product lon Spectra for PFOS Endogenous in Human Sera 04588 2