Document QX87Oo2xe4qkLGMaZjZnqaG36

ARm-om Compound-Specific, Quantitative Characterization of Organic Fluorochemicals in Biological Matrices Kristen J. Hansen*; Lisa A. Clemen; Mark E. Ellefson; Harold O. Johnson (Corresponding author email: kihansen@mmm.com; phone 651-778-6018; fax 651-778-6176) 3M Environmental Laboratory Building 2-3E-09 P.O. Box 33331 St. Paul, MN 55133-3331 Abstract Since the early 1980s, there has been a steady increase in the use of nonvolatile fluorinated organic compounds for a variety of industrial applications. The industrial use of these 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 extraction of extremely low levels of several 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 details of the analytical method for the quantitative analysis of specific organic fluorine containing compounds are described. 004567 i Introduction The unique chemical/physical properties of fluorine make fluorinated organic compounds useful for many commercial applications, and industrial production of these compounds has increased significantly since the early 1980s. Fluorinated organics are used as refrigerants, surfactants, and polymers and as components of pharmaceuticals, 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 energy of the 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 determination of organic fluorine levels in plasma from 106 individuals from five cities in the United States (2). These researchers demonstrated that although levels of 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 liters of plasma and performing nuclear magnetic resonance (NMR) analysis, Guy et al. postulated that the 004568 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 of the organic fluorine in general population blood has been debated and never definitively determined, some have postulated that contamination of 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 route of exposure, researchers agree that such low levels of organic 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 determinations of the 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 method for the analysis of several low-level fluorinated organic compounds in sera and liver tissue is described. After initial extraction of 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 004569 3 product ion upon fragmentation of the molecular ion provides a very selective analysis that is not as likely to be affected by biological interferences. Detection limits and the linear range of the method were determined for four fluorinated organic compounds [PFOA, PFOS, perfluorooctanesulfonylamide (PFOSA), and perfluorhexanesulfonate (PFHS)] in both liver and sera by spiking each 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 and Methods Rabbit and rat sera were purchased from Sigma (St. Louis, MO). HPLC-grade methyl- tert-butyl-ether (MTBE) and methanol were purchased from E.M. Science (Gibbstown, NJ); the tetra-butyl ammonium (TBA) hydrogen sulfate was purchased from Kodak (Rochester, NY); the pH of the TBA solution was adjusted with sodium hydroxide (J.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, 1H, IH, 2H, 2H perfluorooctane sulfonate (THPFOS), was purchased from ICN (Costa Mesa, CA). Extraction Procedure: One half mL of sera, 5 pL of internal 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 to a 15-mL polypropylene tube for extraction. After thorough mixing, 5 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 pm nylon mesh filter into an autovial. Depending upon the species of test animal, the serum extract was typically either colorless or light yellow. 004571 5 For the extraction of liver samples, a liver homogenate of 1 gram of liver to 5 mL of Milli-Q water was prepared. One mL of the 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 preparation of the 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 types of Vacutainers (two labeled "gel and clot activator," two labeled "K3EDTA," and one labeled "no activator") were purchased from Becton Dickinson (Franklin Lakes, NJ). Additional blood collection materials tested consisted of 3-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 surfaces of all 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 004572 6 spiked with analyte and extracted in exactly the same way as the first set to 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, a 1 cm Hypercarb cartridge from Keystone (Bellefonte, PA) was added. Ten pLs of extract were injected onto a 50 x 2mm (5 pm) Keystone Betasil Cig column with a 2 mM ammonium acetate/methanol mobile phase starting at 45% methanol. At a flow rate of 300 pL/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 electrospray 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 summary of transitions monitored. In all cases, the capillary was held between 1.6- 3.2 kV. For PFOA determination, the quantitation ion (m/z=169) corresponds to C 5F 9 '; the product ion m/z=99 corresponds to 004573 7 FSCV for quantitative determination ofPFOS. Quantitation of PFOSA occurs at m/z=78, corresponding to SO2N*; quantitation of PFHS occurs at m/z=80 (SO3'). In the ESMSMS system, the 499 Da.-^ 80 Da. transition can provide a stronger signal than the 499 Da.-^ 99 Da. transition of the PFOS analysis. However, in the analysis of tissue samples collected from some species of animals, an unidentified interfrent was present in the 499 Da.-^ 80 Da. transition. Although this interfrent 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 either a 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 solution of rafFinose in 50/50 ACN/water was infused into the source at 20 pL/hr as a lock mass (503.1612 Da). PFOS, PFOSA, and PFHS were measured at a cone voltage of 70 V; PFOA was measured at a 10-V cone voltage. For analysis of all analytes, the capillary was maintained at 3200 V. Results and Discussion Characterization of the Method A series of experiments, 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. 004574 8 With the exception of PFOA, 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 lower 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 limit of detection 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 limit of detection was calculated. This calculated limit of detection was verified by analyzing a sample that was spiked at that level and extracted. 004575 9 The linear range was determined by analyzing duplicate curves extracted from each matrix over a wide range. Starting with the highest standard, points were removed from the curve until the correlation coefficient for the 1/x 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% of the 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, consisting of HPLC-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 extracts of blood collection supplies were analyzed; none of the target analytes were detected in these extracts. In addition, the Teflon cap liners of glass jars used for reagent storage were extracted with methanol. Low-levels of PFOS and PFOA were detected in some of the extracts of the Teflon liners. These materials were removed from the extraction procedure. Figure 1 compares the results of the multiple response monitoring (MRM) analysis for PFOS in an extraction blank, in unspiked rabbit sera, and in unspiked, commercially available human sera. 004576 10 Identification of Target Analytes The retention times of the analytes extracted from human sera were matched to within 2% of the retention time of standard material spiked into and extracted from rabbit sera. MRM analysis was used for verification of analyte identity. For each analyte except PFOSA, at least two characteristic product ions were monitored, although quantitation was based on the response of a 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 abundances of two or more product ions collected by MRM were confirmed to within 20% of standards as criteria for analyte verification (21). To further verify the identity of the detected analytes, a 30-fold concentrated extraction of one sera sample was prepared. This concentrated extract was used for exact mass determination of all four analytes using high-resolution time-of-flight mass spectrometry. The concentration of the detected analytes were confirmed to within 5 ppm for all target analytes. Figure 2 shows the results of the 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 Quantitation of the analytes was based on comparison of a 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 11 determined from repeat injections of 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 concentration of organic 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 to about 22 ng/mL of organic 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 60 of the 65 samples; PFHS was not detected in one sample. A combination of extraction and analytical methods that do not require chemical derivitization, use small volumes of samples, and are highly sensitive and mass specific were developed for the low-level analysis of several fluorinated organic compounds in sera and liver. Using these methods, samples of human sera collected from biological supply companies were analyzed for four separate fluorochemicals, PFOA, PFOS, PFHS, 004578 12 and PFOSA. Taken together, these fluorochemicals account for 31 ng/mL of organic fluorine in an examination of 65 human sera samples from biological supply companies, consistent with historical reports of total organic fluorine studies. Although this study comprises a relatively small sample set, it does suggest the possibility of a more complete characterization of 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 fraction of the total organic fluorine present is due to the four fluorochemicals quantified in this study. 004579 13 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. 004530 14 References 1 Key, B.D., Howell, R.D., and Criddle, C.S. Fluorinated Organics in the Biosphere. Environ. Sei. Technol. 1997, 31, 2445. 2 Guy, W.S., Taves, D.R., 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 there are Two Forms of Fluoride in Human Serum. Nature 1968, 277, 1051. 4 Taves, D.R. Comparison of `Organic' Fluoride in Human and Nonhuman Serum. Nature 1971, 50, 783. 5 Belisle, J., and Hagen, D.F. Method for the Determination of the Total Fluorine Content of Whole Blood, Serum/Plasma, and Other Biological Samples Anal. Biochem. 1978, 87, 545. 6 Yamamoto, G., Yoshitake, K., Sato, T., Kimura, T., and Ando, T. Distribution and Forms of Fluorine in Whole Blood of Human Male Ana. I Biochem. 1989,182, 371. 7 7 Hagen, D.F.; Belisle, J.; Johnson, J.D.; Venkateswarlu, V. Characterization of Fluorinated Metabolites by a Gas Chromatgraphic Helium Microwave Plasma Detector - The Biotransformation of 1H, lH,2H,2H-Perfluorodecanol to Perfluorooctanoate. Anal. Biochem. 1981, 118, 336. 8 Organofluorine Chemistry Principles and Commercial Applications-, Banks, R.E.; Smart, B.E.; Tatlow, J.C., Eds.; Pelnum Press: New York, 1994. 9 Atmospheric Chemistry, Finlayson-Pitts, B.J., Pitts, J.N. Jr., Eds; John Wiley & Sons: New York, 1986. 10 Belisle, J. Organic Fluorine in Human Serum: Natural Versus Industrial Sources. Science 1981, 212, 1509. 11 Gilliland, F.D., and Mandel, J.S. Serum Perfluorooctanoic Acid and Hepatic Enzymes, Lipoproteins, and Cholesterol: A Study of Occupationally Exposed Men Am. J. Ind. Med. 1996, 29, 560. 12 Griffith, F.D., and Long, J. E. Animal Toxicity Studies with Ammonium Perfluorooctanoate. Am. Ind. Hyg. Assoc. J. 1980, 41, 576. 0045S1 15 13 Kennedy, J.R., and Gerald, L.; Dermal Toxicity of Ammonium Perfluorooctanoate. Toxicol. Appl. Pharmacol. 1985, 81, 348. 14 Kennedy, G.L., Jr., Hall, G.T., Brittelli, M.R., and Chen, H.C. Inhalation Toxicity of Ammonium Perfluorooctanoate. Food Chem. Toxicol. 1986,24, 1325. 15 Hanhijarvi, H., Ophaug, R.H., and Singer, L. The Sex-related Difference in Perfluorooctanoate Excretion in the Rat. Proceedings o f the Societyfor Experimental Biology and Medicine 1982, 171, 50. 16 Venden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.J., and Peterson, R.E. Tissue Distribution, Metabolism, and Elimination of Perfluorooctanoic Acid in Male and Female Rats. J. Biochem. Toxicol. 1991, 6, 83. 17 Venden Heuvel, J.P., Davis, J.W., II, 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 Belisle, J., and Hagen, D.F. A Method for the Determination of Perfluorooctanoic Acid in Blood and Other Biological Samples. Anal. Biochem. 1980, 101, 369. 20 Ylinen, M., Hanhijarvi, H., Peura, P., Ramo, 0. Quantitative Gas Chromatographic Determination of PFOA as the Benzyl Ester in Plasma and Urine. 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 Determination of Sulfonylurea Herbicides in Soil, Anal. Chem. 1996, 68, 3397. 0045S2 16 C om pound Prim ar y ion (D a ) Product ions (D a ) O ptimal Cone Voltaci: (V) O ptimal C ollision Energy (eV) PFOA 413 119, 169*, 219 25 20 PFOS 499 80, 99*, 130 60 45 PFHS 399 80, 99*, 130 60 45 PFOSA 498 78* 60 45 THPFOS 427 80* 60 40 *Product ions were used for quantitation. Table 2. Summary of Primary Ions, Product Ions, and ESMSMS Conditions 004.583 17 A nalyte PFOA PFOS PFOSA PFHS Extraction Efficiency Standard D eviation 101 9 % 93 9 % 95 6 % 85 7 % L imit of D ete c tio n 1.0 ppb 1.7 ppb 1.5 ppb 2.0 ppb Linear Range, EXTRACTED 5-1000 ppb 5-1000 ppb 5-1000 ppb 5-1000 ppb C orrelation C oefficient 0.998 0.995 0.998 0.998 Table 3. Method Characteristics for the Analysis of Specific Organic Fluorochemicals in Sera. (All concentrations are expressed as ng/g.) 004584 18 Analyte PFOA PFOS PFOSA PFHS Extraction Efficiency 87 12 % 100 13 % 56 11 % 71 23 % L imit of D e te c tio n 5.0 ppb 8.5 ppb 3.5 ppb 2.0 ppb L in ea r Range, Extracted 10-1000 5-1000 5-1000 ppb 5-1000 ppb C orrelation C oefficient 0.989 0.991 0.995 0.994 Table 4. Method Characteristics for the Analysis of Specific Organic Fluorochemicals in Liver. (All concentrations are expressed as ng/g.) 004535 19 A nalyte PFOS PFOA PFHS PFOSA Average C o n c e n t r a t io n in S era 33 15 6.6 3* 6.4 5* 1.8 0.3* Range of C o n c e n t r a t io n in S era 5-85 1-13 1-13 <1-2 C oncentration of O rganic Fluorine R epresented 22 4.6 3.7 1.1 * 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 0045S6 20 Figure 2. High Resolution Analysis o f PFOS and PFOSA G045S7 21 Figure 3. Product Ion Spectra for PFOS Endogenous in Human Sera 0045S8 22