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37/f i R & A 6 - 1 ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN PLANT TISSUES WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M Environmental Lab Project No. U2723 AUTHORS: Raymond L. Van Hoven, Ph.D. Jon A. MacGregor, B.S. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: August 28,2001 STUDY COMPLETION DATE: October 22,2001 Submitted to: 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 51 002374 Wildlife International. Ltd. - 2- Project Number 454C-1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY COMPLETION: October 22,2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160,17 August 1989 and OECD Principles ofGood Laboratory Practice (ENV/MC/CHEM (98) 17) with the following exceptions: The stability of the test substance under storage conditions at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Wildlife International, Ltd. tonn-ol DATE SPONSOR APPROVAL: 002075 Wildlife International. Ltd. -3 - Project Number 454C-125 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160,17 August 1989 and OECD Principles ofGood Laboratory Practice (ENV/MC/CHEM (98) 17). The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTTVUY: M atrix Fortification Prparation R aw D ata, Draft Report Final Report DATE CONDUCTED: DATE REPORTED TO: ST U D Y DIRECTOR: MANAGEMENT: August 3 0 ,2 0 0 1 August 3 0 ,2 0 0 1 Septem ber 4 ,2 0 0 1 O ctober 11 , 1 2 and 1 5 ,2 0 0 1 O ctober 1 5 ,2 0 0 1 O ctober 1 6 ,2 0 0 1 O ctober 2 2 ,2 0 0 1 O ctober 2 2 ,2 0 0 1 O ctober 2 2 ,2 0 0 1 H (jS^Lyr'J. James H. Coleman, B.S. Quality Assurance Representative \Q '-o \ DATE 002376 Wildlife International. Ltd._______ -4- Project Number 454C-125 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY DIRECTOR: MANAGEMENT: '/L -- Willard B. Nixon, Ph.I Director o f Analytical Chemistry DATE mm DATE 002377 Wildlife International. L t d . _____________________________ Project Number 454C-125 -5 - TABLE OF CONTENTS Title/Cover Page............................................................................................................................................... 1 Good Laboratory Practice Compliance Statement......................................................................................... 2 Quality Assurance Statement.......................................................................................................................... 3 Report Approval.............................................................................................................................................. 4 Table o f Contents............................................................................................................................................ 5 Summary........................................................................................................................................................... 8 Introduction......................................................................................................................................................9 Objective...........................................................................................................................................................9 Experimental Design.............................................. 9 Materials and Methods.................................................................................................................................... 9 Test Substance.......................................................................................................................................9 Reagents and Solvents........................................................................................................................ 10 Test Systems....................................................................................................................................... 10 Tissue Samples Onion....................................................................................................................................... 10 Alfalfa........................................................................................................ 10 Fruit Samples Tomato.................................................................................................................................... 10 Soybean................................................................................................................................... 11 Analytical Validation Procedures........................................................................................................ 11 Primary and Secondary Stock Solutions............................................................................................. 12 Calibration Standards and Curves...................................................................................................... 12 Matrix Blanks and Matrix Fortifications............................................................................................12 Limit of Quantitation.......................................................................................................................... 12 R esults........................................................................................................................................................... 13 Reagent and Matrix Blank Samples................................................................................................... 13 Method Validation Samples................................................................................................................ 13 Conclusions.................................................................................................................................................... 13 002378 Wildlife International. Ltd.____________________ ___________ Project Number 454C-125 6- - TABLE OF CONTENTS - Continued - References......................................................................................................................................................15 TABLES Table 1 - Method Validation Recoveries for PFOS in Onion.................................................................... 16 Table 2 - Method Validation Recoveries for PFOS in Alfalfa................................................................... 17 Table 3 - Method Validation Recoveries for PFOS in Tomato.................................................................. 18 Table 4 - Method Validation Recoveries for PFOS in Soybean.................................................................19 APPENDICES Appendix 1 Appendix 2 - Protocol............................................................................................................................ 20 Certificate of Analysis.................................................................................................... 29 Appendix 3 - Analytical Method Validation D ata............................................................................... 32 Appendix 3.1 - Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit....33 Appendix 3.2 - Typical HPLC/MS/MS Operational Parameters...........................................................34 Appendix 3.3 - A Typical Calibration Curve for PFO S.........................................................................35 Appendix 3.4 - Example Calculations for a Representative Sample......................................................36 Appendix 3.5 - Ion Chromatogram of a Low-level PFOS Standard.......................................................37 Appendix 3.6 - Ion Chromatogram of a High-level PFOS Standard......................................................38 Appendix 3.7 - Ion Chromatogram o f an Onion Matrix Blank.............................................................. 39 Appendix 3.8 - Ion Chromatogram of a Low-level Onion Fortification Sample....................................40 Appendix 3.9 - Ion Chromatogram of a High-level Onion Fortification Sample...................................41 Appendix 3.10 - Ion Chromatogram of an Alfalfa Matrix Blank............................................................. 42 002379 Wildlife International. Ltd. -7 - Project Number 454C-1 ?S TABLE OF CONTENTS - Continued - Appendix 3.11 - Ion Chromatogram of a Low-level Alfalfa Fortification Sample.................................. 43 Appendix 3.12 - Ion Chromatogram of a High-level Alfalfa Fortification Sample.................................. 44 Appendix 3.13 - Ion Chromatogram o f a Tomato Matrix Blank........................................................... . 45 Appendix 3.14 - Ion Chromatogram of a Low-level Tomato Fortification Sample................................. 46 Appendix 3.15 - Ion Chromatogram of a High-level Tomato Fortification Sample................................ 47 Appendix 3.16 - Ion Chromatogram of a Soybean Matrix Blank.............................................................48 Appendix 3.17 - Ion Chromatogram of a Low-level Soybean Fortification Sample................................ 49 Appendix 3.18 - Ion Chromatogram of a High-level Soybean Fortification Sample............................... 50 Appendix 4 - Personnel Involved in the Study......................................................................................51 002380 Wildlife International. Ltd. - 8- Project Number454C-175 SUMMARY SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 TEST DATES: Study Initiation: August 28,2001 Experimental Start (OECD): August 30,2001 Experimental Start (EPA): August 30,2001 Experimental Termination: October 4,2001 TEST SYSTEM: Tissue Samples (Onion and Alfalfa) and Fruit Samples (Tomato and Soybean) FORTIFIED TEST CONCENTRATIONS: Onion and Alfalfa Tissue Samples (mg a.i./kg) Tomato and Soybean Fruit Samples (mg a.i./kg) 0.0500 0.500 5.00 50.0 0.0500 0.500 5.00 50.0 RESULTS: Samples of tissue matrix (onion and alfalfa) and fruit matrix (tomato and soybean) were each fortified in triplicate with PFOS to reflect nominal concentrations of 0.0500,0.500, 5.00 and 50.0 mg a.i./Kg. The samples were extracted with methanol, agitated, centrifuged, diluted into the instrumental calibration range with dilution solvent (50% methanol : 50% NANOpure water) and analyzed by high performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS). Recoveries of PFOS in onion and alfalfa yielded overall mean percent recoveries of 96.7% (SD = 6.34; CV =6.56%; N = 12) and 94.6%(SD = 4.13;CV =4.37% ;N= 12), respectively. Recoveries ofPFOS in tomato and soybeans yielded overall mean percent recoveries of 90.5% (SD = 5.68; CV = 6.28%; N = 12) and 95.3% (SD = 4.92; CV = 5.16%; N = 12), respectively. Reagent blanks, consisting of solvents, and matrix blank samples, consisting o f unfortified onion, alfalfa, tomato or soybean, were devoid of interfering components. The relative precision (CV) of triplicate fortifications ofPFOS in plant tissue matrix taken through the methodology was typically 5 percent, or better for the 0.500, 5.00, and 50.0 mg a.i./Kg fortification levels. The relative precision at the 0.0500 mg a.i./Kg fortification level, ranged from 2.28 to 11.4 percent. 002381 Wildlife International. Ltd. -9- Project Number 454C-17S INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. Validation samples were fortified with Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) and analyzed with high performance liquid chromatography mass spectrometry (HPLC/MS/MS) to evaluate the performance of a method for the determination o f PFOS in plant tissues. Validation samples were prepared and analyzed between August 30, 2001 and October 4,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454C-125 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective o f this study was to verify the performance of a HPLC/MS/MS methodology for the analyses of PFOS in plant tissues. The validated method will be used in support of definitive plant ecotoxicology tests to be conducted with the test substance. EXPERIMENTAL DESIGN Tissue samples, defined herein as the entire plant system including leaves and stems, and fruit samples, defined herein as the seed-bearing structure o f the flowering plant, were each fortified at four different concentrations in triplicate and analyzed based on HPLC/MS/MS methodology developed at Wildlife International, Ltd. The fortification levels bracketed the anticipated concentrations o f samples from anticipated plant toxicity tests. A reagent blank sample and matrix blank samples, in each matrix were analyzed concurrently to evaluate potential analytical method interferences. A calibration curve was generated from analyses of standard solutions of PFOS with each series of samples analyzed. MATERIALS AND METHODS This study was conducted according to the procedures outlined in the protocol, "Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues" (Appendix 1). Test Substance The test substance was received from 3M Corporation on October 29, 1998, assigned Wildlife International, Ltd. identification number 4675, and stored under ambient conditions. The test substance 002382 Wildlife International. Ltd. -10- Project Number 454C-17S was described as a white powder. It was identified as FC-95 from lot number 217, with an expiration date of August 31, 2006. Information from the most recent Certificate o f Analysis provided by the Sponsor indicated a purity of 86.9% (Appendix 2). Reagents and Solvents All solvents used in this study were HPLC grade or equivalent. All reagents were ACS reagent grade or equivalent. NANOpure water (equivalent to ASTM Type II Designation D1193-91) was used (1). Test Systems Tissue Samples Onion tissue was obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland, on October 2, 200land identified as lot number 09-07-01. Upon receipt, the bulk onion tissue was initially placed in a -4C freezer until further processing. The bulk onion tissue was placed in a cyrogenic (-80C) freezer for approximately 30 minutes, transferred to a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the onion homogenate. The remainder of the homogenized onion was then transferred to a zip-lock bag, and placed into the 4C freezer for use as control matrix in anticipated plant studies. Alfalfa tissue was obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland, on September 21,200l,an d identified as lot number 12-18-01. Upon receipt, the bulk alfalfatissue was placed in a -4C freezer until further processing. The alfalfa was removed from frozen storage and manually chopped using an Ekco 8" chefs knife and plastic cutting board and then returned to the -4C freezer. The frozen tissue was then further homogenized using a Waring blender. An appropriate number of 1.00 g aliquots were weighed from the alfalfa homogenate. The remainder of the homogenized alfalfa was then transferred to a zip-lock bag, and placed into the -4C freezer for use as control matrix in anticipated plant studies. Fruit Samples Tomatoes used were purchased on August 30,2001 from Wal-Mart Super-Center, Milford, Delaware, and identified as lot number 3151 with an expiration date of 8/30/02. The tomatoes were quartered, 02383 Wildlife International. Ltd. - 11- Project Number 454C-PS placed in a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the tomato homogenate. The remainder of the homogenized tomato was then transferred to a 16-oz. French-square bottle and placed into the -4C freezer for use as control matrix in anticipated plant studies. Soybeans were obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland on September 6, 2001, and identified as lot number 05-15-01, with an expiration date of 9/6/02. The soybeans were placed in a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the soybean homogenate. The remainder ofthe homogenized soybean was then transferred to a zip-lock bag, and placed into the -4C freezer for use as control matrix in anticipated plant studies. Analytical Validation Procedures Wildlife International, Ltd. conducted a validation trial using procedures developed at Wildlife International, Ltd. (Appendix 3). Matrix blanks and matrix fortification samples were prepared in tissue samples (onion and alfalfa) and in fruit samples (tomato and soybean). A reagent blank (containing everything except PFOS and matrix) was carried through the entire procedure for each of the four matrices. The tissue samples were extracted with methanol, agitated for a minimum of 30 minutes on a gyratory shaker table at approximately 250 rpm and then centrifuged. The fruit samples were extracted with methanol, agitated for a minimum o f one minute manually and then centrifuged. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection. Concentrations ofPFOS were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil Cu column (50 mm x 2.0 mm, 3-pm particle size) fitted with a Keystone Javelin Cu guard column (20 mm x 2.0 mm). A method flowchart is provided in Appendix 3.1 and the instrument parameters are summarized in Appendix 3.2. U02384 Wildlife International. Ltd. 12- - Project Number 454C-125 Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the test substance (86.9%). A 10.0 mg a.i./mL primary stock solution o f PFOS in methanol was prepared by weighing 1.1508 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (1000,100,10.0,1.00, and 0.100 mg a.i./L) of PFOS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Curves Calibration standards were prepared in 50:50 methanol: NANOpurewater by appropriate dilutions of the 1.00 and 0.100 mg a.i./L stock solutions of PFOS in methanol. Calibration standards ofPFOS, ranging in concentration from 0.400 to 5.00 pg a.i./L, were analyzed with each sample set. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. The calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Appendix 3.3. The concentration of PFOS in the samples was determined by substituting the peak area responses into the applicable linear regression equation (Appendix 3.4). Representative ion chromatograms of low and high calibration standards arc presented in Appendices 3.5 and 3.6, respectively. Matrix Blanks and Matrix Fortifications Selected 1.00-g aliquots of each matrix type were fortified with the appropriate secondary stock solutions of PFOS prepared in methanol using a gas-tight syringe. The matrix blanks for each matrix type were unfortified tissue or fruit. Limit of Quantitation The method limit of quantitation (LOQ) in each matrix was 0.0400 mg a.i./Kg calculated as the product o f the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). 002385 Wildlife International. L t d _______ _____. Project Number 454C-1 -13- RESULTS Reagent and Matrix Blank Samples A reagent blank and three matrix blank samples in each matrix were analyzed to determine possible interferences. No interferences were observed at or above the LOQ (Tables 1,2,3 and 4). Representative ion chromatograms of onion, alfalfa, tomato and soybean matrix blank samples are presented in Appendices 3.7,3.10,3.13 and 3.16, respectively. Method Validation Samples Onion tissue was fortified in triplicate at 0.0500,0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries o f 100, 91.0,97.6, and 98.2, respectively (Table 1). Representative ion chromatograms o f low and high-level fortifications in onion are presented in Appendices 3.8 and 3.9, respectively. Alfalfa tissue was fortified in triplicate at 0.0500,0.500,5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 96.4 94.4 92.8, and 94.8%, respectively (Table 2). Representative ion chromatograms of low and high-level fortifications in alfalfa are presented in Appendices 3.11 and 3.12, respectively. Tomato fruit was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 87.5, 90.6,93.4 and 90.6%, respectively (Table 3). Representative ion chromatograms of low and high-level fortifications in tomato are presented in Appendices 3.14 and 3.15, respectively. Soybean fruit was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 87.9, 95.9, 98.5 and 99.2%, respectively (Table 4). Representative ion chromatograms of low and high-level fortifications in soybean are presented in Appendices 3.17 and 3.18, respectively. The relative precision (CV) of the methodology, as demonstrated by the analysis of triplicate samples was 5 percent, or better for the 0.500,5.00, and 50.0 mg a.i./Kg fortification levels in all testing matrices. The relative precision at the 0.0500 mg a.i./Kg fortification level, ranged from 2.28 to 11.4 percent in the four testing matrices. CONCLUSIONS Onion, alfalfa, tomato and soybean were fortified with PFOS to reflect nominal concentrations of 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg. The samples were extracted with methanol, agitated for the appropriate amount of time, and centrifuged. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol and 50% NANOpurc water. Samples were 002386 Wildlife International. Ltd. - 14- Project Number 454C-19S then analyzed by high performance liquid chromatography mass spectrometry (HPLC/MS/MS). Recoveries o f PFOS in onion yielded an overall mean percent recovery of 96.7% (SD = 6.34; CV = 6.56%; N = 12). Recoveries of PFOS in alfalfa yielded an overall mean percent recovery of 94.6% (SD = 4.13; CV = 4.37%; N = 12). Recoveries of PFOS in tomato yielded an overall mean percent recovery of 90.5% (SD = 5.68; CV = 6.28%; N = 12). Recoveries of PFOS in soybean yielded an overall mean percent recovery o f 95.3% (SD = 4.92; CV = 5.16%; N = 12). Reagent blanks, consisting of solvents, and matrix blank samples, consisting of unfortified plant matrix, were devoid of interfering components. 002387 Wildlife International. Ltd. -15- Project Number 454C-17S REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D 1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. 002388 Wildlife International. Ltd. - 16- Project Number 454C-17s Tablel Method Validation Recoveries for PFOS in Onion Sample Number (454C-125-) Type PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery V R E B -4 Reagent Blank 0 <LOQ2 -- - - VM AB-10 Matrix Blank 0 <LOQ -- _ __ V M A B -11 Matrix Blank 0 <LOQ -- VMAB-12 Matrix Blank 0 <LOQ -- VM AS-37 VM AS-38 VM AS-39 Matrix Fortification Matrix Fortification Matrix Fortification 0.0500 0.0500 0.0500 0.0559 0.0494 0.0446 112 0.0500 100 98.9 SD = 0.00567 89.2 CV= 11.4% VM AS-40 V M A S-41 VM AS-42 Matrix Fortification Matrix Fortification Matrix Fortification 0.500 0.500 0.500 0.437 0.460 0.468 87.3 0.455 91.0 92.0 SD = 0.0161 93.6 CV= 3.54% VM AS-43 VM AS-44 VM AS-45 Matrix Fortification Matrix Fortification Matrix Fortification 5.00 5.00 5.00 4.84 4.86 4.93 96.9 4.88 97.6 97.2 SD = 0.0473 98.7 CV= 0.969% VM AS-46 VM AS-47 V M A S-48 Matrix Fortification Matrix Fortification Matrix Fortification 50.0 50.0 50.0 47.4 50.1 49.7 94.7 49.1 98.2 100 SD = 1.46 99.4 CV= 2.97% Overall Mean = Standard Deviation = CV= __________________________________________________ N = 96.7 6.34 6.56% 12____________________________ 1M easured and Percent R ecovered values w ere calculated using M acQ uan, version 1.6 softw are. M anual calculations m ay vary slightly. 2 T he lim it o f quantitation (LO Q ) w as 0 .0 4 0 0 m g a.i./K g based upon the product o f the low est calibration standard (0 .0 0 0 4 0 0 m g a .i/L ) and the overall dilution factor o f the m atrix blank sam ples (1 0 0 L/K g). 0Q23S9 Wildlife International. Ltd. -17- Project Number 454C-175 Table2 Method Validation Recoveries for PFOS in Alfalfa Sample Number (454C-125-) Type V R E B -3 Reagent Blank VM AB-7 VM AB-8 VM AB-9 Matrix Blank Matrix Blank Matrix Blank VM AS-49 VM AS-50 V M A S-51 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-28 VM AS-29 VM AS-30 Matrix Fortification Matrix Fortification Matrix Fortification V M A S-31 VM AS-32 VM AS-33 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-34 VM AS-35 V M A S-36 Matrix Fortification Matrix Fortification Matrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 -- 0 <LOQ 0 <LOQ 0 <LOQ -- -- -- -- __ 0.0500 0.0500 0.0500 0.0521 0.0477 0.0449 104 0.0482 96.4 95.3 SD = 0.00363 89.7 CV= 7.52% 0.500 0.500 0.500 0.458 0.467 0.490 91.6 0.472 94.4 93.4 SD = 0.0165 98.0 CV= 3.50% 5.00 4.40 5.00 4.74 5.00 4.77 87.9 4.64 92.8 94.9 SD = 0.206 95.3 CV= 4.43% 50.0 47.5 50.0 46.5 50.0 48.3 95.1 47.4 94.8 92.9 SD= 0.902 96.6 CV= 1.90% Overall Mean = Standard Deviation = CV = N= 94.6 4.13 4.37% 12 1M easured and P ercent R ecovered values w ere calculated using M acQ uan, version 1.6 softw are. M anual calculations m ay vary slightly. 1T he lim it o f quantitation (LO Q ) w as 0 .0 4 0 0 m g a.iVKg based upon the product o f the low est calibration standard (0 .0 0 0 4 0 0 m g a.iTL) and the overall dilution factor o f the matrix blank sam ples (1 0 0 L/K g). 002390 Wildlife International. Ltd. - 18- Project Number 454C-125 Table3 Method Validation Recoveries for PFOS in Tomato Sample Number (454C-125-) Type V REB-1 Reagent Blank VM AB-1 VM AB-2 VM AB-3 Matrix Blank Matrix Blank Matrix Blank VM AS-1 VM AS-2 VM AS-3 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-4 VM AS-5 VM AS-6 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-7 VM AS-8 VM AS-9 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-10 VM AS-11 V M A S - 12 Matrix Fortification Matrix Fortification M atrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 -- -- 0 <LOQ 0 <LOQ 0 <LOQ __ -- -- -- __ 0.0500 0.0500 0.0500 0.0493 0.0409 0.0411 98.6 0.0438 87.5 81.9 SD = 0.00479 82.1 CV= 11.0% 0.500 0.500 0.500 0.430 0.480 0.449 85.9 0.453 90.6 95.9 SD = 0.0252 89.8 CV= 5.57% 5.00 4.52 5.00 4.99 5.00 4.50 90.3 4.67 93.4 99.9 SD = 0.277 90.0 CV= 5.94% 50.0 45.9 50.0 44.3 50.0 45.7 91.8 45.3 90.6 88.7 SD = 0.872 91.4 CV= 1.92% Overall Mean = Standard Deviation = 90.5 5.68 CV= 6.28% _______ ___________________________________________ N = 12____________________________ 1M easured and Percent R ecovered values w ere calculated using M acQ uan, version 1.6 softw are. M anual calculations may vary slightly. 1 The lim it o f quantitation (LO Q ) w as 0 .0 4 0 0 m g a.iVKg based upon the product o f the low est calibration standard (0 .0 0 0 4 0 0 m p a.i./L ) and the overall dilution factor o f the m atrix blank sam ples (1 0 0 L/K g). 002391 Wildlife International. Ltd. 19- Project Number 454C -ns Table 4 Method Validation Recoveries for PFOS in Soybean Sample Number (454C-125-) Type V R E B -2 Reagent Blank VM AB-4 VM AB-5 VM AB-6 Matrix Blank Matrix Blank Matrix Blank VM AS-13 VM AS-14 VM AS-15 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-16 VM AS-17 VM AS-18 Matrix Fortification Matrix Fortification Matrix Fortification V M A S -1 9 3 Matrix Fortification V M A S -20 Matrix Fortification V M A S-21 Matrix Fortification VM AS-22 VM AS-23 V M A S-24 Matrix Fortification Matrix Fortification Matrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 - 0 <LOQ 0 <LOQ 0 <LOQ -- -- -- .. 0.0500 0.0500 0.0500 0.0440 0.0449 0.0429 87.9 0.0439 87.9 89.9 SD= 0.00100 85.7 CV= 2.28% 0.500 0.500 0.500 0.487 0.473 0.478 97.3 0.479 95.9 94.7 SD = 0.00709 95.6 CV= 1.48% 5.00 5.01 5.00 4.83 5.00 4.94 100 4.93 98.5 96.6 SD = 0.0907 98.9 CV= 1.84% 50.0 50.0 50.0 48.7 50.0 50.1 100 49.6 99.2 97.4 SD = 0.781 100 CV= 1.57% Overall Mean = Standard Deviation = CV= 95.3 4.92 5.16% __________________________________________________ N = 12____________________________ 1M easured and Percent R ecovered values w ere calculated using M acQ uan, version 1.6 softw are. M anual calculations m ay vary slightly. 1T he lim it o f quantitation (LO Q ) w as 0 .0 4 0 0 m g a.i./K g based upon the product o f the low est calibration standard (0 .0 0 0 4 0 0 m g a.iVL) and the overall dilution factor o f the matrix blank sam ples (1 0 0 L/K g). 3Measured value is the mean of one re-dilution, duplicate injection, 454C-125-19R1, and 19R2. 002392 Wildlife International. Ltd. -20- Appendix 1 Protocol Project Number 454C-125 002393 Wildlife International. Ltd. -21 - Project Number 454C-125 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFO S) IN PLANT TISSUES Environmental Laboratory Request Number U 2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, M innesota 55106 .Wildlife International Ltd 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 May 2 ,2001 002394 Wildlife International. Ltd -22- Project Number 454C-12.5 W ildlife International, Ltd. - 2- ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFO S) IN PLANT TISSUES SPONSOR: 3M Corporation Environmental Laboratory P .O .B ox 33331 S t Paul, M innesota 55133 SPONSOR'S REPRESENTATIVE: M i. Rochelle Robideau TESTING FACILITY: W ildlife International, Ltd. S598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Raymond L. VanHoven, Ph.D. Scientist W ildlife International, Ltd. LABORATORY MANAGEMENT: W illard B. N ixon, Ph.D. Manager o f Analytical Chemistry W ildlife International, Ltd. FOR LABORATORY USE ONLY Proposed Dates: Experimental E xperim ental Start Date: Project N o.: ____________ 1 2 54 54C - ___________ Termination Date: T '3 0 ^ 0 T est Substance N o.: 4675 TestCtcettt^omjJWjhtCtiU.aSwf .Oj StWwi foo .'dttr PROTOCOL NO.: 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002395 Wildlife International. Ltd. -23- Project Number 454C-I7S W ildlife International, Ltd. -3- INTRO DUCTIO N W ildlife International, Ltd. w ill conduct analyses to validate the performance o f a liquid chromatography m ass spectrometry (LC/M S) method for the determination o f Perfluorooctanesulfbnale, Potassium Sah (hereafter referred to as PFOS) in plant tissues and fruit The study w ill be performed at foe W ildlife International, Ltd. analytical chemistry facility in Easton, Maryland. The method developed at W ildlife International, Ltd. w ill be validated by fortifying plant tissues and quantifying foe recoveries o f PFOS. Raw data for a ll work performed at W ildlife Interna tional, Ltd. and a copy o f foe final report w ill be filed by project number in archives located on the W ildlife International, Ltd, site, or at an alternative location to be specified in the final report OBJECTIVE The objective o f this study is to validate the performance o f LC/MS methodology for determination o f PFOS residues in plant tissues and fruits used by W ildlife International, Ltd. EXPERIM ENTAL DESIGN Plant tissues and/or fruits from several species o f plants w ill be fortified with at least four different concentrations and analyzed using LC/MS methodology developed at W ildlife International, Ltd. The method w ill be based on procedures developed at W ildlife International, Ltd. Reagent and matrix blanks anil be analyzed to evaluate potential analytical interferences. One calibration curve w ill be prepared and analyzed with each series o f matrix fortification sam ples. The accuracy, precisian, and lim it o f quantitation o f tire method w ifi be determined MATERIALS AND METHODS T est Substance The test substance w ill be Perfluorooctanesulfbnate, Potassium S a lt Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing W ildlife International, Ltd. written verification that foe test substance has been charactflrired according to GLPs prior to initiation o f the study. The reference material in this study w ill be the test substance. I f written verification o f GLP test substance characterization is not provided to W ildlife International, Ltd., it w ill be noted in the compliance statement o f the final report. PROTOCOL NO.: 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002396 Wildlife International. Ltd. -24- Project Number 454C-125 W ildlife International, Ltd. -AThe Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f foe study. R eagents and Solvents All solvents used in the method or procedure w ill be o f reagent grade or better. All reagents w ill be o f ACS grade or better. Plant Tissues Plant tissues w ill be obtained from onion and alfalfa plants that have no known exposure to the test substance. There are no levels o f contaminants reasonably expected to be present in the plant tissues that are considered to interfere with the purpose or conduct o f the study. Fruits Fruits w ill be obtained horn two species o f plants (tom atoes and soybeans) that have no known exposure to the test substance. There are no levels o f contaminants reasonably expected to be present in the plant tissues that are considered to interfere with the purpose or conduct o f the study. A nalytical V erification Procedures The analytical method to be used w ill be LC/M S based upon procedures developed at W ildlife International, Ltd. The method used w ill be documented in the raw data and summarized in the final report Prior to fire analysis o f analytical sam ples, primary stock sohition(s) w ill be prepared directly from foe test substance. Calibration standards w ill be prepared by appropriate dilution o f foe primary stock soiutkn(s). Prim ary Stock Solution^), C alibration Standards and C urves A minimum o f five calibration standards (o f different concentrations) w ill be prepared and analyzed along with each analysis se t The calibration standard series w ill be injected at the beginning and one standard injected, at a minimum, after every five samples. A minimum o f two injections o f each standard in the calibration standard series w ill be injected with the analysis se t One calibration curve and regression equation w ill be prepared from the series o f calibration standards. PROTOCOL N O .: 454/050201/MV-PT/SUB4S4 Environmental Laboratory Request Number U2723 002397 Wildlife International. Ltd. -25- Project Number 454C-125 W ildlife International, Ltd. -5 - Fortification Stock Solutions Plant tissue and fiu it homogenates w ill be fortified with a stock solu tion s) o f PFOS at levels anticipated to bracket levels in the biocooccntration study. Evaluation o f Interferences One reagent blank and three matrix blanks w ill be analyzed to detect possible interferences. The reagent blank w ill contain everything except the matrix and the test substance. The matrix blanks w ill contain everything except the test substance. V erification A nalyses - M ethod Perform ance M atrix fortifications (spiked plant tissues and fruits or seeds), prepared at known concentrations o f the test substance, w ill be analyzed to determine the recovery o f foe analyte and to evaluate method performance. The anticipated verification series w ill consist o f foe following analyses: SUMMARY OF A TYPICAL VERIFICATION ANALYSIS SCHEME A nalysis l^ p e C on cen tration Number o f T issue Sam ples Reagent Blank (Solvent) 0 (Control) M atrix Blank (Plant tissues) 0 (Control) M atrix Fortifications Level 1 - Low1 Level 2 Level 3 Level 4 -H igh Onion 1 3 3 3 3 3 A lfalfa I 3 3 3 3 3 Total Number o f Analyses 16 16 'M atrix fortifications w ill be prepared at four concentrations. Num ber of F ru it S am p les Tomato Soybean 11 33 33 33 33 33 16 16 . Matrix fortifications w ill represent foe range o f bioconcentration that is anticipated to be found in plant tissue in subsequent effects tests and w ill be selected in consultation with foe Sponsor. PROTOCOL NO .. 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002398 Wildlife International. Ltd - 2 6 - 019876*432 Project Number 454C-1~>s .W ildlife International, Ltd - 6- Individual sam ples (identified by project number and a unique sample identification number) w ill be prepared and analyzed for verification o f the analytical methodology. If difficulties arise in the verification process (e.g. low recoveries or interferences), the Sponsor w ill be notified and the need for additional verification or method development w ill be deter mined through discussion with the Sponsor. Upon com pletion o f the method verification, die Sponsor w ill review the results and authorize use o f the methodology for analysis o f sam ples, or w ill authorize further method development trials. The acceptable range o f recoveries is 70 to 120% o f the nominal concentrations. D ata A nalysis One calibration curve w ill be established for each analytical run. A regression equation o f the concentration versus peak area for the calibration standards w ill be generated. The concentration o f the sam ples w ill be determined by substituting the respective peak area into the regression equation. Statistics to be determined and reported w ill include the mean and standard deviation for each level o f fortification. RECORDS TO B E M AINTAINED Records to be TMiwtint for data generated by W ildlife International, Ltd. w ill indude: 1. A copy o f the signed protocol. 2 . T iW ifirj timn ivt ja rTtw iTatinw n f rtw ~t/iw natyHral tanAmt, if provided by foe Sponsor. 3 . D ates o f initiation and termination o f the te st 4 . Storage conditions lor test substance, analytical standards, and/or sam ples. 3. T est substance and/or analytical standard use log. 6 . Concentration calculations and records o f solution preparation7. Instrument operating conditions and chromatograms. 8. Statistical calculations. 9 . A copy o fth e final report 10. Documentation that the steps in the method were followed. PROTOCOL NO.: 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002399 Wildlife International. Ltd. -27- Project Number 454C-17S ,W ildlife International Ltd. -7 - FINAL REPO RT The report w ill summarize the findings o f the verification, the procedural recoveries obtained, and the methods and instrumentation employed. Upon receipt o f these findings, the Sponsor w ill review the methods and results and evaluate die results for acceptability prior to initiating any fine and/or effects studies. The final report w ill include, but not be lim ited to, the fallowing: 1. Nam e and address o f the facility performing the study. 2. Dates oo which the study w as initiated and completed. 3. Objectives and procedures stated in file approved protocol, including any changes in the original protocol or deviations from the protocol. 4. The test substance and/or analytical standard identification, including name, chemical abstract number or code number, strength, purity, com position, date o f receipt, lot number, storage conditions, physical characteristics, stability and method o f preparation o f test concentrations, if provided by the Sponsor. 3. A detailed summary o f the analytical methodology: A description o f the experimental measurements, example calculations, sample preparation (sam ple weights and dilutions), instrumentation employed, reagents and solvents used, class o f water used, and any major modifications to the method. 6. A description o f circumstances that may have affected the quality or integrity o f the data. 7 . The name o f the Study Director, the names o f other scientists or professionals, and the names o f all supervisory personnel involved in file study. 8. A description o f the transformations, calculations, or operations performed on the data. 9 . The signed and dated reports o f each o f the individual scientists or other professionals involved m the study, i f applicable. 10. The location where raw data and final report are stored at file conclusion o f the study. 11. A statement prepared by the Q uality Assurance Unit listing the dates that study inspections were made and the dates o f any findings reported to the Study Director/Management. PROTOCOL NO.: 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002400 Wildlife International. Ltd. -28- Project Number 454C-125 W ildlife International, Ltd. - 8- CHANGING OF PROTOCOL Planned changes to the protocol w ill be in the form o f written amendments signed by the Study Director and die Sponsor's Representative. Amendments w ill be considered as part o f the protocol and w ill be attached to die final protocol. Any other changes w ill be in the form o f written deviations signed by the Study Director and filed with the raw data. A ll changes to the protocol w ill be indicated in the final report GOOD LABORATORY PRACTICES This study w ill be conducted in accordance w ith Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); and OECD Principles o f Good Laboratory Practice (ENV/M C/CHEM (98) 17). Each study conducted by W ildlife International, Ltd. is routinely examined by the W ildlife International, Ltd. Quality Assurance Unit (QAU) for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. The final report w ill also be reviewed by the QAU. A statement o f compliance with Good Laboratory Practices w ill be prepared for all portions o f the study conducted by W ildlife International, Ltd. The Sponsor w ill be responsible for certification o f compliance with Good Laboratory Practices for procedures performed by other laboratories. Raw data for all work performed at W ildlife International, Ltd. and a copy o f the final report w ill be filed by project number in archives located on the W ildlife International, Ltd. rite, or at an alternative location to be specified in fee final report. PROTOCOL NO.: 454/050201/M V-PT/SUB454 Environmental Laboratory Request Number U2723 002401 Wildlife International. Ltd. -29- Proiect Number 454C-17S Appendix 2 Certifcate of Analysis INTERIM CERTIFICATE OFANALYSIS Revision 1(9/7/00) C en tre A n a ly tica l L ab oratories C O A R eferen ce # : 0 23-018A 3M P roduct: P F O S ,L o t2 1 7 R eferen ce#: SD -018 ________________________ P u rity : 86.9 % ________________________ Test Name Specifications Result P urity1 r _ -w - M 86.9% Appearance Identification NMR M etals (IC P/M S) W hite Crystalline Powder V* % ...V tj.....i ` . .. j . . 3 ,, C onform s P o sitiv e 1. Calcium 2 . M agnesium 3 . Sodium 4 . Potassium 3 3. N ickel 6. Iron 7. M anganese T otal % Impurity (NM R) ** 1 *'> '> t . , % v., * ** * ....................... ,, 1 j TM. * ' j f - i ' . . , ' ' .i v ^ >*'' 0 / ' 4 ,, i 4 * , -5 '<:, <. - - ' > .. .- * 85 !i * 1 A i * * ' 1 -*l* 1. 0.005 w t/w t.% 2. 0.001 w t/w t.% 3. 1.439 w t/w t.% 4. 6.849 w t/w t.% 5. <0.001 w t/w t.% 6. 0.005 w t/w t.% 7. <0.001 w t/w t.% 1.93 w t/w t.% T otal % Impurity (LC /M S) ' * ^ -!>., < ^ J f * */ & .> > `' 8.41 w t/w t.% T otal % Impurity (G C/M S) V V"< -.;v c None Detected ^ JT r /-"V > < > v Related Compounds - POAA R esidual Solvents (TG A) Purity by DSC Inorganic A niens (IC ) 1. Chloride 2 . Fluoride 3 . Bromide 4 . Nitrate 3 . N itrite 6. Phosphate 7 . Sulfate* - ` ..... ' - rt................. 0 3 3 w t/w t.% N one Detected ...1 N ot Applicable3 * <* 1. <0.015 w t/w t% 2. 0.59 w t/w t% 3. <0.040 w t/w t.% 4 . <0.009 w t/w t.% 5. <0.006 w t/w t.% 6. <0.007 w t/w t.% 7. 8.76 w t/w t% O rganic A cid s1 (IC ) 1. TFA 2. PFPA 3. HFBA 4. NFPA ' * * / ... . * " 1. <0.1 w t/w t.% 2 . <0.1 w t/w t.% 3. 0.10 w t/w t% 4 . 0.28 w t/w t% Elem ental Analysis': 1. Carbon 2 . Hydrogen 3 . Nitrogen 4 . Sulfur 3. Fluorine 1. Theoretical Value " 17.8% 2. Theoretical Value = 0% 3. Theoretical Value - 0% 4. Theoretical Value =3.93% 5. Theoretical Value = 60% 1. 12.48 w t/w t% 2. 0.244 w t/w t.% 3. 1.74 w t/w t% 4. 8.84 w t/w t.% 5. 54.1 w t/w t.% COA023-018A Page 1of 3 002402 Wildlife International. Ltd. -30- Project Number 454C-17S INTERIM CERTIFICA TE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% - 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO is not considered an impurity. STFA HFBA NFPA PFPA Trifluoroacetic add Heptafiuorobutyric add Nonofluoropentanoic add Pentafluoropropanoic add ^Theoretical value calculations based on the empirical formula, CiFnS03"K+(MW-538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). / COA023-018A Page 2 of 3 002403 Wildlife International. Ltd. -31 - Project Number 454C-17.5 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The CS value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: _____________________________ ____ ; David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y :_____________________ [______ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 3 002404 Wildlife International. Ltd. -32- Appendix 3 Analytical Method Validation Data Project Number 454C-1?s 002405 Wildlife International. Ltd. -33- Project Number 454C-17S Appendix3.1 Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit Weigh approximately 1.00 gram of homogenized plant matrix per sample into a tared vial. Record weights. For matrix fortification samples, fortify each sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. The matrix blank is unfortified plant matrix. I For each sample, measure 10.0 milliliters of methanol with a Class A volumetric pipette and transfer into the vial. Add 10.0 milliliters of methanol into an empty vial and process as for tire plant samples. The additional sample is the reagent blank. 4 For plant tissue samples, cap vials and place on a shaker table. Allow the samples to shake for a minimum o f 30 minutes at approximately 250 rpm. For the fruit samples, cap vials and shake manually for a minimum of one minute. 4 Centrifuge samples for approximately ten minutes at 2000 rpm. 4 Dilute samples into the calibration range o f the PFOS LC/MS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure water). Add appropriate volume of sample with a gas-tight syringe and bring to volume with dilution solvent. Process matrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level. 4 Ampulate samples and submit for HPLC/MS/MS analysis. 002406 Wildlife International. Ltd. -34- Proiect Number 454C-1 INSTRUMENT: Appendix 3.2 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a PeritinElmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: Keystone Betasil Cu column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin Cig column (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 fiL/minute 70.0% Methanol: 30.0% NANOpure Water containing 0.1% Formic Acid 10.0 pL Approximately 4.0 minutes PFOS MONITORED MASS: 499.0 amu -> 99.1 amu 002407 Wildlife International. Ltd. -35- Appendix3.3 A Typical Calibration Curve for PFOS PFOS 49 9 .0 -> 9 9 .1 No Internal Standard Weighted (1/x) Intercept = 352.9491 Slope = 9618.9219 Correlation Coeff. = 0.99906 Area Project Number 4S4C-17*S 002408 Wildlife International. Ltd. -36- Project Number 454C-!?S Appendix3.4 Example Calculations for a Representative Sample Sample number 454C-125-VMAS-1, nominal concentration of 0.0500 mg a.i./Kg in Tomato Peak Area = 8465 Y-intercept = 2383.6709 Slope =12340.8760 Primary Dilution: Initial Weight (W 0= 1.00 g Original Final Volume (V0= 10.0 mL Secondary Dilution: Initial Volume (VO = 1.00 mL Final Volume (Vf) = 10.0 mL 100 mL 100L Overall Dilution Factor (Vi/Wi x V/VO = -- ~-- = " 7-- (peak area - y-intercept) PFOS at instrument (pg a.i./L) = slope (8465 - 2383,6709) 12340.8760 0.49278 ug a.i. L PFOS in sample (mg a.i./Kg) = PFOS at instrument (pg a.i./L) x overall dilution factor (L/Kg) x conversion factor (0.001 mg/pg) 0.49278 pg a.i. 100L 0,001 mg PFOS in sample (mg a.i./Kg) = L x Kg Pg 0.049278 mg a.i. Kg PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L) nominal x 0.049278 0.0500 x 100 = 98.6% Instrument Software: MacQuan, version 1.6. 002409 Wildlife International. Ltd. -37- Project Number 454P-17S Appendix 3.5 Ion Chromatogram of a Low-level PFOS Standard PFOS 1 STO 0.400 u g a.LA. 4 675A -01 1 0 -2 6 4.98 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. Quant Thres. Min. Width Mutt Width Base. Width RT Win. (secs) Smooth Expected RT 2.0 1.0 12 10 30 10 2 4.37 Area 4336 Height 471 Start Time End Time 3.58 4.04 Integration Width 0.45 Retention Time 3.77 Integration Type CD CD k Tu. Oct 2, 2001 09:49 10Oi 908070' 6050 40 30 20 10 o-l Intensity: 6000 cps 225 28 76 123 155 - | '*-- 1 ~ I' - I ' "I I" T " I 1?8 .. 261 I ` I"1 * 1 - I'" -- I" 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Nominal concentration: 0.000400 mg a.i./L 002410 Wildlife International. Ltd. -38- Project Number 454C-1 Appendix 3.6 Ion Chromatogram of a High-level PFOS Standard PF0S_6 STD 5.00 ug a .IA 4 6 7 5 A-01 ID-31 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Ihres. Quant Hires. Min. Width 2.0 1.0 12 Mutt. Width 10 Base. Width RT Win. (secs) Smooth Expected RT 50 10 2 4.37 Area 4 9 6 2 7 Height 5 1 4 9 Start Time End Time Integration Width Retention Time integration Type 3.53 4.14 0.60 3.77 A -V B Tue, Oct 2, 2001 10:19 intensity: 6000 cps Nominal concentration: 0.00500 mg a.i./L 002411 Wildlife International. Ltd. -39- Appendix 3.7 Ion Chromatogram of an Onion Matrix Blank PFOSJIO 4 5 4C -125- VMAB-11 4.98 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult Width 10 Base. Width RT Win. (secs) Smooth 40 10 2 Expected HT 3.72 Area 0 Height 0 Start Time End Time 0.00 0.00 Integration Width Retention Time 0.00 0.00 Integration Type Wed. Oct 3. 2001 12:55 Project Number 454C-1 intensity: 4000 cps Sample number 454C-125-VMAB-11. The arrow indicates the approximate retention time of PFOS. 002412 Wildlife International. Ltd. -40- Project Number 454C-19.S Appendix 3.8 Ion Chromatogram of a Low-level Onion Fortification Sample PFOS 14 4 5 4C -125- VMAS-38 4.98 In 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Hues. Quant Thres. Min. Width 2.0 0.5 12 Mult. Width 10 Base. Width 40 RT Win. (secs) Smooth Expected RT 10 2 3.72 Area 4605 Height 472 Start Time End Time Integration Width Retention Time 3.57 4.24 0.67 3.80 Integration Type A -BB Wed, Oct 3. 2001 13:19 Intensi! f . 4000 cps 100i 90 80 . 70 60 50 40 30 20 227 10 101120 150 1$9 A . o-l ` I i ^ -- I-- 1 -- I T `" ' r -- T * r > f ^ B T * a i ------- r- -- 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 7 0 3.37 4.04 4.71 Time Sample number 454C-125-VMAS-38,0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. 002413 Wildlife International. Ltd. -41- Project Number 454C-125 Appendix 3.9 Ion Chromatogram of a High-level Onion Fortification Sample PFOS 24 454C -125- V M A S-47 4.98 in 1 period PFOS No Internal Standard U se Area 1: 4 .9 7 MRM, 2 9 8 sc a n s 4 9 9 .0 -> 9 9 .1 Noise Thres. Quant Thres. M ia Width Mult. Width B ase. Width RT Win. (se cs) Sm ooth Expected RT 2.0 0.5 12 10 40 10 2 3.72 Area 1677S Height 1869 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.22 0.67 3.80 A -B 8 Wed, Oct 3. 2001 14:20 100- 9fr 8070 60 SO 40 30 20 10 0+ 30 5064 l i I1 111 41 81 121 0.69 1.36 2.03 intensity: 4000 cps 227 281 Scan 4.71 Time Sample number 454C-125-VMAS-47,50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. 002414 Wildlife International. Ltd. -42- Appendix3.10 Ion Chromatogram of an Alfalfa Matrix Blank PFOSJIO 454C -125- VMAB-8 Tue. Oct 2, 2001 10:43 4.98 in 1 period pros No Internal Standard U se Area 1: 4.87 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult Width 10 Base. Width SO RT Win. (secs) Smooth 10 2 Expected RT 4.37 Area 0 Height 0 Start Time End Time Integration Width Retention Time 0.00 0.00 0.00 0.00 Integration Type Project Number 454C-1 intensity: 6000 cps Sample number 454C-125-VMAB-8. The arrow indicates the approximate retention time o f PFOS. 002415 Wildlife International. Ltd. -43- Project Number 454C-125 Appendix 3.11 Ion Chromatogram of a Low-level Alfalfa Fortification Sample PFOS 9 454C -125- VMAS-49 Thu. Oct 4. 2001 12:39 4.98 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mud. Width 10 Base. Width 40 RTWJn. (secs) 1 0 Smooth 2 Expected RT 3.72 Area 5449 Height 537 Start Time 3.55 End Time Integration Width 4.17 0.62 Retention Time 3.80 Integration Type A - VB intensity: 5000 cps Sample number 454C-125-VMAS-49,0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. 002416 Wildlife international. Ltd. -44- Project Number 454C-17.S Appendix 3.12 Ion Chromatogram of a High-level Alfalfa Fortification Sample P F O S.23 4 5 4C -125- VMAS-34 4.98 tn 1 period pros No Internal Standard U se Area 1: 4.97 MRM. 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult Width 10 Base. Width RT Win. (secs) Smooth 50 10 2 Expected RT 4.37 Area 1 8 6 4 7 Height 1901 Start Time 3.60 End Time Integration Width 4.25 0.65 Retention Time Integration Type 3.89 A - VB Tue. Oct 2. 2001 12:01 intensity: 6000 cps Sample number 454C-125-VMAS-34,50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. 002417 Wildlife International. Ltd. -45- Appendix 3.13 Ion Chromatogram of a Tomato Matrix Blank PFOS_10 454C-1Z5- VMAB-2 Sat, Sep 1. 2001 12:04 4.98 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Hires. 2.0 Quant Hires. 1.0 Min. Width 12 Mult Width 10 Base. Width 40 RT Win. (secs) Smooth 10 2 Expected RT 3.82 Area 8 1 6 Height 79 Start H m e 3.89 Bid Hme 4,04 Integration Width 0.35 Retention Hme 3.80 Integration Type A -B B Proiect Number 454C-1 intensity: 8000 cps Sample number 454C-125-VMAB-2. The arrow indicates the approximate retention time of PFOS. 002418 Wildlife International. Ltd. -46- Project Number 454C-1 Appendix3.14 Ion Chromatogram of a Low-level Tomato Fortification Sample PFOS_12 4 5 4C -125- VMAS-1 Sat, Sep 1, 2001 12:16 4.9B in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2 .0 Quant Three. 1.0 Min. Width Mult. Width Base. Width 12 10 25 BT Win. (secs) 1 0 Smooth 2 Expected RT 3.82 Area 8 4 6 5 Height 972 Start Time End Time Integration Width 3.57 3.99 0.42 Retention Time Integration Type 3.77 A* - BB intensity. 8000 cps Sample number 454C-125-VMAS-1,0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. 002419 Wildlife International. Ltd. -47- Project Number 4 5 4 C -m Appendix 3.15 Ion Chromatogram of a High-level Tomato Fortification Sample PFOS_24 4 5 4C -125- VMAS-11 4.98 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Ttires. 1.0 Min. Width 12 Mutt Width 10 Base. Width 40 RT W in (secs) 1 0 Smooth 2 Expected RT 3.82 Area 24270 Height 2598 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.09 0.54 3.79 A -VB Sat. Sep 1. 2001 13:28 intensity: 6000 cps Sample number 454C-125-VMAS-11, 50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. 002420 Wildlife International. Ltd. -48- Project Number 454C- 17*5 Appendix 3.16 Ion Chromatogram of a Soybean Matrix Blank PFOS 9 454C -125- VMAB-4 Fri. Sep 7, 2001 12:38 4.98 in 1 period pros No Internai Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant Thres. 1.0 Mia Width 12 Mult. Width Base. Width 10 40 RT W ia (secs) 1 0 Smooth Expected RT 2 3.82 Area 0 Height 0 Start Time 0.00 End Time Integration Width Retention Time 0.00 0.00 0.00 Integration Type 10Oi 90 80 70 80 50 40 30 20 10 o-l inlensity: 4000 cps 1 66 B9 109 41 ' 8'l '~ 1 2 1 0.69 1.36 2.03 167 161 2.70 199 232 275 201 ' 241 2 8 1 Scan 3.37 4.04 4.71 Time Sample number 454C-125-VMAB-4. The arrow indicates the approximate retention time of PFOS. U2421 Wildlife International. Ltd. -49- Project Number 454C-1? 5 Appendix 3.17 Ion Chromatogram of a Low-level Soybean Fortification Sample PFOS_12 454C -125- VMAS-13 Fri. S ep 7, 2001 12:56 4.08 in 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .1 Noise Thres. 2.0 Quant H ues. 0.5 Min. Width 12 Mutt. Width 10 Base. Width 60 RT Win. (sees) 1 0 Smooth 2 Expected RT 3.82 Area 4065 Height 4 16 Start Time 3.50 End Time 4.19 Integration Width 0.69 Retention Time 3.87 Integration Type A* - BB lOOl 90 80 70 60 50 40 30 20 10 o4 5"2 I r801 y T1 2- 9-I15--3I 41 81 121 161 0.69 1.36 2.03 2.70 intensity: 4000 cps 231 X202 201 241 3.37 4.04 281 Scan 4.71 Time Sample number 454C-125-VMAS-13, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. 00^422 Wildlife International. Ltd. -50- Project Number 454C-1 Appendix 3.18 Ion Chromatogram of a High-level Soybean Fortification Sample PFO S.26 454C -125- V M A S-24 4.98 In 1 period pros No Internal Standard U se Area 1: 4.97 MRM, 2 9 8 sc a n s 499.0->99.1 Noise Thros. Quant Thres. 2 .0 1 .0 Min. Width Mult. Width B ase. Width RT W in (secs) Sm ooth Expected RT 12 10 40 10 2 3.82 Area 15377 Height 1645 Start Time 3.60 End Time Integration Width Retention Tune Integration Type 4.27 0.67 3.84 A-BB Fri, S ep 7, 2001 14:20 100i 90- 80 70 60 50 40 30 20 10 o4 20 1- r - 39 41 0.69 intensity: 4000 cps 229 75 102 124 81 121 1.36 2.03 200 1S4175XV. 161 201 2.70 3.37 241 4.04 281 Scan 4.71 Time Sample number 454C-125-VMAS-24,50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. - U2423 Wildlife International. Ltd. -51 - Project Number 454C-125 Appendix 4 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management o f this study: 1. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 2. Raymond L. Van Hoven, Ph.D., Scientist 3. Jon MacGregor, B.S., Scientist 4. Frank J. Lezotte, B.S., Chemist 002424