Document 2qkKEJ56Ev4p5ppZnjd957pkp

3M Environmental Laboratory AR 1*26-0026 G) M ethod Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC - Electrospray/M ass Spectrometry Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Manager tGroup Leader --------- - a Technical Reviewer Adoption Date: o j l i h U x Revision Date: ~y ^ h < i Date Date Oilmh Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extraction o f PFOS from Liver Page 1 of 14 001021 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-teri-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 Definitions________________________________________________________________ 3.1 PFOS : perfluorooctanesulfonate (anion of potassium salt) C8F17S03 3.2 PFOSA: perfluorooctane sulfonylamide C8FI7S 02NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8Fi7S 02N(CH2CH3)CH2C 02 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8FI7S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S02N(CH2CH3)H 3.6 M556: C8FI7SO2N(H)(CH2C00H) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions____________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_____________________________________________________________ 5.1 There are no interferences known at this time. 6.0 E q u ip m e n t ____________________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR ETS-8-6.0 Extraction o f PFOS from Liver Page 2 of 14 001022 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 S u ppl ie s a n d M a t e r ia l s__________________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards___________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-iert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction o f PFOS from Liver Page 3 o f 14 001023 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^O /N aH CO^: Weigh approximately 26.5 g of sodium carbonate (N a^O ^ and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction o f PFOS from Liver Page 4 o f 14 001024 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 2-H, 2-H, C8FnS 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 S a m pl e H a n d l in g ______________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l it y C o n t r o l _____________________________________________________________________ 10.1 M atrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. ETS-8-6.0 Extraction o f PFOS from Liver Page 5 o f 14 001025 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C a l ib r a t io n and S t a n d a r d iz a tio n __________________________________________________ 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use Attachment C as an aid in calculating the concentrations of the working standards. Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. ETS-8-6.0 Extraction o f PFOS from Liver Page 6 o f 14 001026 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pi Approx, final cone, of PFOS in liver - Blank 2 0.005 ppm 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.00 ppm 12.0 P r o c ed u r e ____________________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. ETS-8-6.0 Extraction o f PFOS from Liver Page 7 o f 14 001027 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 p.m nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. ETS-8-6.0 Extraction o f PFOS from Liver Page 8 of 14 001028 13.0 Data Analysis and Calculations 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mg) of the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g of Liver x Average density* of homogenate fmg/mL) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations of PFOS and other fluorochemicals in calibration standards using the following equation: uL of Standard x Concentration (ug /mL! = Final Concentration (pg/g or mg/kg) mg L iv er/1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 M e t h o d P e r fo r m a n c e ________________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 P o l l u t io n P r e v e n t io n a n d W a ste M a n a g e m e n t __________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-6.0 Extraction o f PFOS from Liver Page 9 o f 14 001029 16.0 Records 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Tables. Diagrams. Flowcharts, and Validation Data_______________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 References_______________________________________________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction of PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 Affected Documents______________________________________________________ 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date ETS-8-6.0 Extraction o f PFOS from Liver 001030 Page 10 o f 14 Study # Matrix Box # Wk/Day Date Spiked/Analyst ccv MS M SD Surrogate Std approx, ppm actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FC Mix Std approx. 5 ppm actual ppm # FC Mix Std approx. 50 ppm actual ppm # Comments - - - - - B lank L iver H om ogenate: Std # L iver am ount = L iver E xtraction M ethod S pike su rro g ate and S tan d ard m ix. V ortex 15 sec. Pipette 1 m L o f L iver Solution Pipette 1 m L o f f0 .5 M T B A , pH 10. pH = Std. # P ip e tte 2 m L o f 0 .2 5 N a2 C C > 3 /0 .2 5 M N a H C C >3 B u f f e r Std. # D ispense 5m l o f M ethyl-t-B utyl E ther TN -A - Shake 20 m in. Shaker Speed C entrifuge 20-25 m in. C entrifuge Speed R em ove a 4 m L aliauot o f organic layer Put on N itrogen E vaporator to dryness Evaporator T em perature A dd 1.0 m L o f M eth an o l TN -A - V ortex 30 sec. Filter using a 3cc B -D svringe w ith a 0 .2 u m SR I filter into autosam ple vial Cont. Cal. Verifications used the same matrix as for the standard curve. - - - - - - - - _ 2 D ate & Initials Attachment B: MDL/LOQ Values ETS-8-6.0 Extraction o f PFOS from Liver Page 11 o f 16 001031 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb - 1200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS__________________________________________________ Prepared Range of LCR from Range of LCR from Range of Liver range of average ave curve low std low std high std matrix standards curve curve curve curve (ppb) (ng/m L) (ppb) (ng/m L) (ppb) (ng/m L) (ppb) (ng/m L) (ppb) (ng/m L) (ppb) (ng/m L) Rabbit 6.19-1237 12 - 1200 12-1200 6-300 12-300 60 - 1200 LCR from high std curve (ppb) (ng/m L) 60- 1200 Compound: PFOSA Prepared Liver range of matrix standards (ppb) (ng/m L) Range of average curve (ppb) (ng/m L) Rabbit 6.19-1237 12- 1200 LCR from ave curve (ppb) (ng/m L) 12-1200 Range of low std curve (ppb) (ng/m L) 12 - 300 LCR from low std curve (ppb) (ng/mL) 12 - 300 Range of high std curve (ppb) (ng/m L) 60 -1200 LCR from high std curve (ppb) (ng/m L) 60-1200 Compound: PFOSAA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/m L) (ppb) (ng/m L) Rabbit 6.16-1232 12- 1200 LCR from ave curve (ppb) (ng/m L) 30 - 1200 Range of low std curve (ppb) (ng/m L) 30 - 900 LCR from low std curve (ppb) (ng/m L) 60 - 900 Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Attachment B: MDL/LOQ Values ETS-8-6.0 Extraction o f PFOS from Liver Page 12 o f 16 001032 Compound: EtFOSE-OH Prepared Range of Liver range of average matrix standards curve (ppb) (ng/m L) (ppb) (ng/m L) Rabbit 6.17-1235 31 -900 LCR from ave curve (ppb) (ng/m L) 31-900 Range of low std curve (ppb) (ng/m L) N/A LCR from low std curve (PPb) (ng/m L) N/A Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Compound: PFOSEA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/m L) (ppb) (ng/m L) Rabbit 6.17-1235 31 - 1200 LCR from ave curve (ppb) (ng/mL) 31 -1200 Range of low std curve (ppb) (ng/m L) N/A LCR from low std curve (ppb) (ng/m L) N/A Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Compound: M556 Prepared Liver range of matrix standards (ppb) (ng/m L) Rabbit 6.17-1235 Range of average curve (ppb) (ng/m L) 31 - 1200 LCR from ave curve (Ppb) (ng/m L) 60 - 1200 Range of low std curve (ppb) (ng/m L) N/A LCR from low std curve (ppb) (ng/m L) N/A Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Attachment C: Standard Calculations ETS-8-6.0 Extraction o f PFOS from Liver Page 13 o f 14 001033 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All Am't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 All Density g 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Final Final Final cone Final Final Final Std cone cone cone ng/g cone cone cone ng/g ng/g ng/g ng/g ng/g ng/g 5.99 5.99 5.99 5.99 5.99 5.99 12.0 12.0 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 1198 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 All Am't spiked mL 0.005 Validated ranges - approximate concentrations L iver PFOS PFOSA PFOSAA R abbit 5-1000 ppb 5-1000 ppb 5-1000 ppb B ovine E stim ates only, use rabbit values. Rat E stim ates only, use rabbit values. M onkey E stim ates only, use rabbit values. E tF O S E -O H 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction o f PFOS from Liver Page 14 o f 14 001034