Document a4BNvgkVn1VwpZ9EQoVG222BR

AFU2-oo:LH Attachments to April 21, 2000 letter to C. Auer from W. Weppner (Tissue Extraction and Analysis Methods) 1. 3M Environmental Laboratory Method. Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-4.1. Adoption Date: 03/01/99. 2. 3M Environmental Laboratory Method. Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-5.1. Adoption Date: 03/01/99. 3. 3M Environmental Laboratory Method. Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-6.0. Adoption Date: 07/22/99. 4. 3M Environmental Laboratory Method. Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-7.0. Adoption Date: 07/22/99. 5. 3M Environmental Laboratory Method. Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-96.0. Adoption Date: 07/28/99. 6. 3M Environmental Laboratory Method. Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry. Method Number: ETS-8-97.0. Adoption Date: 07/28/99. 000997 3M E n v ir o n m en ta l Labo rato ry M ethod Extraction of P otassium Perfluorooctanesulfonate or O ther Fluorochemical compounds from Serum for Analysis Using H PLC- Electrospray/M ass Spectrometry Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: 0 11 Laboratory Manager fji_______ Group Leader `Z ' / t ? / ? ? Date -flu/S? Date A CLr^tw Technical Reviewer oh/ m I w Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 ETS-8-4.1 Extraction o f PFOS from Serum Page 1 o f 14 000998 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of 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-8-5.1 or other appropriate methods. 3.0 Definitions________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8FI7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F)7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 02N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F 17S 02N(CH2CH3)H 3.6 M556: C8F17S 0 2N(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 Equipment_________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction o f PFOS from Serum Page 2 o f 14 000999 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 S u p p l ie s a n d M a t e r ia l s ___________________________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 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 may 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-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction o f PFOS from Serum Page 3 o f 14 001000 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F,3S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 ION 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. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 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. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^Oj/NaHCC^): Weigh approximately 26.5 g of sodium carbonate (Na^CX,) 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. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 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.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (p-g/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction o f PFOS from Serum Page 4 o f 14 OOlOOl 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C8Fl3S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL of surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 Sample Handling___________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control__________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix 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 the serum following this procedure and use as matrix blanks. See 11.1.4. 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 the control matrix 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 checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. ETS-8-4.1 Extraction o f PFOS from Serum Page 5 o f 14 001002 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 Calibration and Standardization__________________________________________ 11.1 Prepare m atrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 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 standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. ETS-8-4.1 Extraction o f PFOS from Seram Page 6 o f 14 001003 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 Procedure________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-ierr-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction o f PFOS from Serum Page 7 o f 14 001004 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm 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. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations___________________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard fug /mL)_________________ = mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 Method Performance_____________________________________________________ 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 (see 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 check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management________________________ 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-8-4.1 Extraction o f PFOS from Semm 001005 Page 8 o f 14 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 Attachments______________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References_______________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 Affected Documents______________________________________________________ 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions__________________ ;_____________________________________________ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 ETS-8-4.1 Extraction o f PFOS from Serum 001006 Page 9 o f 14 Extraction Worksheet ETS-8-4.1 Study # Matrix Box # Wk/Day DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments ---- -- -- -- -- ---- -- -- Blank Serum Extraction Method Std # -- --- amount = Vortex 15 sec. Pipette Matrix Pipette 1 mL of 0.5 M TBA, pH 10. pH = Volume Std. # mL Pipette 2 mL of 0.25 Na2COy0.25M NaHCO-?buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Centrifuge 20-25 min. Remove a 4 mL aliquot of organic layer Shaker speed: Centrifuge speed: Put on Nitrogen Evaporator to dryness Add methanol Volume Temperature: mL TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um filter into a 1.5 mL autosample vial Cont. Cal. Verifications used same matrix as for std curve. - - - - mL Date & Initials Attachment A ETS-8-4.1 Extraction o f PFOS from Serum 001007 Page 10 o f 14 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 ppb - 1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction o f PFOS from Serum 0O \0O % Page 11 o f 14 Compound: PFOS Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve High curve 1/X 0.991 -9 7 4 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (n g/m L ) 24.8 - 978 4.94 - 248 9 7 .8 -9 7 8 4.94 - 978 LCR from curve (ppb) (n g/m L ) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 LCR from curve (ppb) (n g/m L ) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction o f PFOS from Serum Page 12 o f 14 001009 Compound: EtFOSE-OH Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4 .9 3 -9 7 .6 49.3 - 976 0.993 - 493 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full Range 0.993 - 976 Low Curve High curve 1/X 4.93 - 248 49.3 - 976 0.993 - 976 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (ppb) (n g/m L ) 49.3 - 976 9 .7 6 -9 7 .6 97.6 - 976 9.76 - 976 LCR from curve (PPb) (ng/m L ) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 LCR from curve (ppb) (n g/m L ) 24.8 - 976 9 .7 6 -9 7 .6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary 001010 ETS-8-4.1 Extraction o f PFOS from Serum Page 13 o f 14 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: 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: 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 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 26.3 24.8 25.2 25.8 Surrogate 49.3 50.0 52.4 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 All Am't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit 5.00-1000 5.00-1000 5.00-1000 Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. EtFOSE-OH 5.00-1000 PFOSEA 5.00-1000 M556 5.00-1000 Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction o f PFOS from Serum Page 14 o f 14 001011