Document pe9xNrN0n7MM4VOxR3y3a5DOB

3M Environmental Laboratory IU26-002S M ethod E xtraction of Potassium Perfluorooctanesulfonate or other FLUOROCHEMICAL COMPOUNDS FROM URINE FOR ANALYSIS USING H P L C - Electrospray/M ass Spectrometry/M ass Spectrometry Method Number: ETS-8-96.0 Adoption Date: Author: Lisa Clemen, Glenn Langenburg Revision Date: /\/ j\ Approved By: Laboratory Manager v s f i f i n ------ Group Leader n l fr cluvu!^ Technical Reviewer ih n ? Date Date Date 1.0 Scope and Application____________________________________________________ 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from mine. 1.2 Applicable compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Human, rat, and monkey urine or other fluids as designated in the validation report. Word 6/95 ETS-8-96.0 Extraction of PFOS from Urine Page 1 of 14 001045 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from urine, or other fluids, using an ion pairing reagent and methyl-iert-butyl ether (MtBE). In this method, eight fluorochemicals are extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, M570, perfluorooctanoate (POAA), and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to two m l o f 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 0.5 ml o f methanol, then filtered through a 0.2 pm nylon filter attached to 3 cc plastic syringe into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-97.0 or other appropriate method. 3.0 Definitions________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F 17S 0 3` 3.2 PFOSA: perfluorooctane sulfonylamide CgF 17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F 17S 0 2N(CH2CH3)CH2C 0 2` 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 M556: C8F 17S 0 2N(H)(CH2C 0 0 H ) 3.6 M570: C8F 17S 0 2N(CH3)CH2C 0 0 H 3.7 POAA: perfluorooctanoate C7F 15C 0 0 ` 3.8 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 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 A t this time, it is unknown how the extraction method is affected by potential interferences that may be present such as conjugated fluorochemicals (eg. Glucuronides). Conjugates may become deconjugated during extraction or analysis resulting in a high bias for reported results o f target analytes. ETS-8-96.0 Extraction of PFOS from Urine Page 2 of 14 001046 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 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials____________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable o f 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 o f measuring 2.5 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 glass syringes a minimum o f 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 (N aH C 03), J.T. Baker or equivalent ETS-8-96.0 Extraction of PFOS from Urine Page 3 of 14 001047 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Urine 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 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 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.6 M570 (3M Specialty Chemical Division), molecular weight = 571 8.9.7 POAA (3M Specialty Chemical Division), molecular weight = 452 8.9.8 THPFOS (1-H,1-H, 2-H, 2-H C8F 13S 0 3H) molecular weight = 428 8.9.9 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 10 N 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 o f 10 N NaOH solution into a 100 ml volumetric flask and dilute to volume using MilliQTM water. Store in a 125 ml Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): W eigh approximately 169 g o f TBA into a 1 L volumetric containing 500 ml Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 ml o f 10 N NaOH (While adding the last ml o f 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 = 10.0. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^COj/NaHCOj): W eigh approximately 26.5 g o f sodium carbonate (Na;,C03) and 21.0 g o f sodium bicarbonate (N aH C 03) 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.) ETS-8-96.0 Extraction of PFOS from Urine Page 4 of 14 001048 8.11.3 W eigh approximately 100 mg o f PFOS into a 100 ml volumetric flask and record the actual weight in the Standard Logbook. 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm Og/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. 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 W eigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H, 2-H, C8F I3S 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 o f approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 ml o f surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard o f 100-120 ppm. Record the actual volume transferred in the Standard Logbook. 9.0 S a m p l 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. 9.2 A llow samples to thaw to room temperature prior to extraction. 10.0 Q u a lity C o n tro l_________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot o f 2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 2.0 ml aliquots o f the urine following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f 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 should fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. ETS-8-96.0 Extraction of PFOS from Urine Page 5 of 14 001049 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification per group o f 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 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 o f 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 matrix calibration standards 11.1.1 Transfer 2.0 ml o f urine to a 15 ml centrifuge tube. 11.1.2 Record each sample volume on the extraction sheet. 11.1.3 W hile preparing a total o f twenty-two aliquots in 15 ml centrifuge tubes, m ix or shake between aliquots. 11.1.4 Two 2.0 ml aliquots serve as matrix blanks. 11.1.5 Typically use the standard concentrations and spiking amounts listed in Table 1, at die end o f this section, to spike, in duplicate, two standard curves, for a total of twenty standards, two matrix blanks, and two method blanks. 11.1.6 Refer to validation report FACT-TOX-131, W2067, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.7 Use Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 10 ppb 1500 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. ETS-8-96.0 Extraction of PFOS from Urine Page 6 of 14 001050 Table 1 A pproxim ate spiking amounts for standards and spikes Using 2.0 ml of matrix Working standard pL Approx, final cone, Approx, final cone. (approx, cone.) of analyte in matrix O f analyte in solvent - - Blank Blank 0.500 ppm 5 1.25 ppb 5.00 ppb 0.500 ppm 10 2.50 ppb 10.0 ppb 0.500 ppm 25 6.00 ppb 25.0 ppb 5.00 ppm 5 12.5 ppb 50.0 ppb 5.00 ppm 10 25.0 ppb 100 ppb 5.00 ppm 25 62.5 ppb 250 ppb 50.0 ppm 5 125 ppb 500 ppb 50.0 ppm 7.5 200 ppb 750 ppb 50.0 ppm 10 250 ppb 1000 ppb 50.0 ppm 15 375 ppb 1500 ppb 12.0 P r o c e d u r e ____________________________________________________________________________ 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 2.0 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 sample weight/volume w orksheet.. See A ttachm ent D. The original weight/volume worksheet is included in the study binder. 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 o f standard as described in 11.1, or T able 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 o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 ml methyl-tert-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. ETS-8-96.0 Extraction of PFOS from Urine Page 7 of 14 001051 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are separated. 12.14 Label a fresh 15 ml centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 ml o f the organic layer to this clean 15 ml centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 30 to 60 minutes. 12.11 Add 0.5 ml o f methanol to each centrifuge tube using a graduated pipette. If excessive residue is present, add the methanol and allow the extract to sit for 30 minutes prior to vortexing. 12.17 Vortex mix for 30 seconds. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, fluorochemical components, extraction type, vial file archive number, and analyst(s) performing the extraction. 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 Cap and store extracts at room temperature or refrigerated at approximately 4 C until analysis. 12.21 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 D a t a A n a l y sis a nd C a lc ula tio n s______________________________________________ 13.1 C alculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ml o f standard x concentration o f standard fug /mD____________________ = ml o f standard + ml o f surrogate standard + initial matrix volume (ml) Final Concentration (pg/ml) o f PFOS in matrix 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 M DL report for specific MDL and limit o f quantitation (LOQ) values (see A ttachm ent B). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 M atrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-97.0 for method performance criteria. ETS-8-96.0 Extraction of PFOS from Urine Page 8 of 14 001052 15.0 Pollution Prevention and Waste Management 15.1 Human and monkey sample waste is disposed in infectious biohazard waste containers, all other sample waste is disposed in noninfectious biohazard waste containers. Flammable solvent waste is disposed in high BTU containers. Used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s______________________________________________________________________________ 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 T a b l e s . D ia g r a m s. F l o w c h a r t s, a n d V a l id a t io n D a t e _____________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 17.4 Attachment D, Sample weight/volume worksheet 18.0 R e fe r e n c e s____________________________________________________________________________ 18.1 The validation report associated with this method is FA C T-TO X -131, W 2067. 19.0 A f fe c t e d D o c u m e n t s_________________________________________________________________ 19.1 ETS-8-97.0, "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass Spectrometry/Mass Spectrometry" 20,0 R e v isio n s_____________________________________________________________________________ Revision Number Reason For Revision Revision Date ETS-8-96.0 Extraction of PFOS from Urine Page 9 of 14 001053 Extraction Worksheet ETS-8-96.0 Study # M atrix Box # W k/D av D ateS piked/A nalyst CCV MS M SD S u rro g ate Std A pprox, ppm A ctual ppm # FC -M ix approx. 0.5 p p m actual ppm # FC -M ix approx. 5 ppm actual ppm # FC -M ix approx. 50 ppm a c tu a l ppm # C om m ents ---- -- -- -- -- -- -- -- -- --- -- -- B lan k Std # amount = U rine E xtraction M ethod : V ortex 15 sec. Pipette M atrix , spike w ith appropriate surrogate or FC-M ix Volum e ml P ipette 1 m l o f 0.5 M T B A , pH 10. pH = Std. # Pipette 2 m l o f 0.25 N a2C O y0.25M N aHCO^ buffer Std. # D ispense 5 m l o f m ethyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuge 20-25 min. Centrifuge speed: Rem ove a 4 ml aliauot o f organic layer Put on N itrogen Evaporator to dryness T em perature: Add methanol V olum e ml TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe w ith a 0.2um filter into an autosam ple vial Cont. Cal. Verifications used same matrix as for std curve. - - - - - - - ml D ate & Initials Attachment A ETS-8-96.0 Extraction of PFOS from Urine 001054 Page 10 of 14 MDL/LOQ values for human urine Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) PFOS 4.6 14.7 15 ppb - 1500 ppb (in final MeOH extract) POAA 22.9 72.9 50 ppb - 1500 ppb (in final MeOH extract) PFOSA n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) PFOSAA n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) EtFOSE-OH n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) M556 n/d n/d 25 ppb - 1000 ppb (in final M eOH extract) M570 n/d n/d 25 ppb - 1000 ppb (in final MeOH extract) n/d = not determined NOTE: to calculate MDL, LOQ, and LCR values in ug/ml of urine divide the above values by 4. MDL/LOQ values in rat and monkey urine were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the human urine curves to determine equivalence. Responses in the rat and monkey were similar to the human responses, therefore, their MDL and LOQ are assumed to be similar to the values determined for human urine. If a suitable amount of clean, control matrix is available, samples will be evaluated versus a curve extracted from urine originating from the same species as the specimens. Please see LOQ Summary and MDL study in FACT-TOX-131, W2067 for further information. Attachment B: MDL/LOQ Summary ETS-8-96.0 Extraction of PFOS from Urine Page 11 of 14 001055 Compound: PFOS Prepared range of Human Urine standards (ppb) (ng/ml) Full Range n/d LCR from curve (PPb) (ng/ml) n/d % Recovery Range n/d Low Curve n/d n/d n/d High curve n/d n/d n/d 1/X 2.5ppb - 1500 ppb 15 ppb - 1500 ppb 75-116 Compound: POAA Prepared range of Human Urine standards (ppb) (ng/ml) Full Range n/d LCR from curve (PPb) (ng/ml) n/d % Recovery Range n/d Low Curve n/d n/d n/d High curve 1/X, quadratic n/d n/d 2.5ppb - 1500 ppb 50 ppb - 1500 ppb n/d 86-105 RSD Range n/d n/d n/d +/- 30% RSD Range n/d n/d n/d +/- 30% Attachment B: MDL/LOQ Summary ETS-8-96.0 Extraction of PFOS from Urine Page 12 of 14 0010S6 Ion Pair Standard Curves - Urine Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Box Number: 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 POAA M556 M570 Std cone Std cone Std cone Std cone Std cone Std cone Std cone ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml 0.500 0.501 0.500 0.501 0.499 0.500 0.501 0.500 0.501 0.500 0.501 0.499 0.500 0.501 0.500 0.501 0.500 0.501 0.499 0.500 0.501 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 All Ain't spiked ml 0.005 0.010 0.025 0.005 0.010 0.025 0.005 0.0075 0.010 0.015 All Final vol ml 2.0075 2.0125 2.0275 2.0075 2.0125 2.0275 2.0075 2.0100 2.0125 2.0130 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA M556 Final cone Final cone Final cone Final cone Final cone Final cone ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 1.25 1.25 1.25 1.25 1.24 1.25 2.48 2.49 2.48 2.49 2.48 2.48 6.17 6.18 6.17 6.18 6.15 6.17 12.5 12.5 12.5 12.5 12.4 12.5 24.8 24.9 24.8 24.9 24.8 24.8 61.7 61.8 61.7 61.8 61.5 61.7 125 125 125 125 124 125 187 187 187 187 186 187 248 249 248 249 248 248 372 372 372 372 371 372 M570 Final cone ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 Surrogate Std cone ng/ml 100 Surrogate Final cone ng/ml 125 All Ain't spiked ml 0.0025 Calculated concentrations of standards in methanol extract (0,5 ml final volume) PFOS PFOSA PFOSAA EtFO SE POAA M 556 M 570 Final cone Final cone Final cone Final cone Final cone Final cone Final cone ng/m l ng/m l ng/m l ng/m l ng/m l ng/m l ng/m l 5.00 5.01 5.00 5.01 4.99 5.00 5.01 10.0 10.0 10.0 10.0 10.0 10.0 10.0 25.0 25.1 25.0 25.1 25.0 25.0 25.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 100 100 100 100 100 100 100 250 251 250 251 250 250 251 500 501 500 501 499 500 501 750 752 750 752 749 750 752 1000 1002 1000 1002 998 1000 1002 1500 1503 1500 1503 1497 1500 1503 S u rro g a te Std cone ng/m l 100 S u rro g a te Final cone ng/m l 500 AH A m 't spiked ml 0.0025 Attachment C: Standard Calculation Sheet ETS-8-96.0 Extraction of PFOS from Urine 001057 Page 13 of 14 Prep Date(s): Analyst(s): Sample Matrix: Method/Revision : Target Analyte(s): Sample ID Initial Wt./Vol. g/mL/L Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blank/Identifier: Box: Comments Summary of method: Notes: Attachment D: Weight/Volume Sheet ETS-8-96.0 Extraction of PFOS from Urine 001058 Page 14 of 14