3M Specialty Materials 3M Center St. Paul, MN 55 144 1000 651 733 1110 May 7, 2003 3M BY HAND DELIVERY Wardner G. Penberthy Director, Chemical Control Division Office ofPollution, Prevention& Toxics U. S. Environmental Protection Agency Ariel Rios Building, 7405M 1200 Pennsylvania Avenue, N. W. Washington, D. C. 20460 Re: Follow up Questions Regarding 3M and Dyneon LOl Environmental Monitoring Commitments Dear Mr. Penberthy:

This letter responds to EPA's request in your e mail dated April 30, 2003 for a more detailed description ofthe environmental monitoring commitment contained in the March 13, 2003 3M Letter ofIntent ( LOT). In addition, EPA has requested clarification ofthe plans for continued monitoring ofgroundwater, surface water and other environmental media outlined on page 7, item 3 of the LOT. This letter will describe: 3M's environmental test methodology, monitoring plans by plant, and summary calendar of monitoring plans.

As described in section IT A 3 in the 3M LOT, 3M has already engaged in extensive environmental monitoring, including sampling in the vicinityofits now former PFOA production facility at Decatur, AL; analysis ofserum and liver samples from fish, birds and other wildlife for the presence ofPFOA and other constituents; and sponsoring a Multi City Study in which a variety of environmental compartments were analyzed. These data are available through the OPPTS AR 226 docket.

3M's environmental monitoring commitment in the LOl is to continue sampling at 3M plants in Cottage Grove, MN and Decatur, AL, which were former manufacturing locations ofPFOA and POSF based products. This sampling will include ground water and wastewater monitoring which corresponds with 3M's voluntary commitments and/ or plans established through permits with local regulators.

The environmental monitoring in the vicinity of 3M's Decatur, ALplant will include the current operations ofDyneon, which, as explained in the 3M LOT, is a 3M subsidiary that continues to use APFO, the ammonium salt of PFOA, in the production offluoropolymers. Because Dyneon's U. S. fluoropolymer manufacturing facility is located on 3M's manufacturing site, the environmental monitoring studies to satisfy the LOT commitments will be conducted Wardner G. Penberthy May 7, 2003 Page2 jointly and reported by 3M. Hence, the study methodology, sampling strategy and analytical methods described below are for both 3M and Dyneon. 1 Data will be submitted annually in final report format by the first ofAugust of each year, with the first report in by August 1, 2003. Sampling will take place monthly, quarterly, biannually and biennially, as described in the text below. In light of 3M's phase out of production and significant reduction of use ofPFOA in Decatur, all environmental monitoring under the LOT is anticipated to terminate by December 31, 2006 with a final report in by August 1, 2007.

TEST METHODOLOGY AND VALIDATION: GROUND WATER, SURFACE WATER, AND WASTE WATEREFFLUENT Analytical Test Method: 3M ETS 8 154.1 " Determination of Perfluorinated Acids, Alcohols, Amides, and Sulfonates In Water By Solid Phase Extraction and High Performance Liquid Chromatography/ Mass Spectrometry". See Attachment I. ( Note that this test method supercedes ETS 8 1 15.1 and 155.1, which are referenced in historic test documents.)

Validation: This sampling and analysis method was validated for the collection, extraction, and analytical procedure for the determination ofPerfluorooctanoate ( PFOA) in groundwater, surface water, and drinking water samples. Method accuracy ± 10%. Method precision ± 10%.

Method Summary: Water samples are collected from a site ofinterest and shipped cold to an analytical facility. PFOA/ APFO is extracted from 4OmL water samples using Cl 8 solid phase extraction ( SPE) cartridges. PFOA/ APFO is eluted from the C18 cartridge, using methanol. Separation, identification, and measurement are accomplishedby high performance liquid chromatography/ tandem mass spectrometry ( HPLC/ MS/ MS) analysis. The concentration of each identified component is measured by comparing the MS response ofthe quantitation ion produced by that compound to the MS response ofthe quantitation ion produced by the same compound in an extracted calibration standard ( external standard).

TEST METHODOLOGY AND VALIDATION: FISH Analytical Test Method: 3M ETS 8 23 1.2 " Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices". SeeAttachmentII.

Validation: ISO 17025 Accredited Test Method, ( validation based on FDA guidance documentation " Guidance for Industry Bioanalytical Method Validation, May, 2001). This sampling and analysis method was validated for the collection, extraction, and analytical For other clarification specific to Dyneon, a letter from the Society of Plastics Institute ( SPI) will address EPA's questions concerning the environmental studies at fluoropolymermanuthcturing sites and the questions concerning the Articles of Commerce Test Program in your email to SPI. The current PFOA manufacturers will address the questions concerning the plans for envirorimentaimonitoring at the site of PFOA manufacture in your email to SPI. Wardner G. Penberthy May 7, 2003 Page 3 procedure for the determination ofperfluorooctane sulfonate ( PFOS) in four biological matrices. The method is a performance based method requiring cross validation for the biological matrix application of PFOAIAPFO in fish. Method accuracy ± 5%. Method precision ± 10%.

Method Summary: An amount of sample is prepared ( fluids diluted, tissues homogenized, etc.) at a 1/ 6 dilution, or other dilution using reagent grade water. An aliquot ofthe dilutionlhomogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile ( ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarify the supernatant. The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned C18 SPE cartridge. Finally, the analytes ofinterest are eluted from the SPE cartridge and analyzed by high performance liquid chromatography electrospray tandem mass spectrometry ( HPLCES MS/ MS).

Cottage Grove, MN Groundwater Monitoring To date, numerous groundwater studies have been completed at the site in support ofvarious regulatory programs. These studies have documented that a shallow unconfined groundwater system is the first aquifer beneath the site. A lower confining bed underlies the unconfined aquifer. Natural groundwater flow in the shallow system is generally from north to south and discharges to the Mississippi River along the southern boundary ofthe site. These wells contain the groundwater in the area ofthe plant site and would not represent conditions beyond the site boundaries.

These studies focused on conventional substances and have incorporated fluorochemical analytes only recently. These test results will be included in the upcoming August, 2003 report to EPA under the LOT.

Going forward, five ( 5) existing on site monitoring wells will be sampled and analyzed for PFOA. The selection ofthese wells was based on the site hydrogeologic model and will be representative ofthe site ground water conditions, i. e., both source and non source areas. These wells will be sampled on a semiannual basis during the month ofMayand September commencing in 2003.

Wastewater Effluent Monitoring The site has a multi phased wastewater treatment plant that is used to treat all process wastewaters generated at Cottage Grove. Two ofthe systems treat inorganic wastewaters and the third is an organic, biological treatment system. All ofthe treated process wastewaters from these operations are combined at a single discharge point~ These wastewaters are then combined with non contact cooling and storm water and then discharged to the Mississippi River. Wardner G. Penberthy May 7, 2003 Page 4 Treated process wastewater and non contact cooling/ storm water are discharged under Minnesota NPDES Permit No. MN000149. The designations in the permit for the treated process wastewater, non contact cooling/ storm water, and the combination ofthese two wastewaters are listed in the permit as Outfalls SDOO1, SDOO2, and SDOO3, respectively. In accordance with Section 1.12 ofNPDES Permit, 3M is currently conducting monthly sampling and analysis at Outfall SDOO1 for certain perfluorinated chemicals, including PFOA.

A 24 hour composite sample is collected for duplicate analysis during each sampling event. In accordance with Section 1.12 par. 5 ofthe permit, this monthly analysis at Outfall SDOO1 will be conducted through 2004. The NPDES Permit defines a process to assess the results and allow revision or reduction in the frequencyofmonitoring.

Decatur, AL Groundwater Monitoring To date, numerous groundwater studies have been completed at the site in support ofvarious regulatory programs. The natural groundwater flow in the shallow system is generally from the southwest to the northeast and discharges to the Tennessee River along the boundary of the site. These investigations have involved an extensive characterization ofsite subsurface conditions. Most recently, we conducted a dye trace study that validated the earlier hydrogeological findings and our site conceptual model.

These studies have incorporated fluorochemical analytes only recently. Limited analyses addressing PFOA have been conducted and results will be included in the upcoming August, 2003 report under the LOI.

Going forward eight ( 8) existing on site monitoring wells will be sampled and analyzed for PFOA. The selection ofthese wells was based on the site hydrogeologic model and will be representative of the site ground water conditions, i. e., both source and non source areas. Groundwater samples for PFOA analyses will be collected twice a year, during the June and December sampling events.

Wastewater Effluent Monitoring 3M's Decatur, Alabama manufacturing facilities obtain process water from the City of Decatur Utilities. In addition many ofthe manufacturing operations utilize non contact cooling water, which is obtained from the Tennessee River. All process wastewaters are treated in the site's wastewater treatment facility. The system contains both physical chemical and biological treatment. Process wastewaters are mixed with non contact cooling water prior to discharge to the Tennessee River. The discharge is permitted under Alabama NPDES Permit No. AL0000205. The process wastewater discharge and combined process wastewater/ noncontact cooling water is designated as Outfall 001A and 001, respectively. Wardner G. Penberthy May 7, 2003 Page 5 Sampling will be conducted quarterly. Sampling will be conducted at Outfall 001 and will consist of24 hour composites with duplicate analysis being conducted on the collected sample.

Surface Waters, Sediments, and Fish 3M contracted two previous studies which characterize the concentrations of PFOA and other analytes in surface waters, sediment, and fish in the Tennessee River. As part ofthese efforts, representative samples ofsurface water and sediment were obtained from locations that are upstream, downstream, and in the vicinity of the plant wastewater outfall. Testing was also conducted on a number offish and clams from both upstream and downstream locations. The first study was conducted in 2000 and has been submitted to the EPA ( AR 226). This study report contains detailed descriptions of sampling locations, sampling and analytical methodologies as well as data tabulations and results summaries. The second study was completed recently and this information will be submitted to AR 226 in the near future.

It is 3M's intention to conduct additional sampling to augment the previous studies. These studies will be conducted once every other year with the next set ofsamples being collected in 2004. A project plan for future work will be prepared in the 1st or 2nd quarters of2004. As a part ofthis work, samples will be obtained upstream, downstream, and adjacent to 3M's manufacturing facilities.

Water samples will be collected using standard protocols. Sediments samples will be collected with the use ofa PONAR dredge using the established methodologies. A boat mounted Smith Root type VT A electro shocking device will be utilized for fish collection. Detailed descriptions ofthese sampling protocols and sampling locations will be contained in the work plan.

Monitoring Schedule Below is a schedule ofthe current monitoring plans under the 3M LOT.

Activity 2003 2004 2005 2006 2007 2008 Cottage Grove ground water biannual biannual biannual biannual biannual Cottage Grove effluent monthly monthly quarterly quarterly quarterly Decatur ground water biannual biannual biannual biannual biannual Decatur effluent quarterly quarterly quarterly quarterly quarterly Decatur surface water, sediment & fish once once Report submission by Aug. 1 by Aug. 1 by Aug. 1 by Aug. 1 by Aug. 1 by Aug. 1 Wardner G. Penberthy May 7,2003 Page 6 3M appreciates this opportunity to work with the EPA on this LOT and on our prior, ongoing and future EHS measures for PFOA. We look forward to dialogue with the EPA regarding the content ofthis letter. In addition, 3M plans to review the environmental monitoring data submitted under the LOT with EPA after two years to determine whether any changes to this overall monitoring program would be appropriate based on the data. We anticipate that this review would include discussion ofmedium, frequency ofsampling and testing and location ofsampling.

Sincerely, Please contact one ofus if you have any questions.

Michael A. Santoro, Director Specialty Materials EHS& R 3M Company 651 733 6374 Enclosure DC\ 592269.1 Dr. George H. Millet, Director QEHS Dyneon, a 3M Subsidiary 651 733 5637 3M Environmental Laboratory Method Determination of Perf! uorinatedAcids, Alcohols, Amides, and Sulfonates In Water By Solid Phase Extraction and High Performance Liquid Chromatography/ Mass Spectrometry Method Number: ETS 8 154.1 Adoption Date: 28 Apr 2000 Revision Date: 5 May, 2003 Effective Date: 5 May, 2003 William K. Reagen Manager c2S/~~>`~ 3 Date ETS 8 1 54.1 PaQe I of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Approved By: I Scope and Application This method was validated for the collection, extraction, and analytical procedure for the determination of Pertluorooctane sulfonate ( PFOS), Perfluorooctane sulfonylamide ( FOSA), and Perfluorooctanoate ( PFOA) in groundwater, surface water, and drinking water samples. This method may also be applied to the determination of other periluorinated acids, alcohols, amides, and sulfonates in similar matrices, as long as the defined QC elements are satisfied and with the understanding that the method is not validated for compounds outside thescope of the original protocol This method is based in part on the report " Method of Analysisfor the Determination of Perfluorooctane sulfonate ( PFOS), Perfluorooctane sulfonylamide ( PFOSA), and Pertluorooctanoate ( POAA) in Water" ( see Section 17), as developed and validated by Exygen Research ( formerly Centre Analytical Laboratories, Inc.).

2 Method Summary Water samples are collected from a site of interest and shipped cold to an analyticalfacility. Perfluorinated acids, alcohols, amides, and sulfonates are extracted from 4OmL watersamples using C18 solid phase extraction ( SPE) cartridges. The compounds are eluted from the C18 cartridge, using methanol. Separation, identification, and measurement are accomplished by highperformance liquid chromatography/ tandem mass spectrometry ( HPLC/ MS/ MS) analysis. Highperformance liquid chromatography/ mass spectrometry ( HPLC/ MS) maybe used ifthe defined QC elements are satisfied.

The concentration of each identified component is measured by comparing the MS response of the quantitation ion produced by that compound to the MS response of the quantitation ion produced by the same compound in an extracted calibration standard ( external standard).

3 Definitions 3.1 Analytical Sample Aportion of an extracted Laboratory Sample prepared for analysis.

3.2 Calibration Standard A solution prepared from the Working Standard ( WS) and extracted according to this method. The calibration standard solutions are used to calibrate the instrument response with respect to analyte concentration.

3.3 Duplicate Sample ( DS)

A DS is a separate aliquot of a sample, taken in the analytical laboratory that is extracted and analyzed separately with identical procedures. Analysis of DSscompared to that of the first aliquot give a measure ofthe precision associated with laboratory procedures, but not with sample collection, preservation, or storage procedures.

3.4 Field Blank Control Sample ( FB)

ASTM Type I water placed in a sample container in the laboratory and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation and all analytical procedures. The purpose of the FB is to determine if test substances or other interferences are present in the field environment.

ETS 8 154.1 Page 2 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. 3.5 Field Duplicate ( FD) A sample collected in duplicate at the sametime as the sample and placed under identical circumstances and treated exactly the samethroughout field and laboratory procedures. Analysis of ED compared to that of the first sample gives a measure of the precision associated with sample collection, preservation and storage, as well as with laboratory procedures.

3.6 Field Matrix Spike ( FMS)

A sample collected in duplicate to which known quantities of the target analytes are added in the field at the time of sample collection. Alternatively, the known quantity of target analytes may be added to the sample bottle in the laboratory before the bottles are sent to the field. A known, specific volume of sample must be added to sample container without rinsing. This may be accomplished by making a " fill to this level" line on the outside ofthe sample container. The FMS should be spiked at approximately 50   150% of the expected analyte concentration in the sample. If the expected range of analyte concentrations is unknown, a low and a high spike may be prepared to increase the likelihood that a spike at an appropriate range is made. The FMS is analyzed to ascertain if any matrix effects, interferences, or stability issues may complicate the interpretation of the sample analysis.

3.7 Field Spike Control Sample ( FSCS)

An aliquot of ASTM Type I water to which known quantities of the target analytes are added in the field at the time of sample collection ( at an appropriate concentration to be determined by the project lead) or in the laboratory prior to the shipment of the collection bottles, The FSCS is extracted and analyzed exactly like a sample to determine whether a loss of analyte could be attributed to sample storage and/ or shipment. A low and high FSCS may be appropriate when expected sample concentrations are not known.

3.8 Laboratory Control Sample ( LCS)

An aliquot of ASTM Type I water to which known quantities of the target analytes areadded in the laboratory. Two levels are included, one at the LLOQ ( approx. 25 pg/ mL), the other at a concentration of approx. 100   250 pg/ mL or another concentration to be determined by the project lead. The LCS is extracted and analyzed exactly like a laboratory sample to determine whether the methodology is in control, and whether the laboratory is capable of making accurate measurements at the required method detection limit and higher.

3.9 Laboratory Sample A portion of a sample received from the field for testing.

3.10 Limit of Detection ( LOD)

The LOD is the lowest concentration ofan analyte that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. If required, the LOD may be determined in several ways, including signal to noise ratio and statistical calculations.

3.11 Limit of Quantitation ( LOQ)

The LOQ for a dataset is the lowest concentration ( LLOQ) or highest concentration ( ULOQ) that can be reliably achieved within the specified limits of precision and accuracy during routine operating conditions.

Note: For many analytes, the LLOQ analyte concentration is selected as the lowest non zero standard in the calibration curveto simplify data reporting. Sample LLOQs are matrix dependent 3.12 Matrix Spike ( MS)

ETS 8 1 54.1 Page 3 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. A matrix spike is an aliquot of a sample, to which known quantities of target analytes are added in the laboratory. The MS is extracted and analyzed exactly like a laboratory sample to determine whether the sample matrix contributes bias to the analytical results. Thebackground concentrations of the analytes in the sample matrixmust be determined in a separate aliquot and the measured values in the MS corrected for background concentrations.

3.13 Method Blank An aliquot of ASTM Type I water that is treated exactly like a laboratory sample including exposure to all glassware, equipment, solvents, and reagents that are usedwith other laboratory samples. The method blank is used to determine if test substances or other interferences are present in the laboratory environment, the reagents, or the apparatus.

3.14 Method Detection Limit ( MDL) Determination A MDL is the statistically calculated minimum amount of an analyte that can be measured with 99% confidence that the reported value is greater than zero. One of several processes that may be used to establish a LOD value is found in 40 CFR Part 136 Appendix B.

3.15 Sample A sample is a small portion collected from a larger quantity of material intended to represent the original source material.

3.16 Spiking Stock Standard ( SSS)

Asolution prepared from stock standards used to prepare the working standard.

3.17 Stock Standard ( SS) A concentrated solution of a single analyte prepared in the laboratory with an assayed reference compound. 3.18 Working Standard ( WS)

Asolution of several analytes prepared in the laboratory from SSs and diluted as needed to prepare calibration standards and other required analyte solutions.

4 Warnings and Cautions 4.1 Health and Safety The acute and chronic toxicity of the standards for this method have not been precisely determined: however, each should be treated as a potential health hazard.

Unknown samples may contain high concentrations of volatile toxic ct~ nipounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure.

Thelaboratory is responsible for maintaining a safe work environment and a current awareness of local regulations regarding the handling of the chemicals used in this method. A reference file of material safetydata sheets ( MSDS) should be available to all personnel involved in these analyses.

4.2 Cautions None 5 Interferences ETS 8 154.1 Page 4 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ M~. During extraction and analysis, major potential contaminant sources are reagents and solid phase extraction devices.

All materials used in the analyses shall be demonstrated to be free from interferences under conditions of analysis by running method blanks. Parts and supplies that contain Teflon ® should be avoided due to the possibility of interference and/ or contamination. These may include, but are not limited to: wash bottles, Teflon ® lined caps, autovial caps, HPLC parts, etc.

The use of disposable micropipettes or pipettes to aliquot standard solutions is recommended to make calibration standards and matrix spikes.

6 Instrumentation, Supplies, and Materials Note: Brand names, suppliers, and part numbers are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory performing the analysis.

6.1 Instrumentation Balance, analytical ( display at least 0.000lg), Mettler HPLC/ MS/ MS orHPLC/ MS system, as described in Section 10.

6.2 Supplies and Materials.

Sample collection bottles   LOPE ( e. g., NalgeneTM) narrow mouth bottles with screw cap. Note: Do not use Teflon bottles or Teflon lined caps.

Coolers for sample shipment.

Ice for sample shipment Vacuum pump, BOchi.

Visiprep vacuum manifold, Supelco.

Sep PakVac6cc ( lg) tCl8 cartridges ( part# WAT 036795), Waters. 5OmLdisposable polypropylene centrifuge tubes, \/ WR.

I 5mL disposable polypropylene centrifuge tubes, VWR.

Disposable micropipettes ( 50   I OOiJL, 100   200pL), Drummond.

Class Apipettes and volumetric flasks, various.

Hypercarb drop in guard column ( 4mm) ( part# 844017   400), Keystone.

Stand alone drop in guard cartridge holder, Keystone.

125mL LDPE narrow mouth bottles, Nalgene.

2mL clear HPLC vial kit ( cat# 5181   3400), Agilent/ Hewlett Packard.

Standard lab equipment ( graduated cylinders, disposable tubes, etc.), various.

7 Reagents and Standards Note: Suppliers and catalog numbers are for illustrative purposes only. Equivalent performance may be achieved using chemicals obtained from other suppliers. Do not use a lesser grade of chemical than those listed.

ETS 8 154.1 Page 5 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. 7.1 Chemicals Methanol ( MeOH), HPLC grade, JT Baker, Catalog No. JT9093   2.

Ammonium Acetate, Reagent grade, Sigma Aldrich, Catalog No. A   7330.

ASTM Type I Water, prepared in house.

Sodium Thiosulfate, Reagent grade, JT Baker.

7.2 Standards Potassium perfluorooctane sulfonate Perfluorooctane sulfonylamide Ammonium perfluorooctanoate Others as required.

7.3 Reagent Preparation 250mg/ mL sodium thiosulfate solution   Dissolve 25g of sodium thiosulfate in I OOmL reagent water.

40% methanol wash solution   Measure400mL methanol and adjust volume to I . OL with reagent water.

100mM ammonium acetate solution ( Analysis)   Weigh 7.71g of ammonium acetate and dissolve in 1 . OL of reagent water. Dilute the 100mM solution by a factor of 50 to makethe 2mM ammonium acetate solution used for mobile phase A.

Note: Alternative volumes may be prepared as long as the ratios of the solvent to solute ratios are maintained.

7.4 Spiking Stock Standard ( SSS) Preparation Thefollowing standard preparation procedure serves as an example and maybe changed to suit the needs of a particular study. For example, pL volumes may be spiked into volumetric flasks when diluting stock solutions to appropriate levels.

lOOpg/ mL each PFOS, PFOSA, and POAASSSs   Weigh out 10mg of analytical standard ( corrected for percent salt and purity   i. e., 10 mg C8F17S03K purity 90% = 8.35mg C8F17S03) and dilute to I OOmL with methanol in a I OOmL volumetric flask. Transfer to a I 25mL LDPE bottle or other suitable container. Prepare a separate solution for each analyte. Solutions may be stored in a refrigerator at4 ° ± 2 ° C for a maximum period of 6 months from the date of preparation.

lpg! mL mixed SSS   Add 1. OmL each of the lOOpg/ mL SSSs ( from 7.4.1) to a lOOmL volumetric flask and bring up to volume with methanol. O. lpg/ mL mixed SSS   Add 10. OmL of the 1. Opg/ mL mixed solution ( from 7.4.2) to a lOOmL volumetric flask and bring up to volume with methanol. O. OlpgImL mixed SSS   Add 10. OmL of the 0. lpg/ mL mixed solution ( from 7.4.3) to a lOOmL volumetric flask and bring up to volume with methanol. Storage Conditions   Store all SSSs in a refrigerator at4 ° ± 2 ° C for a maximum period of 6 months from the date of preparation.

7.5 Calibration Standards The following standard preparation procedure serves as an example and may be changed to suit the needs of a particular study, provided the concentrations are calculated correctly.

ETS 8 1 54.1 Page 6 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. 1OO~ Jg/ mLeach PFOS, PFOSA, and POAA stock standard solutions   Weigh out 10mg of analytical standard ( corrected for percent salt and purity) and dilute to 1 OOmL with methanol in a IOOmL volumetric flask. Transfer to a 125mL LOPE bottle or other suitable container. Prepare a separate solution for each analyte. Store solutions in a refrigeratorat 4 ° ± 2 ° C for a maximum period of 6 months from the date of preparation.

lpgimL Working Standard   Add 1. OmL each of the IOOpg/ mLSS solutions ( from 7.5.1) to a lOOmL volumetric flask and bring up to volume with methanol.

O. lpg/ mL Working Standard   Add 10. OmL of the 1 . Opg/ mL mixed solution ( from 7.5.2) to a IOOmL volumetric flask and bring up to volume with methanol.

O. OlpgImL Working Standard   Add l0. OmL of the 0. lpg/ mL mixed solution ( from 7.5.3) to a I OOmLvolumetric flask and bring up to volume with methanol.

Storage Conditions   Store all WSs in a refrigerator at4 ° ± 2 ° C for a maximum period of 6 months from the date of preparation. Calibration Standard   Prepare calibration solutions in ASTM Type I using the following table as a guideline:

Final Concentration of Final Calibration Calibration Standard, Concentration Volume of Standard Volume, mL , pglmL, in ASTM Type I of WS, pg/ mL WS, pL ofASTM Type I Water Water 0.0 0 40 0 0.010 100 40 25 0.010 200 40 50 0.010 400 40 100 0.10 100 40 250 0.10 200 40 500 0.10 300 40 750 0.10 400 40 1000 1.0 100 40 2500 1.0 400 40 10000 1.0 1000 40 25000 Thestandards are processed through the extraction procedure ( Section 11), identical to the laboratory samples. The concentration of the calibration standard in the final extract is equal to 8X the initial concentration, due to the concentration of the standard during the extraction process.

Storage Conditions Store all extracted calibration standards in l5mL polypropylene tubes at 4 ° ± 2 ° C, for a maximum period of two weeks from the date ofpreparation 8 Sample Collection and Handling Note: Sampling equipment, including automatic samplers, must be free of Teflon tubing, gaskets, and other parts that may leach interfering analytes into the water sample. Automatic samplers that composite samples over time should use refrigerated polypropylene samllle containers if possible. Sample bottles should not be rinsed before sample collection.

Labeling: Each sample bottle must display information regarding the collection of that sample, the individual collecting the sample, and any matrix spike that has been added to the sample.

ETS 8 l 54.1 Page 7 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. This includes the volume and concentration of any spiking solution added and the volume and identification of any preservatives added in the field.

Spiking: The spiking scheme will be clearly outlined in the sampling plan, including whetherthe samples will be spiked in the field or in the laboratory prior to the shipment of the bottles to the site. If spiking is to be performed in the field, materials and specific instructions will be included in the sampling kit. Be sure to clearly label each bottle with spiking information if applicable.

TapWater: Open the tap and allow the system to flush until the watertemperature ( 15 ° ± l0 ° C) has stabilized ( usually about two minutes). Adjust the flow to about 500mL/ min and collect samplesfrom the flowing stream.

Ground Water: Purge the well of standing waterusing a pump ~ ra baiFer. Collect the sample directly from the pump or from the bailer.

Surface Water: When sampling from an open body ofwater, fill the sample container with water from a representative area.

Sample Dechlorination: All samplesshould be iced or refrigerated at4 ° ± 2 ° C and kept in the dark from the time of collection until extraction. Residual chlorine should be eliminated by adding 200pL of a 250mg/ mL sodium thiosulfate solution to each tap water sample and associated FB and FSCS ( which may be placed in each bottle before leaving for the sampling site or done in the field.).

Holding Time ( HI): Results of the time/ storage study of all target analytes showed that the three compounds are stable for 14 days in water sampleswhen the samples are dechlorinated and stored as described in the previous section ( see also references in sectioii 17). Therefore, laboratory samples must be extracted within 14 days and the extracts analyzed within 30 days of sample collection. Ifthe HT exceeds 14 days, great care is used when evaluating field spikes to avoid misrepresentation of the sample concentration.

8.1 Field Blanks Process a Field Blank Control Sample ( FB) along with each sample set ( samples collected from the same general sample site at approximately the sametime). At the laboratory, prior to sample collection, fill a sample container with ASTM Type I water, seal, and ship the FB to the sampling site along with the empty sample containers. Return the FB to the laboratory with the filled sample bottles.

When sodium thiosulfate is added to samples, use the sameprocedure to preserve the FB.

8.2 Field Duplicates Collect a Field Duplicate ( FD) for every ten ( 10) samples collected or per each sampling set, if less than 10 samplesare collected.

Separate FDs must be collected for each type of water sample ( ground, tap, etc.) collected.

Collect the FD immediately after the sample.

Preserve, store and ship FD using the sameprocedures as used for the samples.

8.3 Field Spike Control Sample ( FSCS)

A Field Spike Control Sample ( FSCS) mustbe prepared for eachsample shipment If multiple coolers are used to ship a set of samples, each coolermust contain a FSCS.

At the laboratory, fill a sample container with lOOmL of ASTM Type I water. Seal and ship to the sampling site along with the emptysample containers and FBs. Samples may either be spiked in the field or in the laboratory prior to shipment. The method employed should be consistent throughout the study. If the samples are to be spiked in the field, be sure to send appropriate supplies and instructions for the field personnel to follow.

ETS 8 1 54.1 Page 8 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LCIMS. Seal and gently invert the FSCS to mix. Store and ship the FSCS using the same procedures as used for the samples Provide information on sample collection, preservation, shipment and storage. List applicable holding times. Include sample stability and extract storage requirements. Reference the method used for sample preparation, if applicable.

8.4 Field Matrix Spike ( FMS)

A Field Matrix Spike ( EMS) mustbe prepared for each sampling location. One unspiked sample from the same location mustaccompany the EMS to determine endogenous levels in the sample. The samples should be clearly identifiable as being from the same location.

Samples may either be spiked in the field or in the laboratory prior to shipment The method employed should be consistent throughout the study. Ifthe samples are to be spiked in the field, be sure to send appropriate supplies and instructions for the field personne]~ tofollow.

9 Quality Control and Data Quality Objectives Analytical results of the FB, FMS, FD, and FSCS should be evaluated at the conclusion of the study to help interpret the quality of sample data. Analytical results for these control/ duplicate samples must be reported with the sample data.

9.1 Solvent Blanks Solvent blanks are analyzed with each sample set to determine contamination or carryover. Aliquots of methanol represent the solvent used for the standard curve and the sample extraction. Solvent blanks should have area counts that are less than 50% ofthe areacount of the lowest calibration standard.

Solvent blanks should be analyzed prior to and following each calibration curve, each set of system suitability samples, and after no more than 10 unknown sample extracts. If instrument carryover is a problem consecutive solvent blanks may be necessary. In this case the area counts ofthe solvent blanks should return to < 50% of the lowest calibration standard prior to the injection of further standards or samples.

9.2 Method Blanks A method blank consists of an aliquot of ASTM Type I water, equal in volume to the samples, and extracted in the same manner as the samples. At least two method blanks should be prepared and analyzed each day that extractions are performed for a particular study or project. When analyzed the area counts of thesesamples must be less than 50% of the area count of the lowest calibration standard.

9.3 Sample Replicates All samples, including field spikes, trip blanks, etc., should be extracted at least in duplicate, and in triplicate if difficulties were encountered in the sampling and/ or holding conditions of the samples. The relative percent difference ( RPD) of duplicate samples or relative standard deviation ( RSD) should be less than 15% for the precision of sample preparation and analysis to be considered in control.

9.4 Matrix Spike Matrix spikes are prepared for each sample type and analyzed to determine the matrix effecton the recovery efficiency. Matrix spike recoveries should fall within ± 25% of expected values. If the matrix spikes fail, evaluate the lab control spikes. If the LCS are within acceptance criteria there may be matrix issues in the samples. Discuss these in the final report.

ETS 8 I 54.1 Page 9 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Matrix spike duplicates are prepared periodically to measure the precision associated with the analysis. Analyze a matrix spike and matrix spike duplicate ( if prepared) in the same run as the original sample.

Matrix spike and matrix spike duplicate concentrations should fall in the mid range of the initial calibration curveor should be prepared at 1.5 5 times the endogenous concentration of the analyte. Spike concentrations should fall in the low range of the initial calibration curve if extremely low levels are expected. Generally two or more levels are prepared, one in the low range of the curve and one in the mid range. This avoids the need to pie screen unknown samples prior to preparation.

9.5 Laboratory Control Spike Lab control spikes are prepared for each study to ensure recovery of the target analytes. These should be prepared at a minimum of 2 levels and in duplicate or triplicate. Recovery of these samples should be within ± 25% of expected values, and the RPD ( or RSD) be  15%. If recoveries fall outside these limits the samples should be addressed in the final report.

10 Calibration and Standardization 10.1 Instrument Setup Note: In this example, a MicroMass UItimaTM Liquid Chromatography Tandem Mass Spectrometer ( LC/ MS/ MS) is used. Other brands of LC/ MS/ MSs as well as single quadrupole mass spectrometers ( LC/ MS) may be used as long as the method criteria are met. Brand names, suppliers, part numbers, and models are for illustrative purposes only. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meetsthe requirements of this method is the responsibility of the laboratory. The operatormust optimizeand document the equipment and settings used.

Establish the LC/ MS/ MS system and operating conditions equivalent to the following:

Mass Spec: Micromass Ultima ( Micromass)

Interface: Electrospray ( Micromass)

Mode: Electrospray Negative, Multiple Response Monitoring ( MRM)

Harvard infusion pump ( Harvard Instruments), for tuning Computer: COMPAQ Professional Workstation AP200 Software: Windows NT, MassLynx 3.3 HPLC: Hewlett Packard ( HP) Series 1100 HP Quaternary Pump HP Vacuum Degasser HP Autosampler HP Column Oven Note: A 4 x 10mm Hypercarb drop in guard cartridge ( Keystone, part # 844017 400) may be attached on line after the purge valve and before the sample injector port to trap any residue contaminants that may be in the mobile phase and/ or HPLC system.

HPLC Column: Genesis C8 ( Jones Chromatography), 2.1mm x 50mm, 4i. tm ETS 8 154.1 Page 10 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Column Temperature: 35 ° C Injection Volume: l5pL Mobile Phase ( A): 2mM AmmoniumAcetate inASTM Type I water ( See 7.3.1)

Mobile Phase ( B): Methanol met Note: Other HPLC gradients may be used as long as the method criteria are It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions ( e. g. 2.1mm x 30mm) and columns from different manufacturers ( Keystone Betasil C18 etc.) may be used.

Ions Monitored:

PFOA PFOS FOSA 413 169 5.0 499 99 5.2 498 78 5.8 Other product ions may be chosen at the discretion of the analyst~ although m/ z 99 is suggested for PFOS. Use of the suggested primary ion is recommended. Retention times may vary slightly, on a day to day basis, depending on the batch of mobile phaseetc. Drift in retention times is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytical run.

10.2 Tune File Parameters The following values are provided as an example. Actual values mayvary from instrument to instrument. Also, thesevalues may be changed from time to time in order to optimize for greatest sensitivity.

Analyte Dwell, sec Collision Energy, eV Cone, V PFOA 0.2   0.4 10   25 20   30 PFOS 0.2   0.4 30   60 50   80 FOSA 0.2   0.4 20   50 30   60 ETS 8 I 54.1 Determination of Perfluorinated Compounds in Water Using SPE and LCIMS. Page 11 of 17 Time, Percent Mobile Percent Mobile Flow Rate, mm Phase A Phase B mLlmin 0.0 60 40 0.3 0.4 60 40 0.3 1.0 10 90 0.3 7.0 10 90 0.3 7.5 0 100 0.3 9.0 0 100 0.4 9.5 60 40 0.4 13.5 60 40 0.4 14.0 60 40 0.3 Approximate Analyte Primary Ion Product Ion Retention Time ( minutes) Gas Flows Set Cone Gas 150L/ hr Desolvation 700L/ hr Pressures Set Gas Cell 3. Oe   3mbar ETS 8 I54.1 Page 12 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Source Set Capillary 2.6   3.5kV Hexapole I 0.5V Aperture I 0.2V Hexapole 2 0.8V Source Block Temp. 100   150 ° C Desolvation Temo. 250   400 ° C Analyzer Set LMResI 12.5   15. OV HM Res I 12.5   15. OV lEnergy I O. 7V Entrance   2V Exit IV LMRes2 11. OV HMRes2 11. OV lEnergy 2 l. OV Multiplier 650V 10.3 Calibration Curve Analyze the standard curves prior to each set of samples. The validated method specifies that the standard curve should be plotted using a linear fit, weighted 1/ xor unweighted. However, the standard curve mayalso be plotted by quadratic fit ( y = ax' + bx + c), weighted l/ x or unweighted, using suitable software. The calibration curves may include but should not be forced through zero. The mathematical method used to calculate the calibration curveshould be applied consistently throughout a study. Anychange should be thoroughly documented in the raw data.

Ifthe calibration curve does not meet acceptance criteria perform routine maintenanceor prepare a new standard curve ( if necessary) and reanalyze.

For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range. For example, when attempting to quantitate approximately 50 pg/ mL of analyte, generate a calibration curve consisting of the standards from 25 pg/ mL to 1000 pg/ mL ratherthan the full range of the curve ( 25 pg/ mL to 25000 pg/ mL). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards.

High and/ or low points maybe excluded from the calibration curves to provide a better fit over the linear range appropriate to the data or because they did not meet tirejire determined acceptance criteria. Low level curve points should also be excluded if their area counts are notatleasttwice that of the method and/ or solvent blanks. Any curve pointmay be rejected due to a bad injection or failing to meet accuracy requirements of ± 25% ( and ± 30% for the LLOQ). Justification for exclusion of calibration curve points will be noted in the raw data. Aminimum of 6 points will be used to construct the calibration curve.

10.4 Continuing Calibration Verification ( CCV)

Continuing calibration verifications ( CCV) are analyzed to verify the accuracy ofthe calibration curve. Analyze a mid range calibration standard, one of the same standards used to construct the calibration curve, at a minimum after every tenth sample, not including solvent blanks, with a minimum of one persample set. Calibration verification injections must be within ± 25% to be considered acceptable. The calibration curve and the last passing CCV will then bracket acceptable samples. Multiple CCV levels may be used.

10.5 System Suitability A minimum of three system suitability sampleswill be injected at the beginning and end of each analytical run. Typically these samples are run prior to the calibration curve. The system suitability injections must havearea counts with an RSD of~ 5% and a retention time RSD of  2% when evaluated independently.

11 Procedures 11.1 Extraction Scheme Allow samples to equilibrate to room temperature. Thoroughly mix samples by gently inverting the sample bottle. Measure 4OmLof sample into 5OmL polypropylene centrifuge tubes( Spike the Matrix spikes as required*, replace lid and mix well).

Note: * Samples may need to be prescreened to determine an appropriate matrix spike level ( typically 50   150% of sample concentration). Alternatively the samples could be spiked at more than one level, allowing for the inappropriate spike level to be eliminated.

ETS 8 1 54.1 Page 13 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Condition the CI8 SPE cartridges ( Ig, 6mL) by passing approximately IOmLmethanol followed by approximately 5OmL ASTM Type I water( flow rate approximately 2 drop/ sec). Do not let column run dry.

Note: For the following steps, maintain a ` I drop/ sec flow rate. Do not allow the column to run dry at any time. Load the analytical sample onto the C18 SPE cartridge. Discard eluate.

Ten mL of the 40% methanol in water wash mixture is passed through the Cl 8 SPE cartridge to rinse away potential interferences and then discarded. This step must be omitted if perfluorinated compoundswith chain lengths less than C8 are targeted since these will beJost during this wash step.

Elute with exactly 5mL of 100% methanol. Collect eluate into graduated 1 5mL polypropylene centrifuge tubes. This is the target elution fraction ( final volume approximately 4.5 mL as not all of the solvent will leave the SPE column. This will not affect the calculations in any way since the curve is also extracted).

Analyze a portion of the target elution fraction eluent using negative electrospray HPLC/ MS/ MS or HPLC/ MS. Note: Samples are concentrated by a factor of eight during the extraction; Initial Vol = 4OmL ~

Final Vol. = 5mL. Samples are stable at room temperature for at least 24 hours. Analytical samples may be stored in a refrigerator at 4 ° ± 2 ° C until analysis. Standardization of C18 SPE columns   If poor recoveries are observed, it may be necessary to standardize the C18 SPE columns in the following manner before analyzing samples. Use a standard with an analyte concentration between 1000 and 4000 pg/ mL. Repeat the extraction scheme from the beginning up through the eluting with   5mL 100% methanol. After the eluting with   5mL 100% methanol step, collect an additional post elution fraction by eluting with an additional 5mL of 100% methanol. Analyze both fractions by HPLC/ MS/ MS or HPLC/ MS. If the target fraction contains a minimum of 85% of the respective analytes, it may be considered acceptable.

If the wash contains significant standard (> 15%), either the wash volume or percentage of MeOH should be decreased. If the post elution fraction contains significant standard (> 15%), the target elution volume should be increased.

11.2 Sample Analysis Set up analysis sample queue.

Inject the samevolume ( between 5   25pL) of each standard, analytical sample and blank into the instrument.

All sampleswith a concentration> ULOQ must be diluted and reanalyzed. If dilution of the final extract fails to produce acceptable results ( e. g. poor MS recoveries) dilute the original sample and re extract.

12 Data Analysis and Calculations Calculate the analytical sample ( extract) concentration from the standard curve using the following equation:

ETS 8 154. I Page 14 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Extract Concentration, pg/ mL = ( Peak area   intercept)

( slope)

Calculate the percent recovery ofthe FSCS using the following equation:

FSCS % rec. = ( FSCS conc., pci/ mL) x 100 ( Conc. added, pg/ mL)

Calculate the percent recovery of the MSs using the following equaticn:

MS % rec. = ( MS conc., pgfmL   Sample conc., pg/ mL) x 100 ( Conc. added, pg/ mL)

13 Method Performance Note: Anymethod performance parameters that are not achieved must be considered in the evaluation of the data. Nonconformance to any specified parameters must be described and discussed in any reporting of the data.

If criteria listed in this method performance section are not met, maintenancemay be performed on the system and samples reanalyzed, or other actions taken as determined by the analyst. Document all actions in the raw data.

If data are to be reported when performance criteria have not been met, theiiatamustbe footnoted on tables and discussed in the text of the report.

13.1 System Suitability A minimum of three system suitability sampleswill be injected at the beginning and end of each analytical run. Typically thesesamples are run prior to the calibration curve. The system suitability injections musthavearea counts with an RSD of  5% and a retention time RSD of  2% when evaluated independently.

13.2 Quantitation Calibration Curve: The coefficient of determination ( r2) value for the calibration curve must be greater than or equalto 0.990. Each point in the curve must be within ± 25% of the theoretical concentration with the exception of the LLOQ, which may be within ± 30%.

Demonstration of Specificity: Specificity is demonstrated by chromatographic retention time ( within 3% of standard) and the mass spectral response of unique ions.

13.3 Sensitivity Solvent Blanks and Method Blanks: Solvent and method blankarea counts mustbe< 50% that of the lowest standard used in the calibration curve.

Limits of Quantitation ( LOQ): The lower LOQ ( LLOQ) is the lowest non zero active standard in the calibration curve; the peakarea of the LLOQ must be at least 2X that of the extraction blank. By definition, the measured value of the LLOQ must be within 30% of the theoretical value.

ETS 8 I 54.1 Page 15 of 17 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. 13.4 Accuracy CCV Performance: Calibration verification injections must be within ± 25% to be considered acceptable. Thecalibration curve and the last passing CCV will then bracketacceptable samples. MultipleCCV levels may be used.

MatrixSpikes: Matrix spike percent recoveries must be within ± 25% ofthaspiked concentration. If matrix effects are suspected, evaluate the LCS results to determine if a matrix effects are present and if the method is in control based on compliant LCS results. Discuss all results in the analytical report.

13.5 Precision Reproducibility: Reproducibility of the method is defined by the results of duplicate or triplicate analysis of samples. A RPD or RSD of  15% will be considered acceptable.

System Suitability: The system suitability injections musthave area counts with an RSD of  5% and a retention time RSD of  2% when evaluated independently.

14 Pollution Prevention and Waste Management 15 Sample extract waste and flammable solvent is discarded in high BTU containers, and glass pipette waste is discarded in broken glass containers located inthelaboratory.

Records Each data package generated for a study must have the followiriginformationirrcluded: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor ( if applicable), and analyst.

Print the tune page, sample list, and acquisition method to include in the appropriate study folder. Copy these pages and tape into the instrument run log.

Plot the calibration curves as described in this method, then print these graphs and store in the study folder.

Print data integration summary, integration method, and chromatograms and store in the study folder.

Summarize data using suitable software and store in the study folder.

16 Attachments None.

17 References " Method of Analysis for the Determination of Pertluorooctane sulfonate ( PFOS), Perfluorooctane sulfonylamide ( PFOSA), and Perfluorooctanoate ( POM) in Water", E. Wickremesinhe and J. Flaherty, Study Number 023   002, Centre Analytical Laboratories, Inc., State College, Pennsylvania, January 2000.

Validation report for the " Method ofAnalysis for the Determination of Perfluorooctane sulfonate ( PFOS), Periluorooctane sulfonylamide ( PFOSA), and Perfluorooctanoate ( POAA) in Water", E. Wickremesinhe and J. Flaherty, Study Number 023   002, Centre Analytical Laboratories, Inc., State College, Pennsylvania.

ETS 8 1 54.1 Determination of Perfluorinated Compounds in Water Using SPE and LC/ MS. Page 16 of 17 18 Affected Documents None.

19 Revisions Revision Revision Number Revision Number Date Updated to the new format. Changed Title. 7 Section 1: States the validation of3 analytes, removes reference to EPA document that's no longer applicable. Section 2: Provided for the extraction of more than the 3 validated analytes, allows the use of a LC/ MS system, not only the LS/ MS/ MSpreviously mentioned. Section 3: Revised definitions for field matrix spike, field control spike, LLOQ, methodblank, and MDL. Section 5: Reworded the interferences, added recommendation to use disposable pipettes. Section 6: Recategorized and pared down. Section 7: Changed storage time to 6 months. Added more calibration points to the table. Section 8: Addedstatement addressing labeling requirements and spiking procedures. Expanded section 8.8. Section 9: New Section Section 10: Changed some of the parameters in the tables. Allowed for use of different instrumentation. Addedinformation from section 12 of previous version, extensively revised. Section 11 ( section 9 in previous version): Clarification of wash step, stated exact volume of eluate is 5 mL, revised standardization process, removed requirement to use LC/ MS/ MS. Section 12 ( section 13 in previous version: no changes Section 13 ( section 14 in previous version): Extensively rewritten. Section 14 ( section 15 in previous version): no changes Section 15 ( section 16 in previous version): Minorchanges to recording requirements. Section 16 ( section 17 in previous version): Removed attachment. Section 17 ( section 18 in previous version): Removed reference to EPA document that no longer applied to this SOP. Section 18: New section.

ETS 8 I 54.1 Page 17 of 17 Determination of Periluorinated Compounds in Water Using SPE and LC/ MS. 3M Environmental Laboratory Method Solid Phase Extraction and Analysis of Perf! uorinated Acids, Alcohols, Amides, Sulfonates and other Fluorinated Compounds by High Performance Liquid Chromatography/ Mass Spectrometry Method Number: ETS 8 231.2 Adoption Date: 13 November 2001 Revision Date: Upon Signing Effective Date: Upon Signing William K. Reagen Laboratory Manager ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices C/ A~)/ o~ Date Page 1 of 23 Approved By: I 7~ F ~ nt r L ~ 1 14~ r14 da~ ftom ~~

Scope and Application This method describes the extraction of target analytes from various biological matrices using solid phase extraction ( SPE) followed by separation, identification, and quantitation using high performance liquid chromatography mass spectrometly ( HPLC/ MS) or tandem mass spectrometry ( HPLCIMS/ MS). This method has been validated for perfluorooctane sulfonate ( PFOS) in mouse and rat liver and sera using HPLC/ MS/ MS based on the FDA guidance document, " Guidance for Industry   Bioanalytical Method Validation, May 2001". Additionally, the performancebased validations in quail and mallard matrices confirmed that the method can be used in matrices other than the rat and mice matrices already validated, provided the concurrent quality control samples document the acceptability of each data set as shown in 3M Studies E0l 1256 titled " Analytical Phase Report for PFOS: A Reproduction Study with the Mallard" and E01 l245 titled " Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite." 3M Report E02 1042 titled " Method and Validation Report for ` Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS 8 23 1.1" is a compilation of the results ofthese studies.

These procedures are appropriate for perfluorinated acids, alcohols, amides, sulfonates and other fluorinated compounds in any biological matrix provided that quality control sample results meet the precision and accuracy requirements ofthe study.

2 Method Summary This method easily accommodates a significant range of initial sample weights and volumes, which is essential when preparing samples oflimited quantities or when large sample sizes are required to provide low detection limits. When using a 1 gram SPE cartridge, sample amounts typically ranging from 0.02 to 1.0 mLor g are prepared ( fluids diluted, tissues homogenized, etc.) at a 1/ 6, 1/ 10, or 1/ 20 dilution using reagent water. An aliquot ofthe dilution/ homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile ( ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarify the supematant. The supematant is transferred to a clean tube, diluted with water, and passed through apre conditioned C18 SPE cartridge. Finally, the analytes of interest are eluted from the SPE cartridge and analyzed by HPLC/ MS or HPLC/ MS/ MS.

Depending on sample availability and detection limits required, the use of SPE cartridges of different sizes provides flexibility for varying initial sample sizes and final elution volumes. Sufficient quality control samples are incorporated into each study to ensure that precision and accuracyof the study are understood. The required quality control elementsof this performance based method meet the cross validation requirementsbased enthe FDA guidance documents.

3 Definitions 3.1 Dilution A dilution expressed as 1: 5 or 1/ 6 is defined as: 1 mL of sample + 5 mLs of diluent for a total of 6 mLs combined, unless otherwise noted.

3.2 SPE cartridge A column containing an open solvent reservoir, retaining fit, sorbenthed, retaining fit, andliiertip~ The sorbent bed is bonded silica which is designed to selectively retain or elute the compounds of interest depending on the solvent conditions. The compounds of interest can then be separated from difficult matrices and introduced into an appropriate solvent for analysis. SPE cartridges are available with a wide range of sorbent beds in a variety izfsizes, allowing the analyst to process a wide array of sample types andvolumes.

ETS 8 23l. 2 Page 2 of 23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices I ~ ~ 1 1fllL ~ C 1~ SciJ. 4 ~ ~ L L ~~ m I) 5/ O7i2t~) u3 3.3 Reagent grade water Waterwith no detectable concentration( s) ofthe target analyte( s) or less than 1/ 2 the limit of quantitation ( LOQ).

3.4 Quality control sample Sample used to determine extraction precision andaccuracy and to estimate the uncertainty of results generated.

4 Warnings and Cautions 4.1 Health and Safety Warnings Always wear appropriate personal protective equipments such as protective gloves, eye protection, and appropriate clothing when working with biological matrices, solvents, chemicals and instrumentation. For potential hazard information refer to material safety data sheets, packing material, the 3M Environmental Laboratory's Chemical Hazard Review, the 3M Guide to Laboratory Practices or other information as appropriate.

4.2 Cautions Theanalyst must be familiar with the laboratory equipmentandpotential hazards including, butnot limited to, theuse of biological materials, solvents, high temperatures, pressurized gas and solvent lines, high voltage, and vacuum systems. Referto the appropriate equipmentprocedure or operator manual for adilulionalinfbtinatinzraridcaution&

5 Interferences To minimize interferences, Teflon should not be used for sample storage or anypart of instrumentation that comes in contact with the sample orextract.

Co extracted matrix components may enhance or suppress the measured analyte signal in the mass spectrometer. These effects are minimized using this SPE technique, but the precision and accuracy of spike results must be evaluated for possible effectsof co extracted matrix interferences that may be present.

6 Instrumentation, Supplies, and Materials The following instrumentation, supplies, and materials are used while performing this method. Equivalent instrumentation, supplies, andmaterials may be used in place ofthose listed.

6.1 Instrumentation and Equipment Vortexmixer, VWR, VortexGenie 2, or equivalent Ultra Turrax T25 tissue homogenizer with various size blades, or equivalent Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral 1000, IEC, or equivalent Shaker, Eberbach, VWR, or equivalent Balance (+/ 0.1000 g)

Micromass, Quattro II or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source, or equivalent HP! 100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler, or equivalent ETS 8 23l. 2 Page 3 of 23 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices I. ~ :~ L~ ~. 1 ~ i: rt~ cni..~ ~`~ 4o ~ ~

6.2 Supplies and Materials Eppendorfor disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, ( Nalgeneor equivalent), various sizes Volumetric flasks, glass, type A, various sizes 40 mL glass vials ( ICHEM or equivalent)

Plastic sampule vials, ( Wheaton or equivalent), 6 mL ( or other appropriate size)

Centrifuge tubes, polypropylene, various sizes Labels, various sizes Graduated pipettes, glass Syringes, capable ofmeasuring 5 ~. tLto 1000 ~ iL Bottle Top Dispenser( capable ofdispensing 5mL of solvent)

SPE extraction cartridges, for example 1 g, Sep Pak6 cc ti functional C18 ( Waters), or other sizes and adsorbents 75 mL sample reservoir ( or other appropriate size)

Crimp cap glass autovials and caps, crimpers HPLC analytical column: KeystoneBetasil C 18, 2 X 50 mm, 5 tim, or equivalent 7 Reagents and Standards Reagent gradewater, ASTM Type I Milli~ QTM, Kandiyohi purchased drinking water, or equivalent Acetonitrile, HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetate, reagent grade or equivalent Various biological fluids or tissues from supplier or study sponsor for use as control matrix 7.1 Control Matrix Tissue/ Fluid Preparation This method requires that a control matrix be used for the preparation of matrix extracted calibration standards, control spikes, and method blanks. Therefore, an amount of control matrix should be prepared to provide asufficient amount throughout a study. Typically an aliquot of 4OmL is mixed with 200 mL of reagent wateror 40g of matrix is homogenized with 200 g reagent water andused as control matrix. Other amounts may be used to maintain the same dilution ratio as the samples or additional control matrix may be prepared during a study asneeded.

Control matrix is prepared from one of three sources: 1) a study control matrix from a study control received with a sample set; 2) a commercially obtained sample of the same matrix as the study matrix; or 3) asurrogate matrix, also obtained commercially, but of a different type than the study matrix. ( eg. if rat is used to generate calibration curves andQCs for amouse study).

7.2 Reagents Preparation Prepare a 2.0 rnJvI ammonium acetate solution for use as a mobile phase for the liquid chromatograph: Weigh approximately0.300 g arnmonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids are dissolved, and bringto volume using reagent grade water. Store at roomtemperature.

Note: When preparing different volumes or concentrations than those listed for reagents and target analyte standard preparation adjust accordingly.

ETS 8 231.2 Page 4 of 23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices I) u~ re, t may 4a u~ adi a:~ a 4 4a4~ 4am 4 7.3 Target analyte standard preparation Prepare target analyte standard( s) for the matrix extracted calibration curve. Multi component analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations.

Weigh approximately 100 mg of target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol, or other appropriate solvent, for a stock standard of approximately 1000 ppm (~ tg/ mL). The calculated concentration of this stock standard should be corrected for purity and should reflect the fluorochemical anion concentration that is measured.

Dilute the stock solution with methanol for a working standard solution of approximately 50 ppm. Example calculation: l000~ ig/ mLx 5 mL/ 100mL = 50J1g/ mL. Additional dilutions or serial dilutions are made as appropriate.

If surrogate standards or internal standards are part of the study, these stock solutions are prepared using the same procedures outlined above. Additional information regarding the appropriate use of surrogates or internal standards is found in section 9 of this method. Surrogates and internal standards are not required by this method, but may be used for individual studies.

8 Sample Handling It is expected that samples are received at the appropriate conditions and must be kept at the appropriate conditions until the extraction is performed, ( i. e. frozen for biological samples). The condition of samples upon receipt and storage conditions throughout the study should be appropriately documented.

Allow liquid samples and homogenized tissue samplesto thaw/ warm to roomtemperature prior to extraction.

Matrix extracted standards used to calibrate the instrument response for each target analyte are prepared at least once during a study. With each extraction batch, matrix extracted quality control standards must be prepared to verify the extraction efficiency. Alternately, an entire matrix extracted curve may be prepared with each extraction set. Extracted standardsand samplesare stored in capped autovials until analysia.

Extracted standardsand samplesmay be stored at roomtemperature or refrigeralecLatapproximately 4 ° Cuntil analysis can be performed. Ifrequired by the study protocol, 3 post extraction matrix spikes ( i. e. aknown amount of the target analyte spiked into an extracted matrixblank) may be stored with the extracted standardsand samples for evaluation to demonstrate extract stability. Alternately the precision and accuracy ofmatrix extracted quality control samplesmay be used to document analyte stability during the time frameof analysis. Results will be documented in the report.

9 Quality Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot ofmethanol or other appropriate solvent is analyzed periodically throughout each arralyticat sequence as a solvent blank to serve as a check for background concentrations of analyte or cross over contamination between injections. Solvent blanks are not extracted.

9.1.2 Method Blank An aliquot of 1.0 mL of reagent water, or other amount as appropriate, is used as a method biank. Method blanks are extracted and analyzed with each extraction set following this procedure and may or may not be spiked with surrogate depending on study requirements. Method blanks may serve as a measure of analyte background concentrations not attributableto endogenouslevels in the control matrix.

ETS 8 231.2 PageS of 23 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices Ducaamnt may 4a u~ m4.11 ~ arrenL la I 4aVSU. ~ aa 4a4 / ~ 44~ a 9.1.3 Matrix Blank An aliquot of 1.0 mL or 1.0 g of matrix ( diluted or homogenized) is used as a matrix blank. Other amounts are used, as appropriate, in keeping with the amount of sample available for extraction. Matrix blanks are prepared from one of three sources: 1) a study control matrix from a study control received with a sample set; 2) a commercially obtained sample of thesame matrix as the study matrix; or 3) a surrogate matrix, also obtained commercially, but of a different type than the study matrix. ( eg. if rat is used to generate calibration curves and QCs for a mouse study). The matrix to use is the same as the matrix used for the curve. If limited matrix is available, the number of method and matrix blanks may be adjusted, as well as the initial volumes/ weights, and will be notedin the study protocol or in the raw data.

9.1 .3.1 Study control matrix curve  if the study control matrix is used for the curve, preparefour ( 4) matrix blanks using the study control matrix ( two spiked with surrogate and two not spiked). Ifsurrogates are not part ( Ifa study, duplicates are acceptable.

<IP_ADDRESS> Commercially obtained matrix curve   if acommercially obtained matrix is used for the curve, prepare four ( 4) matrix blanks using the same commercially available matrix ( two spiked with surrogate and two not spiked). If surrogates are not partofa study, duplicates are acceptable.

<IP_ADDRESS> Surrogate matrix curve   ifa surrogate matrix is used for the curve, prepare four ( 4) matrix blanks using the same surrogate matrix and prepare four ( 4) matrix blanks using the study control matrix, ifavailable. If study control matrix is not available, prepare four ( 4) matrix blanks using acommercially available matrix of the same type as the study matrix ( two spiked with surrogate and two not spiked). Ifsurrogates are not partof a study, duplicates are acceptable.

9.2 Sample Replicate Samples replicates are prepared accordingto each study protocol, project outline, or as determined by the analyst.

9.3 Surrogate standard If surrogate standard is a component of the study, all samples are spiked with surrogate standard prior to extraction to obtain an appropriate concentration within the target calibration curve, with the exception of blank samples as described in section 9.1.

Typically surrogate standard is spiked into the 1.0 mL or 1.0 g, or other appropriate volume, diluted/ homogenized sample removed for extraction.

9.4 Initial Calibration A minimum of one set ofmatrix extracted standards is prepared and analyzed to establish the relationship of analyte concentration to instrument response for the target fluorochemical analytes. Details are provided in Section 10 ofthis method.

9.5 Quality Control ( QC) Sample Quality control ( QC) samples are used to determine extraction precision and accuracy and to estimate the uncertainty of results generated. Typically 1.0 mL ofthe same matrix used to prepare the initial calibration curve is used for each QC sample, and the target analyte standard is spiked into the 1.0 mL or 1.0 g diluted/ homogenized sample removed for extraction. All spiking should be performed at the same step in the extraction. ( i. e. Ifthe curve is spiked into the 1.0 mL or 1.0 g diluted/ homogenized sample removed for extraction then QC samples, surrogate spike, etc. should also be spiked at that time.)

A minimum of nine ( 9) quality control samples ( QC) will be prepared for each matrix during the course of a study to allow the needed statistical calculations to be made. When study samples are extracted in three or fewer extraction batches, aminimum of three QC samples must be prepared ( one at each level) on each day of sample extraction, and should total nine or more. When 3 or more extraction batches are prepared, fewer than three QC samples may be prepared with each batch provided themethod or protocol QCfrequency requirements are met.

ETS 8 23l. 2 Page 6 of 23 Solid Phase Extraction and Analysis ofFluorochemical Compounds from Various Matrices I. 4yam~ e. m: aa~ 4a ja~ m.$ uaenL io~ 14 day lom ( b' 4a' 244 Thetotal number of QC sampleswithin a study should be at least 5% of thenumber ofunknownsamples or nine total QCs, whichever is greater.

The levels listed below are target guidelines to be used when spiking QC samples and may represent sample concentrations diluted into the range ofthe calibration curve:

Low level: 3X to 5X the lower limit of quantitation ( LLOQ),

Mid level: equivalent to a point near the middle ofthe calibration curve,

High level: 80% of theupper limit of quantitation ( ULOQ)

QC samples extracted with a particular sample are initially analyzed in the same analytical run to demonstrate acceptable quality control for the batch. After the QC samples have been successfully analyzed, the QC samples do not need to be re injected when a subset of samples from a batch are analyzed since the acceptance criteria for the batch have already been met. The extracted calibration curve and continuing calibration verification samples will document the acceptable performance of the instrumentation.

Each QC is expected to show an accuracy 85 115% of expectedand aprecision of 15%. A minimum of 2/ 3 of all QC samples analyzed within an analytical run must meet these criteria. Ifnot, the set must either be re analyzed or reextracted

9.6 Continuing Calibration Verification Sample ( CCV)

One or more initial calibration curve points are re injected after not more than 10 sample injections to verify the continued accuracy ofthe initial calibration curve.

Typically, two CCV sample levels are analyzed after every tenth sample injection. Solvent blanks are not considered samples and are not included as such for determining when QC samples will be analyzed. EachCCV is expected to show an accuracy of 85 115% ofexpected. Ifnot, the set should eitherbe re analyzed or re extracted.

9.7 Matrix Spike ( MS) Sample Matrix spike samples are not a required component ofthis method, but maybeiequireclby protocol or project plan.

If the matrices for the curve are not the same as the sample matrix to be analyzed, or the protocol/ project outline requires matrix spike samples; the specific criteria regarding matrix spikes will be documented in the protocol/ project outline and/ or in a note to file.

9.8 Control Charts Thepercent recovery for each QC sample for each analyte and matrix will be control charted as per SOP ETS 4 26, " Control Charts for Laboratory Analyses".

9.9 Sample Dilution Any sample with an analyte area greater than that of the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range of the curve during analyses and enough sample remains, a post run dilution validation may be performed to verify sample values.

To perform the dilution validation, one sample will be separated into two representative samples ( i. e. two 1.0 mL aliquots for fluid samples or two 1.0 gram amounts for tissue samples, or other amount as determined by the analyst and documented in a note to file) then diluted using two procedures. The first procedure consists of diluting the sample with additional blank matrix prior to extraction ( fluid adding fluid), while the second procedure consists of diluting the extract with solvent post extraction ( methanol extract adding additional methanol solvent).

If the relative percent difference is not within 15% for these two samples, additional testing may be performed to determine which value is a correct representation of the sample concentration as determined by the analyst and documented in anote to file.

ETS 8 231.2 Page 7 of23 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices I.).~ mu a ~ aybe used. ~ ~ 4a d~ : 4a a I.

10 Calibration and Standardization 10.1 Instrument Calibration At least one calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples if available, per study. It will consist of a minimum of nine concentration levels. Additional calibration curves may be extractedon separate sample extraction dates, as detennined by the analyst.

Transfer 1.0 mL of diluted control fluid or 1.0 g of homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the calibration curve. Be sure to vortex mix or shake by hand the control matrix container for approximately 5 seconds between aliquots to ensure a homogenous sample is removed.

Record each standard weight or volume on the weight/ volumes sheet or extraction worksheet, as appropriate.

Four 1.0 mL or 1.0 g aliquots, or other appropriate amount as described in 9.1.2, of control matrix serve as matrix blanks.

A total of 9 standards, four matrix blanks, and fourmethod blanks are prepared in addition to the QC samples and test samples. Thenumber ofstandards andblanks may be adjusted as per section 9.1, oras determinedhy the analyst.

Use Attachment C, or other appropriate form, as an aid in calculating the concentrations of the working standards. Referto section 12 to calculate the actual concentrationofanalyte in each calibration standard and QC sample.

Typically the target analyte standard is spiked into the 1.0 mL diluted fluid or 1.0 g homogenized tissue sample removed for extraction. All spiking should be preformed at the same step in the extraction. ( i. e. Ifthe curve is spiked into the 1.0 mL or 1.0 g diluted/ homogenized sample removed for extraction then QC samples, surrogate spike, etc. should also be spiked at that time.)

Analyze the extracted matrix calibration curve prior to each set of extracts. The curve equation may be determined by quadratic regression analysis, weighted 1 / x, and external standard using the peak areas ofthe target analyte( s) using MassLynx or other suitable software. Other suitable regressions may be used as appropriate.

Any level outside 85%  115% of nominal must be deactivated, and regression re calculated, except the LLOQwhich must be within 20% ofnominal. All levelsmust show a response greater than twice that of the blank. Aminimum of six ( 6) levelsmay be used to plot the curve in anyone set, or the set will bere analyzed.

11 Procedures 11.1 Tissue/ Solid Sample Preparation Obtain tissue/ solid samples.

Cut approximately 1.000 g oftissue (+ 1 0.100 g), or otherappropriate amount, using adissecting scalpel. This partof the procedure is best performed quickly, not allowing the tissue to thaw.

Weigh approximately 1.000 g of solid sample (+ 1 0.100 g).

Weigh the sample directly into ataredplastic sample vial.

Record the weighton the weight/ volume sheet, extraction worksheet, or other appropriate mention.

Return unused tissue/ solid to the appropriate storage conditions afterextractionamounts have beenremoved.

Weigh 5 mL ( or 10 mL) of reagent water into the tared sampule vial containing the tissue/ solid sample then record the weight on the weight/ volume sheet. ( For solid samples that don't require homogenization, skip the remaining steps in this section.)

Homogenize the sample. Put the Ultra Turrax grinder probe in the sample and grind for approximately 2 minutes, or until the sample is homogeneous.

ETS 8 231.2 Page 8 of 23 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices i) oaemem may ~ a ~ aec4 e~ eaa:, 4 1 a 4av~ floe 4aid 44~: 44 Takethe grinder apart and clean it with methanol after each sample. Refer to ETS 9 52 for more information.

If an amount other than 1.000 g ( not within +/ 0.100 g) is removed for an initial weight, adjust the water volume accordingly to maintain a 1/ 6 dilution. ( e. g. if0.5 g is removed for extraction, add a total of2.5 mL ofwater.), or other ratio as documentedin the protocol, the project outline, or a note to file.

11.2 Fluid/ Liquid Sample Preparation Obtain fluid/ liquid sample and allow it to thaw/ warm at room temperature or in lukewarmwater.

Label a 15 niL polypropylene centrifuge tube with the study number, sample ID, extraction date and analyst initials. See attachedworksheet ( AttachmentAor similar worksheet) for documentingthe remaining steps.

Vortex mix the sample for approximately 15 seconds, then transfer 1.0 mL of fluid/ liquid, or other available amount, to a plastic sampulevial, or other appropriate plastic container.

Return unused fluid/ liquid to the appropriate storage conditions after extraction amounts have beenremoved.

Add 5.0 mL of reagent water to the 1.0 mL of fluid/ liquid for a 1/ 6 dilution, or 19 mL of reagent water to the 1.0 mL for a 1/ 20 dilution.

If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilutions as above.

11.3 Tissue and Fluid Sample Extraction After tissue/ solid or fluid/ liquid samples have been prepared according to sections 11 .1 and 11.2, vortex mix or shake by hand the diluted/ homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mLpolypropylene centrifuge tube.

Return unused diluted/ homogenized portions to the appropriate storage condition after extraction amounts have been removed.

Record the volume ofdiluted fluid/ liquid removed on the extraction worksheet, ( AttachmentAor similar worksheet).

Record the weight of the homogenized tissue/ solid ( removed as a 1.0 mL volume or other volume) on the weight/ volume sheet, the extraction worksheet, or other appropriate location.

Spike blanks, samples, and standards, readyfor extractionwith surrogate standard as described hi this method.

Spike each calibration standard matrix with the appropriate amount of standard as described in this method for the calibration curve standardsand each QC sample.

Vortex mix the calibration curve samples and QC samples for approximately 5 seconds.

To each sample and standard, add 5.0 mL of acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds.

Place all samples on the shaker at an appropriate speed for 20 minutes to adequately mix ( a setting of approximately 300 rpm on the models listed in section 6.1).

Remove from the shaker and centrifuge at an appropriate speed for 10 minutes to adequately pellet the precipitate ( a setting of approximately2000 rpm on the models listed in section 6.1).

Add 40.0 mL of reagent grade water to a clean 50 mL centrifuge tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any of the matrix solids into the solution. Cap and mix by inverting several times. In this step the order of addition may be changed ( i. e. the sample maybe put into the centrifuge tube and then the water added).

Attach the reservoir to the SPE cartridgeand attach this reservoir/ cartridgeunitlo a vacuum manifold.

NOTE: When running the vacuum, set the vacuum chamberat approximately 20 kPA  to give an approximate elution flow of 5 7 mL/ min. Flows may vary through cartridges and the kPA may be raised for slow tubes and drying after most have beendrawn down.

Prepare the SPE cartridge by washing twice with approximately 5.0 mL ofmethanol, followed by approximately two 5.0 niL aliquots of water, taking care not to allow the column to run to dryness after each wash.

ETS 8 231.2 Page 9 of 23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices I aa. ama : nay 4 used ~` ~ aem4. I'm ~ days 4c a ( II5/ O7z2~` fi ~

After washing is complete, pour the sample into the reservoir/ cartridge unit and allow all of the liquid to pass through the column to dryness.

Run the vacuum on high for approximately 5 minutes to adequatelydryeach SPE cartridge.

Place a collection 15 mLpolypropylene centrifuge tube under each cartridge and elute with 2.0 mL ofmethanol.

Spike extracted blanks, samples, and standards with internal standard as described in this method.

Label each glass autovial, as appropriate, with the study number, vial file archive number, identification number/ gender/ timepoint ( if applicable) or LIMS number, matrix, final solvent, analyte components ( if needed), extraction type, extraction date, and analyst( s) performing the extraction.

Transfer each eluant to aglass autovial and cap.

11.4 Extract Analysis 11.4.1 Software set up Prior to analysis, a sample list must be created using the following parameters and naming conventions: instrument designator letter, last 2 digits of test year month day, and a letter that will increase through the alphabet with each additional list for that day. Other unique sample list names may be used.

Example Sample List: IYYMMDDaor A020204a I = Initial ofthe instrument name ( A = " Amelia")

YY= Test year ( 02)

MM= Test month ( 02)

DD= Test day( 04)

a = First sample list ( run) of the day ( the next sample list will endwith ` b', the next c,

and so on.)

Filenames are created by using the instrument designator letter, the last 2 digits of the test year month day, and a 3 digit sequential file number that starts with 1 and increases by one for each filename. Other unique filenames may be used.

Example filename: IYYMMDD### or A020204001 I = Initial of instrument name YY = Test year MM = Test month DD = Test day = 3 digit sequential filenumber starting with 1 through999 ( 001)

Also, as part of the sample list, assign a mass spectrometer acquisition method for acquiring data, an inlet file to designate HPLC analytical parameters, a bottle number, an injection volume, and individual sample descriptions.

Typically, analytical batch run sequences begin with a minimum of three system suitability injections, several solvent blanks, and acalibration curve. Theend of the analytical sequenceshould be markedby a closing CCV.

Typically, two CCV samples should be injected after every tenth sample, and all sample extracts must be bracketed by the opening calibration curve and a closing CCV. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be includedas such.

11.4.2 HPLC set up Load the autosampler tray according to the sample list prepared above.

ETS 8 231.2 Page 10 of 23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices a' as 10. used. 4 ceo 14 ~ ftori fl~ ` 7/~~ fl:'~

Download the HPLC inlet file created in section 12.4.1. Record actual conditions in the instrument logbook, or other appropriate location. The following instrument conditions areprovided as an example:

Sample injection volume = 10 IlL Injections/ sample =

Cycle time = 10.0 minutes Flow rate = 300 pL/ min.

Column: Keystone Betasil C18, 2 x 50mm, 5 urn particle size, or equivalent Mobile phase: Solvent A= 2 mM Animonium Acetate in ASTM Type I water Solvent B = Methanol ( HPLC grade)

Solvent gradient:

Time % B 0.00 10%

1.00 10%

5.50 95%

7.50 95%

8.00 10%

11.4.3 Instrument set up The following instrument conditions areprovided as an example:

Drying gas250   400 liters/ hour ES nebulizinggas 10   15 liters/ hour Sourceblocktemperature = 150 ° C Desolvation temperature = 250 ° C These settings may change in order to optimize the response. Please refer to the instrument tune pageand ETS 9 24, " Operation and Maintenance of Micromass ® Triple Quadrupole Mass Spectrometers Fitted With Atmospheric Pressure Ionization Sources," for additional details.

12 Data Analysis and Calculations 12.1 Calculations Ifalternative calculations are used, they will be documented in the raw data.

Calculate the matrix amount contained in the initial dilution using the following equationn Initial Sample Weight ( g) Weight of Tissue Extracted ( g) = . x Weight of HomogenateAliquot ( g) Homogenate Sample Weight ( g)

Initial SampleVolume ( mL) Volumeof Fluid Extracted ( mL) = . x Volume of Fluid Aliquot ( rnL) Diluted Sample Volume( mL)

ETS 8 231.2 Page 11 of23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices I/ oem em a ay ec used if eurrea. fe' 14 ~ ays h'om 43/ 07/ 7403 Calculate actual concentrations ofanalyte in calibration standards using the following equation:

= Spike Concentration ( ug/ mL) x Spiked Amount ( mL) 1000 ng Concentration ( ng/ mL) in Final SPE Eluent Calculate actual concentrationof analyte in fluid ( Ilg/ mL): ( mL) Final SPE Eluent 1 ug ( AR ( ng/ mL eluent) x DF) x Final Eluent Volume ( mL) x Volume of Fluid Extracted ( mL)

Calculate actual concentration ofanalyte in tissue (~ g/ g): = ( gg/ mL) 1000 ng ( AR ( ng/ mL eluent) x DF) x Final Eluent Volume ( rnL) x 1.0 jig = ( jig/ g) Weight of tissue Extracted ( g) 1000 ng AR= Analytical result from MassLynxsummary DF = Dilution factor introduced when sample extract is diluted to fall within the calibration curverange.

Calculate spike percent recoveries using the following equation:

Observed Result   Unspike Sample Result % Recovery =

Spiking Level Calculate relative standard deviation using the following equation:

Standard Deviation Relative Standard Deviation = x 100 Mean Calculate percent deviationusing the following equation: x 100 % Deviation = Calculated Cone.   Expected Cone.

Expected Cone. x 100 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample of the run three ( 3) mid level unextracted calibration standards will be analyzed. The peakarea precision and retention time precision will be monitored at the beginning and the end of the run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD.

If any item of the system suitability fails, system maintenance must be completed prior to running a second set of system suitability samples. The system suitability must pass before starting the calibration. Ifsystem suitability fails at the completion of a run, the sample set must be reanalyzed.

13.2 Uncertainty Estimation The combined uncertainty associated with this method, as determined by the validation results, is as follows: 29% for tissue analyses and 9% for fluid analyses. Referto SOP ETS 12 12, " Estimation ofUncertainty Measurements" and ETS 8 23 1.2 Solid Phase Extraction and Analysis ofFluorochemical Compounds from Various Matrices Page 12 of23 1/ or uncut may be ured, ii' current. ha' I a days from (. 15/ 07/ 2003 the attached uncertainty estimation report ( Attachment E) for additional information on calculating uncertainty for methods.

The uncertainty estimation may change per matrix as data are collected and added to control charts, or as control charts are updated according to ETS 8 26, " Control Charts for Laboratory Analyses".

13.3 Quantitation The coefficient of determination value for the calibration curve, plotted by regression using the peak areas of the analyte( s), must be 0.990 or better.

During the validation it was determined that four curve models adequately describe the concentration response relationship for this method: linear null weighted; linear 1/ x weighted; quadratic null weighted, and quadratic 1/ x weighted. However, it has been determined that quadratic l/ x weighted, external standard will most often be used for sample quantitation.

All active calibration curve points must be within 15% of the theoretical value with the exception of the LOQ point, which may deviate up to 20%.

Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a data range that may be affected by background levelsof the analyte.

Avalid calibration curve must contain at least 6 active points above and including the LOQ.

Ifthe curve cannotmeet these criteria, the sample set must be reanalyzed or reextracted.

13.4 Accuracy and Precision Two thirds of all quality control and continuing calibration verification samples are expected to show an accuracy of 85 115%.

Quality control samplesare expected to show a precisionof 15%.

14 Pollution Prevention and Waste Management Sample waste is disposed of in noninfectious biohazard waste containers.

Flammable solvent waste is disposed ofin high BTU containers.

Glass pipette waste is disposed of in broken glass containers located in the laboratory.

15 Records Complete the extraction worksheetattached to this method, or other applicable worksheet, and store withthe study raw data.

Each page generated for a study must contain the following information ( if applicable): study/ project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor ( if applicable), and analyst. Other information may be added if applicable to the study.

Print the tune page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog, ifapplicable.

Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data.

Print data integration summary, integration method, and chromatograms from MassLynx, and store with the study raw data.

Summarize data using suitable software ( Excel 7.0 or LIMS) and store in the study folder.

Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data.

Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices ~) omui. am clay be mmd if cu ` cut. icr a dams from 1.3/ 0 .2003 16 Attachments AttachmentA: Extraction Worksheet Examples AttachmentB: Sample Weight/ VolumeWorksheet AttachmentC: Calibration Standard Concentration Worksheet AttachmentD: Dilutions Summary Worksheet Attechrnetn E: Uncertainty Estimation Report 17 References 3M report  E0l 1277, " Method Validation for ETS 8 231"

3M Method  ETS 4 26, " Control Charts for Laboratory Analyses"

3M SOP  ETS 12 12, " Estimation ofUncertainty Measurements"

3M Equipment Procedure  ETS 9 24, " Operation and Maintenance of Micromass ® Triple Quadrupole Mass Spectrometers Fitted With Atmospheric Pressure Ionization Sources"

3MEquipment Procedure  ETS 9 52, " Operation andMaintenance of a Tissue Grinder"

FDA guidance document, " Guidance for Industry   Bioanalytical MethodValidation, May 2001"

3Mreport  E0l l256, " Analytical Phase Report for PFOS: AReproduction Study with the Mallard"

3Mreport  E0l 1245, " Analytical Phase Report for PFOS: AReproduction Study with theNorthern Bobwhite"

3M report  E02 l042, " Method and Validation Report for ` Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS 8 23 1.1"

3MStandard Operating Procedure  ETS 12 1, " Analytical Definitions, Abbreviations, Acronyms, and Symbols"

18 Affected Documents None.

19 Revisions Revision Revision Number Revision Description Minor formatting changes. Added detailed information to all sections concerningthe 02/ 18/ 02 extraction procedure, analytical procedure, and calculations. Added attachments and references. 2 The minor changes in this revision will not affect themethod validation results: 10/ 04/ 02 Section 1 added reference to validated method, added clarification to the scope of the method, and removed fish matrix. Section 9.6, 10.1 added text for consistency in spiking calibration curves and quality control samples. Section 9.8 added control chart section.

Section 12 updated calculations and corrected serumfinal concentration units. Section 13.2 added uncertainty estimation. Section 13.3 added text describing which curve model to use during sample analysis. Section 16 added additional extraction sheets and uncertainty estimation report. Section 17 added additional references. Added clarification to text throughout method.

ETS 8 231.2 Page 14 of23 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices 4ocm an i a cc be used ten ant. 3m' 14 days ii em fib a 7/ 2 fr3 Attachment A   ExtractionWorksheet Example 1 Study Number: Prep Date:

Analysts initials: Box#: Method Revision: ETS 8 231 .2 Matrix: Sample Timepoint:

Sample Volume/ Weight Number of diluted Amount and Amount and or sample spike mix surrogate spike Type of column Elution solvent Sample Number description removed used mix used used and lot and volume Comments signature/ Date Blank matrix TN A_________; Amount weighed/ aliguoted: g/ mL 1. Homogenize sample 2. Aliguot 1 mL of diluted matrix into 15 mL polypropylene tube ( record volume or weight accordingly)

3. Spike samples accordingly 4. Add mL of ACN ( TN A to each diluted sample and shake or vortex mix 5. Shake sample for 20 mm ata setting of rpm ( Shaker_~

6. Centrifuge sample for 10 mm at a setting of rpm ( Centrifuge 7. Add 40 mL of water to 50 mL polypropelene centrifuge tube.

8. Decant extract into centrifuge tubes with water 9. Shake sample slightly to ensure proper mixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. Condition column with two washes of   5 mL MeOH ( TN A  donot allow column to goto dryness 12. Wash column with two washes of   5 mL water  do not allow column to goto dryness 13. Filter sample through conditioned column, discarding filtrate 14. Allow column to goto dryness. After dripping stops, draw a high vacuum througltcolumn for at leastñminutes 15. Elute column with solvent ( TN A ) into appropriate 15 mL centrifuge tube 16. Spike samples with uLof internal standard # conc.

17. Transfer sample into appropriately labeled autovial and cap Note: In vacuum steps above setthe vacuum chamber at approximately 20 kPA  this should give approximately 5 7 mL/ min elution flow Flows may vary through cartridges  kPA may be raised for slow tubes and drying after most have been drawn dowriandshut off.

ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices Page 15 of23 I/) uejniem na: 7 Is med fur n'mc. Ioi he 057 s isan ( 73 ts~ n___________

ast________________

~ tooti_________________

I g ,, il~ tiiae9!,===~

`: tieaasnii ,,,_:,, y,,~,,__,

  Attachment A   Extraction Worksheet Example 2 / 1 1 ~ ~

F UI ` 1 htt ~ jr d1 Ii~

~ dUll it! H irvn ii~ th fl I C* iFti~ WSS 1 nsa _

Cd i~ tnW4il,_ y_~_   ttiltltaktil I man _____________

ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices Page 16 of23 I I ` ti'

I L~

~ frTh~ :

; ~ F:_

Cdli htsWlii_~/._,~.,,,,,

5'

as ft______

CdlltàbI. at I I______

Cdii tnt ® Sa I _______ ti________

( diSk ~

tt'I ® Lwt_,,_,_,,,,,,,,

  CdfittMd I I 115' rwra. rn1remçap asaaarak3nrnp%:

~` akacPflwl s c.~ acbak5Nws'wa15M 4nkisIrnna* ~ 4mt' 5) tt$' Dueume~ en ~ ayde used i ~ uueres4 d~ 14 ciayshme () S/ O7/ 2~.%)

Attachment A   Extraction Worksheet Example 3 4, 1 1 j~.

! H h1L4 ~. t d~ In ~ ~ V ht~! 1 ill Ifi d ` Iii IUfl d Ii h h

I t1 Cd3ci~ t~ MtIj __________ tvM~ ________ __________

~ _____

?~ a~ ean, a~,

ta'~~,_~ c__

Cd Cd~ i _______ C_______

° CdoEa1w~ 8 1 Cd W L~ n1d*~_ LL~_

tIe$

rosa___________

a______________

twfll ~ I iii I ~ H It ii P I   ~ "..   f ~ a~ aa~ a ~ f~ 8aa 4, g&~

oi,~ if I f t I p~

a'~\\ ~ \ 88 I :`~ t~   =  t Ot~ F n~ Ii~& s S~ CIltwS~ pfld~ s. t 1t~ ssi ~ ~~ ve SOntcwtt*' tAaa ETS 8 23 1.2 Solid Phase Extraction and Analysis ofFluorochemical Compounds from Various Matrices Page 17 of23 L) c: 4 ~ 1n~ Eu~ tne b~~. scn.( 8u$' reer ~ 4 es. s ~ 4on~ ~/ O7iI) 24 Attachment B  Sample Weight/ Volume Worksheet Prep Date( s): Analyst( s): Sample Matrix: Method/ Revision: Study Number: Equipment Number:

Final Solvent & TN Number:

Sample Initial Water added Volume/ Weight Date omments ID Wt./ Vol. Wt./ Vol. Removed Removed!

gImLIL g/ mL mL/ g Analyst Form Completion Verified By:

ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices Page 18 of23 4ocu~ ne1itmay be used. ii curr'~' Ibr 1~ days ii on I Attachment C: Calibration Standard Concentration Worksheet Prep date( s): Analyte( s): Sample matrix: Method/ revision:

Analyte mix std approx. 0.500 ug/ mL: Standard number: Equipment number: Final solvent and TN#:

Blank Tissue or Fluid/ identifier:

Analyte mix std approx. 5.00 ug/ mL: Analyte mix std approx. 50.0 ug/ mL:

Surrogate std approx. 100 ug/ mL: Actual concentrations ofstandards in the analytemix Analyte Std conc ug/ mL All Amount spiked mL All Final Volume: mL 0.500 0.0015 2.00 0.500 0.0030 2.00 0.500 0.0080 2.00 0.500 0.0160 2.00 0.500 0.0320 2.00 5.00 0.0056 2.00 5.00 0.0080 2.00 5.00 0.0160 2.00 5.00 0.0240 2.00 5.00 0.0320 2.00 5.00 0.0400 2.00 50.0 0.005 2.00 50.0 0.006 2.00 Calculated concentrations of standards in relation to the final 2.0 mL solvent 2.0 mL Final Volume Analyte Final conc. ng/ mL Surrogate Std conc ng/ mL 0.375 100 0.750 Surrogate 2.00 4.00 Final conc nglmL 8.00 14.0 0.500 20.0 40.0 60.0 80.0 100 125 150 ETS 8 23 1.2 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices Page 19 of23 2oclInlen~. may be u. see. ifs ~ 0 m~ t. 14' 14 & a a 15001 bc~ ifr/ 2Ud3 Attachment D: Dilutions Summary Worksheet Study: Dilution Date/ Analyst: Box Number: Solvent/ TN Number: Extraction Date/ Analyst: Matrix/ Timepoint:

SampleNumber or Description Dilutions 1/ 1/ 1/ 1/ 1/ 1/ 1/ Comments Verified By:

Notes: 1/ 10 dilution = I mL of sample + 9 mL of solvent Form Completion Verified By:

ETS 8 23 1.2 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices Page 20 of 23 14ei, 1 neat cu~ U used 1 cur'm if 4y `~ fl c's fro'n 1) 5/ 07/ 2143 ETS 8 231 .1 Uncertainty Estimation Attachment E: Uncertainty Estimation Report A full validation for the use of this method for the determination of PFOS has been performed on rat liver and serum; a partial validation has been performed with PFOS on mouse liver and serum. A method validation report has been issued for these validations, as well as a performance based validation for the analysis of PFOS in mallard and quail liver and serum. This report presents precision and bias evaluations through this method; the results of these evaluations will help in estimating the uncertainty for theuse of this method.

1. Method summary Themethod includes the following procedures:

Weigh subsample Weigh subsample for calibration std Dilute/ Homogenize I Prepare spike std ~.

Measure aliquot Clean up 4~ Final volume j Result 2. Major sources of uncertainty The following describes each ofseveral majorsources of uncertainty indahoratory determinations, the nature of the uncertainty associated with each, and an estimation ofthe extentof the error.

Mass   Balances are used for ° measuring the weight of the sample aliquot for analysis ( using an analytical balance), only for solid matrices ( e. g., liver.) ° measuring the weight of analytical standard used for the preparation of a stock standard solution( using an analytical balance.) As discussed in SOP ETS l2 012, the uncertainty associated with the standard weights can be ignored. Random uncertainties associated with balance measurements were estimated using calibration datafrom one analytical balance:

ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices Page 21 of23 Dilute/ Homogenize Measure aliquot LC/ MS analysis F ,~ r Clean up 4, Final volume .4,

 I LC/ MS calibration i'ocumcl a may be usec. ifcurrent, 14r 1 ~ days I ` 71407/ 2002 Balance ID 118453734 Logbook pages 00062 7  00062 9 Standard deviation 9.58E 04 Square of standard_ deviation 9. l7E 07 n 118 However, the uncertainty associated by the sample and neat standard weighing process is included in the validation studies usingQC samples. No additional factor is needed.

Volume   Volumetric flasks are used for: ° measuring the final volume of spiking solutions. ° measuring the final volume of calibrating solutions. Pipettors are used for: ° measuring the volume ofthe sample aliquot, only for liquid matrices ( e. g., serum.) ° measuring the volume ofwater added before homogenizing. ° measuring the aliquot taken for clean up. ° measuring the final volume of extract. ° measuring volume aliquots of standard solutions in the preparation of spiking solutions. ° measuring volume aliquots of standard solutions in the preparation Dfcalibrating suhrtiuns. As discussed in SOP ETS 12 012, random uncertainties associated with volume determinations were estimated using calibration data from several pipettors:

Equipment type Pipettors Logbook 01054 Dates 4/ 5/ 02  7/ 28/ 02 Standard deviation 1 .33E 02 Square of standard_ deviation 1 .76E 04 n 69 However, the uncertainty associatedby the volume measurements of aliquots and extracts as well as of standard solutions is included in the validation studies using QC samples. No additional factor is needed.

Calibration Standards   Uncertainties associated with the neat standards are not supplied with fluorochemical compounds. Since they are solid compounds, the random uncertainties associated with their purity is considered negligible. No additional factor is needed.

Standardization Process  Thestandardization ( or calibration) process for most instrumental analyses introduces uncertainty because of the inexact nature of the process. With a correlation coefficient of 0.990, the minimum required by the method, the standard error of estimatemay be 10 20%. However, the uncertainty associatedby the standardization process is included in the validation studies usingQC samples. No additional factor is needed.

Extractionand Analysis Process   With recoveries typically being acceptable with an accuracy of 70% to 130%, and with a precision of 15%, it is expected that the greatest contribution to the uncertainties will be associated with the extraction and analysis process. The full validation data generated with PFOS in rat liver and serum were used to estimate this uncertainty, using all the % recoveries obtained during validation, including all sets, whether they met the target recoveries or not. It is assumed to follow a normal distribution.

ETS 8 231.2 Page 22 of 23 Solid Phase Extraction andAnalysis of Fluorochemical Compounds from Various Matrices !.) oaument. cc' 1. e usc 4. il current. 14r ~ I my's fi'nni 05/ 07: 214) 3 Method validation of ETS 8 23 1 Matrix Rat liver Rat serum Standard deviation 2.06E 0l 8.75E 02 Square of standard_ deviation 4.24E 02 7.66E 03 n 116 52 Homogeneity   Not aparameter in the method diagram above, sample homogeneity appears to be a significant effect in the analytical procedure for solid samples ( e. g., liver). For liquid samples, it is expected that this parameter will not be significant. The homogeneity of the liver sub sample is not taken into accountby the recoveries of the QC samples during validation, since spiking occurs after the l g subsample hasbeen taken. However, as discussed in the EURACHEM/ CITAC GuideQUAM: 2000. Pl, it is not practical to measure homogeneity directly. Following the same rationale presented in that document, an uncertainty factor of 0.20 is selected for this parameter. It is assumedto follow a normal distribution.

3. An analysis of the effect of contributing sources on combined uncertainty A summary table ofthecombined uncertainties andexpanded uncertainties ( k = 2) in typical analyses at the 3M Laboratory are therefore:

SUMMARY OF SOURCES OF UNCERTAINTY rat liver rat serum Neat standard negligible negligible Homogeneitystandard deviation 2. OOE Ol

Validation data standard deviation 2.06E Ol 8.75E 02 Homogeneitysquare of standard deviation 4. OOE 02

Validation data square of standard deviation 4.24E 02 7.66E 03 Combined uncertainty 29% 9% Expanded uncertainty 57% 18%

With combined uncertainty, u. a function of the individual uncertainties U,:

And the expanded uncertainty, U:

with k = 2 for 95% confidence interval.

As an example, for rat liver: uc=~ tj Ui 2 U = kxu~

u~= ~ J~ 2.0OE_ Oi) 2+ ( 2X~ 6E   O1) 2 = ~/( 4.00E   02+ 4.24E   02) = 0.287 29%

U = 2 x 0.287 = 0.574 = 57%

ETS 8 23 1.2 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Various Matrices Page 23 of 23