Saltzbart.Ilana@epamail.epa.

gov

11/15/2006 04:16 PM

To Lori E Stephans/AE/DuPont@DuPont

cc Andrea V Malinowski/AE/DuPont@DuPont

bcc

Subject Fifth Extension Request granted in full

History: This message has been replied to.

With this e-mail, EPA grants, in full, DuPont's request for a fifth

extension to reach agreement with EPA on the procedure(s) that DuPont

will use to purify the Fluorotelomer Products, as required by Section

I.E.2.a of Appendix A (conditionally granted by EPA on November 9,

2006)). DuPont will provide EPA with the data by March 7, 2006, and

the parties will reach agreement by March 30, 2006.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.

 

 Ilana 

 Saltzbart/DC/USE 

 PA/US To 

 Lori E Stephans 

 11/09/2006 08:50 <Lori.E.Stephans@USA.dupont.com> 

 AM cc 

 Andrea V Malinowski 

 <Andrea.V.Malinowski@USA.dupont.c 

 om> 

 Subject 

 Conditional Grant for Fifth 

 Extension(Document link: Ilana 

 Saltzbart) 

 

Lori,

In light of the Nov. 11 deadline, this e-mail will serve to

conditionally grant your request for an extension. I will speak with

Cathy regarding the new date that you have proposed and EPA either

accept that date or identify a different date. Thanks.

Ilana Saltzbart
Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.

 

 Lori E Stephans 

 <Lori.E.Stephans 

 @USA.dupont.com> To 

 Ilana Saltzbart/DC/USEPA/US@EPA 

 11/08/2006 04:08 cc 

 PM Andrea V Malinowski 

 <Andrea.V.Malinowski@USA.dupont.c 

 om> 

 Subject 

 Re: deadline Biodegradation 

 SEP-advice requested 

 

IlanaI will be submitting via over night mail tomorrow a 5th request for

extension for agreement to reach a procedure for purification. I hope

the

date I chose will give us plenty of time to complete the path forward

experiments and come to agreement on a procedure. The goal of course

would

be to complete the work as soon as possible.

Please let me know if you have any questions or concerns.

We look forward to talking next week.

Regards,

Lori

(See attached file: CD 2006 30 5th Request for Extension.pdf)

Lori E. Stephans, Ph.D.

Manager, Supplemental Environmental Programs

Rm 4110, Jackson Laboratory

Route 130, Chambers Works

Deepwater, NJ 08023

856-540-3748 (office)

856-540-4077 (fax)
856-341-0255 (cell)

lori.e.stephans@usa.dupont.com

This email message is for the sole use of the intended recipient(s) and

may

contain privileged information. Any unauthorized review, use, disclosure

or

distribution is prohibited. If you are not the intended recipient,

please

contact the sender by reply email and destroy all copies of the original

message.

 Saltzbart.Ilana@e

 pamail.epa.gov

To

 11/06/2006 03:34 Lori E Stephans/AE/DuPont@DuPont

 PM

cc

 Andrea V

 Malinowski/AE/DuPont@DuPont

Subject

 Re: deadline Biodegradation

 SEP-advice requested

Lori, please submit a request for extension and use your best judgment

regarding proposed extension dates.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.
 Lori E Stephans

 <Lori.E.Stephans

 @USA.dupont.com> To

 Ilana Saltzbart/DC/USEPA/US@EPA

 11/03/2006 09:46 cc

 AM Andrea V Malinowski

 <Andrea.V.Malinowski@USA.dupont.c

 om>

 Subject

 deadline Biodegradation

 SEP-advice requested

IlanaIn our path forward agreement dated 08.23.06, the following deadlines

are

expressed:

"The deadline for the parties to reach agreement on the procedure(s)

that

DuPont will use to purify the fluorotelomer products is November 27,

2006.

On or before November 11, 2006, DuPont must provide EPA with all reports

generated pursuant to this proposal and all data associated with the

work

performed pursuant to this proposal."

Since we are not complete even with the equivalency studies, how would

you

like me to proceed with the November 11 and November 27 deadlines.

Shall I

write a 5th request for extension?

November 11 is actually next Saturday and I am in all day site safety

training Tuesday, Wednesday and Thursday. I will need to handle a

formal

request for extension on Monday or Friday.

Thanks,

Lori

Lori E. Stephans, Ph.D.

Manager, Supplemental Environmental Programs

Rm 4110, Jackson Laboratory

Route 130, Chambers Works
Deepwater, NJ 08023

856-540-3748 (office)

856-540-4077 (fax)

856-341-0255 (cell)

lori.e.stephans@usa.dupont.com

This email message is for the sole use of the intended recipient(s) and

may

contain privileged information. Any unauthorized review, use, disclosure

or

distribution is prohibited. If you are not the intended recipient,

please

contact the sender by reply email and destroy all copies of the original

message.

This communication is for use by the intended recipient and contains

information that may be Privileged, confidential or copyrighted under

applicable law. If you are not the intended recipient, you are hereby

formally notified that any use, copying or distribution of this e-mail,

in whole or in part, is strictly prohibited. Please notify the sender by

return e-mail and delete this e-mail from your system. Unless explicitly

and conspicuously designated as "E-Contract Intended", this e-mail does

not constitute a contract offer, a contract amendment, or an acceptance

of a contract offer. This e-mail does not constitute a consent to the

use of sender's contact information for direct marketing purposes or for

transfers of data to third parties.

Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean

 http://www.DuPont.com/corp/email_disclaimer.html

This communication is for use by the intended recipient and contains

information that may be Privileged, confidential or copyrighted under

applicable law. If you are not the intended recipient, you are hereby

formally notified that any use, copying or distribution of this e-mail,

in whole or in part, is strictly prohibited. Please notify the sender by

return e-mail and delete this e-mail from your system. Unless explicitly

and conspicuously designated as "E-Contract Intended", this e-mail does

not constitute a contract offer, a contract amendment, or an acceptance

of a contract offer. This e-mail does not constitute a consent to the

use of sender's contact information for direct marketing purposes or for

transfers of data to third parties.

Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean

 http://www.DuPont.com/corp/email_disclaimer.html

[attachment "CD 2006 30 5th Request for Extension.pdf" deleted by Ilana

Saltzbart/DC/USEPA/US]
Saltzbart.Ilana@epamail.epa.

gov

11/22/2006 04:09 PM

To Lori E Stephans/AE/DuPont@DuPont

cc Lynch.David@epamail.epa.gov

bcc

Subject Next Steps in the Purification Path Forward

For Follow Up: Normal Priority

History: This message has been forwarded.

Lori,

At this time, EPA believes that the most prudent course of action is for

Exygen to perform all of the analytical work for the control experiments

and the sparge for Group 1, Substance 3 and Group 3, Substance 2, under

the terms of the August 23, 2006 Purification Path Forward Agreement.

However, based upon the information and data that EPA has received thus

far pertaining to measuring PFOA in the control systems, EPA believe it

is necessary for Exygen to demonstrate its accuracy in measuring PFOA.

Towards that end, we would like Exygen to propose a design for an

experiment that demonstrates Exygen's accuracy in measuring PFOA.

Exygen should submit this to EPA (through you) by COB Friday, December

1. Because Exygen agreed that it would perform a second round of

equivalence testing at no extra charge to EPA, it is EPA's expectation

that the same term would apply to this experiment to demonstrate

accuracy since EPA is not requesting RAS and Exygen to go through a

second round of equivalence testing.

With regard to spike recovery, EPA does not have sufficient data from

Exygen to conclusively demonstrate that with the SPE procedure, that

Exygen has achieved acceptable spike recovery (i.e., within a range of

75% -- 110%). In order to conclusively demonstrate that Exygen has

achieved acceptable spike recovery, EPA requests either: 1) additional

data from the work that Exygen has already performed or 2) additional

work that clearly demonstrates that Exygen has achieved acceptable spike

recovery.

I will be out of the office until Wednesday, November 29. If you have

any questions pertaining to this e-mail before I return, please contact

David Lynch at lynch.david@epa.gov.

Have a wonderful holiday.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.
RESEARC H

Precise Research.

Proven Results.

November 28, 2006

Lori E. Stephans, Ph.D.

Manager, Supplemental Environmental Programs

Rm 4110, Jackson Laboratory

Route 130, Chambers Works

Deepwater, NJ 08023

Dear Lori:

In response to liana Saltzbart's email dated 11/22/06 concerning Exygen Research's need

to demonstrate accuracy in recovering PFOA from the pot samples by SPE, the following

is proposed:

Exygen proposes to do a 1-day study, free of charge to the EPA, to verify recoveries in

the pot sample solutions. This will satisfy the EPA's request for additional work that

clearly demonstrates that Exygen can achieve acceptable spike recoveries. The

verification study will be performed on the second shipment of pot samples received at

Exygen Research on August 15, 2006. These samples are the ones diluted by Peter

Stchur 1000 times and kept at refrigerated conditions for the entire study (including

shipping).

TLF 10094 El 11184-48A LC/MS/MS (L9440-l)--Exygen Result=Not Detected

TLF 10094 El 11184-48B LC/MS/MS (L9440-2)--Exygen Result = 2.1 ng/mL

TLF 10094 El 11184-48C LC/MS/MS (L9440-3)»Exygen Result = 21 ng/mL

Each sample above will be spiked in triplicate at 3 levels (at -level found in sample, -1/2

level found in sample and ~2x the level found in the sample). The control sample (48A)

will be spiked at the same levels as 48B. A portion of the sample will then be diluted to

40 mL and be put through the SPE extraction and LC/MS/MS analysis procedure listed in

Exygen Method 01M-008-046 Revision 2. This will give 27 spike recovery values to

demonstrate accuracy. If the 27 spike recovery values obtained are between the EPA's

acceptable range of 75-110 % then Exygen will have proven that acceptable spike

recoveries can be obtained using the SPE procedure on the pot samples.

All results and original raw data from the 1-day verification study will be presented in a

report at the conclusion of the trial.

p;0uQ ,^^3058 Research Drive Kjsna

 ^m ^^ College, PA 16801, USA

J: 814.272.1039

F: 814.231.1580

exygen.com
RESEARC H

^.Precise Research.

Proven Results.

TITLE

Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water

Revision 2

AUTHORS

John Flaherty and Karen Risha

DATE ISSUED

August 15, 2005

SPONSOR

DuPont Corporation

P.O. BOX 80302

Wilmington, DE 19880-0302

PERFORMING LABORATORY

Exygen Research

3058 Research Drive

State College, PA 16801

METHOD NUMBER

01M-008-046 Revision 2 (V0001013)

TOTAL NUMBER OF PAGES

28

J^305f

^T^Stal

V

3058 Research Drive

' State College, PA 16801, USA

.7:800.281.3219

F: 814.272.1019

exygen.com
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

MANAGEMENT APPROVAL

6hn Flaherty Date

Vice-President

Exygen Research

^

Richard A. Grazzini, Ph.D. (j*&&*j Date I

President

Exygen Research

Exygen Research Page 2 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

TABLE OF CONTENTS

TITLE 1

MANAGEMENT APPROVAL 2

TABLE OF CONTENTS 3

LIST OF FIGURES 4

1. SUMMARY 5

2. EXPERIMENTAL COMPOUNDS 6

3. CHEMICALS AND SUPPLIES 6

3.1. CHEMICALS 6

3.2. STANDARDS 6

3.3. EQUIPMENT AND SUPPLIES 7

3.4. SOLUTIONS 8

3.5. PREPARATION OF STANDARDS AND FORTIFICATION SOLUTIONS 8

3.5.1. Stock solution 8

3.5.2. Fortification Solutions 9

3.5.3. Calibration Standards 9

3.5.4. Controlling Standard Bias 10

4. METHOD 11

4.1. FLOW DIAGRAM 11

4.2. SAMPLE PROCESSING 11

4.3. SAMPLE PREPARATION 12

4.4. EXTRACTION 12

4.5. QUANTITATION 13

4.5.1. LC/MS/MS System and Operating Conditions (Electrospray) 13

4.5.2. Example MS Operating Parameters 14

4.5.3. Calibration Curve Procedures 15

4.5.4. Sample Analysis 15

4.6. ACCEPTANCE CRITERIA 16

4.7. MASS CALIBRATION CRITERIA 17

4.8. PERFORMANCE CRITERIA 18

4.9. LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ) 18

4.10. TIME REQUIRED FOR ANALYSIS 19

5. CALCULATIONS 19

6. SAFETY 20

7. REFERENCES 20

Exygen Research Page 3 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

LIST OF FIGURES

Figure 1. Calibration curve forPFOA 21

Figure 2. Representative Chromatogram of a 50 ng/L PFOA Standard 22

Figure 3. Representative Chromatogram of a 100 ng/L PFOA Standard 22

Figure 4. Representative Chromatogram of a 250 ng/L PFOA Standard 23

Figure 5. Representative Chromatogram of a 500 ng/L PFOA Standard 23

Figure 6. Representative Chromatogram of 1000 ng/L PFOA Standard 24

Figure 7. Chromatogram of PFOA in Reagent Control Sample 24

Figure 8. Chromatogram of PFOA in a Drinking Water Control Sample 25

Figure 9. Chromatogram of PFOA in a Drinking Water Control Sample

Fortified at 100 ng/L 25

Figure 10. Chromatogram of PFOA in a Stream Water Control Sample 26

Figure 11. Chromatogram of PFOA in a Stream Water Control Sample

Fortified at 100 ng/L 26

Figure 12. Chromatogram of PFOA in a Well Water Control Sample 27

Figure 13. Chromatogram of PFOA in a Well Water Control Sample

Fortified at 100 ng/L 27

Figure 14. Chromatogram of PFOA in a Tap Water Control Sample 28

Figure 15. Chromatogram of PFOA in a Tap Water Control Sample

Fortified at 100 ng/L 28

Exygen Research Page 4 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Reason for revision: Added changes as result of discussions between DuPont and EPA.

1. SUMMARY

This report details a method of analysis for residues of ammonium

perfluorooctanoate (APFO) in water.

APFO forms perfluorooctanoic acid (PFOA) in solution. PFOA is extracted

from water using Cis solid phase extraction (SPE) cartridges. Quantification of

PFOA is accomplished by liquid chromatography/tandem mass spectrometry

(LC/MS/MS) analysis using selected reaction monitoring (SRM). Residues of

PFOA found are mathematically converted and reported as APFO. The chemical

formula of APFO is given in Section 2 of this method.

The anticipated limit of quantitation (LOQ; the lowest fortification specified by

the method which gives adequate recovery according to EPA guidelines) for this

method is 50 ng/L (parts-per-trillion) [or 0.05 micrograms/liter] for APFO.

Quantification is performed using calibration standards prepared in filtered

HPLC water and processed through the extraction procedure, identical to

samples. During the extraction procedure, all samples (including standards,

controls, fortifications etc.) are concentrated by a factor of eight; therefore, the

smallest standard (25 ng/L) injected during the chromatographic run is

equivalent to 200 ng/L (concentrated 8x). The method detection limit (MDL) is

approximately 6 to 15 ng/L depending on the instrument used for analysis. This

value is based on procedures found in 40 CFR Part 136 Appendix B.

This method was developed using stream water, well water, tap water, and

drinking water (bottled water). Typical percent recoveries ± standard deviations

(at 100 and 500 ng/L) for APFO in tap water were 112% ± 4.1%; in well water

were 104% ± 2.3%; in stream water were 108% ± 3.3%; and in drinking water

were 110% ±3.3%.

A representative calibration curve is shown in Figure 1. Representative

chromatograms are shown in Figures 2 to 15.

Exygen Research Page 5 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

2. EXPERIMENTAL COMPOUNDS

The structure of ammonium perfluorooctanoate is given below.

APFO

Chemical Name

Molecular weight

Ammonium Perfluorooctanoate

431

F F F O

O" NH4

F F F F

Note: The PFOA (anion) (formula weight 413) is present in solution and is used

for the assay.

3. CHEMICALS AND SUPPLIES

3.1. CHEMICALS

Chemical

Methanol (MeOH)

Ammonium Acetate

Water

Sodium Thiosulfate

Grade Source

HPLC EMD

Reagent Sigma-Aldrich

HPLC EMD

Reagent JT Baker

Catalog No.

MX0475-1

A-7330

WX0004-1

STANDARDS

Standard Lot Number Purity (%) Source

Pentadecafluorooctanoic 08316DO

Acid

Perfluorooctanoic Acid U14D

96 Aldrich Chem

96 Fluorochem USA

Exygen Research Page 6 of28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

3.3. EQUIPMENT AND SUPPLIES

Equipment Supplier

Balance, analytical (display at least 0.0001 g) Mettler

Vacuum pump Buchi

Visiprep vacuum manifold Supelco

Sep Pak Vac 6 cc (Ig) tC18 cartridges Waters

(part # WAT 036795)

50 mL disposable polypropylene centrifuge tubes VWR

15 mL disposable polypropylene centrifuge tubes VWR

Disposable glass micropipets (50-100 uL, 100-200 Drummond (VWR)

uL)

25 mm diameter glass fibre acrodisc 0.45 u (cat Gelman

#4523)

Class A pipets and volumetric flasks various suppliers

Hypercarb drop-in guard column (4 mm) Keystone

(part #844017-400)

Stand-alone drop-in guard cartridge holder Keystone

125-mL LDPE narrow-mouth bottles Nalgene

HPLC pump (LC10AD) Shimadzu

2 mL clear HPLC vial kit (cat #5181 -3400) HP

Standard lab equipment (graduated cylinders, various suppliers

disposable tubes etc.)

LC/MS/MS and HPLC systems As described in

section 4.5.

Note: Equivalent materials may be substituted for those specified in this method

if they can be shown to produce satisfactory results.

Notes:

1. In order to avoid contamination, the use of disposable labware is highly

recommended (tubes, pipets, etc.).

2. PTFE or PTFE lined containers or equipment, including PTFE-lined

HPLC vials for the HPLC autosampler must not be used.

3. HPLC grade water used during the sample and standard extraction should

be filtered through a Hypercarb guard column using a HPLC pump, as

described in section 4.2. This water is referred to as "filtered HPLC

water", here after in this report.

4. It is necessary to check the solvents (methanol) for the presence of

contaminants by LC/MS/MS before use. Certain lot numbers have been

found to be unsuitable for use.

5. Use disposable micropipets or pipets to aliquot standard solutions to

make calibration standards and sample fortifications.

Exygen Research Page 7 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

3.4. SOLUTIONS

(1) 50 mM ammonium acetate solution is prepared by weighing

3.86 g of ammonium acetate and dissolving in 1 L of HPLC water.

Dilute the 50 mM solution by a factor of 25 to make the 2 mM

ammonium acetate solution used for mobile phase A.

(2) 40% methanol is made by measuring 400 mL methanol and adjusting the

volume to 1 L with filtered HPLC water.

(3) 250 mg/mL sodium thiosulfate solution is made by dissolving 25 g in 100

mL filtered HPLC water.

Note: The aforementioned examples are provided for guidance, alternative

volumes may be prepared as long as the ratios of the solvent to solute ratios are

maintained.

3.5. PREPARATION OF STANDARDS AND FORTIFICATION SOLUTIONS

Analytical standards are used for three purposes:

1. Calibration Standards - These standards are prepared in filtered HPLC

water and are used to calibrate the response of the detector used in the

analysis.

2. Laboratory Control Spikes - These fortifications are prepared at

concentrations corresponding to the LOQ and lOx LOQ and are used to

determine analytical recovery. Laboratory control spikes are prepared in

filtered HPLC water.

3. Matrix Spikes - These fortifications are prepared by spiking into the

field samples at concentrations ranging from 500 ng/L up to 500,000

ng/L. Matrix spikes are used to evaluate the effect of the sample matrix

on analytical recovery.

The absolute volumes of the standards may be varied by the analyst as long as

the correct proportions of solute to solvent are maintained.

3.5.1. Stock solution

Prepare a stock solution of 100 ug/mL of PFOA by weighing out 10 mg

of analytical standard (corrected for purity) and dilute to 100 mL with

methanol in a 100-mL volumetric flask. The stock solution (in 125-mL

LDPE bottles) is to be stored in a refrigerator at 2°C to 6°C and is stable

for a maximum period of 6 months from the date of preparation.

Exygen Research Page 8 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

3.5.2. Fortification Solutions

a. Prepare a fortification standard of 1.0 ug/mL (1000 ng/mL) of PFOA

by adding 1.0 mL of the 100 ug/mL stock solution to a 100-mL

volumetric flask and bring up to volume with methanol.

b. Prepare a fortification standard of 0.1 ug/mL (100 ng/mL) by diluting

10.0 mL of the 1.0 ug/mL solution to 100 mL with methanol in a

volumetric flask.

c. Prepare a fortification standard of 0.01 ug/mL (10 ng/mL) by diluting

10.0 mL of the 0.1 ug/mL solution to 100 mL with methanol in a

volumetric flask.

Example: two hundred microliters of the 10 ng/mL solution spiked into

40 mL of water is equivalent to a 0.05 ppb (50 ng/L) fortification.

Store all fortification standard solutions in a refrigerator (in 125-mL

LDPE bottles) at 2°C to 6°C for a maximum period of 3 months from the

date of preparation, after which time it is necessary to make new

standards using the stock solution. Note also that additional

concentrations may be prepared if necessary.

3.5.3. Calibration Standards

LC/MS/MS calibration standards are prepared in HPLC water filtered

through a hypercarb drop-in guard cartridge. The calibration standards

are processed through the extraction procedure, identical to samples. The

extracted standards may be used for a period of two weeks when stored

refrigerated (at 2°C to 6°C).

The following is a typical example: additional concentrations may be

prepared as needed.

Concentration Fortification Volume of

of Fortification Volume Fortified Control

Solution (ng/mL) (uL) Sample (mL)

0

10

10

10

100

100

100

0

100

200

400

100

200

400

40

40

40

40

40

40

40

Final

Concentration of Calibration

Calibration Standard ID

Standard (ng/L)* (example)

0

25

50

100

250

500

1000

Xcmmddyy-0

Xcmmddyy-1

Xcmmddyy-2

Xcmmddyy-3

Xcmmddyy-4

Xcmmddyy-5

Xcmmddyy-6

* The extracted concentration of the calibration

concentration, due to the concentration of the

(SPE). XC = extracted calibration standard.

standard is equal to 8x its initial

standard during the extraction

A zero standard solution (reagent blank) must be prepared with each set

of standards extracted. This standard is used only to access the reagents

Exygen Research Page 9 of28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

used to prepare the standards and is not included as part of the calibration

curve.

Store all extracted calibration standards in 15-mL polypropylene tubes at

2°C to 6°C, up to two weeks.

3.5.4 Controlling Standard Bias

Prepare a separate stock solution of 100 ug/mL from a different PFOA

neat standard from a different source by weighing out 10 mg of analytical

standard (corrected for purity only) and dilute to 100 mL with methanol

in a 100-mL volumetric flask. The stock solution (in 125-mL LDPE

bottles) is to be stored in a refrigerator at 2°C to 6°C and is stable for a

maximum period of 6 months from the date of preparation.

Prepare a fortification standard of 1.0 jig/mL (1000 ng/mL) of PFOA by

adding 1.0 mL of the 100 ug/mL stock solution to a 100-mL volumetric

flask and bring up to volume with methanol. From that solution, prepare

a fortification standard of 0.1 ng/mL (100 ng/mL) by diluting 10.0 mL of

the 1.0 jig/mL solution to 100 mL with methanol in a volumetric flask.

Prepare a 250 ng/L extracted calibration standard with each set of

extracted standards. Add 100 uL of the 100 ng/mL fortification solution

to 40 mL of hypercarb filtered HPLC water and process through the

extraction procedure, identical to samples.

Note: This standard is referred to as the "check standard"

from this point forward.

The check standard should be included at least once with each set of

extracts analyzed. The check standard should give a response within ±

15% of the average response from the 250 ng/L extracted calibration

standards in the set.

Exygen Research Page 10 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

4. METHOD

4.1. FLOW DIAGRAM

The flow diagram of the method is given below, followed by a detailed

description of each step.

Method Flow Diagram

Measure 40 mL of sample

(fortify samples designated as calibration standards and fresh fortifications)

4

4-

Ci8 Solid-Phase Extraction

Final Volume (5 mL)

LC/MS/MS

4.2. SAMPLE PROCESSING

No sample processing is needed for water samples. However, frozen samples

must be allowed to completely thaw, un-aided, at room temperature. Samples

stored refrigerated should also be allowed to equilibrate to room temperature.

All samples must be thoroughly mixed and any visible solids removed by

filtering the sample through a 25-mm diameter glass fibre acrodisc 0.45 u filter

before being sampled for extraction.

Initial studies show that the method is not effected by the pH of the sample.

Sample pH is recorded prior to extraction.

Control HPLC water, used for the preparation of samples and calibration

standards as well as water used for equilibrating and washing the SPE cartridges,

must be prepared by filtering HPLC water through a hypercarb guard cartridge

using a HPLC pump. Before use, wash the guard cartridge with ~ 25 ml HPLC

grade acetonitrile, then -25 mL of HPLC grade methanol, followed by ~ 25 mL

of HPLC water using the HPLC pump at ~ 2 mL/min flow rate. Then start

collecting the filtered HPLC water eluate from the cartridge. It is recommended

that the cartridge be washed, as described above, after filtering ~ 2 liters of

HPLC water. A flow rate of 2 to 3 mL/min is recommended.

Exygen Research Page 11 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

4.3. SAMPLE PREPARATION

a. Each batch of samples extracted (typically 20 or less) must include at least

one reagent control (method blank using filtered HPLC water) and two

reagent controls fortified at known concentrations to verify procedural

recovery for the batch.

b. Each sample must be extracted in replicate unless the sample is marked as a

field blank. Field blanks do not need to be extracted in replicate.

c. Each sample extracted (except for field blanks) must also be separately

fortified at a known concentration and carried through the procedure to verify

recovery. Matrix spikes should range from 500 ng/L to 500,000 ng/L. If the

sample residue is found to be significantly higher than the level at which the

sample was initially fortified, the sample should be fortified at a higher

concentration (up to 500,000 ng/L) and re-extracted.

4.4. EXTRACTION

1. Measure 40 mL of sample into 50 mL polypropylene centrifuge tubes

(fortify as needed, replace lid and mix well).

Note: For tap water samples and any water samples suspected to

contain chlorine, add 200 juL of a 250 mg/mL sodium thiosulfate solution

to the 40 mL sample and mix well to de-activate chlorine, before

fortification.

2. Condition the dg SPE cartridges (1 g, 6 mL) by passing 10 mL methanol

followed by 5 mL filtered HPLC water (~ 2 drop/sec). Do not let column

run dry.

Note: For the following steps, maintain a ~7 drop/ sec flow rate. Do not

allow the column to run dry at any time.

3. Load sample on conditioned Cig SPE cartridge. Discard eluate.

4. Wash with ~5 mL 40% methanol in water. Discard eluate.

5. Elute with ~5 mL 100% methanol. Collect 5 mL of eluate into graduated

15 mL polypropylene centrifuge tubes (final volume = 5 mL).

6. Analyze samples using electrospray LC/MS/MS.

NOTE: SAMPLES ARE CONCENTRATED BY A FACTOR OF EIGHT DURING THE

EXTRACTION; INITIAL VOL = 40 ML -> FINAL VOL. = 5 ML.

Exygen Research Page 12 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

4.5. QUANTITATION

4.5.1. LC/MS/MS System and Operating Conditions (Electrospray)

Mass Spec: Micromass Quattro Ultima (Micromass)

Interface: Electrospray (Micromass)

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 Quat Pump

HP Vacuum Degasser

HP Autosampler

HP Column Oven

Note: A 4 x 10 mm hypercarb drop-in guard cartridge (Keystone, part # 844017-

400) is 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 Cg (Jones Chromatography), 2.1 mm x 50 mm, 4|ti

Column Temperature: 35° C

Injection Volume: 15 fiL

Mobile Phase (A): 2 mM Ammonium Acetate in HPLC water

Mobile Phase (B): Methanol

Time %A

0.0 60

0.4 60

1.0 10

7.0 10

7.5 0

9.0 0

9.5 60

13.5 60

14.0 60

15.0 60

It may be necessary to adjust the HPLC gradient in order to optimize instrument

performance. Columns with different dimensions (e.g. 2.1 x 30) and also

columns from different manufacturers (Keystone Betasil Cig etc.) could be used,

provided equivalent chromatography is obtained.

Ions monitored:

Approximate

Analvte Mode Transition Monitored Retention Time

PFOA Negative 413 -> 369 ~5 min.

%B

40

40

90

90

100

100

40

40

40

40

Flow Rate CmL/min)

0.3

0.3

0.3

0.3

0.3

0.4

0.4

0.4

0.3

0.3

Exygen Research Page 13 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

The retention times may vary, on a day to day basis, depending on the batch of

mobile phase etc. Drift in retention times (up to ±2 %) is acceptable within an

analytical run, as long as the drift continues through the entire analysis and the

standards are included at the beginning and end of the analytical run.

Note: An alternative LC/MS/MS system may be used once demonstrated to be

equivalent.

4. 5. 2. Example MS Operating Parameters

The following MS operating values are provided as an example only. Actual

values will most likely vary from instrument to instrument. These values may

also change over time even when analyzing samples on the same instrument.

The values are changed to optimize for greatest sensitivity. For example,

hexapole 1 voltage may be set to 2 V; however, as time goes on, hexapole 1

voltage may be increased or decreased. The exact values used each time a set is

analyzed will be documented with each data set.

The mass spectrometer is tuned for the analyte by infusing a ~ 0.2

standard solution of PFOA (at 10 fiL/min, using an infusion pump) via a "T" into

a stream of mobile phase containing 40% methanol and 60% 2mM ammonium

acetate at 0.2 mL/min flow rate. The analyte is initially tuned for the parent ion

and then tuned for the product ion. Once the instrument is tuned, the optimized

parameters are saved as a tune file. This tune file is then used during routine

analysis.

Analvte Dwell (s) Collision Energy (eV) Cone (V)

PFOA 0.2 10 10

Source Set

Capillary 3.0 kV

Hexapole 1 0 V

Aperture 1 0 V

Hexapole 2 0 V

Source Block Temp. 1 00°C

Desolvation Temp. 300°C

Analyzer Set

LMResl 14.0V

HM Res 1 14.0 V

lEnergy 1 IV

Entrance -2 V

Exit 2 V

LM Res 2 14.0 V

HMRes2 14.0V

lEnergy 2 2.0 V

Multiplier 700 V

Exygen Research Page 14 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Gas Flows Set

Cone Gas ~130L/hr

Desolvation -750 L/hr

Pressures Read back

Gas Cell ~3.0e-3mbar

4.5.3. Calibration Curve Procedures

a. Inject the same aliquot (between 10 to 25 uL) of each extracted calibration

standard processed in matrix (ranging from the lowest level standard to the

highest level prepared), into the LC/MS/MS (the final concentrations of these

calibration standards will be equivalent to 8x the concentration of the initial

standard due to the concentration during SPE).

b. Use linear standard curves for quantitation. The zero calibration standard is

not included in the calculation of the calibration curve. Linear standard

curves are generated for each analyte by linear regression using 1/x

weighting of peak area versus calibration standard concentration using

MassLynx 3.3 (or equivalent) software system. Any calibration standard

found to be a statistical outlier by using the Huge Error Test, may be

excluded from the calculation of the calibration curve. However, the total

number of extracted calibration standards that may be excluded must not

exceed 20% of the total number of extracted standards injected.

c. The correlation coefficient (R) for calibration curves generated must be

>0.992 (R2 >0.985). If calibration results fall outside these limits, then

appropriate steps must be taken to adjust instrument operation, and the

standards or the relevant set of samples should be reanalyzed.

A typical calibration curve for PFO A can be found in Figure 1.

4.5.4. Sample Analysis

a. Inject the same aliquot (between 10 to 25 uL) of each standard, sample,

recovery, control, etc. into the LC/MS/MS system.

b. Standards corresponding to at least five or more concentration levels (starting

with the LOQ level or below) must be included in an analytical set (note that

the calibration standards are prepared by spiking filtered HPLC water and

processing similar to actual samples).

c. An entire set of extracted calibration standards should be injected at the

beginning of a set followed by extracted calibration standards interspersed

Exygen Research Page 15 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

approximately every 5-10 samples (to account for a second set of extracted

standards). As an alternative, an entire set of extracted calibration standards

may be included at the beginning and at the end of a sample set. In either

case, extracted calibration standards must be the first and last injection in a

sample set.

d. Inject the check standard once with each set of extracts.

e. The concentration of each sample/fortification/control is determined from the

standard curve, based on the peak area of each analyte. The standard

responses should bracket responses of the residue found in each sample set.

If necessary, dilute the samples to give a response within the standard curve

range.

f. Fortification recoveries falling within 70 to 130% are considered acceptable.

g. The total holding time between sample collection and analysis must not

exceed 14 days. Extracted samples must be stored refrigerated between 2°C

to 6°C until analysis.

h. Field samples in which either no peaks or peaks less than the MDL are

detected at the corresponding analyte retention time will be reported as ND

(not detected). Samples in which peaks are detected at the corresponding

analyte retention time that are less than the LOQ and greater than or equal to

the MDL will be reported as NQ (not quantifiable). All chromatograms are

considered raw data and as such, will be included in the data package with

each report.

The analysis performed during the method development included fortifications at

100 and 500 ng/L of PFOA in well water, tap water, and stream water (collected

locally, State College, PA) and bottled drinking water (from a local grocery

store).

4.6. ACCEPTANCE CRITERIA

The following criteria must be met to ensure the presence of PFOA:

1. Chromatogram must show a peak of a daughter ion at 369 amu from a

parent of 413 amu. The 413 amu parent corresponds to the PFOA anion,

while the daughter ion (369 amu) represents the loss of carbon dioxide.

This transition would not discriminate between linear and branched forms

of PFOA; therefore, both, if present, would be included in the calculation.

If the branched isomer peaks are resolved or partially resolved from the

linear peak, the summed peak areas will be used in the calculation.

Exygen Research Page 16 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

2. Method blanks must not contain PFOA at levels greater than the LOD. If

a blank contains PFOA at levels greater than LOD, then a new blank

sample must be obtained and the entire set must be re-extracted.

3. Recoveries of control spikes and matrix spikes must be between 70-130%

of their known values. If a control spike falls outside the acceptable

limits, the entire set of samples should be re-extracted. If a matrix spike

being used to evaluate an entire extraction set falls outside 70-130%, the

entire set must be re-extracted. If a matrix spike being used to evaluate

an individual sample falls outside 70-130%, the matrix spike along with

its corresponding sample and replicate must be re-extracted.

4. Any calibration standard found to be a statistical outlier by using the

Huge Error Test, may be excluded from the calculation of the calibration

curve. However, the total number of extracted calibration standards that

could be excluded must not exceed 20% of the total number of extracted

standards injected.

5. The correlation coefficient (R) for calibration curves generated must be

>0.992 (R2 >0.985). If calibration results fall outside these limits, then

appropriate steps must be taken to adjust instrument operation, and the

standards or the relevant set of samples should be reanalyzed.

6. The response from the check standard should be within ± 15% of the

average response from the 250 ng/L calibration standard concentrations.

If the standard response falls outside these limits, appropriate steps must

be taken to adjust instrument operation and the set of samples should be

reanalyzed. If the standard response is still outside the ± 15% then

standard preparations should be reviewed, and if warranted, new stock,

fortification and extracted calibration solutions should be prepared. Both

sets of data would then be included in the data package.

7. Retention times between standards and samples must not drift more than

± 2 % within an analytical run. If retention time drift exceeds this limit

within an analytical run then the set must be reanalyzed.

4.7. MASS CALIBRATION CRITERIA

In order to verify mass resolution, a calibration check is performed weekly on the

mass spectrometer using a Nal/Rb/Cs tuning solution. The tuning solution is

infused onto the mass spec using an infusion pump set to a flow rate of 10

uL/min. An MS scan from 80 to 1380 amu is performed on both MSI and MS2.

The calibration is considered acceptable if the following 11 peaks are found

within ± 0.2 amu of their known masses on both MSI and MS2: 84.9, 132.9,

172.8, 322.7, 472.6, 622.5, 772.4, 922.3, 1072.2, 1222.1 and 1372.0. If any of

Exygen Research Page 17 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

the masses are outside the ± 0.2 amu, then the instrument will be recalibrated and

another calibration check performed.

4.8. PERFORMANCE CRITERIA

The following two criteria must be performed as a system suitability test, before

the commencement of analysis when using an instrumentation set-up that has not

been used for this method.

First Criterion:

Run a standard solution on LC/MS/MS corresponding to the estimated LOQ

(50 ng/L) in matrix and obtain a signal to noise ratio of the 413 ->• 369 transition

of at least 9:1, compared to a reagent blank. If this criterion cannot be met,

optimize and change instrument operating parameters (or increase the injection

volume, if appropriate).

Second Criterion:

Run a set of standards of five or more concentration levels, from 1/2 LOQ, up to

the highest concentration level to be included in the analysis. Generate a

calibration curve for PFOA and obtain a linear regression with a coefficient of

determination (R2) of at least 0.985 for PFOA. Once this criterion is met,

samples may be analyzed with standards interspersed.

4.9. LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ)

A Limit of Detection (LOD) and Limit of Quantitation (LOQ) will be reported

for each set of samples analyzed. The LOD is defined using the method blank

(or reagent blank), and is the concentration (as would be expressed in the original

samples) that would correspond to 3 times the peak-to-peak amplitude of the

baseline noise in the vicinity of the PFOA peak. If laboratory or instrumental

background contamination or interference is present, the LOD will be the

concentration that corresponds to three times the amplitude of the background

peak at the retention time of PFOA. The LOQ will be defined as 5 times the

LOD. PFOA levels in the samples below the LOD will be reported as "Not

Detected" (ND). PFOA levels in samples greater than LOD but less than LOQ

will be reported as "Not Quantifiable" (NQ). Values at or greater than LOQ will

be reported as numerical values, rounded to two significant figures. All raw data

will be included in the data package, including peak areas below LOD or LOQ.

LOD/LOQ will be determined, as above, at least quarterly.

Exygen Research Page 18 of 2 8
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

4.10. TIME REQUIRED FOR ANALYSIS

A set of 24 samples (6 standards and 18 samples) can be taken through the

extraction procedure in approximately 2-3 hours by one person. The LC/MS/MS

analysis (standards and 18 samples) will take approximately 7 hours.

5. CALCULATIONS

a. Use Equation 1 to calculate the amount of PFOA found (in ng/L, based on

peak area) using the standard curve (linear regression parameters) generated

by the Mass Lynx software program.

Equation 1:

PFOA found (ng/L) = (Peak area - intercept) x DF

slope

DF = factor by which the final volume was diluted, if necessary.

b. For samples fortified with known amounts of PFOA prior to extraction, use

Equation 2 to calculate the percent recovery.

Equation 2:

Recovery (%) =

[ total analyte found (ng/L) - analyte found in control (ng/L)]

analyte added (ng/L)

Note: Subtract analyte found in control (ng/L) from analyte found (ng/L), if

applicable.

c. If necessary, use Equation 3 to calculate the amount of APFO found (in ng/L)

using the molecular weight ratio of APFO/PFOA:

Equation 3:

MW APFO (431)

APFO found (ng/L) - PFOA Found x

MW PFOA (414)

Exygen Research Page 19 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Note: calculations could also be performed in ppb or ng/mL if concentrations

were assigned in ng/mL instead of ng/L when performing the analysis.

For reporting purposes, field samples in which either no peaks or peaks less than

the MDL are detected at the corresponding analyte retention time will be

reported as ND (not detected). Samples in which peaks are detected at the

corresponding analyte retention time that are less than the LOQ and greater than

or equal to the MDL will be reported as NQ (not quantifiable).

6. SAFETY

The analyst should read the material safety data sheets for all standards and

reagents before performing this method. Use universal precautions when

handling standards and reagents, including working in fume hoods and wearing

laboratory coats, safety glasses, and gloves.

7. REFERENCES

Ellis, D.A.; Martin, J.W.; Muir, D.C.G.; Mabury, S.A. Development of an

 19F

NMR Method for the Analysis of Fluorinated Acids in Environmental Water

Samples. Anal. Chem. 2000, 72, 726-731.

Moody, C.A.; Kwan, W.C.; Martin, J.W.; Muir D.C.G.; Mabury, S.A.

Determination of Perfluorinated Surfactants in Surface Water Samples by Two

Independent Analytical Techniques: Liquid Chromatography/Tandem Mass

Spectrometry and

 19F NMR. Anal. Chem. 2001, 73, 2200-2206.

Exygen Research Page 20 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 1. Calibration curve for PFOA

SompcuwS 1 rang; APFO

CwffidBlrtcf Otntmanatom; 0,991338

Caitjrartcn euro* W.7724 ' X

pofae ffl:n: Ejeemol $W, Am

Dim typo: L'rear, Crtplx Excktda.

5.ec»+-|

1

Rc*pa-acExygen Research Page 21 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 2. Representative Chromatogram of a 50 ng/L PFOA Standard

50pptCl21S39-2

1 £3199-2002 Sm (33,2x2)

21-DeC'19&& 23:34:04

LC/MS/MS unit 6

M.ftM of 3 ChannEls E5-

Area'

1,00 2.CO 3JOO 4.00 S.CO .6.00 7.QO

T\me

Figure 3. Representative Chromatogram of a 100 ng/L PFOA

Standard

tOOpptCl21S9^-3

1*2199-1003 Sm(£G, 2X2)

1 1 , . 1

1.GG

5,t6

21-Dec»1999 23:49:42!

LC/MS/MS unit 6

hCRM«f3Channala£S2.00 ' 3.00 4.00 5.PO 6.00 7.00

5^ Tmne

s.oa

Exygen Research Page 22 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 4. Representative Chromatogram of a 250 ng/L PFOA

Standard

122l99-ZG04Sm(SG.2*2}

100"!

S.09

36.1 tQ-1

1.QO 2,00

11 ,' I ' " I

3,00

22-PecM 99(9 00:05:35

LCJMSJMS unlt€

MRM frf 3 Channels ES

Area

5,00 6.00

Figure 5. Representative Chromatogram of a 500 ng/L PFOA

Standard

500'ppt.Cl21539-5

122-19^2006 Sm{SQ, 2x2)

1,00 2.00

22-Deu-l 999 00:21:22

5.C-9

MRM of 3 Channels ES413>3G9

Area

•4.00 5,00 6,00

i,.. . i .'i^^ypri'i Time

Exygen Research Page 23 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 6. Representative Chromatogram of 1000 ng/L PFOA Standard

10QOp33tCl;21599-5

123199-2006 Sin (SGrass2.)

Ofc37MS|

LCWS/MSlinfce

URM

'I i t I L I If I HP«Y'' ' J ''•;-!-• ' I ! ! l''i"l"<'"»

UaO 2.00 3sCO

413>G69

Affia

«.00 5:OQ 6.00 7.00

j ,!>^-vTimB

Figure 7. Chromatogram of PFOA in Reagent Control Sample

Reagent Corvtrdl

1.QQ ' ' £00

'I'1 I ' '?

3;DO 5-00

LCftfl$/MS urisi $,

MflM of 3 Channel ES*

USBe4

7.00

Timo

Exygen Research Page 24 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 8. Chromatogram of PFOA in a Drinking Water Control

Sample

Drinking water Confrci B

LG/MS/MS unit #

122188-2011 Sro (SO. 2*2) MRM of 3 Channels ES

100-i TM*x 7,39 413 > 369

3*03

Lv*wi

i.' " ' n' • '• < • • -"-I.' -•'i' ' •] i' " ' i' *' ' i' •' ' i' ' ' i •'' ' i' ""*-v 11.111 1 p i 1111 1 Time

1.CO 2.00 3,00 4.gQ &.CO 6.00

Figure 9. Chromatogram of PFOA in a Drinking Water Control

Sample Fortified at 100 ng/L

Drinking water 100 ppt Spfo A

LC/RAS/NIS unit 61

y^M eH 3 Channels ES-

£.09

15477

 I "llSeS

Area

0 i^-j-.-. ,.•!,, , n „. ;,,, . 111 ,-i...,...., .<, c ...,•... i |"'i=^rT771 i^yyn-.'. j... 111 iTr" Time

1,00 2.SO 3.Q& 4jQO S.OO 6.QO 7jCO

Exygen Research Page 25 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 10. Chromatogram of PFOA in a Stream Water Control

Sample

Stream water C&nirol A

121199-1010 Sm(SG, 2x2}

1-Ofti

19-D«-199916;17!4B

LGMS/MS unite

MPM a! 3 Chaniwts ES7,107-16 413> 369

s.oo -CLOT ?.to

Figure 11. Chromatogram of PFOA in a Stream Water Control

Sample Fortified at 100 ng/L

water 100 ppt Spk A

&05

unit 6

MRM of 3 Channels ES*

%i

1.00 2J30 '..9.00 ...; .-4.0.0. 5.0Q "^iTbb ' '' T^hn """^

Exygen Research Page 26 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 12. Chromatogram of PFOA in a Well Water Control Sample

Well Water oantroU 2.

121599-1 mSm (SO, 2x23

lOCh

00:49:361

MRMof3ChaimelaES

Figure 13. Chromatogram of PFOA in a Well Water Control Sample

Fortified at 100 ng/L

WeKWateMQOpptSpkA

tai 593*1118 SratSG,ax2)

10(h

5.15-

2S-413

16-Dec-1999 02:40:11

MRM ol 3 Channels ES413 > 339

1.00 2.00 4.60

Exygen Research Page 27 of 28
Exygen Method No: 01M-008-046 Revision 2 (V0001013)

Figure 14. Chromatogram of PFOA in a Tap Water Control Sample

Tap water Centre! A 23-Oec*19&& 17:34:11

LOWS/MS unit <

122.399^20-11Sm. (35,2X2)

413 > 369

JC^^^^V .„.

'-f]-!"'T"~., r '.' . , t . . I , ' I')," ...,!' " '' !i ''•'. : ' ! ! I ' ' • P^1 I ' • • • I ' , ! • • • , I • I I ,. . ! ! I |i|mS

1,00 2.&D 3rOO 4I..QO 5.00 6.&0 7.00

Figure 15. Chromatogram of PFOA in a Tap Water Control Sample

Fortified at 100 ng/L

12239&-3Q2Q Sm [SG. 2x2)

1QGH .^?L-i ' 413 > 369

t,95c5

Area

,11. 1 !["•,»-.".-.,.,,., |,,.,. , .1...;.. .: --|- ;." , "•"."--'•||.

1

. •.'.. twi . ^. i-Tt-qi^., , u , 111 11.," Tima1

l.DQ • g.QQ 3.00 4.00 S.QO 6.00 7,00

Exygen Research Page 28 of 28
Saltzbart.Ilana@epamail.epa.

gov

12/08/2006 02:09 PM

To Lori E Stephans/AE/DuPont@DuPont

cc

bcc

Subject Comments to Exygen's Proposed Verification Study and 

Method of Analysis

History: This message has been forwarded.

Lori,

EPA has had an opportunity to review Exygen's proposed "verification

study" as described in the November 28, 2006, letter from Karen Risha to

you, and transmitted to me on November 30, 2006. EPA has also had an

opportunity to review Exygen's "Method of Analysis for the Determination

of Perfluorooctanoic Acid (PFOA) in Water, Revision 2 ("Method of

Analysis)".

1. Overall, EPA found the Method of Analysis to be quite good.

However, Exygen should include a mass-labeled extraction surrogate and a

different mass-labeled internal standard.

2. Additionally, EPA seeks clarification from Exygen regarding how the

values for LOQ, LOD, and MDL relate to the calibration standards.

Additionally, please address whether, based upon the calibration

standards, there is the potential for reported concentrations below the

lowest standard concentration.

3. Finally, to eliminate uncertainties, for purposes of the

verification study, EPA has a strong preference that Exygen prepare a

fresh batch of the high, low, and blank controls as described in

DuPont's October 24, 2006 First Progress Report. In particular, Exygen

should follow the Control Sample Recipes in Section 3.1 for the high,

low, and blank. We hope that DuPont will provide any raw materials that

Exygen does not have to avoid any delay in ordering such raw materials.

EPA is providing a table that Exygen should use in reporting the data to

EPA. Exygen should include any additional information in the table that

it would report as standard operating procedure.

(See attached file: Data Quality Summary Table for Verification

Study.xls)

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.
Saltzbart.Ilana@epamail.epa.

gov

12/13/2006 05:35 PM

To Lori E Stephans/AE/DuPont@DuPont

cc chas.simons@exygen.com, jay.bernarding@exygen.com

bcc

Subject Re: Exygen feedback on cost issue.

My client has authorized the additional $2,500 expenditure for Exygen to

prepare the high, low, and blank control systems for the verification

study.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.

 

 Lori E Stephans 

 <Lori.E.Stephans 

 @USA.dupont.com> To 

 Sent by: Ilana Saltzbart/DC/USEPA/US@EPA 

 Lori.E.Stephans@ cc 

 USA.dupont.com 

 Subject 

 Re: Exygen feedback on cost 

 12/08/2006 07:49 issue. 

 PM 

 

Thanks Ilana. I'll give Exygen the Okay to proceed when I get the green

light from you.

----- Original Message -----

From: Saltzbart.Ilana

Sent: 12/08/2006 05:06 PM

To: Lori E Stephans/AE/DuPont@DuPont

Subject: Re: Exygen feedback on cost issue.

Thanks Lori. I'm waiting to hear back from my client regarding the

$2,500, and I'll follow-up with Exygen next week (with a cc to you).
Have a great vacation.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

received this communication in error, please delete the copy you

received, and do not print, copy, retransmit, disseminate, or otherwise

use the information. Thank You.

 Lori E Stephans

 <Lori.E.Stephans

 @USA.dupont.com> To

 Ilana Saltzbart/DC/USEPA/US@EPA

 12/08/2006 04:43 cc

 PM jay.bernarding@exygen.com

 Subject

 Exygen feedback on cost issue.

cc Jay Bernarding Exygen

IlanaHere was Exygen's response to the cost question you posed.

Exygen will agree to perform the verification study described in Karen's

letter of November 28, 2006 at no cost to EPA however, if EPA wants

Exygen

to prepare fresh blank, low control and high control samples in our

laboratory with ingredients provided by DuPont, the charge for this task

will be $2,500 as previously stated.

I hope this will work for EPA. Please let me know if I need to follow

up

with Exygen. Not a problem.

Regards,

Lori

Lori E. Stephans, Ph.D.
Manager, Supplemental Environmental Programs

Rm 4110, Jackson Laboratory

Route 130, Chambers Works

Deepwater, NJ 08023

856-540-3748 (office)

856-540-4077 (fax)

856-341-0255 (cell)

lori.e.stephans@usa.dupont.com

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Saltzbart.Ilana@epamail.epa.

gov

12/28/2006 01:00 PM

To Lori E Stephans/AE/DuPont@DuPont

cc

bcc

Subject Request for DuPont to Resubmit proposed RFPs for 

Characterization and Biodegradation Laboratories

History: This message has been replied to and forwarded.

Lori,

On February 28, 2006, EPA received DuPont's submission of the proposed

RFPs for the Characterization and Biodegradation laboratories. In light

of changes that EPA requested DuPont make to certain documents that are

part of the Panel Administrator package that are also part of the

proposed RFP packages, EPA is requesting that DuPont resubmit the

proposed RFPs with the same modifications that were made to such

documents in the Panel Administrator package. In particular, such

documents include Attachments J, L, and M. Also, please ensure that

the scope of work in each proposed RFP is an exact replication of the

SEP language.

Please resubmit these documents to Tony Ellis on or before February 1,

2006.

Thanks.

Ilana Saltzbart

Attorney-Advisor, US EPA, OECA, OCE

1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460

Phone: 202 564 9935

Fax: 202 564 0035

NOTICE: This communication may contain privileged or other confidential

information. If you are not the intended recipient, or believe you have

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