Document zo0Zj2km5ZkZvyQaw6Xeabjz3
INTERIM REPORT # 1-Analvsis of Public Water Samples
STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur
Monitoring Program
DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792
STUDY DIRECTOR Jaisimha Kesari P.E., DEE
Weston Solutions, Inc. 1400 Weston Way
West Chester, PA 19380 Phone: 610-701-3761
INTERIM REPORT COMPLETION DATE March 8, 2005
PERFORMING LABORATORY Exygen Research
3058 Research Drive State College, PA 16801
Phone: 814-272-1039
STUDY SPONSOR 3M Company
3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374
PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131
Total Pages: 99
Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research.
Principal Investigator Exygen Research
Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc.
Sponsor Representative 3M Company
Exygen Research
Date
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QUALITY ASSURANCE STATEMENT
Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director.
Phase
Date Insnected
Date Reported to Date Reported to
Principal
Exygen Date Reported to
Investigator Management Studv Director
1. Protocol Review
12/21/04
01/03/05
12/22/04
01/27/05
5. Raw Data Review
03/07/05
03/08/05
03/08/05
03/08/05
6. Interim Analytical Report Review
03/08/05
03/08/05
03/08/05
03/08/05
'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the protocol review, and the review of the interim report and associated raw data.
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CERTIFICATION OF AUTHENTICITY
This report, for Exygen Study Number P0001131, is a true and complete representation of the raw data for the study.
Submitted by:
Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039
Principal Investigator, Exygen:
Exygen Research Exygen Research Facility Management:
Date
Date
Study Director, Weston Solutions, Inc.
Sponsor Representative, 3M Company:
Michael A. S Director of Regulatory Affairs
Exygen Research
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STUDY IDENTIFICATION
Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur
Monitoring Program
PROTOCOL NUMBER: EXYGEN STUDY NUMBER: TYPE OF STUDY: SAMPLE MATRIX: TEST SUBSTANCE:
SPONSOR:
STUDY DIRECTOR:
P0001131
P0001131
Residue
Public Water
Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS)
3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144
Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380
STUDY MONITOR:
Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144
PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801
ANALYTICAL PHASE TIMETABLE:
Study Initiation Date:
11/05/04
Interim Analytical Start Date:
02/22/05
Interim Analytical Termination Date: 03/02/05
Interim Report Completion Date: 03/08/05
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PROJECT PERSONNEL
The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study:
Name John Flaherty Karen Risha Paul Connolly Chrissy Edwards Mark Ammerman Amy Sheehan
Title Vice President
Scientist Technical Lead-LC/MS
Technician Sample Custodian Associate Scientist
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TABLE OF CONTENTS
Page TITLE PAGE...................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT......................................................................... 3 CERTIFICATION OF AUTHENTICITY.......................................................................... 4 STUDY IDENTIFICATION............................................................................................... 5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................... 7 LIST OF TABLES.............................................................................................................. 8 LIST OF FIGURES............................................................................................................. 9 LIST OF APPENDICES................................................................................................... 10 1.0 SUMMARY............................................................................................................... 11 2.0 OBJECTIVE...............................................................................................................11 3.0 INTRODUCTION...................................................................................................... 11 4.0 ANALYTICAL TEST SAMPLES..............................................................................12 5.0 REFERENCE MATERIAL........................................................................................12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................13
6.1. Extraction Procedure...............................................................................................13 6.2 Preparation of Standards and Fortification Solutions...............................................13 6.3 Chromatography...................................................................................................... 14 6.4 Instrument Sensitivity...............................................................................................14 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.6 Quantitation and Example Calculation.....................................................................15 7.0 EXPERIMENTAL DESIGN..................................................................................... 17 8.0 RESULTS.................................................................................................................. 17 9.0 CONCLUSIONS........................................................................................................ 17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17
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Table I. Table n.
LIST OF TABLES Page
Summary of PFBS, PFHS and PFOS in Water Samples............................... 19
Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples......... 20
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LIST OF FIGURES Page
Figure 1. Typical Calibration Curve for PFBS in Water................................................22
Figure 2. Extracted Standards of PFBS in Water, 0 ng/L and 25 ng/L, Respectively....23
Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively..........................24
Figure 4. Chromatogram Representing a Public Drinking Water Sample Analyzed for PFBS (Exygen ID: C0061381, Data Set: 022205D)................25
Figure 5. Typical Calibration Curve for PFHS in W ater.............................................. 26
Figure 6. Extracted Standards of PFHS in Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 27
Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 28
Figure 8. Chromatogram Representing a Public Drinking Water Sample Analyzed for PFHS (Exygen ID: C0061381, Data Set: 022205D)................29
Figure 9. Typical Calibration Curve for PFOS in W ater.............................................. 30
Figure 10. Extracted Standards of PFOS in Water, 0 ng/L and 25 ng/L, Respectively ...31
Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively..........................32
Figure 12. Chromatogram Representing a Public Drinking Water Sample Analyzed for PFOS (Exygen ID: C0061381, Data Set: 022205D)................33
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LIST OF APPENDICES
Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" ..............................................................................
Page .34
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1.0 SUMMARY
Exygen Research extracted and analyzed public water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A).
The limit of quantitation for PFBS, PFHS and PFOS in water was 50 ng/L and the limit of detection was 25 ng/L.
PFBS, PFHS and PFOS were not detected at or above 25 ng/L in the water samples.
The average percent recoveries standard deviations for PFBS, PFHS, PFOS in water samples were 158% 9%, 96% 5%, and 99% 8%, respectively.
2.0 OBJECTIVE
The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in public water according to Protocol P0001131 (Appendix A).
3.0 INTRODUCTION
This report details the results of the analysis for the determination of PFBS, PFHS and PFOS in water using the analytical method entitled, "V0001780: Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS."
The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was February 22, 2005, and the analytical termination date for this interim report was March 02,2005.
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4.0 ANALYTICAL TEST SAMPLES
Twenty water samples (Exygen ID C0061381-C0061400) were received on wet ice at 1.2C on February 11, 2005 from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage.
Sample log-in and chain of custody information will be kept at Exygen Research until the conclusion of the study; they will then be located in the raw data package associated with this study. Storage records will be kept at Exygen Research.
5.0 REFERENCE MATERIAL
The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on July, 06, 2000. PFHS was received from 3M at Exygen on January, 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April, 23, 2003.
The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated.
Compound PFBS PFHS PFOS
Exygen Inventory No.. SP0000252 SP0002401 SP0002694
Lot # 101 SE036 430180-1
Purity (%) 96.7 98.6 101.2
Expiration Date 12/04/06 10/18/06 04/23/06
The molecular structures of PFBS, PFHS and PFOS are given below:
PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9S03*K+) Transitions Monitored: 299 - 99 Structure:
FF FF
F S03
FF FF
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PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFoSCh'K^) Transitions Monitored: 399 -- 80 Structure:
F F
F
F F
PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSCVK^) Transitions Monitored: 499 -- 80 Structure:
FFF FFF
6.0 DESCRIPTION OF ANALYTICAL METHOD
The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for this study.
6.1. Extraction Procedure
A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Qg SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray.
6.2 Preparation of Standards and Fortification Solutions
Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in Exygen method V0001780. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standard (corrected for
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purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared.
A set of standards containing PFBS, PFHS and PFOS was prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared:
Cone, of Fort Fort
Solution
Volume
(ng/mL)1
(m-L)
00
10 100
10 200
10 400
100 100
100 200
100 400
1of PFBS, PFHS and PFOS
Volume of Fortified Sample
(mL) 40 40 40 40 40 40 40
Final Cone, of Calibration Std.
(ng/L) 0 25 50 100
250 500 1000
The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation will be kept at Exygen Research until the conclusion of the study; it will then be located in the raw data associated with this study.
6.3 Chromatography
Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 3.5 mins, ~ 10.3 mins, and ~ 12.4 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time.
6.4 Instrument Sensitivity
The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS.
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6.5 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer
Interface: Turbo Ion Spray Liquid Introduction Interface
Computer: DELL OptiPlex GX400
Software: Windows NT, Analyst 1.4.1
HPLC:
Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven
HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 35 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol
Time /min') 0.0 1.0 8.0 10.0 11.0 18.0
Total run time: ~18 min
Flow Rate: 0.3 mL/min
Ions monitored:
%A 65 65 25 25 65 65
Analvte
Mode
PFBS PFHS PFOS
negative negative negative
6.6 Quantitation and Example Calculation
%B 35 35 75 75 35 35
Transition Monitored 299 - 99 399 80 499 -80
Approximate Retention Time
(min) ~3.5 min. ~10.3 min. ~12.4 min.
Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards, except in the case of PFBS, where only five were used; as the 500 ng/L standard results were rejected as outliers. The concentration was determined from the equations below.
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Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program.
Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF slope
Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary.
For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 calculated the percent recovery.
Equation 2: Recovery (%) =
(analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L)
An example of a calculation using an actual sample follows (calculation is for PFOS
only):
Water sample Exygen ID: C0061381 Spk C (Set: 022205D), fortified at 500 ng/L
with where:
peak area
= 155507
intercept
= 548
slope
= 299
dilution factor
=1
ng/L PFOA added (fort level) = 500
amt in corresponding sample = 0 (Not Detected)
From equation 1: Analyte found (ng/L) = fl55507 - 5481 x 1 299
From equation 2: % Recovery
= 518.3 ng/L
= (518.3 - Qppb) x 100% 500
= 104 %
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7.0 EXPERIMENTAL DESIGN
For samples designated as field matrix spikes, PFBS, PFHS and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field.
The samples were extracted in one set. The set consisted of one reagent blank, two reagent blanks fortified at known concentrations, and five sample sites. For each site, a sample, a field duplicate and two matrix field spikes were collected. In the laboratory, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted.
8.0 RESULTS
The PFBS, PFHS and PFOS found in the water samples are listed in Table I. Some peaks were detected in the water samples corresponding to the analyte retention times; however, the peaks detected were all below 25 ng/L.
Fortification recoveries for PFBS, PFHS and PFOS in the water samples are detailed in Table II. The average percent recoveries + standard deviations for PFBS, PFHS, PFOS in water samples were 158% 9%, 96% 5%, and 99% + 8%, respectively.
9.0 CONCLUSIONS
The water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780.
10.0 RETENTION OF DATA AND SAMPLES
When the final analytical report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792.
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TABLES
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Table I. Summary of PFBS, PFHS and PFOS in Water Samples
Exygen ID
Client Sample ID
C0061381
DPWS-PW-0001 -0-040210
C0061381 Rep DPWS-PW-0001 -0-040210*
C0061382 DPWS-PW-0001-0-040210 Dup
C0061385
DPWS-PW-0002-0-040210
C0061385 Rep DPWS-PW-0002-0-040210*
C0061386 DPWS-PW-0002-0-040210 Dup
C0061389
DPWS-PW-0003-0-040210
C0061389 Rep DPWS-PW-0003-0-040210*
C0061390 DPWS-PW-0003-0-040210 Dup
C0061393 C0061393 Rep
DPWS-PW-0004-0-040210 DPWS-PW-0004-0-040210*
C0061394 DPWS-PW-0004-0-040210 Dup
C0061397
DPWS-PW-WTPR-0-040210
C0061397 Rep DPWS-PW-WTPR-0-040210*
C0061398 DPWS-PW-WTPR-0-040210 Dup
C4 Sulfonate PFBS
Perfluorobutanesulfonate
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
Analyte Found (ppt, ng/L) C6 Sulfonate PFHS
Perfluorohexanesutfonate
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
C8 Sulfonate PFOS
Perfluorooctanesuifonate
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
' Laboratory Duplicate ND = Not detected at or above 25 ng/L. NQ = Not quantifiable = Measured concentration between 25 ng/L and the Limit of
Quantitation (LOQ) which is 50 ng/L.
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Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water
Samples
S am ple Description DPWS-PW-0001-0-040210 (C0061381 Spk C, 500 ng/L Spike) DPWS-PW-0001-0-040210 (C0061381 Spk D, 5000 ng/L Spike) DPWS-PW-0001-0-040210 Low Spk 500 ng/L (C0061383, 500 ng/L Spike) DPWS-PW-0001-0-040210 High Spk 5000 ng/L (C0061384, 5000 ng/L Spike)
C 4 S ulfonate PFBS_______________ C 6 S ulfonate PFHS_______________ C8 S ulfonate PFOS
Amount Amt Found Amount
Amt Found Amount
Amt Found Amount
Spiked in Sam ple Recovered Recovery In Sam ple Recovered Recovery In Sam ple Recovered Recovery
(ng/L)
(ng/L)
("fl/L )______ (%>
(ng/L)
("8->______ ! % i _
(ng/L)
(ng/L)______ (%)
500 ND
S42 168
ND
503 101
ND
518 104
5000
ND
7500
150
ND
4540
91
ND
4890
98
500 ND
782 156
ND
510 102
ND
549 110
5000
ND
7470
149
ND
4630
93
ND
4180
84
DPWS-PW-0002-0-040210 (C0061385 Spk E, 500 ng/L Spike)
500 ND
854 171
ND
499 100
ND
513 103
DPWS-PW-0002-0-040210 (C0061385 Spk F, 5000 ng/L Spike)
5000
ND
8030
161
ND
4770
95
ND
4980
100
DPWS-PW-0002-0-040210 Low Spk 500 ng/L (C0061387,500 ng/L Spike)
500
ND
718 144
ND
461 92
DPWS-PW-0002-0-040210 High Spk 5000 ng/L
(C0061388, 5000 ng/L Spike)
5000
ND
8020
160
ND
4830
97
ND 500 100
ND
4570
91
DPWS-PW-0003-0-040210 <00061389 Spk G. 500 ng/L Spike)
500 ND
831 166
ND
493 99
ND
512 102
DPWS-PW-0003-0-040210 (C0061389 Spk H, 5000 ng/L Spike)
5000
ND
7360
147
ND
4500
90
ND
4700
94
DPWS-PW-0003-0-040210 Low Spk 500 ng/L
<00061391,500 ng/L Spike)
500 ND
775 155
ND
485 97
ND
522 104
DPWS-PW-0003-0-040210 High Spk 5000 ng/L
<00061392, 5000 ng/L Spike)
5000
ND
8160
163
ND
5380
108
ND
5730
115
DPWS-PW-00040-040210 <00061393 Spk 1,500 ng/L Spike)
500 ND
854 171
ND
505 101
ND
519 104
DPWS-PW-00040040210 <00061393 Spk J, 5000 ng/L Spike)
5000
ND
7580
152
ND
4550
91
ND
4720
94
DPWS-PW-00040040210 Low Spk 500 ng/L
(C0061395, 500 ng/L Spike)
500 ND
783 157
ND
494 99
ND
547 109
DPWS-PW-00040040210 High Spk 5000 ng/L
<00061396, 5000 ng/L Spike)
5000
ND
8160
163
ND
4990
100
ND
4710
94
DPWS-PW-WTPR0040210 <00061397 Spk K, 500 ng/L Spike)
DPWS-PW-WTPR0040210 (C0061397 Spk L, 5000 ng/L Spike)
DPWS-PW-WTPR0040210 Low Spk 500 ng/L <00061399, 500 ng/L Spike)
DPWS-PW-WTPR-0-040210 High Spk 5000 ng/L <00061400, 5000 ng/L Spike)
500 5000 500 5000
ND 874 175
ND
8070
161
ND 733 147
ND 7630
Average: Standard Deviation:
153
156 9
ND 508 102
ND
4610
96
ND 436 87
ND 4540
Average: Standard Deviation:
91
96 5
ND 526 105
ND
4950
99
ND 438 88
ND 4130
Average: Standard Deviation:
83
99 8
ND * Not detected at or above 25 ng/L (ppt). NQ a Not quantifiable = Measured concentration between 25 ng/L (ppt) and the Limit of
Quantitation (LOQ) which is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values may vary slightly
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FIGURES
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Figure 1. Typical Calibration Curve for PFBS in Water
" O 2 2 Z B 0 IM r.f < 7 F I9 ;U J M > r
f ) A T M lg t ft g . 'Y - 4 3 + 0 1 1 1 7 5 7 - 0 . 0 0 }
A r j, oounte
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Figure 2. Extracted Standards of PFBS in Water, 0 ng/L and 25 ng/L, Respectively
XCCZZZ05-C, 0 ng/L Standard PFBS (Stintimi} Z99/&9.0 amu - sampi* 1 o f 63 from OZZZfOwiff (poak not found)
TlM.aili
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Figure 3.
PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively
Rugenl Control A PFBS(Unknown) 994&9.Q m u umpfe 9 of 53 from 0222050m ff An*: 107counts Hitgftt 976 cps RT: 3.60 min
m2o. SD
M (ID
m o
1
s67
magItsplKI .SO IgA, DP-10- PFIS 0C) 2990900an1-sar* 110TS3102ZO5DNiff m Ana:36itscwi* HtjgltW.cp RT:351all
"I 1 I ' I
9 10 11 12 13 H TRM.ah
IS 16 IT
9 10 TfeM.nii
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Figure 4.
Chromatogram Representing a Public Drinking Water Sample Analyzed for PFBS (Exygen ID: C0061381, Data Set: 022205D)
C0061361 - PFBS (Unknown) Z99.C&9.0 amu *satnpii 15 of 53 from 0ZZZ05D.wiff (peak not found)
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Figure 5. Typical Calibration Curve for PFHS in Water
m Q22ai6Dwar.r(PfH^:-LHar MgnssiM
q QS9$
Area, counts
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Figure 6. Extracted Standards of PFHS in Water, 0 ng/L and 25 ng/L, Respectively
H XCOilZOS O, 0 ngfL Stndfd PFHS (Sttndird) 399.0/80.0 amu - aarnpit 1 of 53 From $22060.wiff (peak not found)
XC02Z2J5-1, Btg/l$ai<Sia>PFHS<ptlCitf() 99 9 iM D IIm - i p * 20f53*C D2Z3SDMiff Aita:79i3oo<i H tlg ltS X .q *T :lO J li
sm-j am
TIB
1027
TM.RIil
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Figure 7.
PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively
Reagent Control A PFHS(Unknown) 399.0/80.0 amu sample 9 of 53 from 0Z220SDj*tff Ana: f 34 counts Height- 1S.6 cps RT: 10.2 min
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Figure 8.
Chromatogram Representing a Public Drinking Water Sample Analyzed for PFHS (Exygen ID: C0061381, Data Set: 022205D)
C0061361 PFHS (Unknown) 3994/60.0 mu simple 16 o f S3 from 0Z22OSDwfff TM An*: 302 count Height 30.S ops RT: 10.3 min
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Figure 9. Typical Calibration Curve for PFOS in Water
TM 0222X0 M*fer.Nt>(PFOS}:'Llit* Rtgrustoi C l/ r iigifg):Y* 2 x+ 5 4 8 (r-D i ^
Ar a. counis
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Figure 10. Extracted Standards of PFOS in Water, 0 ng/L and 25 ng/L, Respectively
XC0222064. Cng/L Stand** PFOS (Standard) 499.0/MV amu aampit 1 of S3 fmmC2Z2060.mfr Ana: 648 counts Haight 3t .1 opt R7:1i.4min
TO)
on
2 400
ZD HE-
0 L - rnmfmmmm-,. 12 3
12.43
5 7 -- r
11 8 ^ * 1 --I-1--I--c-9 10 11 12 13 14 18 16 IT
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Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively
Awgtrtt Control A PFOS (Unknown) 499.OfSO.0 amu (p *a k not found)
9 of 53 from 0222050.wiff
8 *
12 9 i
S6 T
Rtp4ItSpM A, T IflA - FPOS P C ) SBJMDflaBI - p H OTS3OB Q22ZED JMV A n rts e n e u o o ii* Ht g i t i MD. cp RT:i2.*mi
8 T it,nil
11 12 19 14 13 16 IT 12.43
flta g tits p h i i . s m *9/1. of- - FFo s p Q ssnom ani -sanpfc 11orsston DZ2Zi5Diur Ana: 14122)0041 H tig it 1340).cpr RT:l2.4mh
T t.n ii
12.49
TlBt.mli
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Figure 12. Chromatogram Representing a Public Drinking Water Sample Analyzed for PFOS (Exygen ID: C0061381, Data Set: 022205D)
COMtUI PFOS(Unknown) 990/80.0 mu Simple f5 ofS3 from 0222060MifT An: 1261 counts Height 88.2 ops RT: 12. min
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APPENDIX A
Study Protocol P0001131
(Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of
Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water
by LC/MS/MS"
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Exygen Protocol Number: POOO1131
STUDY PROTOCOL
Study Title: Analysis of Perfluorobutanesulfonate (PFBS),
Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small
Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program
Exygen Protocol Number: P0001131
Performing Laboratory: Bxygen Research
3058 Research Drive State College, PA 16801 Phone: (814) 272-1039
Sponsor Representative: Michael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374
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DISTRIBUTION:
1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit
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PROTOCOL APPROVAL
Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program
Exygen Protocol Number: P0001131
APPROVALS
JaisimhatKesan, S' Weston Solutions
Michael A. 3M Comp:
Sponsor Representative
Richard A Exygen Rese:
sident, Facility Management
J .__________
sad, Quality Assurance Unit
Date
7$
Date
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TABLE OF CONTENTS
T IT L E PA G E ............................................................................................................................................................... 1 D IS T R IB U T IO N ......................................................................................................................................................... 2 PROTOCOL A PPRO VAL......................................................................................................................................... 3 TABLE OF C O N TEN TS........................................................................................................................................... 4 IN T R O D U C T IO N ....................................................................................................................................................... 5 TEST M A T E R IA L S ................................................................................................................................................... 5 O B JE C T IV E ................................................................................................................................................................6 TESTIN G F A C IL IT Y .................................................................................................................................................6 S TU D Y DIRECTOR................................................................................................................................................... 7 SPONSOR REPRESENTATIVE...............................................................................................................................7 PR INCIPAL IN V E S TIG A TO R ................................................................................................................................. 7 PROPOSED EXPER IM EN TA L START A N D TE R M IN A TIO N D A T E S ..........................................................7 ID E N T IF IC A T IO N A N D JUSTIFICATION OF TH E TEST S Y S T E M ..............................................................8 SAMPLE PROCUREMENT, RECEIPT A ND R E T E N T IO N ...............................................................................8 SAMPLE ID E N T IF IC A T IO N ...................................................................................................................................9 A N A L Y T IC A L PROCEDURE S U M M A R Y ...........................................................................................................9 V E R IFIC A TIO N OF A N A L Y T IC A L PROCEDURE.............................................................................................9 M E TH O D FOR CONTROL OF B IA S ......................................................................................................................11 STA TISTIC A L M E T H O D S .......................................................................................................................................11 GLP S T A T E M E N T .....................................................................................................................................................11 R EPO R T.......................................................................................................................................................................11 SAFETY A N D H E A L T H ........................................................................................................................................... 12 A M E N D M EN TS T O PR O TO CO L...........................................................................................................................13 D A T A RECORD K E E P IN G ......................................................................................................................................13 Q U A L IT Y A SSU R A N C E.......................................................................................................................................... 14 R ETEN TIO N OF D A T A A N D A R C H IV IN G ......................................................................................................... 14 A PPEND IX I, A N A L Y T IC A L M E TH O D S ............................................................................................................. 15
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INTRODUCTION
The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program.
The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research.
TEST MATERIALS
The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M.
PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4 F9 SO3 I O Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 --> 99 Structure:
PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CiFuSOa'K4') Lot Number: SE036 Purity: 98.6%
Transitions Monitored: 399 -> 80 Structure:
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PFOS
Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFiySOalC) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -> 99 Structure:
OBJECTIVE
The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods:
V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS"
V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS"
V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS"
V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS"
V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS"
V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS"
V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
TESTING FACILITY
Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039
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STUDY DIRECTOR
Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com
SPONSOR REPRESENTATIVE
Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374
PRINCIPAL INVESTIGATOR
John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john.flaherty@exygen.com
PROPOSED EXPERIMENTAL START AND TERMINATION DATES
It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report.
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IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM
The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum
The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study.
The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area.
SAMPLE PROCUREMENT, RECEIPT AND RETENTION
Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study.
Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at -10C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at <, -10C.
The receipt and processing o f the samples will be documented in the final report and raw data associated with the study.
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SAMPLE IDENTIFICATION
Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number.
Sample storage conditions and locations will be documented throughout the study.
ANALYTICAL PROCEDURE SUMMARY
References: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic
Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers Slimmed into total PFBS, total PFHS, and total PFOS found.
VERIFICATION OF ANALYTICAL PROCEDURE
A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette.
For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample
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collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias.
For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias.
For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias.
Low and high spiking levels for each matrix are defined below:
M atrix
Low Spiking Level
High Spiking Level
Water
500nR/L
5000 ng/L
Soil
4ng/g
40 ng/g
S edim ent Fish Clams
Vegetation Small Mammal Liver
4 ng/g
10ng/g
lOng/g
10ng/g 10ng/g
40 ng/g
100ng/g 100ng/g 100ng/g 100ng/g
Small Mammal Serum
10 ng/mL
100 ng/mL
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Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report.
METHOD FOR CONTROL OF BIAS
Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications.
STATISTICAL METHODS
Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable.
GLP STATEMENT
All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative.
REPORT
A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and
o f the testing facility.
A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records).
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The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management.
A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page.
Description o f the instrumentation used and operating conditions.
All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level.
Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms.
All circumstances that may have affected the quality or integrity of the data will be documented in the report.
Locations where raw data and the final report are to be archived.
Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative.
All applicable requirements for reporting o f study results as per 40 CFR 792.185.
SAFETY AND HEALTH
Laboratory personnel will practice good sanitation and health habits.
Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s).
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AMENDMENTS TO PROTOCOL
All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by die Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be die date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative.
DATA RECORD KEEPING
Records to be maintained include the following (as appropriate):
Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study
Chromatograms- All chromatograms will contain the following:
Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run.
Additionally, fortifications will include the amount o f analyte added and the sample number of the sample that was fortified.
Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL).
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As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions.
QUALITY ASSURANCE
The QA Unit o f Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative.
RETENTION OF DATA AND ARCHIVING
All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor.
The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research.
Exygen will obtain permission from the study director before discarding or returning samples.
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APPENDIX I
ANALYTICAL M ETHODS
V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS"
V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS"
V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS"
V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS"
V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS"
V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS"
V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
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Exygen Protocol Number: P000113!
ANALYTICAL METHOD Method Number: V0001780
Method of Analysis for the Determination of Perfloorooctanolc A dd (PFOA) in Water b yL C /M S /M S
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College, PA 16801
Approved By:
_CrAka______
Paul Connolly
1
Technical Leader, LC-MS, Exygen Research
/fu L c f
/Z*JoohhnFFllaahhterty / ' VViicce* OPmrebsiident, Operations, Exygen Research
<0ItiAW
Date
Exygen Research
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ExygesRuM tch
M ethod Number VOOO1780
ANALYTICAL m e t h o d
Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LOMS/MS
1.0 Scope
Iliis method it to be employed for the isolation and quantitation of perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water.
2.0 Safety
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
3.0 Sample Requirement
3.1 At least 40 mL of test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room
temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3000 rpm for ->5
minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for this
project.
4.0 Reagents and Standards
4.1 Water - HPLC grade 4.2 Methanol-HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma*Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volume injector capable of injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS).
5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centriftigc tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-]00uL, 100-200uL). 5.7 125>mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg) tCIS SPE cartridges.
Page 2 of ?
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Page 51 o f 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygenResearch
Method Number V001780
I ANALYTICAL METHOD
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS
$.12 SPB vacuum manifold. S.13 Centrifuge capable o f spinning 50 mL polypropylene tubes at 3000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SOmm, 5^ (P/N: 82505-052130)
6.2 Temperature: 30C 6.3 Mobile Phase (A): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program:
Time fmin) 0.0 1.0 8.0 20.0 22.5
65 6$ 25 25 65
Flow Rate % B (mUminl
35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 1S pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes.
The above conditions are intended as guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 nvz.
The above conditions am intended as a guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water.
Alternate volumes may be prepared.
Exygen Research
Page 18 of 65
Page 52 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
ExygenRssttrch
Method Number VOOOJ7SO
[________________________ ANALYTICAL METHOD________________________
Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Water by
LOMS/MS
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution of~ !00 (LgfotL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPB bottle. 9.1.2 A 10 pgtaL fortification solution of PFOA is prepared by bringing IU mL of the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPB bottle. 9.1.3 A 1.0 pg/mL fortification solution ofPFOA is prepared by bringing !<> m Lofthe 10 pg/mL solution to a final volume oflOO with methanol in a 125 mL LDPB bottle. 9.1.4 A 0.1 pgfaiL fortification solution ofPFOA is prepared by bringing 10 m Lofthe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPB bottle. 9.1.5 A 0.01 pg/mL fortification solution ofPFOA is prepared by bringing 10 mL o f the 0.1 pg/raL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock ind fortification solutions are to be stored in a refrigerator at approximately 4'C and are stable for a maximum period of 6 months from the date o f preparation.
9.2 Standard Calibration Solutions
9.2.1 9.2.2
LC/MS/MS calibration standards are prepared in HPLC water. The calibration standard# are processed through the extraction procedure, identical to samples.
Tbe following is a typical example: additional concentrations may be prepared as needed.
Final
Concentration Fortification Volume of Concentration of Calibration
of Fortification Volume Fortified Control Calibration
Standard ID
Solution (oeb) L) Santole (mL) Standard (mu>* (examole)
0 0 40
0 XCmmddyy-0
10 100 40 10 200 40
25 XCmmddyy*!
so XCmmddyy*2
10 400 40
100 XCmmddyy-3
100 100 40
250 XCmmddyy*4
100 200 100 400
40 40
500 XCmmddyy-5 1000 XCmmddvY-6
* The extracted concentration of the calibration standard is equal to 8* its initial
concentration, due to the concentration o f the standard during the extraction (SPE).
XC " extracted calibration standard.
Paye 4 oJ '
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
B xygu Retttrch
Method Number V00017ft0
ANALYTICAL METHOD
Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS
9.2.3 9.2.4 9.2.5
A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15*mL polypropylene tubes st 2C to 6'C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as
10.0 Batch Set Up
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified st known concentrations (lab control spike) to verify procedural recovery for the batch.
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project
11.0 Sample Extraction
11.1 Measure 40 mL o f sample or s portion of sample diluted to 40 mL with water into 50 mL polypropylene centriAige tubes (fortify as needed, replace lid and mix well).
11.2 Condition the C SPE cartridges (! g, 6 mL) by pasting 10 mL methanol followed by 5 mL o f HPLC water (>2 drop/sec). Do not let column run dry
11.3 Load sample on conditioned Cts SPE cartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol. Collect 5 mL o f eluate into graduated
15 mL polypropylene centrifiige tubes (final volume 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set
12.3 An entire set of extracted calibration standards must be included at the beginning and at the end of s sample set. Extracted standards must he interspersed between every 5*10 samples. As an alternative, an entire set of extracted calibration standards may be injected st the beginning of a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set of extracted standards). In either case, extracted calibration standards must be the first and last injection in s sample set.
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peak area
Pa* } of 7
Page 20 of 65
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Page 54 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen Research
MethodNumber V0001780
ANALYTICAL METHOD
Method o f Analysis for the Determination ofPerftuorooctanoic Acid (PFOA) in Water by LC/MS/MS
versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be ftnther diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss of carbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted.
133 Recoveries o f 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 o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from die calculation of the calibration curve However, the total numb o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected.
13.5 The correlation coefficient (R) for calibratimi curves generated must be 20.992 (R3 20.985). If calibration results fall outride these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed.
13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed.
14.0 Calculations
14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/L) - (Peak area - intercept) x DF slope
DF " factor by which the final volume was diluted, if necessary.
Page 6 of ?
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Page 55 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number POOO1131
Bxygto RtHtreh
MMbodNumber VO00I780
ANALYTICAL METHOD
Method of Analyst for tbe Determination o f PerfluoroocUnoic Acid (PFOA) in Water by LC/MS/MS
14.2 For samples fortified with known amounts of PFOA prior to extraction, use tbe following equation to calculate tbe percent recovery.
Recovery (%) "
f total analytefound (ng/L) - analyte found in control (ng/L)l . analyteadded (ng/L)
Exygen Research
Pig* 7 o f/
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Page 56 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V0001781
Method of Analysis for die Determination o f Perfluorooctuolc Acid (PFOA) Id Soil b y LC/MS/MS
Analytical Totting Facility:
Exygen Research 3058 Research Drive State College, PA 16801
Approved By:
T U . C_iL
Exygen Research
Total Page: 7 Page 23 o f 63
Page 57 of 99
Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygeo Protocol Number: P0001131
E x y graltM N ffh
Method Number V000178J
ANALYTICAL METHOD
Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
1.0 Scope
This method ii to be employed for the isolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in soil.
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 At least 15 g o f test sample for extraction. 3.2 No sample processing is needed for toil samples. 3.3 Samples stored refiigerated should be allowed to equilibrate to room
temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for (his
piqject.
4.0 Reagents sod Standards
4.1 Water-HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma*Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS).
5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 SOmL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (SO-lOOuL, 100*200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath.
Page 2 o f 1
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen Reacarch
Method Number V00178I
_______________________ ANALYTICAL METHOD________________________
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
5.14 Wrist-action shaker. 5.15 Centrifuge capable of spinning SOmL polypropylene tubes al 5000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130)
62 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program:
Time Imml
0.0 1.0 8.0 20.0 22.5
65 65 25 25 65
Flow Rate % B fmUminl
35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area-external standard calibration curve. 6.8 Run Time: - 23 minutes.
The above conditions are intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 m/z for PFOA.
The above conditions are intended as a guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0154 g of ammonium acetate to 1000 mL o f water.
Alternate volumes may be prepared.
Page 3 of ?
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Page 59 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOO1131
Exygen Research
MethodNumber V000I78I
ANALYTICAL METHOD
Method o f Analytic for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a atock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical ctandard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 p^m L fortification solution of PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution of PFOA is prepared by bringing 10 m Lofthe 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution ofPFOA is prepared by bringing ID mL o f foe 1.0 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA ia prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period of 6 months from the date o f preparation.
9.2 Standard Calibration Solutions
9.2.1 9.2.2
LC/MS/MS calibration standards are prepared in HPLC water The calibration standard are processed through the extraction procedure, identical to sample. The following is a typical example; additional concentrations may be prepared as needed.
Final
Concentration Fortification Volume of Concentration of Calibration
ofFortification Volume Fortified Control Calibration
Standard ID
Solution (mb) (PL) Samole (mL) Standard foot)* iexaniDlc)
0 0 40
0 XCmmddyy-0
10 100 40 10 200 40
25 XCmmddyy*1 50 XCmmddyy-2
10 400 40
100 XCmmddyy-3
100 100 40
250 XCmmddyy-4
100 200 100 400
40 40
500 XCmmddyy-5 1000 XCmmddvY*6
* The extracted concentration of foe calibration standard is equal to 8x its initial
concentration, due to the concentration o f the ctandard during the extraction (SPE).
XC " extracted calibration standard.
Page 4 o f 7
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Page 60 of 99
Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygra Rtaureh
MethodNumber VOOO178]
| ANALYTICAL METHOD
Method o fAnalyst tor the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
9.2.3 9.2.4 9.2.5
A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standard in 13*mL polypropylene tubes at 2C to 6*C, up to two weeks.
Alternate volumes and concentrations o f standards may be prepared as needed.
10.0 Batch Set Up
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using 5 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to vertfy procedural recovery for the batch.
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project.
11.0 Sample Extraction
11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well).
11.2 Add 5 mL o f methanol and shake on a wrist action shaker for *-15 minutes. 11.3 Transfer the tubes to an ultrasonic bath and sonicate for -15 minutes. 11.4 Bring the volume up to 40 mL with water in the 50 mL polypropylene
centriflige tube. 11.5 Centriftige for -1 0 minutes at -3000 rpm. 11.6 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by 5 mL of HPLC water ( - 2 drop/tec). Do not let column run dry I t.7 Load (decant) the sample on the conditioned C n SPE cartridge. Discard
eluate. 11.8 Elute with -5 mL 100% methanol. Collect 5 mL o f eluate into graduated
15 mL polypropylene eaitriAige tubes (final volume - 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set.
12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set Extracted standards must be interspersed between every 5*10 samples. As an alternative, an entire set of
Pate 5 of?
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOO1131
ExygenRoaewch_________________________________________ Method Number V00017SI
I
.. ANALYTICAL METHOD
~
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LOMS/MS
extracted calibratton standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to acoount for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for foe analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed.
Acceptance Criteria
13.1 Chromatogram must show a peak of a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter km (369 amu) represents the loss o f carbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted.
13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f foe calibration curve. However, the total number of extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected.
13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or foe relevant set of samples should be reanalyzed.
13.6 Retention times between standards and samples must not drift more than i 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed.
Page 6 r 7
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Exygen Research
Page 62 of 99
Interim Report #1 -Analysis o f Public W ater Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygenResearch
Method Number VOOO1781
ANALYTICAL METHOD
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS
14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/L) - fPtllt ire - imercml) x DF slope
DF - factor by which foe final volume waa diluted, if necessary.
14.2 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery.
Recovery ( H ) -
[total analytefound(ng/L) - analyte found in control (ng/L)] ^ analyteadded (ng/L)
14.3 Use the following equation to convert the amount o f PFOA found in ng/L to ng/g(ppb).
PFOA found (ppb) - fPFOA found fne/L) x volume extracted <0.0401 ample weight (5 g)
14.4 Use foe following equation to calculate the amount of PFOA found in ppb based on dry weight
PFOA found (ppb) dry weight - PFOA found (ppb) x (100% / total solids/*/)]
Pig*7 of 7
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Page 63 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ANALYTICAL METHOD Method Number V0001782
Method o f Analysis for the DetermladoD of Perfluorooetsnoic Acid (PFOA) io Sedimeol by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College. PA 16801
Approved By:
Cj L
Paul Connolly
1
Technical Leader. LC-MS, Exygen Research
lO h h M Date
a /M d u y Aoha FUherty ' f Vice President, Operations, Exygen Research
Date
Exygen Research
Total Pages: 7 Page 30 of 65
Page 64 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygra Research
Method Number V0001782
| ANALYTICAL METHOD
|
Method o f AnalyiU for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS
1.0 Scope
This method is to be employed for foe isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a undem Mass Spoctrometric Detector (LC/MS/MS) in sediment.
2.0 Safety
2.1 Always observe aafo laboratory practices. 22 Consult the appropriate MSDS before handling any chemical for proper safety
precaution.
3.0 Sample Requirement
3.1 At least 30 g o f test sample for extraction. 3.2 No sample processing ia needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room
temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for tills
project
4.0 Reagents end Standards
4.1 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid -Sigma-Aldrich
5.0 Instrument and Equipment
3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5-200 pL connected to a tandem Maas Spectrometer (LC/MS/MS).
3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable of reading to 0.00001 g. 3.4 SOmL disposable polypropylene centriftige tubes. 3.3 15 mL disposeble polypropylene centrifuge tubes. 5.6 Disposable micropipets (SO-lOOuL, 100-200uL). 5.7 125-tnL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial test. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 oc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold.
Page 2 o d
Page 3! of 65
Exygen Research
Page 65 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exygen Research
Method Number VOOO1782
I ANALYTICAL METHOD
\
Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment bv
LC/MS/MS
'
5.13 Vortexer. 5.14 Wrist-action ahiker.
5.15 Centriflige capable of spinning 50 mL polypropylene tubes at 3000 rpm.
6.0 Chrorastographic System
6.1 Analytical Column: FtuophaaeRP (Keystone Scientific), 2.) mm x SOmm. Sp (P/N: 82505-052130)
6.2 Temperature: 30*C 6.3 Mobile Phaae (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program:
Time (mini 0.0 1.0 8.0 20.0 22.5
ZA 65 65 25 25 65
Flow Rate
Z M fmL/min) 35 0.3 35 0.3
75 0.3
75 0.3 35 0.3
6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes.
The above conditions a n intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System 7.1 Mode: ElectrOspny Negative MRM mode, monitoring 413 - t 369 m/z for
Tbe above conditions are intended aa a guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water.
Page 2 of 7
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Page 66 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen P ro to co l N um ber: P 0 0 0 1 131
B*y|<a Rwwrch
Method Number V0001782
I ANALYTICAL METHOD
Method o f Analysis for the Determination o f Perfiuorooctanoic Acid (PFOA) in Sedimem by LC/MS/MS
8.2 Extraction Solution
8.2.1 1% acetic add in water is prepared by adding 10 mL of acetic acid to 1000 mL o f water.
Alternate volumes may be prepared.
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing to mL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution of PFOA is prepared by bringing 10 mL of foe 10 pg/raL solution to a final volume oflOO with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution of PFOA ia prepared by bringing 10 mL o f foe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f foe 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from foe date ofpreparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f foe 0.1 pg/mL fortification solution.
9.2.2 The following ia a typical example: additional concentrations may be
Concentration
of Fortification Soluti] fao/mL)
too 100 too 10 5 2
Volume
(mL) 10 5 2 10 10 10
Diluted to (mL)
100 100
100 100 100 100
Final Concentration
fng/mL) 10.0 so 20 1.0 o.s 0.2
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Exygen Study No.: P0001131
E x ygen P rotocol N um ber: P 0 0 0 1 131
Exyfu& M tticfc
Method Number V00QP82
| ANALYTICAL METHOD
|
Method o fAnalyse for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS
9.2.3 Store all calibration atandarda in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to aix months.
9.2.4 Alternate volumes and concentrations o f atandarda may be prepared as needed.
10.0 Batch Set Up
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch.
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project
11.0 Sample Extraction
11.1 Weigh 5 g of sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well).
11.2 Add 35 mL of 1% acetic acid, cap, vortex and shake on a wrist action shaker for-4 0 minutes.
11.3 Centrifuge the tubes at ~3000 rpm for -20 minutes. 11.4 Condition foe Cu SPE cartridges (l g, 6 mL) by passing 10 mL methanol
followed by 20 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) foe sample on the conditioned C u SPE cartridge. Discard
eluate. 11.6 Add 20 mL o f methanol to the todimeot left in the bottom of the 50 mL
centrifuge tube. Cap, vortex md shake on a wrist action shaker for -30 minutes. 11.7 Centrifiigc the tubes at -3000 rpm for -2 0 minutes. 11.8 Decant foe methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column with 4 mL o f methanol Collect the eluate and add it to foe eluate collected in step 11.8. 11.10 Condition a second Cu SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -200 mL o f water and load on the second conditioned SPE cartridge. 11.12 Elute with -S mL 100% methanol Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifuge tubes (final volume " 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS.
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
BxygeaRewch
Method Number V0001783
| ANALYTICAL METHOD
|
Method o f Analysis for die Determination ofPerfluorooctanoic Acid (PFOA) in Sediment bv
LC/MS/MS
'
12.0 Chromatography
12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to st least five or more concentration levels must be included in an analytical set.
12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set of calibration standards may be injected at die beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample set.
12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Author diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f catbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire act must be re-extracted.
13.3 Recoveries o f control spikes and matrix spikes must he between 70*130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70* 130% should be evaluated by the analyst to determine if re-extraction is warranted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number of extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected.
13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 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.
Page of?
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygcaRcM uch
MethodNumber V0001782
ANALYTICAL METHOD
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS
13.6 Retention timei between standards and samples must not drift more than 4 % within an analytical ran. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed.
14.0 Calculations
14.1 Use the following equation to calculate the amount o f PFOA found (in ng/ml, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ngftnL) - (Peak area - intercept) x DF slope
DF - factor by which the final volume w u diluted, if necessary.
14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery.
Recovery (%) -
| total analyte found (ng/mL) - analytefound in control (ng/mL)j j]QQ analyteadded (ng/mL)
14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng'gfppb)-
PFOA found (ppb) - fPEQA found fae/mL) x final volume fS mL)l sample weight (3 g)
14.4 Use the following equation (if necessary) to calculate the amount of PFOA found in ppb based on dry weight
PFOA found (ppb) dry weight - PFOA found (ppb) x (100% / total sohds(%)]
PlfC7 of 7
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOO1131
ANALYTICAL METHOD Method Number V0001783
Method of Aiafyils for the Determination of Perfluorooctanoic Acid (PFOA) in Fish aod Clams by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College PA 16801
Approved By.
v U C -Jl.
Paul Connolly
>
Technical Leader, LC-MS, Exygen Research
q / m d / _______
John Flaherty
' Vice President, Operations, Exygen Research
Date
r
Date
Exygen Research
Total Pages: 8 Page 37 o f 65
Page 71 o f 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOOl 131
Exygen Ham ich
Method Number V0001783
AMa LYTICAL m e t h o d ________________________
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
1.0 Scope
This method it to be employed for the isolation and quantitation o f perfluoiooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectnunetric Detector (LC/MS/MS) in fish and clams.
2.0 Safety
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely
precautions.
3.0 Sample Requirement
3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a
food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project
4.0 Reagents sod Standards
4.1 Water-HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Aacoibic add - Reagent grade 4.10 Dimethyidichlorosilane- Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Pcrfluorooctanoic Acid - Sigma-Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up io 2 solvents equipped with variable volume injector capable o f injecting 5-200 pL connected to a tandem Mats Spectrometer (LC/MS/MS).
5.2 A device to collect raw data for peak integratimi and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g.
Pag 2 of8
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygM Rttcaich
Method Nunfcer V0001783
I ANALYTICAL METHOD
Method of Analysis fix' the Determination of Perfloorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
5.4 Rotary evaporator.
5.5 Tissumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centriftige tubes. 5.8 15 mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50*100uL, 100*200uL) 5.10 125-mL LDPB narrow-mouth bottles. 5.11 2 mL clear HPLC vial Idt. 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057177). 5.15 Wrist action shaker. 5.16 Centrifuge capable of spinning 50 mL polypropylene tubes at 2000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: FluopbaseRP (Keystone Scientific). 2.1 mmx 50 mm. 5m (P/N: 82505-052130)
6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program:
Tima fmint
0.0 1.0 8.0 20.0 22.5
65 65 25 25 65
Flow Rate fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 pL (can be increased to aa much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes.
The above conditions are intended as a guide and may be changed in order to optimize the HPLC system.
Page 3 o f8
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen fceicirch
Method Num ber V0OO1783
ANALYTICAL METHOD
Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Gams by LC/MS/MS
7.0 MS/MS System
7.1 Mode: Electroepray Negative MRM mode, monitoring 413- 369 m.7 for PFOA.
The above conditions ate intended as a guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 toM ammonium acetate in water is prepared by adding 0.154 g of ammonium settate to 1000 mL o f water.
8.2 Extraction Solutions
8.2.1 8.2.2
2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL o f mediano). 30% Diratthyldichlorosilane in toluene is prepared by bringing 3 mL ofdimtthyklichloroailane to s final volume of 10 mL with toluene.
Alternate volumes may be prepared.
9.0 Standard Preparation 9.1 Standard Stodc/Fortification Solution
9.1.1 9.12 9.1.3 9.1.4 9.1.3
Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrocted for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m Lofthe 1.0 pg/mL solution to a final volume of 100 with methanol in a 123 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pgftnL solution to a final volume of 100 with methanol in a 123 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation.
Page 4 of S
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exyfen Rewvch
Method Number V0001783
I a n a ly tica l m eth o d
Method o f Analysis for the Determination o f Perfhiorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
9.2 Standard Calibration Solutions
9.2.1 9.2.2
LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/raL fortification solution. The following ia a typical example: additional concentrations may be prepared aa needed.
Concentration
Final
of Fortification Volume
Diluted to
Concentration
Solution (ua/mL) (mL)
(mL)
(MitelL)
1.0 5.0 100
0.05
1.0 2.5 too
0.025
1.0 1.0 too
0.01
0.05 10 100
0.005
0.025 10 100
0.002S
0.1 10 100
0.001
0.005
10
100
0.0005
9.2.3 Store all calibration atandards in 125-mL LDPE narrow-mouth bottles
at 2C to 6C, up to aix months.
9.2.4 Alternate volumes and concentrations of standards may be prepared as
needed.
10.0 Batch Set Up
10.1 Each bitch o f samples extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch.
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project.
11.0 Sample Extraction
11.1 Weigh 5 g o f frozrti sample into 50 mL polypropylene centrifuge tubes (fortify aa needed, replace lid and mix well).
11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for -15 minutes 11.3 Place the tubes in a freezer for -1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g
carbon, and l g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL of acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the t25 mL pear-shaped flasks by rinsing with the 30% dimethyldichloroailane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry die flasks completely before use, either by air-drying or with a stream of nitrogen.
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exygen Research
Method Number VOOOI7U
ANALYTICAL METHOD
Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
11.7 Centriftige the 50 mL polypropylene tubes containing sample at -2000 rpm for-1 0 mutates.
11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluatc in the 125 mL silanized pear-shape flask.
11.9 Add 10 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the ftozen flu phase using a tisaumizer for -30 seconds and rinse the tissumizer with -10 mL of acetonitrile into the tube.
11.10 Shake die sample again for -10 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm
for-10 minutes.
11.13 Decant the extract onto the same SPE column. Collect the eluate into the tame pear-shaped flask and combine with the eluent from the initial extraction.
11.14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the same pear-duped flask.
11.15 Add 3-4 drops of t-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 409C).
11.Id Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve.
11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow alt analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set.
12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards mutt be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards), in either case, calibration standards must be the first and list injection in u sample set
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system.
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exygen Reaorcb
M ethod Number VOOO783
A N A L Y T IC A L m e t h o d
Method o f Analysis for die Determination ofPerfluoroocttnoic Acid (PFOA) in Fish and Gams by LC/MS/MS
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be fiirther diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.S ppb, then a new blank sample mutt be obtained and the entire set must be re-extracted.
13.3 Recoveries ofoootrol spikes and matrix spikes must be between 70-130% of their known valuee. If a control spike fails outside the acceptable limits, the entire set o f samples should be re-extracted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o fdie total number o f standards injected.
13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). If calibration results foil outside these timits, then appropriate steps must bo taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed.
13.6 Retention timet between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds (his limit within an analytical run then die set must be reanalyzed.
14.0 Calculations
14.1 Use the following equation to calculate the amount of PFOA found (in ng/mL, based on peek area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/mL) (Peak area - intercept) slope
14.2 Use the following equation to convert the amount of PFOA found in ng/mL to ng/g (ppb).
PFOA found (ppb) - fPFQA found (ng/mL) x final volume (mU x DPI sample weight (g)
DF * factor by which the final volume was diluted, if necessary.
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Exygen Research
Page 77 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exygen Research
Method Number V0001?8J
ANa LYTIC.U. m e t h o d
Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS
14.3 For samples fortified with known amounts of PFOA prior to extraction, use the following equation to calculate the percent recovery.
Recovery (% )-
[ total analytefound (ng/g) analyte found in control (ng/g)] analyteadded (ng/g)
Exygen Research
Page 8 uf#
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Page 78 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001 B l
ANALYTICAL METHOD Method Number: V0001784
Method of Analysis for the Determination o f Perflnorooctanolc Acid (PFOA) lo Vegetation by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College, PA 16801
Approved By:
"vLA C J lL
Paul Connolly
'
Technical Leader, LC-MS, Exygen Research
-- tafr&M
Date
Jo h n Flaherty Vice President, Operations, Exygen Research
Date
Exygen Research
Total Pages: 7 Page 45 of 65
Page 79 of 99
Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exyfcn ftem rch
Method Number VOObt 784
A N A LYTICAL m e t h o d
Method o f Analyst for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
1.0 Scope
This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation.
2.0 Safety
2.1 Always obecrve safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 At least 20 g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a
food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project
4.0 Reagents and Standards
4.1 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NH* - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L*Ascorbic add - Reagent grade 4.10 Dimethyklichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich
3.0 Instrument and Equipment
3.1 A high performance Liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volume injector capable of injecting 3-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS).
3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g.
P a g * 2 u t'7
Page 46 o f 65
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOOl 131
ExyfnRMMxcb
Method Number V0001784
ANALYTICAL METHOD
Method o fAnalysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 50 mL disposable polypropylene centrifuge tubes. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50*lOOuL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial kit. 5. i l Diapoaable pipettes. 5.12 Autopipettea (100-1000 pL and 10-100 pL), with disposable tips. 5.13 SPE tubes (20mL) (Supelco oat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifbge capable o f spinning SOmL polypropylene tubes at 2000 ipm.
6.0 Chromatographic System
6.1 Analytical Column: Fluophaac RP {Keystone Scientific), 2.1 minx 50 mm, 5p (P/N: 82505-052130)
6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program:
Tima (mini
0.0 1.0 8.0 20.0 22.5
LA 65
65 25 25 65
Flow Rate (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Rim Time: ~ 23 minutes.
The above conditions are intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --369 m/z for PFOA.
Page 3 of 7
Page 47 of 65
Exygen Research
Page 81 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen Rtseaicb
Method Number V0001784
ANALYTICAL METHOD
Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
The above conditions are intended as a guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water.
8.2 Extraction Solutions
8.2.1 8.2.2
2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic add in 100 mL o f methanol.
3 0tt DimethytdichlorosUane in toluene it prepared by bringing 3 mL ofdimethyldichloroeilane to a final volume o f 10 mL with toluene.
Alternate volumes may be prepared.
9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution
9.1.1 Prepare a stock solution o f 100 pg/mL of PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL wiih methanol in a 123-mL LDPE bottle.
9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pg/raL solution to a final volume o f 100 with methanol
in a 125 mL LDPE bottle. 9.1.3 A0.1 pgfaiL fortification solution ofPFOA it prepared by bringing 10
mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final votume of 100 with methanol in a 125 mL LDPE bottle. 9.1.5 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 6 months from the date of preparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution.
Page 4 I *
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen Research
001Method Number VQ 784
ANALYTICAL. M E T H O D
Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
9.2.2 The following is a typical example: additional concentrations may be prepared as needed.
Concentration of Fortification Solution fua/mL)
1.0
Volume
(mL) 5.0
Diluted to (mL)
100
Final Concentration
(wt/mL) 0.05
1.0 2.5 100
0.025
1.0 1.0 100
0.01
0.05 0.025 0.1 0.005
10 10
to 10
100 100 100 100
0.005 0.0025 0.001 0.0005
9.2.3 Sum all calibration standards in 125-mL LDPE narrow-mouth bottles
at 2*C to 6*C, up to six months.
9.2.4 Alternate volumes and concentrations o f standards may be prepared as
needed.
10.0 Batch Set Up
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lah control spike) to verify procedural recovery for the batch
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project.
11.0 Sample Extraction
11.1 Weigh 5 g of frozen sample into SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well).
11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for -15 minutes. 11.3 Centrifuge the 50 mL polypropylene tubes containing sample at 2000 rpm
for 10 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in aequence with 2 g florisil, 2 g silica gel. 2 g
carbon, and 1 g LC-NHj. Condition the columns with 20 mL of methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by nosing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying or with a stream o f nitrogen. 11.7 Decant foe extract on to a conditioned SPE column fitted inside the mouth of the pear-stuped flask. Collect the eluate in the 125 mL silanized pear-shape flask.
Pige 5 o f "
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygea Research
Method Number VOOGl 784
I a ^ a Ly t ic a i. m e t h o d ~
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
11.8 Add 20 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. 11.9 Shake die sample again for -10 minutes on a wrist-action shaker. 11.10 Centriflige the 50 mL polypropylene tubes containing sample at -2000 rpm
for-5 minutes. 11.11 Decant the extract onto die same SPE columa Collect the eluate into the
same pear-shaped flask and combine with the eluent from (he initial extraction. 11.12 Repeat step 11.8 through 11.11 again.
11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C).
11.14 Make die final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-stuped flask and swirl to mix/diasolve.
11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS.
12.0 Chromatography
12.1 Inject the same amount of each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set.
12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f enacted standards). In either case, extracted calibration standards must be the first and last injection in a sample set.
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide.
P*g*6of 7
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOO1131
ExygmHesrnrcli
Method Number V000I7B4
AXa LVTICAL m e t h o d
I
Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be re-extracted.
13.3 Recoveries o f 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 o f samples should be re-extracted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve.
However, the total number o f calibration standards that could be exetuded must not exceed 20% o f the total number o f standards injected. 13.5 The correlstion coefficient (R) for calibration curves generated must be
20.992 (R1 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than
4 % within an analytical run. If retention time drift exceeds this limit within
an analytical run then the set must be reanalyzed.
14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by die Maas Lynx software program:
PFOA found (ng/mL) * fPh Kam intercept! slope
14.2 Use the following equation to convert foe amount o f PFOA found in ng/mL to ng/g(ppb).
PFOA found (ppb) - (PFOA found (ng/mL) x final volume fmD x DF1 sample weight (g)
DF factor by which foe final volume was diluted, if necessary.
143 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery.
Recovery (%)
[total analyte found (ng/g) analyte found in control (ng/g)] ^ ^ analyteadded (ng/g)
Page 7 ot '
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P001131
ANALYTICAL METHOD Method Number: V0001785
Method of Analysis for the Determination of Perfloorooctaaolc Acid (PFOA) In Small Mammal Liver by LC/MS/MS
Analytical Testing Facility:
Exygen Research
3058 Research Drive State College, PA 16801
Approved By:
* ~ v ^ --X . c -- l X t j _________
Paul Connolly
I
Technical Leader, LC-MS, Exygen Research
o /s
Jb6hnFFllaaherty / r Vice Predsiedent, Operations, Exygen Research
_
Date Date
Exygen Research
Total Pages: 7 Page 52 of 65
Page 86 of 99
Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOO1131
ExygraRettnch
Method Number VOW1785
| ANALYTICAL MTHOD
Method ofAnalysis for tbe Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
1.0 Scope
Thi* method it to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in small mammal liver.
2.0 Safety
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 At least S g of test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a
food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. Alternately, if there is an insufficient amount o f sample (-less than 5 g), then no processing it necessary and the sample can be used as supplied. 3.3 Sample collection procedures will be specified in the sampling plan for this project
4.0 Reagents and Standards
4.1 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate-A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-AIdrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 3 solvents equipped with a variable volume injector capable of injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS).
5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (50-1OOuL, 100~200uL). 5.7 125*mL LDPE nanow*mouth bottles. 5.8 2 mL clear HPLC vial kit.
Page 2u p
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number; P0001131
ExyicoX etevch
Method N umber VOOOI785
| ANALYTICAL METHOD
Method ofAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
5.9 Dispotable pipettes. 5.10 Autopipettee (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waten Sep Pak Vac 6 cc (lg) tCl8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemizer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable of spinning 15 mL polypropylene tubes at 3000 rpm.
6.0 Chromatographic System
6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130)
61 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water
Mobile Phase (B): Methanol Gradient Program:
Time (min)
0.0 1.0 8.0 20.0 22.5
&A 65 65 25 25
65
Flow Rate % B CmUmin)
35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 1S pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area-external standard calibration curve. 6.8 RunTime: - 2 3 minutes.
The above conditions tre intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 m/z for PFOA.
The above conditions are intended as a guide and may be changed in order to optimize the MSMS system.
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOO1131
Exygen Research
Method Number VOQG1785
I a n alytical m e t h o d
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 tnM ammonium acetate in water is prepared by adding 0. i S4 g o f ammonium acetate to 1000 mL o f water.
Alternate volumes may be prepared.
9.0 Standard Preparation
9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 yg/mL fortification solution of PFOA is prepared by bringing I mL o f the 100 p^m L solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution of PFOA is prepared by bringing 10 mL o f die 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator ai approximately 4C and are stable for a maximum period of 6 months from die date of preparation.
9.2 Standard Calibration Solutions
9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution.
9.2.2 The following is typical example: additional concentrations may be prepared as needed.
Concentration
Final
of Fortification Volume
Diluted to
Concentration
Solution fnt/mL) (mL)
(mL)
(na/mL)
100 5.0 100
5.0
100 2.0
100
2.0
100 1.0 100
1.0
5.0 10 100
0.5
2.0 10 100
0.2
1.0 10 100
0.1
9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles
at 2* to 6*C, up to six months.
9.2.4 Alternate volumes and concentrations o f standards may be prepared as
needed.
Plge4 of 7
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
Exyfea Rcaurch
Method Number V0001785
| ANALYTICAL METHOD
Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
10.0 Batch Set Up
10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch
10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project.
11.0 Sample Extraction
11.1 Weigh 1 g o f sample into a 50 mL polypropylene centrifuge tubes <fortify as needed, replace lid and mix well)- Note that alternate weights o f liver may be measured depending on the sample size available for use.
11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuetnizer for-1 minute. 11.4 Transfer t mL o f foe sample using a disposable pipette into a 15 mL
disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for -2 0 minutes on a wrist-action shaker. 11.6 Centrifuge foe tubes at -3000 rpm for -5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35
mL o f water. 11.8 Condition the Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by 5 mL o f HPLC water (~ 2 drop/soc). Do not let column run dry 11.9 Load the sample on conditioned Cu SPE cartridge. Discard eluate. 11.10 Elute with -2 mL o f methanol. Collect 2 mL o f eluate into a graduated
15 mL polypropylene centriftige tube (final volume - 2 mL). 11.11 Analyze samples using electrosprey LC/MS/MS.
12.0 Chromatography
12.1 Inject the tame amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set.
12.3 An entire set o f calibration standards must be included at the beginning and at the end of a sample set. Standards must be interspersed between every 5-1o samples. As an alternative, an entire set of calibration standards may he injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set of standards). In either case, calibration standards must be foe first and last injection in a sample set
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using t/x weighting o f peak area
Page 5 of 7
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
ExygeaReacarch____________________________________________ Method Number V0001785
I
.................. ANALYTICAL METHOD
.........
Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
veraus calibratimi standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Simple response should not exceed standard responses. Any samples that exceed standard responses should be ftirther diluted and reanalyzed.
13.0 Acceptance Criteria
13.1 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.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA i t levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted.
13.3 Recoveries o f 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 o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted.
13.4 Any calibration standard found to be a statstica) outlier by using the Huge Error Test, may be excluded from die calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number of standards injected.
13.5 The correlatimi coefficient (R) for calibration curves generated must be 0.992 (R1 0.985). If calibration resulti foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed.
13.6 Retention time between standards and samples must not drift more than 4 % within an analytical ran. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed.
14.0 Calculations
14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program:
PFOA found (ng/mL) ( P f g a m - intercept1) x DF x aliquot factor slope
DF factor by which the final volume was diluted, if necessary. Aliquot fc e to r-10
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: POOOl 131
Exyim Research
M ethod Number V0001785
ANALYTICAL m e t h o d
Method of Analysis for the Determination ofPerfluoroocUnoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS
14.2 For ample* fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate foe percent recovery.
Recovery (%) -
[ total analytefound (ngftnL) - analyte found in control (ng/roL)] analyte added (ng/mL) '
14.3 Use the following equation to convert the amount of PFOA found in ng/mL 10 ng/g (p|*).
PFOA found fppb) - fPFOA found (ng/mL) x final volume fmL)l sample weight (g)
Exygen Research
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Page 92 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOO1131
ANALYTICAL METHOD Method Number V0001786
Method of Aaalysis for the Determination of Perfluorooctaoolc Acid (PFOA) in Small Mammal Senna by LC/MS/MS
Analytical Testing Facility:
Exygen Research 3058 Research Drive State College, PA 10801
Approved By:
Paul Connolly
I
Technical Leader, LC-MS, Exygen Research
a /#
John Flaherty / Vice Preceidaliit, Operations, Exygen Research
mlu>to\[
Date
Dtue
Exygen Research
Total Pages: 7 Page 59 of 65
Page 93 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study No. : P0001131
Exygen Protocol Number: P0001131
ExygenResearch
MethodNumber V0001786
I a n a ly tic a l m e t h o d
Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Senun by LC/MS/MS
1.0 Scope
This method is to be employed for the isolation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum.
2.0 Safety
2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety
precautions.
3.0 Sample Requirement
3.1 At least 1raL o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum
samples must to allowed to completely thaw to room temperature before use. 3 J Sample collection procedures will be specified in the sampling plan for this
project.
4.0 Reagents and Standards
4.1 Water-HPLC grade 42 Methanol-HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich
5.0 Instrument and Equipment
5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS)
5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centriflige tubes. 5.5 15 mL disposable polypropylene centriflige tubes. 5.6 Disposable micropipets (50-100uL, 100-2Q0uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexcr.
Page 2 of ?
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
ExygenRewvck
Method Number V000I786
I .. ANALYTICAL m e t h o d
Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS
5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm
6.0 Chromatographic System
6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505*052130)
6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program:
Time (min) 0.0 1.0 8.0 20.0 22.5
%A 65 65 25 23 65
Flow Rate
% B fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3
6.6 Injection Volume: 15 |iL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes.
The above conditions are intended as a guide and may be changed in order to optimize the HPLC system.
7.0 MS/MS System
7.1 Mode: Electrospray Negative MRM mode monitoring 413 -- 369 m/z for PFOA.
The above conditions ire intended is guide and may be changed in order to optimize the MSMS system.
8.0 Preparation o f Solutions 8.1 Mobile Phase
8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g of ammonium acetate to 1000 mL of water.
Alternate volumes may be prepared.
Page 3 o f f
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Interim Report #1-Analysis of Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: POOO1131
ExygtnR--arch________________________________________ Method Number V000J786
I ANALYTICAL METHOD Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS
9.0 Standard Preparation
9.1 Standard Stoek/Foitification Solution 9.1.1 Prepare a stock solution o f -100 ug/mL of PFOA by weighing 10 nig o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 p^m L fortification solution of PFOA is prepared by bringing t mL o f the 100 pgtoL solution to a final volume of 100 with methanol in a 12S mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 1U mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125mLU)PE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation.
9.2 Standard Calibration Solutions
9.2.1 9.2.2
LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed.
Concentration ofFortiAcaticm Volume
Diluted to
Final Concentration
Solution (oaftaL) (mL)
(mL)
(nt/mL)
100 S.0 100
5.0
100 2.0 100 too 1.0 100 3.0 10 100
2.0 1.0 0.5
2.0 10 100
0.2
1.0 10 100
0.1
9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles
at 2*C to 6C, up to six months.
9.2.4 Alternate volumes and concentrations of standards may be prepared as
10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated contra! and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project.
4 of 7
Il
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygsn Research
Method Number V000178*
I
ANALYTICAL METHOD
...........
Method o f Analysis for the Determimtion o f Per/luorooctanoic A dd (PFOA) in Smell Mammal Serum by LC/MS/MS
11.0 Sample Extraction
11.1 Meacure 1 mL o f sample into a SOmL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes of serum may be measured depending on the sample size available for use
11.2 Add water to the sample for a final volume o f 20 mL. Cap tightly 11.3 Vortex for-1 minute. 11.4 Transfer 1 mL o f the sample using a disposable piped into a IS mL
disposable ccntriftige tube. 11.5 Add 5 mLofacetooitrile and shake for--20 minutes on a wrist-action shaker 11.6 Centrifuge the tubes at -3000 rpm for -5 minutes. 11.7 Decant the supernatant into a $0 mL disposable centrifuge tube and add 35
mL o f water. 11.8 Condition the Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol
followed by 5 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cis SPE cartridge. Discard eluate. 11.10 Elute with -2 mL of methanol. Collect 2 mL o f eluate into a graduated
15 mL polypropylene centrifuge tube (final volume - 2 mL). 11.11 Analyze samples using electroepray LC/MS/MS.
12.0 Chromatography
12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples.
12.2 Standards of PFOA coiTesponding to at least five or more concentration levels must be included in art analytical aet.
12.3 An entire set o f calibration standards must be included at the beginning and ai the end of a sample set. Standards must be interspersed between every 5-1<> samples. As an alternative, an entire set of calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set.
12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using MaasLynx 3-3 (or equivalent) software system.
12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed.
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Interim Report #1-Analysis o f Public Water Samples
Exygen Study No.: P0001131
Exygen Protocol Number: P0001131
Exygen Research
Method Number VQQ0I786
ANALYTICAL METHOD
Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS
13.0 Acceptance Criteria
13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ton (369 amu) represents the loss o f carbon dioxide.
13.2 Method blanks must not contain PFOA at levels greater than the LOQ if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted.
13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If control spike falls outside the acceptable limits, (he entire set o f samples should be re*cxtracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted.
13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standard that could be excluded must not exceed 20% o fthe total number o f standards injected.
13.3 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 0.983). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed.
13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed.
14.0 Calculations
14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using foe standard curve (linear regression parameters) generated by foe M us Lynx software program:
PFOA found (ng/mL) " /Peak area - intercept! x DF x aliquot factor slope
DF " factor by which the final volume w u diluted, if necessary. Aliquot factor - 20
14.2 For aamples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery.
Recovery (%)
[ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ ^
analyteadded (ng/mL)____________________________ Pag 4 of7
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Page 98 of 99
Interim Report #1-Analysis o f Public Water Samples
Exygen Study N o.: P0001131
Exygen Protocol Number: POOO1131
Exyten Reaerrch
Method Number V0Q0I7S6
I ANaL yTICAI. METHOD
Method o f Analyaia for the Determination o f Perfluorooctanolc Acid (PFOA) in Small Mammal Serum by LC/MS/MS
14.3 Uie the following equation to convert the amount o f PFOA found in ngtm L to ppb.
PFOA found (ppb) - [PFOA found fnn/ml Av Bnl volume (mLtl aample volume (mL)
Exygen Research
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