Document Y9jmNrzoX24xwXEX8eenDd6D
1
RESEARCH
Precise Research. Proven Results.
AR226-1745
m
a
Analytical Report
I
3M EnvironmentalTechnology and Safety Services
II Analysis of Perfluorooctanesulfonate in Mallard and Quail Egg Yolk
Exygen Report No. 023-070
m
Testing Laboratory
a
Exygen Research
3058 Research Drive
State College, PA 16801
1
Requester
William K. Reagen, Ph.D. 3M Environmental Technology and Safety Services
Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331
PAGE 1 OF 5
F 814.272.1019
exygen.com
1 lntroduction
Results are reported for the analysis of total perfluorooctanesulfolnate (PFOS) in mallard and quail egg yolk samples. Also reported is the PFOS contained in each of the three fractions of egg yolk: very low density lipoprotein (VLDL), phosvitin, and lipovitellin. This study was conducted in accordance with the analytrcalphase protocolgiven in Attachment E.
2 Sample Receipt
The samples were submitted in plastic bags, labeled with magic marker and typed print. A copy of all sample log-in information is presented in Attachment A
Twelve mallard egg yolk samples and twelve quail egg yolk sample:; were received on 06/01/02. The samples were shipped frozen on dry ice via Federal Express ( FedEx). The twelve mallard yolk samples were combined and homogenized into one sample. The same was done for the twelve quail yolk samples. The samples were stored frozen from lime of receipt until analysis. Four mallard albumen and shell membrane samples and four quail albumen and shell membrane samples were also received on 06/01/02. These sampl'es were not analyzed as part of this study.
3 Methods - Analytical and Preparatory
3.1 Sample Preparation 3.1.1 Total PFOS Content
A 1.0 g sample was used for the extraction procedure. Twenty-five milliliters of methanol were
- - added to the sample. The sample was placed on a wrist-action shaker for 15 minutes and then
centrifuged at 2000 rpm for 10 minutes. A five milliliter aliquot of the sample was taken and 0.5 g of carbon was added. The sample was shaken then allowci!d to sit. Each sample was filtered and analyzed by LC/MS/MS electrospray.
3.1.2 PFOS Content in Yolk Fractions
A 5.0 g sample was used for the fractionation. Ten milliliters of ;ii solution containing 0.67 M
- MgS04,1 mM phenylmethanesulfonylfluoride (PMSF), and 2 pM leupeptin were added to the
sample. The sample was shaken for 2 minutes and then the suspension was transferred to a centrifuge tube. The sample was centrifuged at 200,000 x g at 4C for 24 hours. The sample was then separated into 2 segments: the very low density lipo~irotein(VLDL) and the high density fraction (HDF).
The VLDL was dissolved in a solution containing20 mM Tris-HCI, 1!jOmM NaCI, 0.2 mM EDTA, 1 rnM PMSF, and 5 pM leupeptin. The HDF was resuspended in (a solution containing 0.45 M
MgS04,1 mM PMSF, and 2 pM leupeptin. Both VLDL and HDF fractions were centrifuged at 200,000 x g at 4C for 16 hours. The VLDL was then dissolved in a solution containing 20 mM Tris-HCI, 150 mM NaCI, 0.2 mM EDTA, 1 mM PMSF, and 5 pM leupeptin and stored at 4C. The HDF was dissolved in a solution containing 0.45 M MgSO,I, 1 mM PMSF, and 2 pM
PAGE 2 OF 5
leupeptin and then two volumes of cold water was added dropwise ,and then stored overnight at 4C.
The HDF supernatant (lipovitellin) was decanted and diluted with cold water and stored overnight at 4C. The HDF precipitate (phosvitin) was dissolved in a solution containing 0.4 M MgS04, 1 mM PMSF, and 2 pM leupeptin and then diluted with cold water and stored overnight at 4C. The lipovitellin and phosvitin were then recovered by centrifugation at 106,000 x g at 4C for 1 hour and dissolved in a solution containing 1 M NaCI, 5 mM Tris-HCI, 1 mM PMSF, and 2 1M leupeptin.
All of the resulting precipitates (-1.0 g) from each fraction were used for the extraction
- - procedure. Twenty-five milliliters of methanol were added to the sample. The sample was
placed on a wrist-action shaker for 15 minutes and then centrifuged at 2000 rpm for 10 minutes. A five milliliter aliquot of the sample was taken and 0.5 g of carbon was added. The sample was shaken then allowed to sit. Each sample was filtered 'and analyzed by LC/MS/MS electrospray
3.2 Sample Analysis by LC/MS/MS
In High Pressure Liquid Chromatography (HPLC), an aliquot of extract is injected and passed
through a liquid-phase chromatographic column. Based on the a,ffinity of the analyte for the
stationary phase in the column relative to the liquid mobile phase, the analyte is retained for a characteristic amount of time. Following HPLC separation, mass spectrometry provides a rapid
and accurate means for analyzing a wide range of organic compoimds. Molecules are ionized, fragmented, and detected. The ions characteristic of the compound is observed and quantitated
against standards. Each sample was analyzed in duplicate.
An HP1000 system interfacedto a Micromass Quattro Ultima system was used to analyze the sample extracts. A gradient elution through a Jones Chromatography Genesis C-8 50 x 2.1 mm x 4pm column was used for separation.
The following gradient was performed:
Mobile Phase (A): Mobile Phase (B):
iime
0.0 0.4
1 .o
7.0 7.5 9.0 9.5 14.0
2mM Ammonium Acetate in Type I Watcer Methanol
- %A
- %B
60
40
60
40
10
90
10
90
0
100
0
100
60
40
60
40
The following parameters were used for operation of the mass spectrometer:
Parameter Ionization Mode Polarity Transitions Monitored
Setting Electrospray Negative 499->99
PAGE3OF5
Gas Temperature Drying Gas (N2)
350C
7.0 Umin
4 Analysis
4.1 Calibration
A 6-point calibration curve was analyzed at the beginning, throughout, and at the end of the analytical sequence for PFOS. The calibration points were preparmefdor PFOS at 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 ng/mL. The instrument response versus the concentration was plotted for each point. Using linear regressionwith l l x weighting, the slope, ,y-interceptand coefficient of
determination (P) were determined. A calibration curve is acceptakile if ? 2 0.990.
For the results reported here,'calibration criteria were met. The calibration curves are included in the raw data in Attachment C.
4.2 Surrogates
Surrogate spikes are not a component of the analytical method.
4.3 Laboratory Control Spikes
Laboratory control spikes in the analytical set were prepared by adding a known concentration of the analytes to control egg yolk samples. Laboratory control spikes are used to assess method accuracy. The laboratorycontrol spikes must show remveries between 70-130% or the data is rejected. For the results reported here, the spikes were within the acceptable range.
4.4 Matrix Spikes
Matrix spikes were not included with this study.
4.5 Sample Related Comments Duplicate injections were performed for all sample analyses.
Please see Attachment B for a detailed listing of the analytical re!t;ults. Results are reported in parts per billion (ng/g) for PFOS.
Samples are disposed of one month after the report is issued unless otherwise specified. All electronic data is archived on retrievable media and hard copy reports are stored in data
folders maintained by Exygen. Hardcopy data is stored for a minimum of five years. 3M
Environmentalwill be notified 30 days prior to the disposal of hardcopydata.
PAGE40F5
7 Attachments
7.1 Attachment A: Chain of Custody
7.2 Attachment B:Analytical Results
7.3 Attachment C: Raw Analytical Data 7.4 Attachment D: Study Personnel 7.5 Attachment E: Analytical Phase Protocol
a
8 Signatures
PAGE 5OF5
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See Original Looin
Exygen Research
Sample Login Report
Study Number: Protocol:
023-070 NA
EGG YOLK
NA
EGGYOLK
.
NA
RICK K 6-4-02
RICK K 6-4-02
Page 1 of 1
FREEZER 8 FREEZER 8
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Composite of samples: 201551-201 562
Composite of samples: 201 563-201 574
Verified By/Date:
4-y-uz
Printed 6/4/2002
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Exygen Research
Sample Login Report
I I Studv Number:
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Protocol:
023-070 NA
Page 1 of 3
0201551 E01-1379-31882 6-1-02 21115
EGG YOLK
NA
0201552 E01-1379-31883 6-1-02 21:15
EGG YOLK
NA
0201553 E01-1379-31884 6-1-02 21:15
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NA
0201554 E01-1379-31885 6-1-02 21:15
EGG YOLK
NA
0201555 E01-1379-31895 6-1-02 21115
EGG YOLK
NA
0201556 E01-1379-31896 6-1-02 21115
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NA
0201557 E01-1379-31897 6-1-02 21115
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NA
0201558 E01-1379-31898 6-1-02 21:15
EGG YOLK
NA
0201559 E01-1379-31939 6-1-02 21115
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NA
0201560 E0l-1379-31943 6-1-02 2135
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0201561 E01-1379-31947 6-1-02 21115
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0201562 E01-1379-31951 6-1-02 21115
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0201568 E01-1378-31655 6-1-02 21:15
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LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
LAWRENCE 0 6-3-02
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Verified By/Date:
d-2-02
Printed 6/3/2002
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Exygen Research
Sample Login Report
Study Number: 023-070
-
Protocol:
NA
Page 2 of 3
6-3-02
0201570 E01-1378-31657 6-1-02 21115
EGG YOLK
NA
LAWRENCE 0
FREEZER 8
6-3-02
0201571 E01-1378-31694 6-1-02 21:15
EGG YOLK
NA
LAWRENCE 0
FREEZER 8
6-3-02
0201572 E01-1378-31698 6-1-02 21:15
EGG YOLK
NA
LAWRENCE 0
FREEZER 8
6-3-02
0201573 E01-1378-31702 6-1-02 21:15
EGG YOLK
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LAWRENCE 0
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6-3-02
0201574 E0l-1378-31706 6-1-02 21115
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6-3-02
0201575 E01-1379-31940 6-1-02 21~15 EGG ALBUMEN
NA
LAWRENCE 0
FREEZER 8
6-3-02
I 1 I I I 1 I 0201576 E01-1379-31944 6-1-0221:15
EGGALBUMEN
NA
LAWRENCE 0 6-3-02
FREEZER 8
0201577 E01-1379-31948 6-1-02 21:15
EGG ALBUMEN
NA
LAWRENCE 0 6-3-02
FREEZER 8
0201578 E01-1379-31952 6-1-02 21115
EGG ALBUMEN
NA
LAWRENCE 0 6-3-02
FREEZER 8
0201579 E01-1378-31695 6-1-02 21115
EGG ALBUMEN
NA
LAWRENCE 0 6-3-02
FREEZER 8
0201580 E01-1378-31699 6-1-02 21115
EGG ALBUMEN
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LAWRENCE 0 6-3-02
FREEZER 8
0201583 E01-1379-31942 6-1-02 21:15
EGG SHELL
NA
LAWRENCE 0
FREEZER 8
MEMBRANE
6-3-02
0201584 E01-1379-31946 6-1-02 21:15
EGG SHELL
NA
LAWRENCE 0
FREEZER 8
MEMBRANE
6-3-02
0201585 E01-1379-31950 6-1-02 21115
EGG SHELL
NA
MEMBRANE -
LAWRENCE 0 6-3-02
FREEZER 8
0201586 E01-1379-31954 6-1-02 21115
EGG SHELL
NA
LAWRENCE 0
FREEZER 8
MEMBRANE
6-3-02
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Verified By/Date:
6-3-0~
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Printed 6/3/2002
m
0201587 0201588 0201589 0201590
E01-1378-31697 E01-1378-31701 E01-1378-31705 E01-1378-31709
6-1-02 21:15 6-1-02 21:15 6-1-02 21115 6-1-02 21:15
Exygen Research
Sample Login Report
Study Number: 023-070
Protocol:
NA
EGG SHELL
NA
LAWRENCE 0
MEMBRANE
6-3-02
EGG SHELL
NA
LAWRENCE 0
MEMBRANE
6-3-02
EGG SHELL
NA
LAWRENCE 0
MEMBRANE
6-3-02
EGG SHELL
NA
LAWRENCE 0
MEMBRANE
6-3-02
FREEZER 8 FREEZER 8 FREEZER 8 FREEZER 8
Page 3 of 3
F-DRY ICE-A F-DRY ICE-A F-DRY ICE-A F-DRY ICE-A
Stored in FREEZER 8 until Log-In.
Stored in FREEZER 8 until Log-In.
Stored in FREEZER 8 until Log-In.
Stored in FREEZER 8 until Log-In.
- - I_-. I -
-I ,
Printed 6/3/2002
513 1l2002 Project: E01-1379
.
3M ENVIRONMENTAL LABORATORY
CONTRACT LABORATORY WORK ORDER BY SAMPLE Contract Lab@): EXYGEN
1 of 2
Requester: Robideau, Rochelle R (0002-03E-09)
Completion Date:
Department: 502180 Site Source:
Project Lead: Rwhelle R. Robideau
Project Number:
Phone Number: 65 1-778-7065
Date Received: 101412001
Email Address: inobideau@mmm.com
a- Project Description:N. Bobwhite Reproduction (Eggs)
Ship Date: Comments:
/
NOTE: MISC SERV = Protein separation to be conducted by Exygen Research.
3M Sample
~
~~~~
E01-1379-3 1882
Analysis Code
MI SC-SERV
Sampled Date Sample Description
~
61 131200 1
10 DDm a.i. 230 H Yolk
Analvtical Method
. Comoonenls
Misc. Services
Misc. Services
~~~
~~
Analvsis Due Date
6130i2002
EO 1-1379-3 1883
Analvsis Code
MISC-SERV
- EO 1 1379-31884
Analysis Code
MI SC-SERV
E01-1379-3 1885
Analvsis Code
MISC-SERV
EO 1-1379-3 1895
Analysis Code
MISC-SERV
EO 1-1379-31896
Analysis Code
MISC-SERV
6/13/200 1
10 DDm a.i. 232 H Yolk
Analvtical Method
Comoonenls
Misc. Services
Misc. Services
611 31200 1
10 m m a i . 234 H Yolk
Analvtical Method
ComDonenB
Misc. Services
Misc. Services
611 31200 1
10 DDm a.i. 236 H Yolk
Analvtical Method
Comwnenls
Misc. Services
Misc. Services
~~
~
~
~~
611312001
10 DDm a.i. 229 I Yolk
Analvtical Method
' Comoonenls
Misc. Services
Misc. Services
6/13/2001
10 m m a.i. 231 I Yolk
Anahtical Method
Componenls
Misc. Services
Misc. Services
/
AnalvsiiDue Date
613012002
Analvsis Due Date
613012002
/ .
Analvsis Due Date
6130l2002
Analvsis Due Date
613012002
./-
Analysis Due Date
613 012002
E01-1379-31897
Analysis Code
MI SC-SERV
E01-1379-31898
Analysis Code
MISC-SERV
E01- 1379-31939
Analwis Code
MISC-SERV
611312001
10 m m a.i. 233 I Yolk
Analvtical Method
ComDonenB
Misc. Services
Misc. Services
611312001
10 Dom a.i. 235 I Yolk
Anahtical Method
Comoonenb
Misc. Services
Misc. Services
6/21/2001
10 DDm a.i. 226 J Yolk
Analvtical Method
CornDonenis
Misc. Services
Misc. Services
Analysis Due Date
613 012002
.
Analvsis Due Date
613012002
Analvsis Due Date
6l3Ol2002
-
513 1l2002
3M ENVIRONMENTAL LABORATORY
CONTRA(JT LABORATORY WORK ORDER BY SAMPLE
2 of2
Project: E01-1379 (cont.)
3M Sample
~
~~
E01-1379-3 1940
Analvsis Code
Sampled Date Sample Description
6/2 112001
10 Dum a.i. 226 J Albumen
Analytical Method
Corn ponen b
MISC-SERV
Misc. Services
Misc. Services
Contract Lab@):EXYGEN
,,
Analysis Due Date
6/30/2002
EO 1-1379-31942
Analvsis Code
MISC-SERV
6/21/2001
10 uDm a.i. 226 J Shell Membrane
Analvtical Method
Cornoonen ts
Misc. Services
Misc. Services
Analysis Due Date
613012002
EOI-1379-3 1943
Analvsis Code
MISC-SERV
6/2 1/2001
10 Dum a.i. 230 J Yolk
Analytical Method
Cornponen b
Misc. Services
Misc. Services
Analysis Due Date
613012002
E01-1379-3 1944
Analvsis Code
MISC-SERV
612112001
10 uDm a.i.230 J Albumen
Analytical Method
' Cornponenb
Misc. Services
Misc. Services
Analysis Due Date
6/30/2002
EO 1- 1379-31946
Analysis Code
MISC-SERV
612 11200 1
10 DDm a.i. 230 J Shell Membrane
Analvtical Method
Corn oonen b
Misc. Services
Misc. Services
Analysis Due Date
613012002
EO 1-1379-31947
Analvsis Code
PFOS
- EO 1 1379-31948
Analvsis Code
PFOS
612 112001
10 Dum ai. 232 J Yolk
Analytical Method
Corn oonenb
PFOS by ESMS
PFOS
6/21/2001
10 uum a.i. 232 J Albumen
Analytical Method
Corn oonen b
PFOS by ESMS
PFOS
Analys,is Due Date 613012002
Analysis Due Date
613 012002
E01-1379-3 1950
Analwis Code
PFOS
E01- 1379-31951
Analpis Code
PFOS
E01 -1 379-3 1952
Analvsis Code
PFOS
E01-1379-31954
Analwis Code
PFOS
6/21/2001
10 DDm a.i. 232 J Shell Membrane
Analvtical Method
Components
PFOS by ESMS
PFOS
6/21/2001
10 Dum a.i. 234 J Yolk
Analvtical Method
Cornoonenb
PFOS by ESMS
PFOS
612 112001
10 Dum a.i. 234 J Albumen
Analvtical Method
Cornoonen ts
PFOS by ESMS
PFOS
612 1I200 1
10 DDm a.i. 234 J Shell Membrane
Analytical Method
Cornoonenb
PFOS by ESMS
PFOS
Analysis Due Date
613012062
Analysis Due Date
613012002
Anal& Due Date
613 012002
Analysis Due Date
6/30/2002
-
-
3M Environmental Labor
Form 38778 - PWO
Shipping Addrear: 3M Bidg 2-3E-09 935 BushAvenue
E??? Company
Robideau, Rochelle R 3M
U
City, State, Zip Telephone#
, h S , 778 70hS
Special Instructions andlor Specific Regulatory Requirements:
(method. limit of detection. reporting units, etc.)
Item I #I
Client Sample Identification
I 1. b0 m m a.i. 230 H Yolk
2. 10 ppm a.i. 232 H Yolk
3. 10 ppm a.i. 234 H Yolk
4. 10 ppm a i 236 H Yolk
5. 10 ppm a.i. 229 I Yolk
6. 10 ppm a.i. 231 I Yolk
7. 10 ppm a.i. 233 I Yolk
8. 10 ppm a.i. 235 I Yolk
9. 10 ppm a.i. 226 J Yolk
I (10ppm a.i. 226 J Albumen
~__
~
FAX #
1
Date Available
~ Date Due
]Contract Lab
\I
w+,G - N7- w- r -----
I&YGd I I I I I
I
Analysis Reauested:
Complete below. Attach any associated information.
I
131882 31883 31884 31885 31895 31896 31897 31898 31939
131940
Time
d Sampled
k/13/01 I
I
b/13/01
b/13/01
b/l3/Ol
5/13/01
5/13/01
5/13/01
5/13/01
,5/21/01
b/21/01 I
I
Matrix/ Media
z
-0
I-
Original~ AccompanyinQSamples
Last Page - Originator
See Reverse Side for Instructions
a Environmental Laboratory ,, Chain of Custody /Request for Laboratory Analytical
R
3M Env. Lab Project #
I
(method. limit of detedion. reporting units.-elc.)
I
I
I Preservatives:
I I
I
I
I
I
I
I
I
I
I
I
Analysis Requested:
I
Completebelow. Attach any associated Information.
Item #,
Client Sample Identification
1. ho DDm a.i. 230 J Albumen
2. bo ppm a.i. 230 J Shell Membrane
3. 10 ppm a.i. 232 J Yolk
4. 10 ppm a.i. 232 J Albumen
5. 10 pprn a.i. 232 J Shell Membrane
13. 10 ppm a.i. 234 J Yolk
7. 10 ppm a.i. 234 J Albumen
8. 10 ppm a.i. 234 J Shell Membrane
9.
10. I
. __
3M IlMS#
b1944
131946 31947 31948 31950 31951 31952 31954
Date
Time
Sampled Sampled
k/21/01 I
I
b/21/Ol I
I
1/21/01
4/21/01
5/21/01
5/21/01
5/21/01
5/21/01
Matrix/ Media
I
I
-
I
i
Page 3 0 f 2 _
Original- A m p a n y - Samples
Last Page - Originator
See Reverse Side for l n s t r u d i i o
513 112002
3M ENVIRONMENTAL LABORATORY
C O N T R A m LABORATORY WORK ORDER BY SAMPLE
1 of2
Project: E01-1378
Contract Lab(s): EXYGEN
Requester: Robideau, Rochelle R (0002-03E-09)
Completion Date;:
.
Department: 502180 Site Source: Project Number: Date Received: 10/4/2001
w' Project Description: Mallard Reporduction (Eggs) I
Ship Date:
Comments:
Project Lead: Rochelle R. Robideau Phone Number: 651-778-7065 Email Address: i:rrobideau@mmm.com
. /
NOTE: MISC SERV = Protein separation conducted by Exygen Research.
3M Sample E01-1378-31638
Analysis Code
MISC-SERV
EO 1-1378-3 1639
Analvsis Code
MI SC-SERV
Sampled Date Sample Description
611 31200 1
10 uurn a.i. 230 H Yolk
Analvtical Method
. ComDonents
Misc. Services
Misc. Services
611 31200 1
10 uurn a.i. 232 H Yolk
Analvtical Method
Componenb
Misc. Services
Misc. Services
~~
~
Analvsis Due Date
613012002
/ .
Analysis Due Date
613 012002
~~
-
EO 1-1378-3 1640
Analvsis Code
MISC-SERV
EO 1-1378-3 1641
Analwis Code
MISC-SERV
6l13l2001
10 uum a.i. 234 H Yolk
Analvtical Method
Cornmnen b
Mise. Services
Misc. Services
6l13l200 1
10 Dum a.i. 236 H Yolk
Analvtical Method
Cornmnenb
Misc. Services
Misc. Services
Analvsis Due Date
. 613012,002
Analysis Due Date
613012002
-
E01-1378-31654
Analvsis Code
MISC-SERV
~
~~
~~
EO 1- 1378-31655
Analysis Code
MISC-SERV
61 131200 1
10 DDm a.i. 229 I YOk
Analvtical Method
CornDonen tr
Misc. Services
Misc. Services
~
~
~~
6113/2001
10 Dum a.i. 231 I Yolk
Analvtical Method
Components
Misc. Services
Misc. Services
Analvsis Due Date
6f3012902
Analvsis Due Date
613012002
E01-1378-3 1656
Analvsis Code
MISC-SERV
61 131200 1
10 Dum a.i. 233 I Yolk
Analvtical Method
CornDonen ls
Misc. Services
Misc. Services
Analysis Due Date
613 Of20_02
E01-1378-3 1657
Analysis Code
MISC-SERV
EO 1-1378-31694
Analysis Code
MISC-SERV
6113f2001
10 uurn a.i. 235 I Yolk
Analvtical Method
CornDonen b
Misc. Services
Misc. Services
6121f2001
10 uum a.i. 230 J Yolk
Analvtical Method
CornDonen b
Misc. Services
Misc. Services
Analvsis Due Date
6130f2002
Analysis Due Date
6l3012002
513 112002
3MENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE
2 of2
Project: E01-1378 (cont.)
3M Sample
Sampled Date Sample Description
EO1-1378-31695
Analysis Code
MISC-SERV
6/2112001
10Dum a.i. 230J Albumen
Anahtical Method
ComDonents
Misc. Services
Misc. Services
Contract Lab@):EXYGEN .,,
Analysis Due Date
613012002
-
EOI-1378-31697
Analwis Code
MISC-SERV
6/2112001
10mrn a.i. 230J Shell Membrane
Analytical Method
Comaonenb
Misc. Services
Misc. Services
Analysis Due Date
61300QQ2
E0l-1378-31698
Analvsis Code
MISC-SERV
612l/2OO1
10Dum a.i. 232J Yolk
Analytical Method
Components
Misc. Services
Misc. Services
Analysis Due Date
613012002
EO1-1378-31699
Analwis Code
MISC-SERV
612 11200 1
10mma.i. 232J Albumen
Analytical Method
Componen b
Misc. Services
Misc. Services
Anal& Due Date
613012002
EO 1-1378-31701
Analysis Code
MISC-SERV
6/2lROO1
10mrn a.i. 232J Shell Membrane
Analytical Method
Com ponen ls
Misc. Services
Misc. Services
Analysis Due Date
613012002
EO 1-1 378-31702
Analwis Code
PFOS
612 11200 1
10o m a.i. 234J Yolk
Analvtical Method
Componenls
PFOS by ESMS
PFOS
/
Analvsis'Due Date
6130l2002
EO 1- 1378-31703
Analvsis Code
PFOS
E01-1378-31705
Analysis Code
PFOS
612 11200 1
10uDm a.i. 234J Albumen
Analvtical Method
ComDonen b
PFOS by ESMS
PFOS
~~
~~
6121I200 1
10 m m a.i. 234 J Shell Membrane
Analytical Method
ComDonents
PFOS by ESMS
PFOS
Analysis Due Date
613 012002
/ .
Analysis Due Date
6130l2002
EO 1- 1378-31706
Analwis Code
PFOS
E01-1378-31707
Analysis Code
PFOS
612 11200 1
10DDm a.i. 236J YOk
Analvtical Method
Componen b
PFOS by ESMS
PFOS
6 / 2 112001
10DDm a.i. 236J Albumen
Analvtical Method
Components
PFOS by ESMS
PFOS
Analysis Due Date
613012002
, .
Analysis Due Date
613012002
EO1-1378-31709
Analysis Code
PFOS
6/21/2001
10m m a.i. 236 J Shell Membrane
Analvtical Method
Com aonenls
PFOS by ESMS
PFOS
Analysis Due Date
613012002
.
Form 38778 ~ PWO I
Project IDlProject Name
I
Shipping Address:
Telephone:
3M Bidg 2-3E-09
Sample Receiving: (651) 778-4948
Template #
335 Bush Avenue St Pau1,MN 55106
Alternate: (651) 778-6751
FAX: (651) 7786176
. 5 IDrnind I b a '",C". L I - Y Dept. # (main)
Mailing Addressnnn7 -nqF-ns
City, State, Zip
cT lTelephone# h51 778 7nhs
Soecial instructions andlor Specific Re-gulator-yReq.uirements:
(method. limit of detection. reporting units. e1c.l
FAX ##
I I
II
I I
Anialysis Requested:
Temperature:
'C
Other Assoclaled CoCs
Page I_ O*f
0 Received on Ice
Original -AccompanyingSamples
Last Page - Originatw
See Reverse Side for Instructions
3M Environmental Labor
Form 30770 - PWO
Shipping Address: 3M Bldg 2-3E-09 935 Bush Avenue St Pau1,MN 55106
Telephone: Sample Receiving:(651) 778-4
' Analysis Requested:
Complete below. Attach any associated Information.
, Samole Condition UDon Receipt:
Temperature:
'C
0 Acceptable 0 Other:
0 Received on Ice
Copier IO
Page>
01
Original -AccompanyingSampler
Commenls:
Last Page - Originalor
See ReverseSide for lnslruclions
I I
~
II
RESEARCH
Precise Research. Proven Results.
Sample "Condition Upon Receipt" Form
Protocol #
A/#
Exygen Study # 023 - 0 70 Date & Time Received 6-/ d 3
27LN .
Condition of Samples /-Pr, 2cr - #
1
Temporary Storage Location rI - P
1
Initials & Date
i,/
4 -/-f?z
I
May 24, 200213 T: 800.2812219
F: 814.272.1 019
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Exygen Research
Sample Login Report
I Protocol:
NA
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6-5-02
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I
Page 1 of 1
!Markets #33.State
I
Printed 6/5/2002
*,-j ;
. - .- THANK YOU FOR SHOPPIING
P .I
WEIS MARKETS 4! 33 STATE COI.LEGE, PA
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THIS IS AN EXACT COPY OF THE ORIGINAL DOCUMENT."
,p .)
d-5-(:j i
DATE--,-
..
lm
RESEARCH
Precise Research. '.. Proven Results.
Summary of PFOS Residue Found (ppb) in Combined Mallard Egg Yolk Samples
Sponsor
Analyte Found
Fort Level
Recovery
ID na (Reagent Control)
(ppb) NQ
-IlPPb)
(%) -
na (Reagent Control)"
NQ
na (Reagent Control)
NQ
na (Reagent Control)"
NQ
na (Matrix Control)
NQ
na (Matrix Control)" na (Matrix Control) na (Matrix Controlp
na (LCS)
NQ
- NQ - NQ
108
100
108
na (LCS)
111
100
111
na (LCS)
103
100
103
na (LCS) **
100 53600
100
100
**A
52300
- ** Dup
52600
- ** DupA
52500
-
NQ = Result is above the detection limit of - 0.5 ppb but below the quantitiition limit of 10 ppb.
A Duplicate Injection
**composite of samples E01-1378-31638,31639,31640,31641,31654,31655,31656,3165'7,31694,31698,31702,31706
.
F 814.231.1580
RESEARCH Precise Research.
Proven Results.
Summary of PFOS Residue Found (ppb) in Combined Quail Egg Yolk Samples
Sponsor
ID
na (Reagent Control)
Analyte Found
(ppb) NQ
Foirt. Level l(ppb)
Recovery
(%) -
na (Reagent Control)'+ na (Reagent Control) na (Reagent Control)A na (Matrix Control) na (Matrix Control)A na (Matrix Control) na (Matrix Control)A
na (LCS) na (LCS)
- NQ
NQ
-- NQ
NQ
- NQ - NQ - NQ
104
100
104
102
100
102
na (LCS)
106
100
106
na (LCS) **
109 75000
100
109
**A
74300
** Dup
48700
** DupA
49900
-
NQ = Result is above the detection limit of - 0.5 ppb but below the quantitation limit of 10 ppb.
A Duplicate Injection
*+composite of samples E01-1379-31882,31883,31884,31885,31895,31896,31897,318!~~8,31939,31943,31947,31951
F 814.231.1580
In
RESEARCH
Precise Research. Proven Results.
1
Summary of PFOS Residue Found (ppb)
I
in Mallard*/Quail** Egg Yolk Fractions
- Sponsor
Analyte Found
Fort. Level
Recovery
I
ID
(ppb)
(ppb)
("/O)
na (Reagent Control)
ND
na (Reagent Control)A
ND
na (Reagent Control)
ND
na (Reagent Control)A
ND
na (Matrix Control)-VLDL
13.5
na (Matrix Control)-VLDLA
14.3
na (Matrix Control)-Phosvitin
NQ
na (Matrix Control)-PhosvitinA
NQ
na (Matrix Control)-Lipovitellin
NQ
na (Matrix Control)-LipovitellinA
NQ
*-Mallard VLDL
41 800
*-Mallard VLDLA
42500
*-Mallard Phosvitin
3600
*-Mallard PhosvitinA
3580
*-Mallard Lipovitellin *-Mallard LipovitellinA
891 0 8830
-
**-Quail VLDL
39700
- **-Quail VLDLA
**-Quail Phosvitin
39900 815
**-Quail PhosvitinA
838
**-Quail Lipovitellin
1300
**-Quail LipovitellinA
1340
-
- ND = Result is below the detection limit of 0.5 ppb.
- NQ = Result is above the detection limit of 0.5 ppb but below the quantitirtion limit of 10 ppb.
A Duplicate Injection
+ composite of samples E01-1378-31638,31639,31640,31641,31654,31655,31656,31657', 31694,31698,31702,31706
*+composite of samples E01-1379-31882,31883,31884,31885,31895,31896,31897,318SI8,31939,31943,31947,31951
F 814.231.I580
E
n
3058 Research Drive State college, PA 16801
RESEARCH
T: (814) 272-1039 F: (814) 231-1580
STUDY PERSONNEL LOG
I
PROTOCOL NA
EXYGEN STUDY 023-070
1
PRINTED NAME
SIGNATURE
1 INITIALS
.1
1
1
II ill
June 28,2001/1
ANALYTICAL PHASE PROTOCOL
ANALYTICAL PHASE TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM
MALLARD AND QUAE EGG YOLK FOR ANALY5;IS USING HPLC-
ELECTROSPRAY/MASS SPECTROMETRY
SPONSOR 3M Environmental Technology and Safety Services
Building 2-3E-09 PO Box 3333 1
St. Paul, MN 55133-3331
DATA REQUIREMENTS Analy-tical Method Requirements
PERFORMING LABORATORY Exygen Research (Exygen) 3058 Research Drive Stiite College, PA 16801 Phone 814-272-1039
Exygen Research
Page 1 of 39
Title: EXTRACTION OF POTASSIUM PERFLUOROOCTPiNESULFONATE! FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAYMASSSPECTROMEiiTRY ,
TABLE OF CONTENTS Page
TABLE OF CONTENTS.......................................................................................................... 2 1. PURPOSE...................................................................................................................... 3 2. REFERENCEMATERIAL ............................................. ........................................... 3
3. SPONSOR................................................. ........................ ...........................................4
4. TESTING FACILITY (PERFORMINGLABORATOItY...................................... 4 5. PROPOSEDEXPERIMENTALTIME-FRAME...................................................... 4 6. SAMPLEPROCESSING, STORAGEAND IDENTIFICATION..........................4 7. ANALYTICALMETHOD......................................................................................... 5 8. EXPERIMENTALDESIGN....................................................................................... 5 9. RECORDS.................................................................................................................... 5 10. DATA AND REPORT ..................................................... ........................................... 6 11. COSTS................................................................................ ........................................... 6 12. PROTOCOL APPROVAL..........................................................................................7 APPENDIX: ANALYTICAL METHODS ................................. ........................................... 8
Exygen Research
Page 2 of 39
Title:
EXTRACTION OF POTASSIUM PERFLUOROOCTANliWJLFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAYMASS SPECTROMlXRY
1. PURPOSE The purpose of this study is to analyze mallard and quail egg yolk samples and their fractions for residues of perfluorooctanesulfonate (PFOS) using methods entitled, "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yo`lk" and "Determination of Peduorooctanesulfonate in Egg Membrane, Albumen and Yolk by L C M S M S." Both methods can be found in the Appendix.
2. REFERENCE MATERIAL
The following analytical standard will be used:
Test Material PFOS
Lot Number
215
Tl3D
Chemical name and structure of the compound is presented below.
PFOS Chemical Name: IUPAC Name:
CAS Number: Molecular Weight:
Perfluorooctanesulfonat e 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt 2795-39-3
499 (C8Fi7S03-)
0
0
m e : The neutral molecule and standard from which the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [C817S0&], molecular weight 538.
A record of test and reference substance receipt, storage conditions, and a record of use will be maintained at Exygen. Forms documenting chain-of-custody and shipping records for tracking of the test substances will be included as part of the raw data package.
Exygen Research
Page 3 of 39
All standarddtest substances and any prepared solutions must be identified with a unique label or number on the container or cross-referenced to the container.
HAZARD INFORMATION
A current MSDS for the chemical(s) used in this study will be maintained at the testing facility.
3. SPONSOR
3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331
Sponsor Representative: Bill Reagen
4. TESTING FACILITY / PERFORMING LABORA'rORY
Exygen Research 3058 Research Drive State College, PA 16801
Project Manager: Emily R. Decker, Exygen
5. PROPOSED EXPERIMENTAL TIME-FRAME
Analytical Start Date Analytical Termination Date Report Issued
June 3, 2002 June 21,2002 July 3 1, 2002
6. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION
All mallard egg yolk samples received will be combined into one composite sample using a blender. The same process will also be used for all of the quail egg yolk samples.
Each sample will be assigned a unique sample identification number at Exygen, which will be used for tracking and identification of the samples. The samples will be stored in a tempemme-monitored freezer, maintained at < -10" C, except. when removed for extraction and analysis as described in the method. The: samples will be kept isolated fiom the test substance di.iring storage.
Sample receipt and storage location and conditions during the study will be: documented. All samples and any resulting sample extracts will be identified. with a unique label or sample number. Such identification will be either on the: container or cross-referenced to the container.
Exygen Research
Page 4 of 39
7. ANALYTICAL METHOD
The composite mallardquail egg yolk samples will be ctxtracted and analyzed first according to the analytical method titled "Determination of Periluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LCMSMS" to determine the total amount of PFOS in each sample. Then the mallardquail composite yolk samples will be separated into three liactions using the method "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yolk." Each fraction will then be extracted and analyzed according to the method "Determination of Peffluorooctanesulfonate in Egg Membrane, Albumen imd Yolk by LCMSMS".
8. EXPERIMENTAL DESIGN Samples obtained by 3M Environmental will be shipped to Exygen Research for analysis. Samples will be extracted and analyzed at Exygen according to method, "Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LCMSMS." `
Methods to control bias will include assay of untreated control samples, fortification of untreated control samples to obtain rc;.:coverydata, and replicate analysis of fortifed samples to provide an indication of reproducibility. Fortification will be made to the matrix prior to extract ion.
The average recovery and relative standard deviation of the fortified samples will be calculated. If necessary, apply a standard test for outliers. If an outlier exists, then it may be excluded from the statistical analysis. Also, the average residue found and standard deviation for each matrix will be cailculated.
9. RECORDS
Records to be maintained include, but are not liimited the following (as appropriate) :
1. Sample tracking sheet(s) 2. Sample receipt records, storage history, and chains of custody 3 . History and preparation of standards (stock, fortification, calibration) 4. Description of any modifications to the method 5. Instrument run sheets, bench-sheets or logs 6. Analytical data tables 7. All chromatographic and instrumental conditions 8. Sample extraction and analysis dates 9. A complete listing of study personnel, signatures and initials 10. Chronological presentation of all study correspond'ence 11. Any other data necessary for the reconstruction of the study
All chromatograms will contain the following: a. Sample identification, date, arrow or other indication of the area of interest, and injection number corresponding to the run.
Exygen Research
Page 5 of 39
I
1 b. Additionally, fortifications will include the fortification level of the analyte.
c. Analytical standard chromatograms will additionally include the concentration (e.g., pg/ml, ng/mL, ppb, ppt, etc.).
Each data set will contain information on tempera.tures, flow rates, column parameters, gases, instrument parameters, and instrument type, etc.
10. DATA AND REPORT
1. All raw data and the original signed protocol will be maintained in the study file. This data includes the laboratory notebooks, imalytical standard solution preparation, sample chain of custody sheets, sample work sheets, chromatograms, calibration curves, and any other alppropriate data generated.
2. A report will be issued by Exygen at the completicrn of the study according to the Sponsor's specifications. The report contents should include, but not limited to: 1. Objectives and procedures stated in the protoco I 2. Analytical and statistical methods used 3. Reference materials identified by name, lot, purity, and other characteristics 4.Name of performing laboratory and analytical start and termination dates 5. Tables containing all applicable data 6. All chromatographic and instrumental conditioris 7. A complete listing of Exygen study personnel
11. COSTS The total cost for performance of the study will be $30,000.00.
Exygen Research
Page 6 of 39
12. PROTOCOL APPROVAL
II
Project Manager, Exygen
Sponsor, 3M
/&gzif/&
Bill Reagen
m
Exygen Research
Page 7 of 39
APPENDIX: ANALYTICAL MFTEIODS A, "Determination of Perfluorooctanesulfonatein Egg Membrane, Albumen and Yolk by LC/MS/MS"
B. "Lipovitellin, Phosvitin, and Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail Egg Yolk"
*
Exygen Research
Page 8 of 39
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- TITLE
Determination of Perflu rooctanesulfonatein Egg Membrane. ..;lbumenand Yolk by
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AUTHORS
Emily Stauffer and John Flaherty
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D A IS~SUED
Janhary 2,2001
SPONSOR
3M Environmental Laboratory Building 2-3E-09 PO Box 33331
St. Paul, MN 55133-3331 . ..
PERFORMING LABORATORY ,
. .. . Centre Analytical Laboratories, Inc. (Centre:)
3048 Research Drive State College, PA 16801
-C1"NTRESTUDY NUMBER
023-015
.. .
CENTRE METHOD NUMBER .
OOM-023-015
f.
_TOTAL NUMBER OF PAGES
..
28
..
.-
..
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Exygen Research
Page 9 of 39
Centre Method No: 00M-023-015
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MANAGEMENTAPPROVAL
HI
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1: As per 4d CER 792.3,method development is not required to be conducted in compliance
i
with Good Laboratory Practices. However, the wo$ Was in conformancewith applicable
r.
standard operating procedures and general GLP regulations.
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3)blOI
111
Date
Principal Investigator.
Centre Analytical Laboratories,,h c .
/5ohnFlaherty
1
Laboratory Manager
.
Date
Centre Analytical Laboratories, Inc.
!
.-
4-rttCtjl-O(
1111
Date
- Centre Analytical Laboratories, Inc.
Ill
insor Represhtative . ?!MEnvbnmental Laboratory .
..
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Exygen Research
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. Centre Method No: OOh-023-015
TABLE OF CONTENTS
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1. SUMMARY................... ........*........................................................ 6
2. EXPERIMENTAL'COMPOUNDS.........+. ............................... :...............7
3. CHEMICALS AND SUIF'PLLES ..................I..... ......".........;..............7
3.1. CmhuCALs ................................................................................................................................................ 7 3.2. ~ANDARD.9........................................................................................... 1.................................... .;.............7 33. EQUIPMENT SuppuEs.............................................................. :......................................................... 8 3.4. SOLUTION.S....;....................................................................................................................................8....... 35. PREPARATIOONFST~(:K. FORTIFICATIONA.NDCuremnQNSOLUTION...S..................................... ;.....9
3.5.1. Stock Solutwn......................................... .............................. ......................................................... 9 3.5.2. FortificationSolutions............................................... ....-..............................................................9.. 3.5.3. &libration Stanthirds..................................................................................................................... 9
4. METHOD............ ............................................................. ............... 10
. .
4.1. 4.2.
FSALMOPWLDEPIRAOGCRE.A.S..M.S..I..N.......G..........................................................................................................
.................................................................... .....................................................................
1010
4.3. SAMPLE-mON, ............................................................................................................................. 10
4.3.1 Egg Membrane......................................... .................................... ..................................................11
4.3.2. Egg Yo& md.4a-n .................................................................................................................. 11
4.4. 4
.
ANALYSrS 4.1.. L ~
BYWLcSs/yMstse/mMsan..d....O...p..e..r..a..t.i.n...g...C...o...n..d...i.t.i.o...n..s...(.T...u...r.b...o..l.o...n..r..p...r.t..i.y..).....................................................:......................,...............1122
1
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4.4:2. Example TuneFile Parameters ............................. ....................................... :........... 13
4.4.3. CalibmtionProcedures.......................................................;......................................................... I3
4.4.4. Sample Analysk................................................................................................................................ 14
45. pERIT)RMMa a m ........................................................................................................................ 15
I
4.6. TIMEREQUIRED ANALYSIS................................................................................................... :...........16
5. .CALCULATIONS............................... I........-...".................................!.......16
6; SAFETY............... ..........................................i....-..................................... 17
TABLES........................................................................................................................ 18
F'IGURES..................... -................................;..-..........................'.. 22
.. Centre Analytical Laboratories: Inc. Study +Y 023-015
Page 3
Exygen Research
Page 11 of39 .
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=
LIST OF T U L E S
6 TABLE? suMMARy OF RIscovERlEs FAR PFos cpmEGG . MEMBRANE................................ .....i.......................................................... 19 !Ic.
f,'
TABLE
II:
.
MSUEMMBMRAARNYEO..F....R...E....C...O...V....E...R...I...ES
FO:..R....P...F...O...S....I.N.....M....A...L...L...A...R...D.....E...G...G........
19
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. TABLEq:SUMMAR.YOF RECOVERIES FOR PFOS IN QUAILEGG YOLK..r................ :...........................................................................
20
TABLE IV:SuMMA,R.YOF RECOVERIES FOR PFos IN MALLAN) EGG
1I111
. YOLK ............:................. ............................................................................ 20
llll
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1l1'11
TABLE V S w Y OF R I X O a R E S FOR PFOS IN QUAIL EGG
, I
. . ALBUMEN...................................................................................................... 2.1 . 1,
2
.
' TABLEv1:SUMMARY OF RIXOVERIES FOR PFOS INMALLARD EGG
I
AWBUMEN............................... ..................................................................... 21
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111
111
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Centre Analytical Labontories, Inc. Study # 023-015
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Centre Method No: OOM-023-015
LIST OF FIGURES
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Figure 1: Representative Chromatogramof a Quail Egg Membrane Control................23
* Figure 2: RepresentativeChromatogramof a Maliard Egg hkmbrane Control............23 ! Figure 3: RepresentativeChrornatogramofa Quail Egg Yolk Control......................... 24
Figure 4 Representative Chrotnatogramofa MallardEgg yolk Control........... 24 Figure 5: RepresentativeChromatogramof a Quail Egg Albumen Control..................25
Figure 6: Rep-ksentativeChrornatogramofa Mallard Egg Albumen Control ..............25
Figure
7:
R10epnrgelsgen(ptap.tbiv).e...C...h...r.o..r..n..a..t.o...g..r.a..m....o...f..a....Q...u..a..i.l..E...g..g....M....e..m...b...r.a..n..e...C...o...n..t.r..o..i..F...o..rtified
at 26
Figure 8: Representative Chrornatogkm of a Mallard Egg Membrane Control Fortified at
. 10.0nglg (ppb)................................................................. ............ .......26
- Figure 9: Representative Chrornato,k of a Quail Egg Yolk Control Fortified at
. 1O.D nglg.(p.pb)............. ............................ i.................................................... '27
Figure.10: Representative Chromatogram of a Mallard Egg Yolk Control Fortified at
. . 10.0 ngg (ppb)....................... ..................... ..................................................
27
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Figure.11: Representative Chromatogram ofa Quail Egg Albumen Control Fortified at ,.
..
. 10.0nglg (ppb).............................. :............................................................... 28
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Egufe 12: Representative Chromatogram of a Mallard Egg Albumen Control.Fortifiedat
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10.0 nglg (ppb) .............................................................................................. 28 I t
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C&h Method No: OOM-023-015 i
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1. SUMMARY
. -
1
.This document details perfluorooctanesulfonate
a method (PFOS) in
of egg
analysis for rhe residual determination of
membrane, albumen and yolk. The chemical
formula of the analyte is given in Section 2 of this method.
i, 1I
Perfluorooctanesulfonatc is exbracted from each matrix with methanol (MeOH). For egg membrane samples, the methanol extract is Passed through a membrane filter. For egg albumen and yolk samples, a 0.5g of carbon is added to an aliqupt of the methanol
extract. Quantification of PFOS is accomplishedby liquid chromatography/tandem mass spectrometry( L c I M S / M S ) analysisusing selected readtion monitoring,(SM).
', I
For this method the LOQ is 10 nglg (parts-per-billion). .This is based on stydiks
conducted with egg membrarie, albumen, and yolk control samples during method development.
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Centre Method No: OOM-023-015
2. EXPERIMENTALCOMPOUNDS '.
I .. The molecular structyreof PFOd is given below:
I
PFOS
Molecular weight: 499 (CsF17S03-7
Chemical Name : Pe:rfluorooctanesulfonate
W A C name :1-Octaneziulfonicacid,1,1,2,2,3,3,4,4,55,6,6,7,7,8,8,8-
..
heptadecaifluoro-potassium salt
,
CAS # : 27'95-39-3 I
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Note: The n e u d molecule and standard f o F which PFOS (anion)is derived
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from is perfluorooctanesulfonatepotassium salt [CSFI~SO~(Km]olecular
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weight 538). .
.3. CHEMICAIS.&~ SUPPLIES . .
3.1. CHEMICALS - . .
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Chemical
. . Grade
Source
CatalogNo. '
Carbon
. 1201400
Supelco
572 10-U
- - Methanol (Meow
Ammonium Acetate
HPLC . (VWR) J . T . B e e r fi9093-2
Reagent
Aldrich Cher@cal 111-87-5
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Water
Type I
Centre
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Vype I water = electrical resistivity,.minimum of 16.67MQ-cm at 25"C, from a
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Labconcow waterprg workstation, bged'on ASTM classification) ..
..
- 3.2. STANDAkDS
Standard Grade
Test Control
. Source .
I
Reference Number
PFOS , Analytical
TCR-000 E746
3M Environmental Laboratory, Si. Paul, h4N
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Centre Method No: OOM-023-015
3.3. EQULPP~EANMT)S U P P ~ S
' EQUIPMENT
. SOURCE
Balance, 5 place analytical
Mettler
Balance, 1place top-loading Mini Bead Beater-8 Wrist-action shaker
Mettler
Bio Spec Products :
Burrell
..
- Bench-top centrifuge
Minicentrifuge -1201 50 mL disposable polypropylene centrifuge tubes
IEC VWR VWR
Stainless steel beads (cat # 11079)
Bio Spec Products ,
Micro-centrifuge tubes
VWR
Disposable pipettes, test tubes etc.
VWR
15-mL disposable polypropylene centrifuge tubes
VWR
Whamanm Anotopm Filter (cat # 6809-1022)
VWR ' .
I*
. 2-mL E p p e n d o P microcentrifugetubes (cat# 22-
'VWR
j
36-335-2)
8
.
2-mL clear HPLC vial kit (cat # 5181-3400)
HP
Standardlab equipment (class A pipettes'and
volumetric flasks,graduatedcylinders,etc.)
LC/MS/MS and HPLC!systems
ASdescribedin .
Section4.4.1. :.
..
1. In order to avoid contamination, the use of disposable labware is c
highly recommended (containers, tubes, pipettes, etc.). 9
2.
'THePfLloCn voi-raltecfalposn,-slihnoeudldcnoonttabieneursseodr.
equipment, .
including
teflo.n-lin. ed.
I
4. It is necessrq to check the solvents (methanol) for the presence of ' contaminants by LC/MS/Ms before use. Certain lot numbers have been found to be'unsuitable for use.
5. . Use disposable rhicnipipettes or pipettes to aliquot standard solutions I and when preparing standards and samples for extraction.
6. .Equivalent :materials may be.substituted for those specified in this method. However, the use' of carbon from Supelco is strongly
. recommended
3.4. SOLUTIONS
.-
i. 50 mM ammonium acetate solution: Dissolve 3.85 g of ammonium
I
acetate in 1L of ASTM w e I water. Store in an appropriate glass bottle
at room tempcxature for up to 1 year.
2. 2 m M ammonium acetate solution: Dilute 40 of the 50 & '
. &onium acetate solution in a liter of ASTM type I wate;, for mobile
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Centre Analytical Labontorics, Inc. !Study # 023-015 .
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Centre Method No: OOM-023-015 ' S
phase A. Store: in an appropriate glass bottle at room tempexhre for up to ltyear.
m: I
The voluines shown are provided for guidance; alternative volumes!
1.
may be prepared.
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3.5. TION ON OF STOCK, FORTIFICATIOANND, C A ~ Z B ~ T I O N
SOLUTIONS ,
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Analytical stkdards an: prepared for two purposes. They are used to fortify untreated simples in order io determine analytical recovery and to calibrate the
response oithe detectorused in the analysis.
The analyst may vary the absolute volumes of the standards as long as the correct proportions of solute to solventare maintained. The solutions cited below are given as an.example; alternative concentrations may be prepared if needed. -
3.5.1. Stock Solutbn
&pare stock solution of PEOS. at 100 pg/mL by weighing out 10.0 mg of
analyticdstandard (corrected for percent salt). Adjust final volume to 100 mL
with methanol in a 100-mL volumetric flask. Store this stock soiution (in
125-mL LDPE bottles) in a refrigerator at 2C to 6OC for a maximum period .
of 6 mon,bs from the date of preparation.
. -.
3.5.2. Fortij7catio.hSolutions
a. 1.0 u&nL FortijScation Solution - Pipette 1.0 mL of the 100 p g / k stock
. solution into a 100 mL. volumetric flask. Bring up to. volume with-'
methanol. .
b. . O ( ) i t i c a t i o n Solution - Pipette 10.0 d of the'1.0 pg/d
fortification solution.into a IOO-mL volumetric flask and 6ring up to
volume with me'thanol..
C. 0.01 u p / d Fortification Solution - Pipette 10.0 mL of the.0.1 p g / d
fortificiition solution into a 100-d volumetric flask and bring up to
volume with methanol.
*.
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Store-all fortification standard solutions (in 125-mL LDPE bottles) in a
refrigerator at 2C to 6C for a maximum period of 6 months from the date 1
.of preparation.
3.5.3. Calibration Standatdr
Prepare six LCM3,iMS calibration standards in methanol via dilution of the -
0.1 p g / d and 0.01 pg//mLfortification so~utions.
Centre Analytical Labor&ories,Inc. Study # 023-015
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Centre Method No: OOM-023-015
vo. 1 ,o. 1
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0.01
0.01
0.01
2
100
0.002
1
100
0.001
5
100
0.0005
1
100
0.0001
0.5
100
0.00005
Store all calibration Standard solutions (in 125-mL LDPE bottles) in a refrigeratorat 2C to 6C for a maximum period of 6 months from the date of preparation.
4. METHOD
-4.1.'n o w DIAGRAM . .
The flow diagram of the. method is given below, followed by a detailed
descriptionof each step.
. L e 'Weigh 1g of matrix (0.-lgfor membrane), Fortify if needed - 1.
..
. Extract withMe'OH
~
3.
c
C:arbon clean up (except membrane)
L
Filter .
J
Dilution
,.
Lc/MSIM4s analysis I
4.i. SAMPLE PROCESSING
-All samples are received frozen and will be kept frozen.@elow-10 "C)until time of the extraction.
4.3..SAMPLEEXTRAC~ION -
NOTE: All egg sample masces were separated prior to arrival at Centre. .
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Centre Method No: 00M-023-015:
4.3.1 Egg Membrane
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a. Allow sample to thaw.
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b. Weigh 0.1 g (+ 0.005g) of sample into a 2 - a EppendorTM tube. Record
f\'
the weight to the nearest 0.0001g. Fortify untreated control samples at this
point for determinationof method recovery.
I
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C. Add 1 IILof MeOH and three stainless steel beads, niplace lid tightly and.
- homogenize with bead beater set at position 7 (-2500 rpm) for 5 minutes.
Centrifuge the tubes with mini-centrifuge at 2,000 $m for 2,minUtes,'
I
then carefully transfer supematant to a new 2-mL-EppendorPM tube,
I
filtering with Whatmanm AnOtopm 10 filter if necessary.
, d. Mi aaknega05.4Xmd~iloutfimonetbhyantroalnussfimng-hdgisp0.o1samblLemoficfrioltpraipteetttoesH(1P0L0C-2v0ia0lps~a)n.d.
il '
e. Stom the rest of the extract in a refrigerator at approximately 2C to 6C.
I
for future re-dilution or re-injection. I
4.3.2. .Egg Yolk a i d Albumen
,
a Alloy sample to thaw.
..
b. Weigh 1.0 g (2 0.05g) of sample into a 50-mL disposable polypropylene centrifuge tube. Record the weight to the nearest 0.0001g. Fortify untreated control samples at this point for .deterniination of method: recovery.
C.
Add 25
shaker f
mL
or 15
- of MeOH, replace lid tightly.
minutes. Centrifuge tubes at
and st&e
2,000 rpm
-on a wrist-action
for 10 minutes..
1
d. Transfer about 5 mL of extract into a 15-mL disposable polypropylene . centrifuge tube, add 0.5 g cai-bon. replace lid tightly, arjd shake by hand for about 10 seconds.
- e. Let the tubes sit for 2 minutes, Carefullytransfer -2.0 mL, of the sample ,
into a 1 0 - d . disposable syringe barrel connected to a Whatmanm . Anotopm 10 filter. Filter SWple into two HPLC viis, one for injection
. and one for storage in a refrigeratorat approximately2C to 6C for future =-dilution or re-injection.
Centre Analyticid Laboratories, Inc. !Study # 023-015
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Mass Spec:
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p~ sCIEX API 3000,Biomolecular Mass Analyzer
Interface: .
S ( X XTurbolon SprayLiquid IntroductisnInterface
i.
Zarvard infusion pump ;
!
Computer. ' Power Macintosh G3
softwarc: . . ~ PI: Sciexhalyst 1.1
Windows NT
HPLC:
Hlewlett Packard (HP) Series 1100
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HP Quat Pump.
HP Vacuum Degasser
HP Autosampler .
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HP Column Oven .
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HPLC Column: Genesis Cg (Jones Chromatograihy), 2.1 mm x 50 F , 4p .
ColumnTemp.: 3!i0 C
.,
biection Vol.: 10pL
Mobile Phase (A): 2 &AImmonium Acetate in ASTMrype I water
Mobile Phase (B): Methanol
. Flow Rate:
0.3 d m i n .
,w 9bA
0
60
. 1.0
0
7.0
0
1.5
40
. 11.0
40
- % B
40 100
100 60 . . 60 :
. :.c . .
p
,.
[:
t -1
!.
It may .be necessary to adjust the HPLC gradient in order to optimize
instrument performance. Columns with different dimensions (e,g., 2.1 mm x
4[,
30 mm) and columns from differentmanufacturers,(Keystone Betasil CIe8tc.)
!
can be used, provided equivalent chromatographyis obtained.
..
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Ions monitored
Approximate
'
Analvte
PFQS .
Mode
negative
+ Transition Monitored 499 99
Retention Time (min) 4.20
-
.,
- . On a day-to-day basis, the retention times may vary slightly depending-on the '
batch of mobile phase, etc.
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4.4.2.'Example Tune File Parameters
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The following values are provided as 8n example. Actual values may vary
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from instrument to instrument. A h ,these values may be changed from time
I1
to time in order to optimize for greatest`sensitivity.
'
The mass spectrometer is tuned using a 0.5pghL PFOS solution,prepared via
1
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dilution of the stock solution in methanol. The solution is infused (using a
`T'connector) at 10 W m i n into a 0.2 mUmin stream of mobile phase
consisting of 40% methanol and 60% 2 mM ammonium acetate. The analytes
are initially tunqd for the parent ion and then tuned for the product ion. Once
III
the instrument is tuned, the optimized parameters are saved as a "tune file".
!.I
This tune fileis then used during routine analysis. The tuning procedure may
I.
be repeated as'necessq to ensure Optimal Sensitivity.
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Controls
Set
IS-Iodpray
46TO
.
OR-Orifice
-6 1.O
RNG-FocusRing
-270.0
QO-Quad 0 Rod Offset
10.0
144-Inter quad 1lens
9.3
STBtubbies-
15.0
RO1-Quad1 Rod Offset .
9.3
IQ2-Inter quad 2 lens
20.0
R02-Quad 2 rod offset
84.0
. .c
. ST3-Stubbies
. 100.0
R032.Quad 3 rod ol`fset
86.0
DF-CEM Deflection Plate
300.0
CEM-Channel Electron Multiplier
2400.0
i
x
Gas R>ws
:NebulizerGas Curtain Gas Collisiorl Gas . . TISTemperature
~
. .-Set
1
.12
. 13
.4
350'C
3.4.3. Calibration ;Procedures
a. Inject the same v,olume(between 10 to 20 &) of each calibration standard . (prepared in MeOH) into the LC/MS/MS. .
b. Use linear, l/x weighted standard curves for quantification. Linear . stgdarcl curves are generated for each set by linear regression using the
.
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appropriate sofiware system. Any cqbration standards fdling outside f , 30%. based on its calculated concentration, must be excluded from the
calibration curv~:. However, the total number of calibration standards th; may be excluded must not exceed 30% of the total number of stand=&
injected.
c. The correlation coefficient (r) for ,calibration curves generated must be
I'
20.9925 (220.985). If calibration results fall outside 'these limits, then
appropriate steps should be taken to adjust instrument operation, and the
relevant set of simples must be reanalyzed.
;
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4.4.4. SampleAnalysis i
a. Inject the same volume used for the calibration standards (between IO to
25 pL)of each sample, fortification,control, etc. into the LC/MS/MS.
b. Standards correspondingto at least Si.x concentrationsmust be included in
I
an analytical set.
c. Inject an entire :ret of standards (six) at the beghing of the run and inject
standards interspersed about every 3-10 samples. All sample injections must be bracketed by standard injections (see Section4.5).
*.
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d. Each set of samples analyzed (not to exceed 25) must include at least one reagent control (method blank). one ASTM Type I water blank, at least one matrix control, b d two matrix confrol samples fortified at known concentrations and carried through the procedure to verify recovery.
..
e. All samples must be analyzed with duplicate injections.
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Note:
ansllysis performed -during method development included '
fortifications at 10,50 and 250 ndg (ppb) for the mdilyte.
1.
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f. The concentration of each sample; fortification,contiol, etc. is determined iI,
from the.standard curve based on the peak area of the anaiyte in all
standards injected during a .set. ' The standard responses must bracket
responses of thr: residue found in the sample set. If necessary, dilute the
samples &d re-:analyzeto give a fisponse within the standard curve range.
,
g. Fortifications that bracket the highest residue expected in each treated . sample Vjill be included with each sample set. If residues <are found
. ' outside these limits, additional fortifications will be .included in a
. subsequent sample'set to establish e a t method recoveries are available for the,analyteof interest at concentrationsexceeding those in treued samples.
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Centre Method No: 00M-023-015
I h. Fortification recoveries &thin 60 to 130% are acceptable for fortifications at the LOQ level. Recoveries between 70 to 120% are acceptable f0.r fortifications at levels greater than $e LOQ. Failure to meet these criteria . requires an investigatioi Of cause and a full reanalysis of the affected
1P samples.
j i. Samples in which no vaks are detected at the corresponding analyte retention times will be reported as ND (not detected). Samples in which peaks are detected at the kOITeSponding analyte retention times but are less
than the lowest standard will be ported as NQ (not quantifiable).
j. If samples are not loaded on the hstrument to be analyzed'theday they are
extracted,samplesmust be stored.refrigeratedat approximately ZoCto ij0c
until analysis and analyzed preferably within a week.
Recoveries from' method development for all matrices can be found in
Tables I-VI.
..
4.5. PERFoRMANcEc-
. .. ..
The f&wiig two crit:&a must be met before the initial analysis of samples, especially when using different instrumentation set-ups than those cited in this method.
First Criterion- Inject a standird solution on the LCIMSIMS corresponding to:t h e
estimated LOQ (10 ppb LOQ kequivalent to a standaid solution of 0.2 ng/mL)
and obtain a signal to noise ratio Of at least 9:1 relative'to the reagent blank. If
this criterion cannot be met; optimize
change .instrument operating
parameters.
- Second Criterion Inject a set of'standardsranging from at or below the LOQ, up
to the highest concentra.tion leyef. Generate a calibration curve for theanalyteand
I
obtain a linear regression with a:co$icient of determination (r2)of at least 0.985 . . ,
for the analyte. Once: this criterion has been demonstrated, samples may be
analyzed with standardsinterspersed.
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4.6. TIME REQUIRED FOR ANALYSIS
6
,
A set of 14 samples can be taken through the extraction procedure in
I
approximately three hours by one person. The LCMSMS analysis (8-10
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standards and 14 sample:;) will take approximately6 hours.
I
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5. CALCULATIONS
m
.5.1 ANA~TEFOUND:
Calculate the amount of analyte found (in ng/mL, based on pe& m a ) using he standard curve generated ;by the MacQuan ~ o f t w mp r o m using Equation 1.
. Equationl:
Anaiyte found ( n g / d ) = b e a k area - intercept)
slope.
5.2 COMFONEN&TSI.K)UECONCENTRATION
., . ,
t. .:
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. Determine the component residue concentrationusing Equation 2 ..
Equation2: .
..
Residue found (ng/g) = (analyte found (nglmL) x DF x Fv (IT&))
sample weight (g)
Where DF= dilution factor and Fv = final volume
a:nglg = ppb
1
5.3 PERCENTRECOVERY
Calculate the peient recavery for samples fortified with known amounts of
analytes prior to extraction, from Equation 3.
.-I
- Equation3: . I.-
(nglg found - averageng/g found in control)
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Recovery (%) =
@gadded .
XlOO
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i
6. SAFETY
* I,
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There are no unusual hazards associated With this method. The analyst should
11
t' read the material safety thta sheets for dl Tagents before performing this method
Normal laboratory precautions should be taken.
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TABLES
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Table I: Summary of Recoveries for PFOS in Quail Egg Membrane
Sample ' 0003798 Matrix Blank A
F o i Level' (ppb) 0
0003798 Matrix Blank I
0
0003798 Matrix Blank C
0
0003798 Spk A
10
0003798 Spk B
10
0003798 Spk C
10
0003798 Spk D
50
0003798 Spk E
50
0003798 Spk F
50
0003798 Spk G
250
0003798 Spk H
250
0003798 Spk I
250 AVERAGE
STANDARD DEVIATION RELATIVE STANDARD DEVIATION
%Recovery
NA NA
NA 88 95 92 92 95 96
93 95
93 93
2
8
.
3
Table II: Summary of Recoveries for PFOS in Mallard Egg Membrane
-
_.
Sample
Fort Level
% Recovej
(ppb)
0003802 Matrix Bla& A
0
NA
0003802MatrixBlankB .
0
NA
0003802 Matrix Blank C '
0
NA
0003802 Spk A
10
106
0003802 Spk B .
10
122
0003802 Spk C
10.
106
0003802 spk D
50
96
0003802 Spk E
50
96
0003802 Splc F
50
90
0003802 Spk G
250
93
0003802 Spk H
250
92
0003802 Spk I
. 250
93
AVERAGE:
99
. STANDARDDEVIATION:
10
RELAT[VE STANDARDDEVIATION.
10
NA = Not Applicable
.
. I.
!
' I)
!
j ..
'
C
..
.
: !
.
I 'I
1 .;
I
. Centre Analytical Laboratories. Inc. Study 11 023-015
Exygen Research
Page 1.9
Page27 of 39 .
1 I
i II
.
a
Centre Method No: 00M-023-015
. Table,III: Summary of Recoveries :orPFOS in Quail Egg Yolk
Sample
0003795 Matrix Blank A 0003795 Matrix Blank B 0003795 Matrix Blank C
0003795 Spk A 0003795 Spk B 0003795 Spk C 0003795 Spk D GUO3795 Spk E
0003795 Spk F
0003795 SRk G 0003795 Spk H
Fort. Level
I
. *
(n&)
o
0
0
10
. 10
'
10
50 I
'
50
50 I ' ' 250
. 250
9b R e ~ ~ v e r y a
I
!
NA NA NA 69.
68, 70 72 80 83 85 ' 89
0003795 Spk I
250
88
AVERAGE:
7.8-
.STANDARDDEVIATION
RELAmrE STANDARDDEVIATION
-9 11
Table IV: Summary of Receveries for PFOS in Mallard Egg Yolk
Sample -
0003799 Matrix Blank A . 0003799 ~ a t r i xBlank B , 0003799 Matrix Blank C
0003799Spk.A . 0003799 Spk B 0003799 Spk C 0003799 Spk D 0003799 SpkE 00037.99 Spk F 0003799 Spk G 0003799 Spk H 0003799 Spk .I
Fort Level
(nglg) 0 0. 0
' 10
10
. 10
50
*
50
50
250
250
250
AVERAGE:
% Recovery
NA NA NA 83 82 1OQ 85 .
8 8 ~.
90
87. 85 ' 88 88'
STANDARD DEVIATION
5- .
aUm/'ESTANDARDDEVIATION: . 6 .. .-
!
NA = Not Applicable
..
: I ..
. Centre Analytical Labontories, Inc. Study # 023-015
Page20 .
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Exygen Research
Page 28 of 39
I IN
i
!
..
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Centre Method No: 00M-023-015
.
. Table V: Summary of Recoveries for PFOS in Quail Egg Albumen
. . Sample I
Fort. Lcvel
46 Recovery '
I
- I
(ppb)
0003196Matrix Blank A
0
NA
!
0003796 Matrix Blank B
0
NA
j;
0003796 Matrix Blqnk C
0
NA
I
a03796 Spk A
10
79
I(
v 3 7 9 6 Spk B
10 .
0003796 Spk C
10
93
88 -
0003796 Spk D
50
96
0003796 Spk E
50
95
0003796 Spk F
50
90
0003796 Spk G
250 .
97
0003796 Spk H
.250
102 "
0003796 Spk I
250 AVERAGE:
102
94 .
STANDARD DE'VIATION:
7
. REI.,AlWE STANDARD DEVIATION:
7
Table VI: Summary of Recolveriesfor PFOS in Mallard Egg Albumen
Sample .
-- Fort.Level (ppb)' ~
: 0003800MatrixBlankA '
0
0003800 Matrix Blank B
0..
0003800MatrixBlank C .
0
0003800 Spk A , '
10
- 0003800SpkB
10
0003800 Spk C
10
- 0003800 Spk D
50
0003800Spk E
50
Ob03800 Spk F .
50 .
0003800 Spk G
250
0003800 Spk H
250
0003800 Spk I
250
..
AVERAGE:
STANDARD DEVIATION: '
RELATIVE STANDApD DEVIATION:
46 Recovery
NA NA NA ' . 93 85 85 96
100
100 . 98 102 .
- 101 '
96 7 7
. NA = Not Applicable
.. Centre Analytical Labonton&, Inc. Study k 023-015
Exygen Research
. ! Page 21
Page 29 of 39
.
II
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Centre Method No: OOM-O&-015
s
.
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FIGURES
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. C C N ~A~nCalytical Laboratories.INC.Study # 023-015
. Exygen Research
Page 22
Page 30 of 39
. , , .. . - , ___________,____._._... .... . . .. '' - ' .- .---. -. .. -.--.-..-...---. ---- ----.-.__ _ ____
-. S
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6
' Centre Method No: OOM-023-015
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Figure 1: Repraentatiye Chromatogram of a Quail EggMembrane Control
!
. 11
1luAk 0.000 OUO~I00
a:u:3r 111
lu 1 1.61 ; 1.11
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' Figure 2: Representative Chromatogram of a Mallard Egg Membrane Control
.-. . .
11'1
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11
-1
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Centre AnalyticalLaboratories, IIIC.Study # 023-015
Exygen Research
-Page 23 Page 31 of 39
. .-. ... - .
III
I
Centre Method No: OOM-023-015
. Figure 3: Representative Chromatogram of a Quail Egg,Yolk Control
!
I. m
la
P
rn 1
,la
1.n
u
1.11
1
u
t; II
1t.1 IK
7.11 dr
14
0.1
0.01
' s.w
4.w
1.1)
.n
m Y
UI
!
.-
- Figure 4: Representative Chromatolgram of a Mallard Egg Yolk Control
t :
II
T :
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Centre h l y t i c a l Laboratories, Inc:. Study # 023-015
Exygen Research
Page 24
Page32of39 .
. lllll Centre MethodNO:OOM-023-015
Figure 5: Representative Chromatogramof a Quail Egg Albumen'Control
tn. U n
Y
w
MI
:1I
7
7
L
1
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Ill1 I
1
Figure 6: Representative Chroma1:ogramof a Mallard Egg Albumen Control
I u(
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. Centre Analytical Laboratories, Inc. Study # 023-015 ., .
Exygen Research
Page 25
Page 33 of 39
I1
I
. Centre Method No: 00M-023-015
S'
1111
Fipre 7: Representative Chromatogram of a Quail EggMembrane Control Fortified at 10 ng/g (ppb)
.
11
I
I
.-
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'Figure8: Representative Chromatogram of a Mallard Egg Membrane Control Fortified at 10.0 ng/g (ppb)
, .--
I I.6i
1.11
;1
11.0 a u
1.10 mia 0.10
1.01
.
1.oa k.03
3.M
L
ru
R
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Centre Analytical Laboratories,Inc. Study # 023-015
.Exygen Research
Page 26 Page34of39
c
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Centre Method No: 00M-023-015 ;
Figure 9: Representative Chromatogram of a Quail Egg Yolk Control
Fortified at 10.0 ng/g @pb) .
.
Il1
.
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' Figure 10: -RepresentativeChromatogram of a Mallard Egg Yolk Control
Fortified at 10.0 nglg (p:pb)
I II
. .z
..
..
lI. T1U IIN ' i
1-66
1.11
Y
I . 1
10.I sac l.Il..Ln 8.20 0.81 ;
u
m ' w
1.00 .
Y
4.00
1.00
(I(
lo mI
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II
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Centre Analytical Laboratories, Inc. Study # 023-015
Exygen Research
! . .
..
Page 27
Page35 of39 .
Ill I * ! Centre Method No: OOM-023-0151 b
- Figure 11: Representative Chromatogram of a Quail Egg Albumen Control
Fortified at 10.0 ng/g (ppb)
..I
m
I i I
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i! .
, I
in i
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u u u u I I I.!
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:
Figure 12: Representative Chromatogram of a Mallard Egg Albumen Control Fortified at 10.0 ng/g (p:pb)
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Centre Analytical Laboratories. Inc. Study #,02.3-015 . .
Exygen Research
,
Page 28
Page 36 of 39
1
Lipovitellin, Phosvitin, a n e w Low from Chicke9n6 a[1n9d6J0a]pandesSetiQfauniaiiiei t gaIg.,::
Density g
Lipo'pr.otein
(VLDL) 5
. Isolation
- d
!
(May, 1994) *
I
1) Break open egg, separate yolk, and roll yolk on a moist paper towel to remove adhering albumen.
2) Puncture yolk membrane and let yolk drain into a graduated cylinder,
3) Dilute 1 volume of egg yolk with 2 volumes of a solution containing 0.67 M MgS04, 1 mM phenylmethanesulfonylfluoride(PMSF), and 2'pM
leupeptin.
II
4) Cover cylinder tightly with Parafilm@Mand mix well by gently inverting
several times.
5) Using a 20 gauge needle arid 12 cc syringe, transfer the suspension to Beckman polyallomer Quick-Seal@tubes (tube size will depend on amount of yolk prepared and rotors available).
IIIII
6 ) Seal tubes, place into appropriate rotor, and centrifuge at 200,000 x g at 4 O C .for 2 4 hours.
7 ) Following centrifugation, 4 layers will be evident in the tubes (see
diagram at right): A, a firm layer of yellow gel (VLDL); B, a clear colorless solution; C, a viscous yellow solution grading to a firm A-
IIII
pellet at the bottom; D, a fluffy yellow suspension (VLDL).
Layers B and C constitute the high density fraction (HDF)
and layer A and the suspended material in D constitute the
(very) low density fraction.
C
8 ) Using a 20 gauge needle and 12 cc syringe, make t w o punctures in the
r
top of the tube and withdraw layer D as well as several milliliters
below the level of layer A until layer B is reached.
9) With a scalpel, cut the tube above the level of the remaining liquid. The
yellow upper pellet of the tube (VLDL) is then redissolved in a solution
containing 20 mM Tris-HCI (pH 81, 150 mM NaCI, 0.2 mM EDTA, 1
mM PMSF, and 5 pM leupeptin. The lower pellet (high density .
fraction, HDF; Layer C) should be gently washed with, and then
I
resuspended in, a solution containing 0.45 M MgS04, 1 mM PMSF,
'Ill
Exygen Research
Page 37 of 39
c
2
. and 2 pM leupeptin. Both VLDL and' HDF solutions are then centrifuged at 200,000 x g at 4 "C for 16 hours.
10) Following centrifugation of the VLDL solution, the upper pellet (VLDL) is . . re-dissolved in a minimal amount of 20 mM Tris-HCI (pH 8), 150 mM NaCI, 0.2 mM EDTA, 1 rriM PMSF, and 5'vM leupeptin, filtered (0.45 pM), sodium azide added to a final concentration of 1 mM (i.e., dilute stock solution 1000 XI,aliquoted, and stored at 4 "C. All of the VLDL preparation is now completed. The lower pellet (Layer C) of the HDF tube is first re-dissolved in a solution Of 0.45 M MgS04, 1 mM PMSF, and 2 pM leupeptin. Then, two volumes of cold "e-pure" water are
added to the HDF solution dropwise, with stirring, at 4 "C (thus, MgS04 concentration is now 0.15 MI. The solution is then left
standing (without stirring) at 4 "C overnight.
11) On the following morning, is yellow gelatinous precipitate should be adhered to the bottom of the flask. Decant (and save) the supernate (see step 12), then dissolve the precipitate in a solution of 0.4 M MgS04, 1 mM PMSF, and 2 pM leupeptin. Add one volume of cold "e-pure" water dropwise (thus solution is diluted to 0.2M MgS04) and let stand overnight (without stirring) a t 4 "C. On the following morning, the precipitate I(phosvitin1is recovered by centrifugation at 106,000 x g at 4 "C for 1 hour and the resulting pellet is dissolved in
buffer containing 1 M NaCI, 5 m M Tris-HCI (pH 7.81, 1 mM PMSF, 2 pM leupeptin, and sodiurn azide added to a final concentration of 1
mM (i.e., dilute stock solution 1000 XI. Aliquot and store at 4 "C.
12) The "supernate" (0.15 M IlrlgSO4; see step 11) should be decanted,
diluted with two volumes of cold "e-pure" water (final concentration of 0.05 M M g S 0 4 ) dropwise with stirring at 4 " C , and allowed to
stand overnight (without stirring) at 4 "C. The resulting white
precipitate (lipovitellin) is then recovered by centrifugation at 106,000 x g at 4 "C for 1 hour and dissolved in buffer containing 1 M NaCI, 5 mM Tris-HCI (pH 7.81, 1 mM PMSF, and 2 pM leupeptin, and sodium azide added to a final concentration of 1 mM (i.e., dilute stock
solution 1000x). A final protein concentration of 8-10 mglmL is
desired.
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