Document OEQdor8DxEBmG6KJQRXeLk50K

tW g;en RESEARCH ^P.Prreecciisse Research. Proven Results. 185 g Analytical Report 3M Environmental Technology and Safety Services Analysis of Perfluorooctanesulfonate in Mallard and Quail Egg Yolk Exygen Report No. 023-070 Testing Laboratory Exygen Research 3058 Research Drive State College, PA 16801 Requester William K. Reagen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PACE 10F 5 ^ ^ 3 0 5 8 Research Drive M State College, PA 16801, USA ^ T : 800.281.3219 F : 814.272.1019 exygen.com 001308 T Introduction 'M0 i l l S l i l l S P I S Results are reported for the analysis of total perfluorooctanesulfonate (PFO S) 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 analytical phase protocol given 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 samples 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 samples 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. T h e 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. T he sample was shaken then allowed to sit. Each sample was filtered and analyzed by LC/M S/M S electrospray. 3.1.2 PFOS Content in Yolk Fractions A 5.0 g sample was used for the fractionation. Ten milliliters of a solution containing 0.67 M MgSQ, 1 m M phenyfmethanesulfonylfluoride (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 lipoprotein (VLDL) and the high density fraction (HDF). The VLDL was dissolved in a solution containing 20 m M Tris-HCI, 150 m M NaCI, 0.2 m M EDTA, 1 mM PMSF, and 5 pM leupeptin. The HDF was resuspended in a solution containing 0.45 M M g S 0 4, 1 m M PMSF, and 2 pM leupeptin. Both VLDL and H D F 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 m M NaCI, 0.2 m M EDTA, 1 mM PMSF, and 5 pM leupeptin and stored at 4C. The HDF was dissolved in a solution containing 0.45 M MgSO*, 1 mM PMSF, and 2 pM PAGE2 OF5 leupeptin and then two volumes of cold water was added dropwise and then stored overnight at 4C. The H D F supernatant (lipovitellin) was decanted and diluted with cold water and stored overnight at 4C . The H D F precipitate (phosvitin) was dissolved in a solution containing 0.4 M M g S 0 4, 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 m M Tris-HCI, 1 m M PMSF, and 2 pM l&upeptin. 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 affinity 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 compounds. 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 H P1000 system interfaced to 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. T he following gradient was performed: Mobile Phase (A): 2m M Ammonium Acetate in Type I W ater - Mobile Phase (B): Methanol T im e %A %B 0.0 60 40 4ti 0.4 60 40 1.0 10 90 7.0 10 90 7.5 0 100 >< 9.0 0 100 9.5 60 40 14.0 60 40 1 The following parameters were used for operation of the mass spectrometer: 1 Parameter Ionization Mode Setting Electrospray Polarity Negative Transitions Monitored 499->99 PAGE3 O f 5 Gas Temperature Drying Gas (N2) 350C 7.0 L/min 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 prepared for P FO S 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 regression with 1/x weighting, the slope, y-intercept and coefficient of determination (r2) were determined. A calibration curve is acceptable if r2 > 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. T he laboratory control spikes must show recoveries 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 RelatedComments Duplicate injections were performed for all sample analyses. 5 Data Summary Please see Attachment B for a detailed listing of the analytical results. Results are reported in parts per billion (ng/g) for PFOS. 6 Data/Sample Retention 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 Environmental will be notified 30 days prior to the disposal of hardcopy data. PAGE 4 OF5 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 8 fly R./Decker, Principal Investigator O iln . f t ) _________ /foh n M. Flaherty, Vice-President ^ S jo T - Date Date PAGE5 OFS COI s IIIIIIII I Exygen ID Client Sample 1 0201613 (Composite) 0201614 (Composite) See Original Login See Original Login Exygen Research Sample Login Report Study Number: 023-070 Protocol: NA EGG YOLK EGG YOLK NA NA RICK K 6-4-02 RICK K 6-4-02 Page 1 of 1 I FREEZER 8 FREEZER 8 See Original Login See Original Login Composite of samples: 201551-201562 Composite of samples: 201563-201574 O .5 Verified By/Date: Printed 6/4/2002 l ft t ft ft Exygen Research Sample Login Report Study Number: 023-070 Protocol: NA Page 1 of 3 ] _________________________ I Exygen ID Client Sample ID . Received - - \ ; Matrix.--.'.'!"; 6y:;v 0201551 E01-1379-31882 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201552 E01-1379-31883 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRYICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201553 E01-1379-31884 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until 6-3-02 Log-In. 0201554 E01-1379-31885 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201555 E01-1379-31895 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201556 E01-1379-31896 6-1-02 21:15 EGG YOLK NA LAWRENCE O ' FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201557 E01-1379-31897 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201558 E01-1379-31898 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201559 E01-1379-31939 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201560 E01-1379-31943 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201561 E01-1379-31947 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201562 fiOfHr.r.'i E01-1379-31951 f~A4 i n a 6-1-02 21:15 EGG YOLK EGG VOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. MA ! awrfwcf n CRPP7PRO F-DRY IOE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201564 E01-1378-31639 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201565 E01-1378-31640 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 T-DRYICE-A Stored In FREEZER 8 until 6-3-02 Log-In. 0201566 E01-1378-31641 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201567 E01-1378-31654 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICEA Stored in FREEZER 8 until 6-3-02 Log-In. o *s 0201568 E01-1378-31655 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 'r* Verified By/Date: 4. - 2 -oZ Printed 6/3/2002  ft ft Exygen Research Sample Login Report Study Number: 023-070 Protocol: NA Page 2 of 3 ] _________________________ I Exygen ID Client Sample ID ' 'R e c e iv e d " ' J Matrix Submitted B y |f 0201569 E01-1378-31656 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201570 E01-1378-31657 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201571 E01-1378-31694 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201572 E01-1378-31698 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201573 E01-1378-31702 6-1-02 21:15 EGG YOLK NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201574 E01-1378-31706 6-1-02 21:15 EGG YOLK NA LAWRENCE O. ' FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 untH 6-3-02 Log-In. 0201575 E01-1379-31940 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201576 E01-1379-31944 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201577 E01-1379-31948 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201578 E01-1379-31952 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201579 E01-1378-31695 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201580 E01-1378-31699 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until Log-In. 0201581 EQ1-1378-31703 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 PRFF7ER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201582 E01-1378-31707 6-1-02 21:15 EGG ALBUMEN NA LAWRENCE O 6-3-02 FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. 0201583 E01-1379-31942 6-1-02 21:15 EGG SHELL MEMBRANE NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201584 E01-1379-31946 6-1-02 21:15 EGG SHELL MEMBRANE NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until 6-3-02 Log-In. 0201585 E01-1379-31950 6-1-02 21:15 EGG SHELL MEMBRANE NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until 6-3-02 Log-In. 0201586 E01-1379-31954 6-1-02 21:15 EGG SHELL NA LAWRENCE O FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until n MEMBRANE 6-3-02 Log-In. M Verified By/Date: 9 t Printed 6/3/2002 ft 1 Exygen ID -C lientS am ple ID -=. 0201587 E01-1378-31697 6-1-02 21:15 0201588 E01-1378-31701 6-1-02 21:15 0201589 E01-1378-31705 6-1-02 21:15 0201590 E01-1378-31709 6-1-02 21:15 EGG SHELL MEMBRANE EGG SHELL MEMBRANE EGG SHELL MEMBRANE EGG SHELL MEMBRANE Exygen Research Sample Login Report Study Number: 023-070 Protocol: NA NA LAWRENCE O 6-3-02 NA LAWRENCE O 6-3-02 NA LAWRENCE O 6-3-02 NA LAWRENCE O 6-3-02 Page 3 of 3 ] _________________________ I KBS FREEZER 8 FREEZER 8 FREEZER 8 FREEZER 8 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. UA W* ' Verified By/Date; Printed 6/3/2002 5/31/2002 Project: EOl-1379 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE Contract Lab{s): EXYGEN Requester: Robideau, Rochelle R (0002-03E-09) Department: 502180 Site Source: Project Number: Date Received: 10/4/2001 Project Description: N. Bobwhite Reproduction (Eggs) Completion Date: Project Lead: Rochelle R. Robideau Phone Number: 651-778-7065 Email Address: nTobideau@mmm.com Comments: NOTE: MISC SERV - Protein separation to be conducted by Exygen Research. 3M Sample E01-1379-31882 Analysis Code MISC_SERV Sampled Date Sample Description 6/13/2001 10 DBm a.i. 230 H Yolk Analytical Method gmpopents Mise. Services Mise. Services E01-1379-31883 Analysis Code MISC_SERV 6/13/2001 10 DDm a.i. 232 H Yolk Analytical Method Comoonents Mise. Services Mise. Services E01-1379-31884 Analysis Code MISC_SERV 6/13/2001 10 DDm a.i. 234 H Yolk Analytical Method CtUnpopcnt; Mise. Services Mise. Services E01-1379-31885 Analysis ode M1SC_SERV 6/13/2001 10 DDm a.i. 236 H Yolk Analytical Method Components Mise. Services Mise. Services E01-1379-31895 Analysis Code MISCSERV 6/13/2001 10 DDm a.i. 229 I Yolk Analytical Method Comnonente Mise. Services Mise. Services E01-1379-31896 Analysis Code MISCSERV 6/13/2001 10 DDm a.i. 231 lY olk Analytical Method Comoonents Mise. Services Mise. Services E01-1379-31897 Analysis Code M1SC_SERV 6/13/2001 10 DDm a.i. 233 I Yolk Analytical Method Comnonenb Mise. Services Mise. Services E01-1379-31898 Analysis Code MISC_SERV 6/13/2001 10 pom a.i. 235 I Yolk Analytical Method Comnonenls Mise. Services Mise. Services EOI-1379-31939 Analysis Code MISCSERV 6/21/2001 10 DDm a.i. 226 J Yolk Analytical Method gpiponenls Mise. Services Mise. Services Analysis Due Date 6/30/2002 s' Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 S' Analysis Due pate 6/30/2002 Analysis Due Date 6/30/2002 s' Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 Analysis Due Dpte 6/30/2002 Analysis Due Date 6/30/2002 5/31/2002 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE 2 of 2 Project: E01-1379 (cont) Contract Lab(s): EXYGEN 3M Sample Sampled Date Sample Description EOI-1379-31940 Analysis Code MISC_SERV 6/21/2001 10 ppm a.i. 226 J Albumen Analytical Method Componenti Mise. Services Mise. Services Analysis Due Pate 6/30/2002 EOI-1379-31942 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 226 J Shell Membrane Analytical Method Components Mise. Services Mise. Services Analysis Due Dat? 6/30/2002 E01-1379-31943 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 230 J Yolk Analytic M tlM ponente Mise. Services Mise. Services Analysis Due Date 6/30/2002 E01-1379-31944 Analysis Code MISC_SERV 6/21/2001 10 ppm a.i. 230 3 Albumen Analytical Method Components Mise. Services Mise. Services Analysis Due Pate 6/30/2002 EOI-1379-31946 Analysis Code MISC_SERV 6/21/2001 10 pom a.i. 230 J Shell Membrane Analytical Method Components Mise. Services Mise. Services Analysis Due Date 6/30/2002 E01-1379-31947 Analysis Code PFOS 6/21/2001 10 ppm a.i. 232 J Yolk Analytical Method Components PFOS by ESMS PFOS Analysis Due Date 6/30/2002 E01-1379-31948 Analysis Code PFOS EOI-1379-31950 Analysis Code PFOS 6/21/2001 10 pom a.i. 232 J Albumen Analytical Method Components PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 232 J Shell Membrane Analytical Method Components PFOS by ESMS PFOS Analysis Due Date 6/30/2002 Analysis Due Date 6/30/202 EOI -1379-31951 Analysis ode PFOS 6/21/2001 10 ppm a.i. 234 J Yolk Analytical Method Components PFOS by ESMS PFOS Analysis Due Date 6/30/2002 E01-1379-31952 Analysis Code PFOS 6/21/2001 10 ppm a.i. 234 J Albumen Analytical Method Components PFOS by ESMS PFOS Analysij Due Date 6/30/2002 E01-1379-31954 Analysis Code PFOS 6/21/2001 10 ppm a.i. 234 J Shell Membrane Analytical Method Comoonenti PFOS by ESMS PFOS Analysis Dpo Date 6/30/2002 I 3 M Environmental Laboratory Shipping Address: 3M Bldg. 2-3E-09 935 Bush Avenu* SI. Paul, MN 55106 Telephone: S m irks Receiving: ($51)778-4948 A ltw nsls: (SSI) 77S4763 FAX: (651)778-9178 \ \ \ Report Results ,, to: Contact Name R0bideau. Rochelle R Company 3M Mailing Addressnnn7n:!Fnq City, State, Zip Telephone # j e i 778-7065 Special Instructions and/or Specific Regulatory Requirements: (method, limit of detection, reporting units, etc.) ft Chain of Custody /Request for Laboratory Analytical Project ID/Project NameXT D-----___________________________ Template# " r ~ ~ 'T iKahte^ort Due Date Project Lead p ^ i,,, ii< P P ^ V .^ ,.. 1ntemal Due Date Dept. # (main)_______ c m i o n ______________________ ^ :iass/Job/Project # Date Available Date Due Contract Lab FAX# w n n s.A n fi._____________________ v' Preservatives^, 5808 ft ft ft 3M Env Lab Project # For Internal Use Only E 01-1379 r d * v 'Cc M 7 * o - It e ]G> ) Hex Analysis Requested: Complet* below. Attach any associated Information. Total Number of Containers None ....... s--'iai,2- Other Item # Client Sample Identification 1. SOppm a.i. 230 H Yolk 2. 10 ppm a.i. 232 H Yolk 3M Date LIMS# Sampled 31882 5/13/01 31883 5/13/01 Time Sampled VOCs zoXrt <Ow0 X " Matrix/ Media Enter the number of containers of each ? * (Enter a n X h die box below to indkata request} 1 3. 10 ppm a.i. 234 H Yolk 31884 5/13/01 4. 10 ppm a.i. 236 H Yolk 31885 5/13/01 5. 10 ppm a.i. 2291 Yolk 31895 5/13/01 6 . 10 ppm a.i. 2311 Yolk 31896 5/13/01 7. 10 ppm a.i. 233 I Yolk 31897 5/13/01 8. 10 ppm a.i. 235 I Yolk 31898 5/13/01 9. 10 ppm a.i. 226 J Yolk 10. 10 ppm a.i. 226 J Albumen 31939 5/21/01 31940 5/21/01 il< f' O1i inr np b iu a' .yi:: ^(tem # ^->n-anlTi:Sirh-eHl1.-M---e-m--b-r-a-n-e---------- -----------__ / 2 s ~\Rejjiquisbed by/Affilaton -T31ir9v4a2__ 6/21/01 _f,m\ icyi---Tme -- - -- Collector1* signature: Date . Shipped Via: . j __ * L \ ; feceivd by/AfRIiatioti Time Date PS. % t ' / Z o ( D * ' f-i-O l. Z /'f -/-Z rtl.r JC r* Sample Condition Upon Receipt: Temperature: OtherAssociatedCoCs: c O/Z 'A M V O Acceptable 0 Other O Received on Ice Copies to: t/ Comments: % TO Pag - l Of Original - Accompanying Sample la s t Page - Originator S e a R e v e r s a S iria fnr lntnu**r Environmental Laboratory Form 38778 - PWO Shipping A d d m i: 3M Bldg. 2-3E-09 935 Bush Avmus St Paul, MN 55106 Talaphona: Sam pla Rcalving: (881) 778*4948 Alternate: (881) 778-8783 FAX: (851)778-8178 \ Contact Name R nhidea,, R n c h e i i e r Company .. -v. *taor - ' Matting City, State, Zip a: Telephone # i 651 778-7065 Special Inatvuctlona and/or Specific Regulatory Requlrementa: (method, limit of detection, reporting units, etc.} t 1 1 1 ft 1 1 Chain of Custody /Request for Laboratory Analytical Project 1D/Project NameXT D,,u,,,u;,,, P a -- ________________ Template# Finaffiepoit Due Date Projed Lead PnViirtr.ni. Internal Due Date P e p ta O r g in )________ 5Q218Q __________ Class/Job/Project # FAX# 1(6.51)328^136 Preservatives: ft ft 5809 ft 8 a 3M Env, Lab Project # For Internal Use Only__ E 01-1379 Analysis Requested: Comptt balow. Attach any aaaociatad Information. mo $ Item # Client Sample Identification 1. 10 ppm a.i. 230 J Albumen 2. 10 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 ppm a.i. 232 J Shell Membrane 6. 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. Collected by (print): : Item# H S ~ \ ^Bhquished by/Affiliatioji * t-i 3M Date LIMS# Sampled 31944 5/21/01 31946 5/21/01 31947 5/21/01 31948 5/21/01 31950 5/21/01 31951 5/21/01 31952 5/21/01 31954 5/21/01 Time Sampled Matrix/ Media Enter the number of containers of each -- -- -- (Enter an 'X in the box beto* to indicate reouest} jL v Collector's signature: Time ;t Shipped Via: : r-E *L - tip p Received by/Affiliation s-j-a z Time T-l 1f Date i Chain of Custody J***. **s Ir~.X~ Sample Condition Upon Receipt: Temperature: 'C Other Associated CoCs: O Acceptable 0 Other: O Received on Ice J_____________________________ r> n r # - _ Comments: ________________ % *C ---------------------------------------------------------------------------------- /& U L _ JD Paga s y Of X - Original - Accompanying Sample Last P ^ a - Originator S aa R e v a r ia Siri far (natniHinne 5/31/2002 Project: EOl-1378 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE Contract Lab(s): EXYGEN Requester: Robideau, Rochelle R (0002-03E-09) Department: 502180 Site Source: Project Number Date Received: 10/4/2001 Project Description: Mallard Reporduction (Eggs) Ship Date: Completion Date: Project Lead: Rochelle R. Robideau Phone Number: 651-778-7065 Email Address: rrrobideau@mmm.com Comments: NOTE: MISC SERV = Protein separation conducted by Exygen Research. 3M Sample H01-1378-31638 AnalvsisCode MISC_SERV Sampled Date Sample Description 6/13/2001 10 nom a.i. 230 H Yolk Analytical Method Comoonents Mise. Services Mise. Services EOl-1378-31639 AnalvsisCode MISC_SERV 6/13/2001 10 ppm a.i. 232 H Yolk Analytical Method Comoonenls Mise. Services Mise. Services E01-1378-31640 AnalvsisCode M1SC_SERV 6/13/2001 10 ppm a.i. 234 H Yolk Analytical Method Comnonenti Mise. Services Mise. Services E01-1378-31641 AnalvsisCode M1SC_SERV 6/13/2001 10 ppm a.i. 236 H Yolk Analytical Method Comnonenti Mise. Services Mise. Services E01-1378-31654 AnalvsisCode MISC_SERV 6/13/2001 10 ppm a.i. 229 I Yolk Analytical Method Comppnnf} Mise. Services Mise. Services E01-1378-31655 AnalvsisCode M1SC_SERV 6/13/2001 10 ppm a.i. 231 I Yolk Analytical Method Component; Mise. Services Mise. Services E01-1378-31656 AnalvsisCode MISC_SERV 6/13/2001 10 ppm a.i. 233 I Yolk Analytical Method Component! Mise. Services Mise. Services EO1-1378-31657 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 235 I Yolk Analytical Method Components Mise. Services Mise. Services EOI-1378-31694 Analysis Cod MISC_SERV 6/21/2001 10 ppm a.i. 230 J Yolk Analytical Method Compopenb Mise. Services Mise. Services Analysis Due Date 6/30/2002 s' Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 Analysis Due Pate 6/30/2002 Analysis Due Date 6/30/2002 Analysis Due Pate 6/30/2002 Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 5/31/2002 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE 2 of 2 Project: E01-1378 (cont) Contract Lab(s): EXYGEN 3M Sample Sampled Date Sample Description m E01-1378-31695 Analysis Code MISC_SERV m E01-1378-31697 Analysis Code m MISC_SERV 6/21/2001 10 ppm a.i. 230 J Albumen Analytical Method ontOi)ents Mise. Services Mise. Services 6/21/2001 10 com a.i. 230 J Shell Membrane Analytical Method Comnonents Mise. Services Mise. Services Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 E01-1378-31698 m Analysis Code M1SC_SERV 6/21/2001 10 ppm a.i. 232 J Yolk Analytical Method Comoonenb Mise. Services Mise. Services Analysis Due Date 6/30/2002 m EOI-1378-31699 Analysis Code MISC_SERV m EOI-1378-31701 Analysis Code MISC_SERV 6/21/2001 10 ppm-a.i. 232 J Albumen Analytical Method Componenti Mise. Services Mise. Services 6/21/2001 10 ppm a.i. 232 J Shell Membrane Analytical Method Comoonenb Mise. Services Mise. Services s' Analysis Due Pate 6/30/2002 Analysis Due Date 6/30/2002 EOI-1378-31702 Analysis Code PFOS m EOI-1378-31703 Analysis Code PFOS E01-1378-31705 Analysis Code PFOS 6/21/2001 10 ppm a.i. 234 J Yolk Analytical Method Comoonenb PFOSbyESMS PFOS 6/21/2001 10 ppm a.i. 234 J Albumen Analytical Method Comoonenb PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 234 J Shell Membrane Analytical Method Comoonenb PFOS by ESMS PFOS Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 s' Analysis Due Date 6/30/2002 EOI-1378-31706 Analysis Code PFOS *41 EOI-1378-31707 Analysis Code PFOS *1 E01-1378-31709 Analysis Code 1 PFOS 6/21/2001 10 ppm a.i. 236 J Yolk Analytical Method Comoonenb PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 236 J Albumen Analytical Method omponenb PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 236 J Shell Membrane Analytical Method Comoonenb PFOS by ESMS PFOS Analvsjs Ppy paty 6/30/2002 s' Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 0013 3M Environmental Laborat Form 38778 *PWO Shipping Address: IM Bkig. 2-3E-09 335 Bush Avenus St Paul, MN 55106 Telephone: Sample Recalvlng: (651) 77B-4M8. Alternate: (8S1) 778-6753 FAX: (651) 7784178 a Contact Name Robidean Rochelle R 3 ' Company S ac vaoao.. " Mailing Addressnnn-, City, State, Zip Special Instructions and/or Specific Regulatory Requirements: (method, limit of detection, reporting units, etc.) Item # Client Sample Identification 1. 10 ppm 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. 2291Yolk 6. 10 ppm a.i. 2311Yolk 7. 10 ppm a.i. 233 I Yolk 8. 10 ppm a.i. 235 I Yolk s. 0 ppm a.i. 230 J Yolk 10. 10 ppm a.i. 230 J Albumen * 1 AAa Chain of Custody /Request for Laboratory Analytical Project ID/Project Name,, n ___ J __iT, Template # 'filial Report Due Date Project Lead Dept. # (main) P apEaHa P PrkVtt/fAAll Internal Due Date ------03180------------------------ Class/Job/Project # Date Available Date Due '' Contract Lab Preservatives^ 4Mj ozXfl O<XNw8 5. s 3M Date Time LIMS# Sampled Sampled 31638 i/13/01 31639 i/13/01 31640 i/13/01 31641 i/13/01 31654 i/13/01 31655 i/13/01 31656 i/13/01 31657 5/13/01 31694 5/21/0! 3T553 5/21/01 J1AU-7 rTTTTTn--- 1 Matrix/ Media Enter the number of containers of each 1 VOCs None Total Number of Containers A 5810 3M Env/Lab. Project # E 01-1378 r iZ & * iv * * " 0L*m k u * Analysis Requ ested: Comp etaM o w , Attach any m i loclated Informat!o rL r (Enter an *X* in the box below to indicata reaueati A an, 1 -- 1 i------------------------------- -- -- LJiample Condition Upon Receipt: O Acceptable C Other: t /Temperature: * Other Associated CoCs. *sAL. t r i ` &------ 'C Uj h , Q l O Received on Ice Copies to: /v M 6 w fn w ift l ` ,, -1 > O ri/unA l . A riV u n n s n u in n Cai / J Comments: i ft ft ft ft eport Results to: 1 - VOCs Total Number of Containers 3M Environmental Laboratoi 3M BWb 2-3E-09 935 Bush Avenue St Paul. MN 55106 S*mpl (Ucilvlng: Alternate; (651) 7784753 FAX: {651) 776-617 ^\ \ Chain of Custody /Request for Laboratory Analytical 5811 P r ie c t ID/Project N a m e ^ , , ^ _______________ ______________ Template # r 1Wat Report Due Date Project Lead Dept.# (main) P n rU i-p 1ntemal Due Date (:iass/Job/Project # 3M Env. Lab Project # - i j E01-1378 Contact Name Robideau. Rochelle R Company Date Available Mailing A d d r e s ^ ^ p g g Date Due City. State, Zip a Contract Lab Telephone# ____, ^ 77R-7n/5______________________ FAX# i ^ i > T 7e . i ; n r ; _______________________ Special Instructions and/or Specific Regulatory Requirements: (method. limit of detection, reporting unis, etc.) v' 1 P re se rv a tiv e s: 3 y (, 0 S T K f ' i t tfts u J g Hi r A nalysis Requested: Completo below . Attach any associated inform ation. Item Client Sample Identification # 1. I0 ppm a.i. 232 J Albumen 2. I0 ppm a.i. 232 J Shell Membrane 3. 0 ppm a.i. 234 J Yolk 4. I0 ppm a.i. 234 J Albumen 5. IOppm a.i. 234 J Shell Membrane 6. I0 ppm a.i. 236 J Yolk 7. IOppm a.i. 236 J Albumen 8. IOppm a.i. 236 J Shell Membrane b\ 10. T>3. Collected by (print): /B 1{~~\JItem# Relinquished by/Affiliation ki-fo (hoijAAjM o C ' H " o 3M UMS# 31699 31701 31702 31703 31705 31706 31707 31709 Date Sampled i/21/01 1/21/01 1/21/01 i/21/01 i/21/01 i/21/01 i/21/01 i/21/01 Time Sampled n oo 23 ZX .l zCo Matrix/ Media Enter the number of containers of each ! 1 hi $ \P (T (Enter an X in the box betow to n o c e te reaueat) * 3J \\j \1/ Collector's signature: Time . Date ;Shipped Via: S/ji/ti- rCv'trA Received by/Affiliation J , , \ >. < r-i-o z J /Time Dat zn f S-i-oz , Sample Condition Upon Receipt: O Acceptable O Other ^Temperature: c O Received on Ice f. O ,e ? c T X C* * ~ 'o * l* r Copies to: -C O Qr `1________________________________ 'f-'- .-- \ ' ,, " 7 (ltd1 L Exveen m# RESEARCH >Precise Research. Proven Results. Sample "Condition Upon Receipt" Form Protocol# Exygen Study # JL O-S - C7 l.l *, fr 0 Date & Time Received - / - 03. / / J j ' Condition of Samples /- - /? _______ Temporary Storage Location / - - Y Initials & Date ^ -/->2- Waybill # SCl JJU A __ & d d ) . Comments: S e r/iy rd a ji d / j ,u&/-_J?mi / i , /ft/C >./- z - Q :W V O R D \prucess-login\S am pleC ondiaonoaR cceipt.doc M a y 24. 2002/3 3058 Research Drive State College, PA 16801, USA VT: 800.281.3219 F: 814.272.1019 exygen.com ( - 0 . 3 E 0 w U'1 ' i i I t i I I ** *A**ft*BI A 0201615 NA 6-5-02 12:00 Exygen Research Sample Login Report Study Number: Protocol: 023-070 NA Page 1 of 1 EGG NA LAWRENCE O FREEZER 8 REFRIGERATED Purchased at Weis 6-5-02 Markets #33, State College, PA______ o * : Verified By/Date: r Printed 6/5/2002 r 6 ^ / : L ^ O ... w ei3! ^ ^ THANK YOU FOR SHOPPING UEIS MARKETS 33 STATE COLLEGE, PA. Itex *UQ EGG5 L * TAX Cash .00 w n- . CHANGE BAL Price .79 F .79 1.00 .21 b/0S/02 12-29 PM 003.3,02 .C150- HS R5K-! US HQU.QUR UEIS..SH0PPER5 CLUB CAN SAUE VO `S EUERYDAY UEIS CLUB.MEMBERS HAUE THE POWER TO SAUE MORE jie ir an exact copy of \E ORIGINA*- document. > 0 DATE - S ' * Z* Summary of PFOS Residue Found (ppb) in Combined Mallard Egg Yolk Samples Sponsor ID na (Reagent Control) na (Reagent Control)* na (Reagent Control) na (Reagent Control)A na (Matrix Control) na (Matrix Control)* na (Matrix Control) na (Matrix Control)* na (LCS) na (LCS) na (LCS) na (LCS) ** Dup ** DupA Analyte Found (ppb) NQ NQ NQ NQ NQ NQ NQ NQ 108 111 103 100 53600 52300 52600 52500 Fort. Level <PPb) 100 100 100 100 m - - Recovery (%) 108 111 103 100 . . - - 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 E01-137B-31638, 31639, 31640, 31641, 31654, 31655, 31656,31657, 31694, 31698, 31702, 31706 J& 3,03055f8 Research Drive State College, PA 16801, USA VJ : 814.272.1039 F: 814.231.1580 PWOPn rr\m -- Analytical. ^ Report Summary of PFOS Residue Found (ppb) in Combined Quail Egg Yolk Samples Sponsor ID na (Reagent Control) na (Reagent Control)* na (Reagent Control) na (Reagent Control)* na (Matrix Control) na (Matrix ControljA na (Matrix Control) na (Matrix Control)* na (LCS) na (LCS) na (LCS) na (LCS) * **A ** Dup ** DupA Analyte Found __________ EE}________ NQ NQ NQ NQ NQ NQ NQ NQ 104 102 106 109 75000 74300 48700 49900 Fort. Level (ppb) -I - - 100 100 100 100 - - - - Recovery (%) 104 102 106 109 - 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, 31898, 31939, 31943, 31947, 31951 ^ ^ 3 0 5 8 Research Drive ^ jk S ta te College, PA 16801 ^ T : 814.272.1039 I F : 814.231.1580 Gkv\/o=>nri'rry> /-j * Summary of PFOS Residue Found (ppb) in Mallard'/Quail** Egg Yolk Fractions Sponsor ID na (Reagent Control) na (Reagent Control)A na (Reagent Control) na (Reagent Control)A na (Matrix Control)-VLDL na (Matrix Controi)-VLDLA na (Matrix Control)-Phosvitin na (Matrix Control)-PhosvitlnA na (Matrix Control)-Lipovltellln na (Matrix Control)-Lipovitellin* ' -Mallard VLDL '-Mallard VLDLA -Mallard Phosvitin -Mallard Phosvitin* -Mallard Lipovitellin -Mallard Lipovitellin* -Quail VLDL ' -Quail VLDL* -Quail Phosvitin ' -Quail Phosvitin* ' -Quail Lipovitellin -Quail Lipovitellin* Analyte Found (ppb) ND ND ND ND 13.5 14.3 NQ NQ NQ NQ 41800 42500 3600 3580 8910 8830 39700 39900 815 838 1300 1340 Foil. Level (ppb) .1 Recovery (%) ND s Result is below the detection limit of - 0.5 ppb. 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 EOI-1378-31638, 31639, 31640, 31641, 31654, 31655,31656, 31657, 31694,31698, 31702, 31706 ** composite of samples E01-1379-31882, 31883, 31884, 31885, 31895, 31896, 31B97, 31898, 31939,31943,31947, 31951 x .3058 Research Drive mstSatalte College, PA 16801, USA VJ : 814.272.1039 f: 814.231.1580 ov\/npn r n m :/* G -0 1 3 . 3058 Research Drive State College, PA 16801 T: (814) 272-1039 F: (814) 231-1580 PROTOCOL STUDY PERSONNEL LOG NA_________ EXYGEN STUDY O Vt ^O | PRINTED NAME Prvt-U l . Db o a t if \ \ \ 'O pJ \ __ SIGNATURE INITIALS c& > /2 ,/c .. ........... _ v %<6\ ._____________ x \ \ _\ \ \ June 28, 2001/1 ANALYTICAL PHASE PROTOCOL ANALYTICAL PHASE TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANI'.SULFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROME TRY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 DATA REQUIREMENTS Analytical Method Requirements PERFORMING LABORATORY Exygen Research (Exygen) 3058 Research Drive State College, PA 16801 Phone 814-272-1039 Exygen Research Page 1 o f 39 4M M Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS ________ _____________________________________________________________ Page TABLE OF CONTENTS......................................................................................................... 2 1. PURPOSE....................................................................................................................... 3 l 2. REFERENCE MATERIAL......................................................................................... 3 3. SPONSOR......................................................................................................................4 4 TESTING FACILITY / PERFORMING LABORATORY...................................... 4 5 PROPOSED EXPERIMENTAL TIME-FRAME.......................................................4 6 SAMPLE PROCESSING, STORAGE AND IDENTIFICATION.......................... 4 7. ANALYTICAL METHOD...........................................................................................5 8. EXPERIMENTAL DESIGN........................................................................................ 5 9. RECORDS...................................................................................................................... 5 10. DATA AND REPORT..................................................................................................6 IT COSTS............................................................................................................................. 6 12. PROTOCOL APPROVAL............................................................................................ 7 APPENDIX: ANALYTICAL M ETHODS........................................................................... 8 Exvgen Research Page 2 o f 39 G0 1 3 : <4 EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY 1. PURPOSE The purpose o f 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 Yolk" and "Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS." Both methods can be found in the Appendix. 2. REFERENCE MATERIAL The following analytical standard will be used: Test Material PFOS Lot Number 215 Purity (%) TBD Chemical name and structure o f the compound is presented below. PFOS Chemical Name: IUPAC Name: CAS Number: Molecular Weight: Perfluorooctanesulfonate 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 (CgFnSOs- ) O C 8F 17S II o O Note: The neutral molecule and standard from which the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [C8F17SO3K], molecular weight 538. A record o f 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 o f 39 All standards/test 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 LABORATORY 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 31, 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 o f 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 o f the samples. The samples will be stored in a temperature-monitored freezer, maintained at < -10 C, except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during 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 o f 39 7. ANALYTICAL METHOD The composite mallard/quail egg yolk samples will be extracted and analyzed first according to the analytical method titled "Determination o f Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS" to determine the total amount o f PFOS in each sample. Then the mallard/quail composite yolk samples will be separated into three fractions 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 o f Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS". 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 LC/MS/MS." ' Methods to control bias will include assay o f untreated control samples, fortification o f untreated control samples to obtain recovery data, and replicate analysis o f fortified samples to provide an indication o f 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. I f 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 caJculated. 9. RECORDS Records to be maintained include, but are not limited the following (as appropriate): 1. Sample tracking sheet(s) 2. Sample receipt records, storage history, and chains o f 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 o f 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 o f the area of interest, and injection number corresponding to the run. Exygen Research Page 5 of 39 001337 b. Additionally, fortifications will include the fortification level o f 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 temperatures, 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, iinalytical standard solution preparation, sample chain o f custody sheets, sample work sheets, chromatograms, calibration curves, and any other appropriate data generated. 2. A report will be issued by Exygen at the completion 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 protocol 2. Analytical and statistical methods used 3. Reference materials identified by name, lot, purity, and other characteristics 4. Name o f performing laboratory and analytical start and termination dates 5. Tables containing all applicable data 6. All chromatographic and instrumental conditions 7. A complete listing o f Exygen study personnel 11. COSTS The total cost for performance o f the study will be $30,000.00. Exygen Research Page 6 o f 39  12. PRO TO CO L APPROVAL m Project M anager, Exygen ' _______________________ - t j z E n ^l/D e Scientist ck e(rI Date il il Sponsor, 3M Bill Reagen Date Exygen Research Page 7 of 39 c e lli APPENDIX: ANALYTICAL METHODS A. "Determination o f Perfluorooctanesulfonate in 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 o f 39 TITLE Determination of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by * LC/MS/MS i * ALTPQRS Emily Stauffer and John Raherty ' D A tE ISSUED January 2,2001 SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 53133-3331 PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 CENTRE STUDY NUMBER 023-015 CENTRE METHOD NUMBER OOM-023-015 TOTAL NUMBER OF PAGES 28 Exygen Research Page Centre Method No: Q0M-023-015 ,I i MANAGEMENT APPROVAL As per 4d CFR 792.3, method development is not required to be conducted in compliance . with Good Laboratory Practices. However, the work was in conformance with applicable standard operating procedures and general GLP regulations. I bmi Stauffer jlkK Date Principal Investigator Centre Analytical Laboratories, Inc. ./John Flaherty / / Laboratory Manager Centre Analytical Laboratories, Inc. Date -M Jlfl-0\ Richard j^Gjptizzini, PhD. President- Date Centre Analytical Laboratories, Inc. onsor Representative 3M Environmental Laboratory Centre Analytical Laboratories. Inc. Study#023-015 Exygen Research Page 2 Page 10 o f 39 Kl m 11 Centre Method No: OOM-023-015 TABLE OF CONTENTS TITLE PAGE- MANAGEMENT APPROVAL. .2 TABLE OF CONTENTS. LIST OF TABLES_____ .3 \ .4 LIST OF FIGURES. . 1. SUMMARY___ .6 2. EXPERIMENTAL COMPOUNDS. .7 3. CHEMICALS AND SUPPLIES 3.1. Chemicals___.'..................... ~ ..................................................- ......................................... 7 3.2. St a n d a r d s ....................................... 33. Equipment and Supplies-----3.4. Solutions., ..................- ........................- ..................................;............7 .............................. ........................................... 8 .................................................................................. 8 3 3 . PREPARATION OF STOCK. FORTIFICATION, ANDCAUBRATIQN SOLUTIONS...................................... .....;.....9 3.5. 1. Stock Solution..........-............ ----......... ------ ....................................... 9 3.5.2. F ortification Solutions---------------------------- --~ ...... ..........- ........................ :...........................9 3.5.3. C alibration Standards........... -- ............................................. -- -............. 9 4. METHOD. .10 4.1. Flow Diagram........ 42 . Sample Processing.. ..... ......10 ..............10' 4 3. Sample extraction., 4.3.i Egg Membrane.... 4 .3 .2 . E g g Y o lk a n d A lb u m e n ...... ............... 10 _____// .............. 11 4.4. Analysis by LC/MS/MS..................................--- --- .......................... .......................... 12 4 .4 .i ..LC/M S/M S System and O perating C onditions (T urbolonspray).................................. /2 4.4.2. E xam ple Tune F ile P aram eters.--.-- ----------------- ---....................'............... 13 4.4.3. C alibration P rocedures.... .......... -....... -......... -- ............................... ........ 13 4.4.4. Sam ple A nalysis....... -........... -............ ........ ... ..................................... M 4 3 . P e r fo r m a n c e Cr it e r ia ..................................................................................................................................................13 4.6. T im e R e q u ir e d fo r a n a l y s is ................- .................... .................. - ........................ - ..........................-- --------- 16 5. CALCULATIONS.:. .16 6. SAFETY. .17 TABLES. FIGURES 18 ___ 22 Centre Analytical Laboratories, Inc. Study #023-015 Page 3 Exygen Research Page 11 of 39 0013 1 'i 1* [Ilf Centre Method No: 00M-023-015 HI LIST OF TABLES m 1 TABLE 1 SUMMARY OF RECOVERIES f 6 r PFOS IN QUAIL EGG MEMBRANE............................... '.............................................................. 19 i UN TABLED: SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG MEMBRANE............................... .................................................................. 't ai TABLE HI: SUMMARY OF RECOVERIES FOR PFOS IN QUAIL EGG 1 YOLK ................................ 20 TABLE IV: SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG mi YOLK.................................... v .............................. V................................................... ;....... 20 TABLE V: SUMMARY OF RECOVERIES FOR PFOS IN QUAIL EGG . ALBUMEN.......................... il 21 TABLE VL SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG ALBUMEN................................. 21 ni 1 i ! Centre Analytical Laboratories, Inc. Study #023-015 Exygen Research > I b 1 ! Page 4 Page 12 of 39 G013-V4 C entre M ethod N o: 0OM-O23-O15 LIST OF FIGURES Figure 1: Representative Chromatogram of a Quail Egg Membrane Control................ 23 Figure 2: Representative Chromatogram of a Mallard Egg Membrane Control............ 23 Figure 3: Representative Chromatogram of a Quail Egg Yolk Control......................... 24 Figure 4: Representative Chromatogram of a Mallard Egg Yolk Control..................... 24 Figure 5: Representative Chromatogram of a Quail Egg Albumen Control.................. 25 Figure 6: Representative Chromatogram of a Mallard Egg Albumen Control.............. 25 Figure 7: Representative Chromatogram of a Quail Egg Membrane Control Fortified at 10 ng/g (ppb)...................................................................................................26 Figure 8: Representative Chromatogram of a Mallard Egg Membrane Control Fortified at 10.0 ng/g (ppb)................ ............................................................................... 26 Figure 9: Representative Chromatogram of a Quail Egg Yolk Control Fortified at 10.0 ng/g (ppb)......... - ........................... ............................................. '...... 27 Figure-10: Representative Chromatogram of a Mallard Egg Yolk Control Fortified at 10.0 ng/g (ppb).................................... ..... v................................................. 27 Figure.il: Representative Chromatogram of a Quail Egg Albumen Control Fortified at . 10.0 ng/g (ppb)............................. ................................................................ 28 Figure 12: Representative Chromatogram of a Mallard Egg Albumen Control Fortified at 10.0 ng/g (ppb).............................................................................................. 28 I. I l Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 5 | Page 13 of 39 G0 13 C enfre M ethod No: Q0M-023-015 1. SUMMARY This document a method of analysis for the residual determination of perfluorooctancsulfonate (PFOS) in egg membrane, albumen and yolk. The chemical formula of the analyte is given in Section 2 of this method. Perfluorooctanesulfonaie is extracted 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 accomplished by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using selected reaction monitoring, (SRM). i For this method the LOQ is 10 ng/g (parts-per-billion). This is based on studies conducted with egg membrane, albumen, and yolk control samples during method development. C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Exygen Research Page 6 Page 14 C entre M ethod N o: OOM-023-015 2. EXPERIMENTAL COMPOUNDS . ti I The molecular structure of PFOS> is given below: PFOS Molecular weight: 499 (CsFnSr") 0 C8F,tS -- K+ il 1 Chemical Name: Perfluorooctanesulfonate IUPAC name :l-Octanesulfonic acid,l,1,2,2,33,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-potassium salt CAS#: 2795-39-3 Note: The neutral molecule and standard form whichPFOS (anion) is derived from is perfluorooctanesulfonate potassium salt [C1F 17SO3K] (molecular weight 538). 3. CHEMICALS AND SUPPLIES 3.1. Chemicals - Chemical Grade Source Carbon Methanol (MeOH) Ammonium Acetate' Water 120/400 HPLC Reagent TX2 i .Supelco (VWR) J. T. Baker Aldrich Chemical Centre I i / p c i =w a t e r c i c b u i i t t J x c a i a u v u / , m i m u i u u i u i io .o / jVlS>4-Cjn a t LabconcoTM waterpro workstation, based on. ASTM classification) Catalog No. 57210-U JT9093-2 111-87-5 3.2. Standards Standard PFOS Grade Analytical Test Control Reference Number TCR-OOO746 Source 3M Environmental Laboratory, St. Paul, MN Centre Analytical Laboratories, Inc. Study # 023-015 Page 7 Exygen Research Page 15 o f 39 c o i : * '' 7  III a i a. hi i \ i w| 11 m m Centre Method No: OOM-023-015 3.3. E quipm ent and Supplies equipm ent SOURCE Balance, 5 place analytical Mettler Balance, 1 place top-loading Mettler Mini Bead Beater-8 Bio Spec Products Wrist-action shaker Bench-top centrifuge Bunell IEC Mini-centrifuge -1201 50 mL disposable polypropylene centrifuge tubes 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 WhatmanTM AnotopTM Filter (cat # 6809-1022) VWR . 2-mLEppendorfTM microcentrifuge tubes (cat# 22- VWR 36-335-2) 2-mL clear HPLC vial kit (Cat # 5181-3400) HP Standard lab equipment (class A pipettes and volumetric flasks, graduated cylinders, etc.) . LC/MS/MS and HPLC systems As described in - Section 4.4.1. * Note; 1. In order to avoid contamination, the use of disposable labware is highly recommended (containers, tubes, pipettes, etc.). 2. Teflon or teflon-lined containers or equipment, including teflon-lined HPTC. vial caps, should not be used. 4. It is necessary to check the solvents (methanol) for the presence of contaminants by LC/MS/MS before use. Certain lot numbers have been found to be unsuitable for use. 5. Use disposable micropipettes or pipettes to aliquot standard solutions 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 1. 50 mM ammonium acetate solution: Dissolve 3.85 g of ammonium acetate in 1 L of ASTM type I water. Store in an appropriate glass bottle at room temperature for up to 1 year. 2. 2 mM ammonium acetate solution: Dilute 40 mL of the 50 mM ammonium acetate solution in a liter of ASTM type I water, for mobile Centre Analytical Laboratories, Inc. Study#023-015 Page 8 Exygen Research Page 16 of 39 0013 C entre M ethod No: OOM-023-015 phase A. Store in an appropriate glass bottle at room temperature for up to lyear. Note-' The volumes shown am provided for guidance; alternative volume^ may be prepared. ` *. 3.5. Preparation of Stock, Fortification, and calibration Solutions kI 1 Analytical standards are prepared for two purposes. They are used to fortify untreated siamples in order to determine analytical recovery and to calibrate the response of the detector used in the analysis. The analyst may vary the absolute volumes of the standards as long as the correct proportions of solute to solvent are maintained. The solutions cited below are given as an example; alternative concentrations may be prepared if 3.5.1. Stock Solution Prepare stock solution of PEOS at 100 pg/mL by weighing out 10.0 mg of analytical-standard (corrected for percent salt). Adjust final volume to 100 mL with methanol in a 100-mL volumetric flask. Store this stock solution (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period ' of 6 months from the date of preparation. 3.5.2. Fortification Solutions a- 1.0 ue/mL Fortification Solution - Pipette 1.0 mL of the 100 pg/mL stock -solution into a 100 mL volumetric flask. Bring up to. volume withmethanol. '/ ' ' b. QJ MS/tnL Fortification jSal,mtifl,n - Pipette lO.O mL of the LO pg/ml fortification solution into a 100-mL volumetric flask and ring up to volume with methanol.. c- 0-01 pg/mL Fortification Solution - Pipette 10.0 mL of the.0.1 pg/mL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. 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 of preparation. 3.5.3. Calibration Standards Prepare six LC/M5/MS calibration standards in methanol via dilution of the 0.1 pg/mL and 0.01 pg/mL fortification solutions. C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 9p 4 g e i r iv l ! ! ^ \I ; i: Exygen Research Page 17 of 39 G O ia m tit j ! m C entre M ethod No: OOM-023-O15 This is a typical example; additional concentrations may be prepared as I needed ' * Initial Cone. (us/mL) 0.1 .0.1 0.1 0.01 0.01 0.01 Volume (mL) 5 2 1 5 1 0.5 Diluted to (mL) 100 100 100 100 100 100 Final Cone. (ue/mL) 0.005 0.002 0.001 0.0005 0.0001 0.00005 Store all calibration standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. 4. METHOD 4.1. Flow Diagram The flow diagram of the method is given below, followed by a detailed description of each step. Weigh 1 g of matrix (O.-lg for membrane). Fortify if needed i Extract with MeOH ' i Carbon clean up (except membrane) I' FUter 4 Dilution LC/MS/M^S analysis 4.2. Sample Processing j All samples are received frozen and will be kept frozen'(below -10 C) until time of the extraction. j. | i 1 | i i I 4.3. S a m ple extraction NTE: All egg sample matrices were separated prior to arrival at Centre. > Centre Analytical Laboratories, Inc. Study # 023-015 Page 10 Exygen Research Page 18 of 39 C h O i' C entre M ethod No: OOM-023-O5 4J3.1 Egg Membrane i a. Allow sample to thaw. * b. Weigh 0.1 g ( 0.005g) of sample jnto a 2-mL EppendorfTM tube. Record the weight to the nearest O.OOOlg. Fortify untreated control samples at this point for determination of method recovery. c. Add 1 mL of MeOH and three stainless steel beads, replace lid tightly and. homogenize with bead beater set at position 7 (-2500 rpm) for 5 minutes. 1 Centrifuge the tubes with mini-centrifuge at 2,000 rpm for - 2; minutes,' then carefully transfer supernatant to a new 2-mL.EppendorfTM tube,, filtering with WhatmanTM AnotopTM 10 filter if necessary. d. Make a 5X dilution by transferring 0.1 mL of filtrate to HPLC vials and adding 0.4 mL of methanol using disposable micropipettes (100-200pL). ; e. Store the rest of the extract in a refrigerator at approximately 2C to 6C for future re-dilution or re-injection. 4.3.2. E gg Yolk and Album en a. Allow sample to thaw. b. Weigh 1.0 g ( 0.05g) of sample into a 50-mL disposable polypropylene centrifuge tube. Record the weight to the nearest O.OOOlg. Fortify untreated control samples at this point for -determination of method recovery. c. Add 25 m L of MeOH, replace Hd tighdy, and shake on a wrist-action shaker for 15 minutes. Centrifuge tubes at - 2,000 rpm for - 1 0 minutes. d. Transfer about 5 mL of extract into a 15-mL disposable polypropylene centrifuge tube, add 0.5 g carbon, replace lid tightly, arid shake by hand for about 10 seconds. e. Let the tubes sit for - 2 minutes, carefully transfer - 2.0 mL of the sample into a 10-ml disposable syringe barrel connected to a WhatmanTM AnotopTM 10 filter. Filter sample into two HPLC vials, one for injection and one for storage in a refrigerator at approximately 2C to 66C for future re-dilution or re-injection. C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 P age 11 I 0 C entre M ethod No: OOM-023-015 I 4.4. Analysis by LC/MS/MS 4.4.1. LC/MS/MS System and Operating Conditions ( f iirbolonspray) * Mass Spec: PE SCIEX API 3000,Biomolecular Mass Analyzer Interface: SGEXTurboIon Spray Liquid Introduction Interface Harvard infusion pump , Computer: ' Power Macintosh G3 Software: . . , PESciexAnalyst 1.1 WindowsNT HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump. HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Genesis Cs (Jones Chromatography), 2.1 mm x 50 mm, 4ji Column Temp.: 35 C Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTMtype I water Mobile Phase (B): Methanol . Flow Rate: 0.3 mL/min. Time 0 1.0 7.0 1JS 11.0 1 lA 60 . ` 0 0 40 40 %B 40 100 100 60 60 : 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 30 mm) and columns from different manufacturers (Keystone Betasil Cu etc.) ccaann be used, provided equivalent chromatography is obtained. Ions monitored: Analyts PFOS . Approximate Mods Transition Monitored Retention Time (mini negative 499 99 4.20 On a day-to-day basis, the retention times may vary slightly depending on the batch of mobile phase, etc. - J * i" C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Page 12 Exygen Research Page 20 of 39 ( ` .A.5 * C entre M ethod No: OOM-023-015 4.4.2. Example Tune File Parameters The following values are provided as an example. Actual values may vary from instrument to instrument. Also, these values may be changed from time to time in order to optimize for greatest sensitivity. 1 The mass spectrometer is tuned using a 0.5pg/mL PFOS solution, prepared via dilution of the stock solution in methanol. The solution is infused (using a "T" connector) at 10 pl/m in into a 0.2 mL/min stream of mobile phase consisting of 40% methanol and 60% 2 mM ammonium acetate. The analytes are initially tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved as a "tune file". This tune fil is then used during routine analysis. Th tuning procedure may be repeated as necessary to ensure optimal sensitivity. Controls IS-Iospray OR-Orifice RNG-Focus Ring QO-Quad QRod Offset IQ I- Inter quad 1 lens STTStubbies' ROl-Quadl Rod Offset IQ2-Inter quad 2 lens R02-Quad 2 rod offset ST3-Stubbies R03-.Quad 3 rod offset DF-CEM Deflection Plate CEM-Channel Electron Multiplier Set -4000.0 -61.0 -270.0 10.0 9.3 * 15.0 9.3 20.0 84.0 10.0 86.0 300.0 2400.0 Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature . gel 12 13 4 350i 4.4.3. Calibration Procedures a. Inject the same volume (between 10 to 20 pL) of each calibration standard (prepared in MeOH) into the LC/MS/MS. b. Use linear, 1/x weighted standard curves for quantification. Linear . standard curves are generated for each set by linear regression using the C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Page 13 Research Page 21 Centre M ethod No: 0OM-O23-O15 appropriate software system. Any calibration standards falling outside 30% based on its calculated concentration, mast be excluded from the calibration curve. However, the total number of calibration standards that may be excluded must not exceed 30% of the total number of standards injected. c. The correlation coefficient (r) for .calibration curves generated must be 0.9925 (1^20.985). If calibration results fall outside these limits, then appropriate steps should be taken to adjust instrument operation, and the relevant set of samples must be reanalyzed. *i 4.4.4. Sample Analysis 1. .1 a. Inject the same volume used for the calibration standards (between 10 to 25 pL) of each sample, fortification, control, etc. into the LC/MS/MS. b. Standards corresponding to at least six concentrations must be included in an analytical set. c. Inject an entire set of standards (six) at the beginning of the run and inject standards interspersed about every-5-10 samples. All sample injections must be bracketed by standard injections (see Section 4.5). d g arb 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, and two matrix control samples fortified at known concentrations and carried through the procedure to verify recovery. e. All samples must be analyzed with duplicate injections. N ote: The analysis performed -during method development included fortifications at 10,50 and 250 ng/g (ppb) for the analyte. f. The concentration of each sample, fortification, control, etc. is determined from the standard curve based on the peak area of the analyte in all standards injected during a set The standard responses must bracket responses of the residue found in the sample set. If necessary, dilute the samples and re-analyze to give a response within the standard curve range. g. Fortifications that bracket the highest residue expected in each treated sample will 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 that method recoveries are available for the analyte of interest at concentrations exceeding those in treked samples. C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Page 14 Il II 1 1 I 1 II II II 1I! II 1 Centre M ethod No: 00M -023-015 h. Fortification recoveries within 60 to 130% are acceptable for fortifications at the LOQ level. Recoveries between 70 to 120% are acceptable for fortifications at levels greater than the LOQ. Failure to meet these criteria ' requires an investigation of cause and a full reanalysis of the affected samples. a i. Samples in which no peaks are detected at the corresponding analyte retention times will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention times but are less ,,than the lowest standard will be reported as NQ (not quantifiable). j. ,,Background levels of analyte found in control/biank samples that correspond to values below the LOQ, but are still quantifiable, will be used to correct fortification recoveries. ' j. If samples are not loaded on the instrument to be analyzed the day they are extracted, samples must be stored refrigerated at approximately 2C to 6C until analysis and analyzed preferably within a week. Recoveries from method development for all matrices can be found in Tables I-VI. 4.5. Performance Criteria The following two criteria must be met before the initial analysis of samples, especially when using different instrumentation set-ups than those cited in this ' method. * . j- First Criterion - Inject a standard solution on the LC/MS/MS corresponding to the estimated LOQ (10 ppb LOQ is equivalent to a standard 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 and- change .instrument operating parameters. Second Criterion - Inject a set of standards ranging from at or below the LOQ, up to the highest concentration level. Generate a calibration curve for the analyte and obtain a linear regression with a coefficient of determination (r2) of at least 0.985 for the analyte. Once this criterion has been demonstrated, samples may be analyzed with standards interspersed. 1 Centre Analytical Laboratories, Inc. Study#023-015 Page 15 Exygen Research Page 23 of 39 o o ia .:" . Centre M ethod No: OOM-023-015 4.6. Time Required for Analysis A set of 14 samples can be taken through the extraction procedure in approximately three hours by one person. The LC/MS/MS analysis (8-10 standards and 14 samples) will take approximately 6 hours. 5. CALCULATIONS 5.1 Analyte Found: Calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve generated by the MacQuan software program using Equation 1. Equation 1: Analyte found (ng/mL) = (peak area - intercept) slope- 5.2 Component Residue Concentration . Determine the component residue concentration using Equation 2 Equation 2: . Residue found (ng/g) = ^ a ly te found_(ng/mL) xD FxFV (mL)) sample weight (g) Where DF = dilution factor and FV = final volume lisle: ng/g = ppb 5.3 Percent Recovery Calculate the percent recovery for samples fortified with known amounts of analytes prior to extraction, from Equation 3. Equation 3: Recovery (%) = (ng/g found - average ng/g found in control) ng/g added xlOO C e n tre A n a ly tic a l L a b o ra to rie s . In c . S tu d y # 0 2 3 -0 1 16P a g e i i Exygen Research Page 24 of 39 C entre M ethod No: OOM-023-O15 6. SAFETY I There are no unusual hazards associated with this method. The analyst should, read the material safety data sheets for all reagents before performing this method. Normal laboratory precautions should be taken. I I I C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Exygen Research Page 17 Page 25 I I C entre M ethod No: OOM-023-015 I I TABLES i I. C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Exygen Research Page 18 Page 26 of 39 IR m * I I I I m \ \ 14 in til I C entre M ethod No: OOM-023-015 Table I: Summary of Recoveries for PFOS in Quail Egg Membrane Sample Fort Level' (ppW 0003798 Matrix Blank A 0003798Matrix Blank B 0 0 0003798 Matrix Blank C 0003798 Spk A 0003798 SpkB 0003798Spk C 0 10 10 10 0003798 SpkD 0003798 SpkE 0003798 SpkF 0003798 Spk G 0003798 SpkH 0003798 SpkI 50 50 50 250 250 250 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: %Recovery NA NA NA 88 95 92 92 95 96 93 95 93 93 2 3 Table H: Summary of Recoveries for PFOS in Mallard Egg Membrane Sample Fort. Level (ppb) 0003802Matrix Blank A 0 0003802Matrix Blank B 0003802 Matrix Blank C 0003802 Spk A 0003802SpkB 0 0 10 10 0003802 Spk C 0003802 Spk D 0003802 SpkE 0003802 SpkF 0003802Spk G 0003802 SpkH 10 50 50 50 250 250 0003802 SpkI 25 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: %Recovery NA ` NA NA 106 122 106 96 96 90 93 92 93 99 10 10 . - NA = Not Applicable Centre Analytical Laboratories, Inc. Study#023-015 Exygen Research Page 19 Page 27 of 39 G` C entre M ethod No: OOM-023-OI5 Table. IK: Summary of Recoveries for PFOS in Quail Egg Yolk Sample 0003795 Matrix Blank A 0003795 Matrix Blank B 0003795 Matrix Blank C 0003795 SpkA 0003795 SpkB 000379S SpkC 0003795 SpkD 0003795 SpkE 0003795 SpkF 0003795 Spk G 0003795 SpkH 0003795 SnkI Fort. Level ____________ ( ja m _____________ ' 0 0 0 10 10 10 50 , 50 30 , 250 . 250 250 AVERACE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: %Recovery 1 NA NA NA 69. 68, 70 72 80 83 85 89 88 78 9 11 Tabl IV: Summary of Recoveries for PFOS in Mallard Egg Yolk Sample 0003799Matrix Blank A 0003799 MatrixBlankB . 0003799 MatrixBlank C 0003799 Spk 0003799 SpkB 0003799 SpkC 0003799 SpkD 0003799 SpkE 0003799 SpkF 0003799 SpkG 0003799 SpkH 0003799Snk I Fort. Level W e)____________ 0 0 0 10 10 10 50 50 50 250 250 250 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: * Recovery NA NA NA 83 82 10Q 85 88- . 90 8785 88 88' 5 "6 . NA = Not Applicable C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Exygen Research Page 20 Page 28 of 39 li Centre Method No: 00M-023-015 Table V: Summary of Recoveries for PFOS in Quail Egg Albumen m . Simple i Fort Level %Recovery i i ____________ M ______________ 0003796Matrix Blank A 0 NA m 0003796 Matrix Blank B 0003796Matrix Blank C 0 0 NA NA 0003796 SpkA 10 79 0003796 SpkB 10 . 93 H 0003796 SpkC 10 88 0003796 SpkD 50 96 0003796 Spk E 50 95 0003796 SpkF 50 90 0003796 Spk G 250 97 0003796 Spk H 250 102 0003796 Snkl 250 102 fl AVERAGE: 94 STANDARD DEVIATION: 7 RELATIVE STANDARD DEVIATION: 7 if Table VI: Summary of Recoveries for PFOS in Mallard Egg Albumen Sample Fort Level % Recovery 1 -______ (PPbl--____________ : 0003800Matrix Blank A ' 0 NA 0003800Matrix Blank B 0 NA 0003800Matrix Blank C o NA 0003800 Spk A . 10 93 . 0003800SpkB 10 85 0003800 SpkC 10 85 0003800 Spk D 0003800SpkE 50 50 . 96 100 0503800 SpkF 50 . 100 0003800 Spk G 250 98 0003800 Spk H 250 102 0003800Spk I . 250 AVERAGE: 101 96 STANDARD DEVIATION: 7 * RELATIVE STANDARD DEVIATION: 7 NA Not Applicable Centre Analytical Laboratories, Inc. Study 8023-015 M Exygen Research ill Page 21 Page 29 of 39 fO C entre M ethod N o: OOM-O3-015 FIGURES ! C e n tre A n a ly tic a l L a b o ra to rie s , In c . S tu d y # 0 2 3 -0 1 5 Exygen Research Page 22 Page 30 of 39 I'M C entre M ethod No: OOM-023-015 Figure 1: R epresentative Chrom atogram o f a Q uail E gg M em brane Control I H II II Ilf Figure 2: Representative Chromatogram of a Mallard Egg Membrane Control I% Centre Analytical Laboratories, Inc. S t u d y # 0 2 3 - 0 1 5 I Exygen Research I -Page 23 Page 31 o f 39 Il C entre M ethod No: 00M -023-015 Figure 3: R epresentative C hrom atogram o f a Q uail E gg .Yolk Contro] Figure 4: Representative Chromatogram of a Mallard Egg Yolk Control Centre Analytical Laboratories, Inc. Study # 023-015 il Exygen Research Page 24 Page 32 of 39 o n t 3 .: Centre Mtethod No; 00M-O23-015 - Figure 5: R epresentative Chrom atogram o f a Q uail E gg A lbum en C ohtrol !r fii t. e Figure 6; Representative Chromatogram of a Mallard Egg Albumen Control I* Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 25 I1 Page 33 o f 39 * Centre M ethod No: Q0M -023-015 * Figure 7: Representative Chromatogram of a Quail EggMembrane Control Fortified at 10 ng/g (ppb) , i , i m m IM II Figure 8: Representative Chromatogram of a Mallard Egg Membrane Control Fortified at 10.0 ng/g (ppb) M Centre Analytical Laboratories, Inc. Study # 023-015 m Exygen Research m Page 26 Page 34 of 39 0013 C entre M ethod No: OOM-023-015 * Figure 9: Representative Chromatogram of a Quail Egg Yolk Control Fortified at 10.0 ng/g (ppb) Figure 10: Representative Chromatogram of a Mallard Egg Yolk Control Fortified at 10.0 ng/g (ppb) NNiill itoti: bui -1O2/11.1IWo/c* Ac*. TImi AM IMUSMi fcw iN fittati Uh AniMUl ORNICMtMmtti U f . Utk: N toijlt: N . NU I>Uol N* MJ. titiai N TU. UUt: 8m lilit iit KTt T1m li..inf \ M.I Mm M.20l Hi lM.Ml 42..0000 to tie. Tn*i RiteunttlN TImi felfkt: Ititi TImi tM TlM i MMUl 4.52 t i l lM CM 44..401 til Centre Analytical Laboratories Inc. Study # 023-015 Exygen Research Page 27 Page 35 of 39 001 3. . C entre M ethod N o: OOM-023-015' * Figure 11: Representative Chromatogram of a Quail Egg Albumen Control Fortified at 10.0 ng/g (ppb) I* tupi Uhi U Figure 12: Representative Chromatogram of a Mallard Egg Albumen Control Fortified at 10.0 ng/g (ppb) '0 Centre Analytical Laboratories, Inc. Study # 023*015 Exygen Research Page 28 Page 36 of 39 0013 H >11 1 1 'll ll II lit ll 1 ll IN ` * I mi pee*' Lipovitellin, Phosvitin, an Very Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail gg Volk TBmardl and C o o k r fl* 96 [1960] and Stifani e t a !., JBC 265:882-888 [1990]) (May. 1994) * 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 leupeptirv. 4) Cover cylinder tightly with ParafilmM and mix well by gently inverting several times. 5| Using a 20 gauge needle and 12 cc syringe, transfer the suspension to Beckman polyallomer Quick-Seat tubes (tube size will depend on amount of yolk prepared and rotors available). 6) Seal tubes, place into appropriate rotor, and centrifuge at 2 00,00 0 x g at 4 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 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. 8) Using a 20 gauge needle and 12 cc syringe, make two punctures in the 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 8), 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 g ently washed with, and then resuspended in, a solution containing 0.45 M MgS04, 1 mM PMSF, j: ! Exygen Research Page 37 of 39 2 and 2 pM leupeptin. Both VLDL and HOF solutions are then centrifuged at 2 0 0 ,0 0 0 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 mM PMSF, and 5 `pM leupeptin, filtered (0.45 pM), sodium azide added to a final concentration of 1 mM (i.e., dilute stock solution 1000 x), 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 MgSO^, 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 M). The solution is then left standing (without stirring) at 4 #C overnight. 11) On the following morning, a 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 .2 M MgSO^ and let stand overnight (without stirring) at 4 C. On the following morning, the precipitate (phosvitln) is 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 mM Tris-HCI (pH 7.8), 1 mM PMSF, 2 pM leupeptin, and sodium azide added to a final concentration of 1 mM (I.e., dilute stock solution 1000 x). Aliquot and store at 4 C. 12) The "supernate" (0.15 M MgS04.; see step 11) should be decanted, diluted with two volumes of cold "e-pure" water (final concentration of 0.05 M MgSO.4.) dropwise with stirring at 4 "C, and allowed to stand overnight (without stirring) at 4 C. The resulting white precipitate (lipovitelHn) 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 7.8), 1 mM PMSF, and 2 pM leupeptin, and sodium azide added to a final concentration of 1 mM (i.e., dilute stock solution 1000 x). A final protein concentration of 8-10 mg/mL is desired. Exygen Research Page 38 o f 39 001 v \T e : u , i k)? F K o i-'v 4 * - V U C >L IXo c A t7 o O E C rC r Y ou^ tXL.ore'' |:i. w /o.fc7M A4^JOY i \ CCiv^r. <. 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