Document nRDbxZmBEXXrjVYEwxGOBX4R

. Precise Research. . Proven Results. 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 PAGE 10F 5 College, PA 16801, USA F: 814.272.1019 exygen.com 1 Introduction Results are reported for the analysis of total perfluorooctanesulfonate (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 analytical phase protocol given in Attachment E. m 2 Sample Receipt The samples were submitted in plastic bags, labeled with magic marker and typed print. A copy m 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 M 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. 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. 3.1.2 PFOS Content in Yolk Fractions A 5.0 g sample was used for the fractionation. Ten milliliters of a solution containing 0.67 M M g S 0 4, 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 lipoprotein (VLDL) and the high density fraction (HDF). The VLDL was dissolved in a solution containing 20 mM Tris-HCI, 150 mM NaCI, 0.2 mM 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 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 M g S 0 4, 1 mM PMSF, and 2 pM PAGE 2 OF5 ieupeptin and then two volumes of cold water was added dropwise and then stored overnight at 4C. The HDF supernatant (llpovitellin) 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 M gS 04, 1 mM PMSF, and 2 pM Ieupeptin 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 pM Ieupeptin. 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 HP1000 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. The following gradient was performed: Mobile Phase (A): Mobile Phase (B): 2mM Ammonium Acetate in Type I W ater Methanol Time 0.0 0.4 1.0 7.0 7.5 9.0 9.5 14.0 %A 60 60 10 10 0 0 60 60 %B 40 40 90 90 100 100 40 40 The following parameters were used for operation of the mass spectrometer: Parameter Ionization Mode Polarity Transitions Monitored Setting Electrospray Negative 499->99 PAGE 3 OF5 Gas Temperature Drying Gas (N2) 350C 7.0 L/min 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 prepared for 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 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. The 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 Related Comments 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. Data/Sampfe 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 m 7.1 Attachment A: Chain of Custody m 7.2 Attachment B: Analytical Results 7.3 Attachment C: Raw Analytical Data m 7.4 Attachment D: Study Personnel 7.5 Attachment E: Analytical Phase Protocol h m 8 Signatures m ( Efaity R./t)ecker, Principal Investigator * m y<iohn M. Flaherty, Vice'fresident m m t |s|o2 Date 7A M Date M HI m PAGE 5 OF5 m 1 ft ft ft Exygen ID Client Sample ID 0201613 (Composite) 0201614 (Composite) Received . See Original Login See Original Login Mat'ix EGG YOLK EGG YOLK Exygen Research Sample Login Report Study Number: 023-070 Protocol: NA Submitted By NA . NA Logged in By/Date RICK K 6-4-02 RICK K 6-4-02 Page 1 of 1 1__________________________ 1 Initial Loc. FREEZER 8 FREEZER 8 Condition of Somplos Com monks See Original Login Composite of samples: 201551-201562 See Original Login Composite of samples: 201563-201574 Verified By/Date: Prlnted 6/4/2002 B ExygenID Client Sample ID 0201551 E01-1379-31882 Received 6-1-02 21:15 0201552 E01-1379-31883 6-1-02 21:15 0201553 E01-1379-31884 6-1-02 21:15 0201554 E01-1379-31885 6-1-02 21:15 0201555 E01-1379-31895 6-1-02 21:15 0201556 E01-1379-31896 6-1-02 21:15 0201557 E01-1379-31897 6-1-02 21:15 0201558 E01-1379-31898 6-1-02 21:15 0201559 E01-1379-31939 6-1-02 21:15 0201560 E01-1379-31943 6-1-02 21:15 0201561 E01-1379-31947 6-1-02 21:15 0201562 E01-1379-31951 6-1-02 21:15 02015S3 E01-1373-31636 5-1-02 21:15 0201564 E01 -1378-31639 6-1-02 21:15 0201565 E01-1378-31640 6-1-02 21:15 0201566 E01-1378-31641 6-1-02 21:15 0201567 E01-1378-31654 6-1-02 21:15 0201568 E01-1378-31655 6-1-02 21:15 Matrix EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK pnrx Yfi! i< EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK Exygen Research Sample Login Report Study Number: Protocol: 023-070 NA Submitted By NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Logged in By/Date LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 Page 1 of 3 i-------------------- ----- n-----Initial Loc. Condition of Samples Comments FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. ' FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 FRFF7FR a F-DRY ICE-A F-DRY ICE-A Stored in FREEZER 8 until Log-In. Ff.-veH jn FRFF7FR 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. Verified By/Date: Printed 6/3/2002 isi Exygen ID Client Sample ID 0201569 E01-1378-31656 Received 6-1-02 21:15 0201570 E01-1378-31657 6-1-02 21:15 0201571 E01-1378-31694 6-1-02 21:15 0201572 E01-1378-31698 6-1-02 21:15 0201573 E01-1378-31702 6-1-02 21:15 0201574 E01-1378-31706 6-1-02 21:15 0201575 E01-1379-31940 6-1-02 21:15 0201576 E01-1379-31944 6-1-02 21:15 0201577 E01-1379-31948 6-1-02 21:15 0201578 E01-1379-31952 6-1-02 21:15 0201579 E01-1378-31695 6-1-02 21:15 0201580 rvorv* co \ E01-1378-31699 CAt l 070_0 7f1/3 6-1-02 21:15 C_4_fJO 04"1c 0201582 0201583 0201584 0201585 0201586 E01-1378-31707 E01-1379-31942 E01-1379-31946 E01-1379-31950 E01-1379-31954 6-1-02 21:15 6-1-02 21:15 6-1-02 21:15 6-1-02 21:15 6-1-02 21:15 Exygen Research Sample Login Report Study Number: Protocol: 023-070 NA Page 2 of 3 ___________________________ 1 Matrix EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG YOLK EGG ALBUMEN EGG ALBUMEN EGG ALBUMEN EGG ALBUMEN EGG ALBUMEN EGG ALBUMEN /\ | pi rackj EGG ALBUMEN EGG SHELL MEMBRANE EGG SHELL MEMBRANE EGG SHELL MEMBRANE EGG SHELL MEMBRANE Submitted By NA NA NA NA NA NA NA NA NA NA NA NA KJA NA NA NA NA NA Logged in By/Date LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O . 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 1 A,\_A_/pCKJpC r\ 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 Initial Loc. ' Condition o f Samples Comments FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. ' FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-in. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 CDCC7CDfi F-DRY ICE-A c ,n p y 1HF-A Stored in FREEZER 8 until Log-In. in CDCC7PP Q Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored In FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. FREEZER 8 F-DRY ICE-A Stored in FREEZER 8 until Log-In. Verified By/Date: Printed 6/3/2002 1i Exygen 10 Client Sample ID 0201587 E01-1378-31697 0201588 E01-1378-31701 0201589 E01-1378-31705 0201590 E01-1378-31709 Received 6-1-02 21:15 6-1-02 21:15 6-1-02 21:15 6-1-02 21:15 Exygen Research Sample Login Report Study Number: Protocol: 023-070 NA ^U'MU'IWUL -------------------- ik 11 11 1v1 Matrix Submitted By EGG SHELL MEMBRANE NA EGG SHELL MEMBRANE NA EGG SHELL MEMBRANE NA EGG SHELL MEMBRANE NA Logged in By/Date LAWRENCE 0 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 LAWRENCE O 6-3-02 Page 3 of 3 I Initial Loc. FREEZER 8 FREEZER 8 FREEZER 8 FREEZER 8 Condition of Samples Comments F-DRY ICE-A Stored in FREEZER 8 until Log-In. F-DRY ICE-A Stored in FREEZER 8 until Log-In. F-DRY ICE-A Stored in FREEZER 8 until Log-In. F-DRY ICE-A Stored in FREEZER 8 until Log-In. Verified By/Date; Printed 6/3/2002 5/31/2002 Project: E01-1379 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE Contract Lab(s): EXYGEN 1 of 2 Requester: Robideau, Rochelle R (0002-03E-09) Department: 502180 Site Source: Project Number: Date Received: 10/4/2001 Project Description: N. Bobwhite Reproduction (Eggs) Ship Date: 5 `( 3 l / D t - ------ Completion Date: Project Lead: Rochelle R. Robideau Phone Number: 651-778-7065 Email Address: tTrobideau@mmm.com Comments: NOTE: M1SC 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 ppm a.i. 230 H Yolk Analytical Method ComDonents Mise. Services Mise. Services E01-1379-31883 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 232 H Yolk Analytical Method Comoonenls Mise. Services Mise. Services E01-1379-31884 Analysis Code MISC_SERV 6/13/2001 10 DDm a.i. 234 H Yolk Analytical Method Comoonenb Mise. Services Mise. Services E01-1379-31885 Analysis Code MISC_SERV 6/13/2001 10 DDm a.i. 236 H Yolk Analytical Method ComDonents Mise. Services Mise. Services E01-1379-31895 Analysis Code MISC_SERV 6/13/2001 10 D D m a.i. 229 I Yolk Analytical Method Components Mise. Services Mise. Services E01-1379-31896 Analysis Code MISC_SERV 6/13/2001 10DDma.i. 2 3 1 1 Yolk Analytical Method ComDonents Mise. Services Mise. Services E01 -1379-31897 Analysis Code MISC_SERV 6/13/2001 10 DDm a.i. 233 I Yolk Analytical Method ComDonents Mise. Services Mise. Services E01 -1379-31898 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 235 I Yolk Analytical Method Components Mise. Services Mise. Services E01-1379-31939 Analysis Code MISCSERV 6/21/2001 10 DDm a.i. 226 J Yolk Analytical Method ComDonents Mise. Services Mise. Services s' - Analysis Due Date 6/30/2002 s' Analysis Due Date 6/30/2002 Analvsis Due Date 6/30/2002 s' Analvsis Due Date 6/30/2002 Analysis Due Date 6/30/2002 s' Analvsis Due Date 6/30/2002 Analvsis Due Date 6/30/2002 Analvsis Due Date 6/30/2002 Analysis Due Date 6/30/2002 s' 5/31/2002 3MENVIRONMENTAL 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 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 226 J Albumen Analytical Method Comoonents Mise. Services Mise. Services Analvsis Due Date 6/30/2002 E01-1379-31942 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 226 J Shell Membrane Analytical Method Comoonents Mise. Services Mise. Services Analvsis Due Date 6/30/2002 E01-1379-31943 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 230 J Yolk Analytical Method Comoonents Mise. Services Mise. Services Analysis Due Date 6/30/2002 E01-1379-31944 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 230 J Albumen Analytical Method Comoonents Mise. Services Mise. Services Analvsis Due Date 6/30/2002 EOI-1379-31946 Analvsis Code MISC_SERV 6/21/2001 10 ppm a.i. 230 J Shell Membrane Analytical Method Comoonents Mise. Services Mise. Services Analysis Due Date 6/30/2002 E01-1379-31947 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 232 J Yolk Analytical Method Components PFOS by ESMS PFOS Analvsis Due Date 6/30/2002 E01-1379-31948 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 232 J Albumen Analytical Method Comoonenb PFOS by ESMS PFOS Analysis Due Date 6/30/2002 E01-1379-31950 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 232 J Shell Membrane Analytical Method Comoonents PFOS by ESMS PFOS Analysis Due Date 6/30/20(12 EOI-1379-31951 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 234 J Yolk Analytical Method Comoonents PFOS by ESMS PFOS Analvsis Due Date 6/30/2002 E01-1379-31952 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 234 J Albumen Analytical Method Components PFOS by ESMS PFOS s' Analyse Due Date 6/30/2002 E01-1379-31954 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 234 J Shell Membrane Analytical Method Comoonents PFOS by ESMS PFOS Analysis Due Date 6/30/2002 3 M Environmental Laboratory Shipping Address: 3M Bldg. 2-3E-09 935 Bush Avenue SI. Paul, MN 55106 Telephone: Sample Receiving: (651) 778-4948 Alternate: (681) 778-8753 FAX: (651) 778-8178 \\ \ Chain of Custody /Request for Laboratory Analytical Project ID/Project NameXT Template # ----- f D------ ____________________________ -KafiTe>ort Due Date Project Lead Dept. # (main) Rochelle P . Robideau nternal Due Date 3lass/Job/Project # 5808 -- _ 3M Env. Lab Project # For Internal Use Only E01-1379 Report Results to: Contact Name R o b ideau. Rochelle R Company 3M Date Available f M, Mailing Addressnnn 7_n -,p_ng Date Due City, State, Zip Teiephone#___ l 6s1 7 7 8.7Qfi4___________________________ FAX# i / n T 7 -< n is ___________________________ Contract Lab Special Instructions and/or Specific Regulatory Requirements: v' (method, limit of detection, reporting units, etc.) Preservatives^; \ ozf X O* <Nn X oo>IA z0 l o !o S 2 II |5 , 'Ca isf7fcO'" loOCl fc"*- ) Analysis Requested: Complete below. Attach any associated information. F C aIX Item # Client Sample Identification 1. 10 p pm a.i. 2 3 0 H Y o lk 2. 10 ppm a.i. 232 H Y o lk 3. 10 p pm a.i. 234 H Y o lk 4. 10 p pm a.i. 23 6 H Y o lk 5. 10 p pm a.i. 2 2 9 1 Y o lk 6. 10 ppm a.i. 2 3 1 1 Y o lk 7. 10 p pm a.i. 233 1 Y o lk 8. 10 ppm a.i. 235 I Y o lk 9. 10 p pm a.i. 22 6 J Y o lk 10. 10 ppm a.i. 226 J Albumen 3M LIMS# Date Sampled 31882 S/13/01 31883 5/13/01 31884 5/13/01 31885 5/13/01 31895 5/13/01 31896 5/13/01 31897 5/13/01 31898 5/13/01 31939 5/21/01 31940 5/21/01 Time Sampled Matrix/ Media Enter the number of containers of each 1 (Enter an 'X' in the box below to Indicate request) Relinquished by/Affiliation -31943 6/21/01 Time Date f ` Shipped Via: i teceived by/Affiliation ............ Time Z /'/ Date Sample Condition Upon Receipt: Temperature: Other Associated CoCs: 'C O Acceptable 0 Other: O Received on Ice f I v Page - l Of . 2 * Original - Accompanying Samples l/ Last Page * Originator Comments: & TO try y [jrtP 'J See Reverse Side for Instructions Report Results to: None Other Total Number of ^pontainers 'I s i ft 1 f t ft 3M Environmental Laboratory Form 30778 - PWO Shipping Address: 3M Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Telephone: Sample Receiving: (651) 778-4948 Alternate: (651) 778-6753 FAX: (651)778-6176 \ Contact Name Rnhideail Rochelle R Company 3M Chain of Custody /Request for Laboratory Analytical Project ID/Project NameXT _________________ Template # Finaffieport Due Date Project Lead Dept. # (main) ?nrb#*11r 1? PrtVtiHpaii Internal Due Date Class/Job/Project # 50 21 80----------------------------- Date Available Mailing A d d ress^ ^.n ^F .-O O City, State, Zip Telephone#__ j 65i 778-7065________________________FAX# 1 Special Instructions and/or Specific Regulatory Requirements: v (method, limit of detection, reporting units, etc.) ' Date Due Contract Lab ________________________ P re s e rv a tiv e s : * 5809 3M Env. Lab Project # E01-1379 , 1/. y> f Y iy JD t f X `I Q ;7v 2 c c M l UU: Analysis Requested: Complete below. Attach any associated Information. n o XZ * OCXNO o(A O> $ 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. 3M LIMS# 31944 31946 31947 31948 31950 31951 31952 31954 Date Sampled 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 Time Sampled Matrix/ Media Enter the number of containers of each (Enter an 'X' in the bux below to indicate reouest) * * * \J/ 10. Collected by (print): 'D>* Ot3+Of-)* . Item # xTT o c `to SoZ linquished by/Affiliatio Time M :y > pate Sample Condition Upon Receipt: Temperature: Other Associated CoCs: _ O Acceptable O Other: u rceceivea on ice __ _^__ Copies to: d c y -- ----------------------------------------------------------- x_Page Of Original - Accompanying Samples -v //Z /Z . Last Page - Originator Collector's signature: Shipped Via: Received by/Affiliation -- Comments: jz> <57 ^ 6^ See Reverse Side for Instructions Tim e; -H i Date 5/31/2002 Project: E01-1378 3M ENVIRONMENTAL LABORATORY CONTRACT LABORATORY WORK ORDER BY SAMPLE Contract Lab(s): EXYGEN 1 of 2 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 E01-1378-31638 Analysis Code MISC_SERV Sampled Date Sample Description 6/13/2001 10 oom a.i. 230 H Yolk Analytical Method . ComDonents Mise. Services Mise. Services E01-1378-31639 Analysis Code MISCSERV 6/13/2001 10 ppm a.i. 232 H Yolk Analytical Method Comoonents Mise. Services Mise. Services E01-1378-31640 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 234 H Yolk Analytical Method Components Mise. Services Mise. Services E01-1378-31641 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 236 H Yolk Analytical Method Comoonents Mise. Services Mise. Services E01-1378-31654 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 229 I Yolk Analytical Method Components Mise. Services Mise. Services E01-1378-31655 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 2 3 1 1 Yolk Analytical Method Comoonents Mise. Services Mise. Services E01-1378-31656 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 233 I Yolk Analytical Method Comoonents Mise. Services Mise. Services E01-1378-31657 Analysis Code MISC_SERV 6/13/2001 10 ppm a.i. 235 I Yolk Analytical Method Components Mise. Services Mise. Services E01-1378-31694 Analysis Code MISCSERV 6/21/2001 10 ppm a.i. 230 J Yolk Analytical Method Comoonents 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 Date 6/30/2002 Analysis Due Date ' 6/30/2002 Analysis Due Date 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 m E01-1378-31695 Analvsis Code MISC_SERV Sampled Date Sample Description 6/21/2001 10 ppm a.i. 230 J Albumen Analytical Method Components Mise. Services Mise. Services Analysis Due Date 6/30/2002 EOI-1378-31697 Analvsis Code m MISC_SERV 6/21/2001 10 ppm a.i. 230 J Shell Membrane Analytical Method Comoonents Mise. Services Mise. Services Analvsis Due Date 6/30/202 E01-1378-31698 m Analvsis Code MISC_SERV < E01-1378-31699 Analvsis Code MISC_SERV m E01-1378-31701 Analvsis Code m MISC_SERV 6/21/2001 10 ppm a.i. 232 J Yolk Analytical Method Comoonents Mise. Services Mise. Services 6/21/2001 10 ppm a.i. 232 J Albumen Analytical Method Comoonents Mise. Services Mise. Services 6/21/2001 10 ppm a.i. 232 J Shell Membrane Analytical Method Comoonents Mise. Services Mise. Services Analysis Due Date 6/30/2002 s'- Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 EOI-1378-31702 m Analysis Code PFOS m E01-1378-31703 Analysis Code PFOS E01-1378-31705 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 234 J Yolk Analytical Method Comoonents PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 234 J Albumen Analytical Method Comoonents PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 234 J Shell Membrane Analytical Method Comoonents PFOS by ESMS PFOS y Analysis Due Date 6/30/2002 Analysis Due Date 6/30/2002 y Analvsis Due Date 6/30/2002 E01-1378-31706 Analvsis Code PFOS *+i E01-1378-31707 Analvsis Code PFOS 6/21/2001 10 ppm a.i. 236 J Yolk Analytical Method Comoonents PFOS by ESMS PFOS 6/21/2001 10 ppm a.i. 236 J Albumen Analytical Method Comoonents PFOS by ESMS PFOS Analysis Due Date 6/30/2002 y Analvsis Due Date 6/30/2002 E01-1378-31709 l Analvsis Code PFOS 6/21/2001 10 ppm a.i. 236 J Shell Membrane Analytical Method Comoonents PFOS by ESMS PFOS Analysis Due Date 6/30/2002 Report Results to: None 7s , Other Total Number of Containers | ft 1 3M E n v iro n m e n ta l L a b o ra to ry , Form 38778 - PWO Shipping Address: 3M Bldg. 2-3E-09 335 Bush Avenue St. Paul, MN 55106 Telephone: / X\ j /Sample Receiving: (651) 778-4948/ J X Alternate: (651) 778-6753 FAX: (651) 778-6176 \ Contact Name RohiHea,, p r e l l e R Company ' Chain of Custody /Request for Laboratory Analytical "" Project ID/Project Name* ,, ____rT7___________^ Template # iftrial Report Due Date Project Lead Dept. # (main) Rochelle P Pobideau cm ion Internal Due Date Class/Job/Project # ' "" Date Available Mailing Addressn n n 9 f n p_nQ Date Due City, State, Zip Telephone# 1651 778-7065 Special Instructions and/or Specific Regulatory Requirements: (method, limit of detection, reporting units, etc.) Contract Lab FAX* 1/A4n 778.fi176____________________ v7 P reservatives^ 5810 3M Env. Lab Project # For Internal Use Only E01-1378 r r i rvV;1V C {AtC M t ix & C>I V A nalysis Requested: Complete below. Attach any associated information. 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. 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. iinV/ ppili u.i. -D\J JTv1~vniiv, 10. 10 ppm a.i. 230 J Albumen oRaeretrtjy (print): ~ ' 10 p^mr ^ .i. 22 J Y o lk O3 o 9 i\= d t* c u Sample Condition Upon Receipt: O Acceptable O Other: Temperature: 'C O Received on Ice Oth^Assoda^dCoCs ^ ICC T lfft** C cr `.S-------------------------- c i /Page L Original * Accompanying Samples Xozo * OOXN) Oin >O 3M LIMS# 31638 31639 31640 31641 31654 31655 31656 31657 31694 31695 Date Sampled i/13/01 i/13/01 i/13/01 i/13/01 i/13/01 i/13/01 5/13/01 5/13/01 5/21/01 5/21/01 Time Sampled Matrix/ Media Enter the number of containers of each _L r A. (Enter an 'X* in the box below to indicate request) 3 1 6 9 8 (5/21/01 Time 3>^A Collector's signature: Date Shipped Via: 5/3f L ^ ^icceeiiveai byy//Aiirffili;ation ________ S-S-Q Z Time Date z / ' r <f-/-OZ / \J Comments: Last Page - Originator See Reverse Side foe Instructions 3M Environmental Laboratory Chain of Custody /Request for Laboratory Analytical 3M Bldg 2-3E-09 St935 Bush Avenue Paul, MN 55106 Sample Receiving: (651) 778-4 Alternate: (651) 778-6753 ^ FAX: (651) 778-6176 \\ n /T7Project ID/Project NameKinl, _ i ____A __ ___^ #Template v~`'f?irial Report Due Date #Project Lead Dept. (main) cm ion nternal Due Date PobHrmi 1 #;iass/Job/Project Robideaua ' Contact Name Rochelle R to3\. V) Company Date Available * Mailing A d d re s i^ ^ ^ ft^ p _0Q tato~o City, State, Zip Date Due DC FAX# rnsm _ai A____________Telephone#___, 778-7065________________________ vSpecial Instructions and/or Specific Regulatory Requirements: Fki(method, limit of detection, reporting units, etc.) 1 77 * Contract Lab P re s e rv a tiv e s : 5811 3M #Env. Lab P roject For Internal Use Only E01-1378 /oh ,0 V 7- Kf` <(Tjbc ftsu rO Analysis Requested: Complete below. Attach any associated information. Total Number of Containers #Item Client Sample Identification 1. 10 ppm a.i. 232 J Albumen 2. 10 ppm a.i. 232 J Shell Membrane 3. 10 ppm a.i. 234 J Yolk 4. 10 ppm a.i. 234 J Albumen 5. 10 ppm a.i. 234 J Shell Membrane 6. 10 ppm a.i. 236 J Yolk 7. 10 ppm a.i. 236 J Albumen 8. 10 ppm a.i. 236 J Shell Membrane C7. 10. Coilected by (print) o O3Kat3f- Item # -*------|1- o `Cc5 .Oc Relinquished by/Affiliation Sample Condition Upon Receipt:_____ O Acceptable O Other: Temperature: 07 i T X w C 'C O Received on Ice (^ r A r Copies to: ... ------------------------------------ Page Original - Accompanying Samples 3M MS# 31699 31701 31702 31703 31705 31706 31707 31709 Date Sampled /21/01 i/21/01 i/21/01 i/21/01 i/21/01 i/21/01 i/21/01 i/21/01 Time Sampled OZzn O zCNO O>10 0 None i 0 0 Matrix/ Media Enter the number of containers of each _L X. * (Enter an *X* in the box below to indicate request) 1 yj/ * VJ \V N1/ Time Date Collector's signature: Shipped Via: Received by/Affiliatlon Time z /// Date ^L { ltd Last Page - Originator Comments: See Reverse Side for Instructions Proven Results. Sample "Condition Upon Receipt" Form Protocol# Exygen Study # MA. 0 3 3 - 7 ,( O'?? .0 Z' Date & Time Received - / - J. X Condition of Samples D ru ic ,* - Temporary Storage Location / -- Y Initials & Date ^ -/->2. Waybill # ? 3 < /? / o / 3 / f t e J A i S ) Comments: d 'd n u e . / V a r i e r . /f/K - /* - / - * Q:\VVORD\process-login\SampleConditiononReceipt.doc May 24, 2002/3 3058 Research Drive ^ S' tSatai te College, PA 16801, USA VT: 800.281.3219 F: 814.272.1019 exygen.com KI I ft ft ExygenID Client Sample ID 0201615 NA Received. 6-5-02 12:00 EGG Exygen Research Sample Login Report Study Number: Protocol: 023-070 NA Page 1 of 1 L o g ^ B y /D a te - I R S P a p illo n of-Samples P ^ ^ r n m e n t s T ^ NA LAWRENCE O FREEZER 8 REFRIGERATED Purchased at Weis 6-5-02 Markets #33, State College, PA Verified By/Date: r ; C Printed 6/5/2002 L^J : i - '. - v - U 1 w ei 3 k . THANK YOU FR SHOPPING WEIS MARKETS 33 STATE COLLEGE, PA. Ite *UQ EGGS L : * TAX .... .00 Cash CHANGE th- : BAL Pr ice .79 F .79 1.00 .21 6/0.5/9.2. 12:29 PM 0033 02 C 150 . H 5 . ......... = ASK-US HOU 0R UEIS. SHOPPERS CLUB CAN SAUE VOLI $ $ 'S EUERYDAY ... WEIS CLUB MEMBERS HAUE THE POWER TD SAUE MORE T H IS IS AN EXACT CO PY O F THE O R IG IN A L D O C U M E N T." Ay nATE HI ini m m Report-- Analytical ^ 1 Summary of PFOS Residue Found (ppb) in Combined Mallard Egg Yolk Samples Sponsor ID na (Reagent Control) na (Reagent Control)A na (Reagent Control) na (Reagent Coritrol)A na (Matrix Control) na (Matrix Control)A na (Matrix Control) na (Matrix Control)A na (LCS) na (LCS) na (LCS) na (LCS) ** A ** Dup ** DupA Analyte Found __________ (EEb)________ NQ NQ NQ NQ NQ NQ NQ NQ 108 111 103 100 53600 52300 52600 52500 Fort. Level (ppb) 100 100 100 100 - 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 -1378-31638, 31639, 31640, 31641, 31654, 31655, 31656, 31657, 31694, 31698, 31702, 31706 ^ L 3 03055f8i Research Drive # m s' tSatatte College, PA 16801, USA VJ: 814.272.1039 F: 814.2 3 1 .1 5 8 0 Summary of PFOS Residue Found (ppb) in Combined Quail Egg Yolk Samples Sponsor IP na (Reagent Control) na (Reagent Control)A na (Reagent Control) na (Reagent Control)A na (Matrix Control) na (Matrix ControljA na (Matrix Control) na (Matrix Control)A na (LCS) na (LCS) na (LCS) na (LCS) ** **A Dup ** DupA Analyte Found (PPb) NQ NQ NQ NQ NQ , NQ ' NQ NQ 104 102 106 109 75000 74300 48700 49900 Foirt. Level (ppb) Recovery (%) 100 104 100 102 100 106 100 109 -- -- -- -- 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 0,35058 Research D rive State College, PA 16801, USA Y,T: 814.272.1039 F: 814.2 3 1 .1 5 8 0 L Report-- Analytical Y Summary of PFOS Residue Found (ppb) in Mailard'/Quail" Egg Yolk Fractions Sponsor Analyte Found Fort. Level Recovery * ID (PPb) (ppb) (%) na (Reagent Control) ND na (Reagent Control)A ND m na (Reagent Control) ND na (Reagent Control)* ND na (Matrix Control)-VLDL 13.5 m na (Matrix Controi)-VLDL* 14.3 na (Matrix Control)-Phosvitin ' NQ na (Matrix Control)-Phosvitin* NQ m na (Matrix Control)-Lipovitellin NQ na (Matrix Control)-LipovitelIin* NQ ` -Mallard VLDL 41800 ` -Mallard VLDLA 42500 ` -Mallard Phosvitin 3600 ` -Mallard Phosvitin* 3580 m ` -Mallard Lipovitellin 8910 ` -Mallard Lipovitellin* 8830 " -Quail VLDL 39700 m ` ` -Quail VLDL* 39900 " -Quail Phosvitin 815 - " -Quail Phosvitin* 838 - " -Quail Lipovitellin " -Quail Lipovitellin* 1300 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 quantitation 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, 31898, 31939, 31943, 31947, 31951 UN m ,3058 Research D rive X- # m S tSatatte College, PA 16801, USA YJ: 814.272.1039 F: 8 1 4 .2 3 1 .1 5 8 0 o w a o n r~r\m 3058 Research Drive State College, PA 16801 T: (814) 272-1039 F: (814) 231-1580 STUDY PERSONNEL LOG PROTOCOL NA_________ EXYGEN STUDY O V t ^O June 28, 2001/1 ANALYTICAL PHASE PROTOCOL ANALYTICAL PHASE TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY 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 of 39 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 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 11. COSTS..................................................................................................................... 6 12. PROTOCOL APPROVAL...................................................................................... 7 APPENDIX: ANALYTICAL METHODS.......................................................................8 Exvgen Research Page 2 o f 39 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM MALLARD AND QUAIL EGG YOLK FOR ANALYSIS USING HPLCELECTROSPRAY/MASS SPECTROMETRY 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 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 of the compound is presented below. PFOS Chemical Name: IUPAC Name: CAS Number: Molecular Weight: Perfluorooctanesulfonate 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8- heptadecafluoro-, potassium salt 2795-39-3 499 (C8F17S031 O II C8Fi7|S---- O' o Note: The neutral molecule and standard from wliich the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [C8F17SO3K], 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 wil l 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 LABORA TORY 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 of 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 of Perfluorooctanesulfonate in Egg Membrane, Albumen and Yolk by LC/MS/MS" to determine the total amount of 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 of 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 of untreated control samples, fortification of untreated control samples to obtain recovery data, and replicate analysis of fortified 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 calculated. 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 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 correspondence 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 o f 39 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 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, analytical standard solution preparation, sample chain of 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 protoco1 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 conditions 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 Project Manager, Exygen EScni^elnjt/iDstecke(Jr Sponsor, 3M Bill Reagen Date Date y 02- Exygen Research Page 7 o f 39 APPENDIX: ANALYTICAL METHODS A. "Determination of 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 li TITLE mi Determination of Perfluorooctanesulfonate in Egg Membrane, Ajlbumen and Yolk by * . LC/MS/MS I il AUTHORS Emily Stauffer and John Flaherty M D A tE ISSUED il January 2,2001 SPONSOR 0 3M Environmental Laboratory Building 2-3E-09 a POBox 33331 St. Paul, MN 55133-3331 PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 0 CENTRE STUDY NUMBER 023-015 CENTRE METHOD NUMBER 00M-023-015 . TOTAL NUMBER OF PAGES ' 28 1 II Exygen Research ml Page 9 of 39 Centre Method No: 0OM-023-01S . MANAGEMENT APPROVAL !| * , As per 40 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 perating procedures and general GLP regulations. I 3jo|o[ Smily Stauffer Date Principal Investigator . Centre Analytical Laboratories, Inc. ' O .L /y ? /. U st yioh n Flahertvy y / Laboratnoryv Manager . Centre Analytical Laboratories, Inc. M r Date Centre Analytical Laboratories, Inc. iobideau ffonsor Representative 3M Environmental Laboratory Centre Analytical Laboratories, Inc. S:udy # 023-015 Exygen Research Page 2 Page 10 Ill I ill II Centre Method No: OOM-023-015 TABLE OF CONTNTS TITLE PAGE. MANAGEMENT APPROVAL------TABLE OF CONTENTS_______ a i.2 LIST OF TABLES______________________________ ;________________________ 4 LIST OF FIGURES___ :______________________ ___________________ :...._____ 1. SUMMARY. 2. EXPERIMENTAL COMPOUNDS------------------ ------------------- '.---- ;_______ 7 3. CHEMICALS AND SUPPLIES------------------- .....----------------------- ----------- 7 3.1. Ch e m ic a ls ................ 7 3.2. Sta n d a r d s ............... ............................................................................... 7 3.3. Eq u ipm en t an d Su pplies........................................................... ...........................................................8 3.4. So lu tio n s................. .................................................................... 8 3.5. Preparatio n o f Sto ck, Fo r tific atio n , an d Ca lib r atiq n So lu t io n s ...................................... .......... 9 3.5.1. S tock So lu tio n ..............................................................- ............ :.............................................................9 3.5.2. F ortification Solutions................................................ .-- ........ '.................................. :......................... 9 3.5.3. C alibration S ta n d a rd s................................................. 9 4. METHOD. .10 4.1. Flow D ia g r a m ........... ..10 4.2. Sam ple Pro cessing ... ..10' 4.3. Sa m p le e x t r a c t io n ... ..10 4.3.1 E gg M e m b ra n e ..... ..11 4.3.2. Egg Yolk and Albumen . .11 4.4. An alysis b y L C /M S/M S....... .................................. ........................ ....:................ 12 4.4.1. LC /M S/M S System a n d Operating C onditions (T urbolorupray) ..................................................... 12 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..................................................................... 14 4 5 . Performance CRnERiA............ .................................................................................................................. .. 4.6. T im e Req uired for a n a ly s is ....................................................................................................... :.............16 5. CALCULATIONS.:------------------------------ 16 6; SAFETY.....!---------------- 17 TABLES............................................... 18 FIGURES_____ _______________________________ ________ _____ .......______ 22 Centre Analytical Laboratories, Inc. Study # 023-015 Page 3 Exygen Research Page 11 of 39 Centre Method No: 00M-023-015 LIST OF TABLES . TABLE! SUMMARY OF RECOVERIES f6 r PFOS IN QUAIL EGG . ' MEMBRANE................................ '.................. .............................................. 19 TABLE II: SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG MEMBRANE................................ .................................................................... i. TABLE HI: SUMMARY OF RECOVERIES FOR PFOS IN QUAIL EGG 1 YOLK................................. 20 TABLE IV: SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG Y o l k ............. 20 TABLE V: SUMMARY OF RECOVERIES FOR PFOS IN QUAIL EGG ' . ALBUMEN....... .................. v.......................................................................... 21 TABLE VI: SUMMARY OF RECOVERIES FOR PFOS IN MALLARD EGG ALBUMEN.................................. 21 Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 4 Page 12 Centre Method No: 0OM-O23-O15 LIST OF FIGURES . * Figure 1: Representative Chromatogram of a Quail Egg Membrane Control............... 23 4 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).............- .................................................................................. '.'21 Figure-10: Representative Chromatogram of a Mallard Egg Yolk Control Fortified at 10.0 ng/g (ppb).............................................;.......................................... ........27 Figure.ll: 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 Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 5 Page 13 of 39 Il II . ` Centt-e Method No: 00M-023-015 II ' 1. SUMMARY II This document details a method of analysis for -the residual determination of ^ perfluorooctanesulfonate (PFOS) in egg membrane, albumen and yolk. The chemical | . formula of the analyte is given in Section 2 of this method. ,' Perfluorooctanesulfonate is extracted from each matrix with methanol (MeOH). For egg g _ 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 ' i' '' . ; For this method the LOQ is 10 ng/g (parts-per-billion). This is based on studies j conducted with egg membrane, albumen, and yolk control samples during method m i . development. '' ' - III 1 III II I- Centre Analytical Laboratories, Inc. Study # 023-015 i IH Exygen Research II Page 6 Page 14 of 39 Centre Method No: 0OM-O23-O1S 2. EXPERIMENTAL COMPOUNDS i The molecular structure of PFO^ is given below: PFOS . Molecular weight: 499 (CsFnSCb- ) 1 0 1 c8f17s -- ' K+ ' Chemical Name : Perfluorooctanesulfonate .' IUPAC name :1-Octanesulfonic acid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8- ' heptadecafluoro-potassium salt CAS # : 2795-39-3 . '' Note: The neutral molecule and standard form which.PFOS (anion) is derived from is perfluorooctanesulfonate potassium salt [C8FnS03K] (molecular weight 538). ' -. 3. CHEMICALS AND SUPPLIES . . * '* 3.1. Chemicals . ' . Chemical ' . Grade Source . Catalog No. Carbon Methanol (MeOH) Ammonium Acetate' Water 120/400 Supelco HPLC (VWR) J. T. Baker Reagent Aldrich Chemical Type I Centre 57210-U JT9093-2 111-87-5 ,- (Type I water = electrical resistivity, minimum of 16.67 Mil-cm at 25C, from a LabconcoTM waterpro workstation, based on ASTM classification) .P ' 3.2. Standards Standard Grade Test Control - Source . _________ ______ ________ Reference Number _______________________ PFOS Analytical TCR-00017-46 3M Environmental . . . ' Laboratory, St. Paul, MN Centre Analytical Laboratories, Inc. Study # 023-015 Page 7 Exygen Research Page 15 of 39 ill l ll 11 I i i m Centre Method No: OOM-023-015 3.3. E quipm ent a n d Supplies EQUIPMENT SOURCE Balance, 5 place analytical Mettler Balance, 1 place top-loading > Mettler Mini Bead Beater-8 Bio Spec Products Wrist-action shaker Burrell Bench-top centrifuge IEC Mini-centrifuge -1201 VWR 50 mL disposable polypropylene centrifuge tubes 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 Whatman^1AnotopTM 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 HPLC 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 . So l u t io n s . -. 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  11 m i 10 I Dll . ` 1 Centre Method No: OOM-023-015 phase A. Store in an appropriate glass bottle at room temperature for up to lyear. , I Note: The volumes shown are provided for guidance; alternative volumes' may be prepared. ' 3.5. Pr e pa r a t io n o f S t o c k , F o r tificatio n, a n d Ca l ib r a t io n S o l u t io n s iI 1 Analytical standards are prepared for two purposes. They are used to fortify untreated samples 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 needed. - 3.5.1. S tock S o lu tio n ' 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 (Smonths from the date of preparation, .; 3.5.2. F o rtific a tio n S olutions a. 1.0 ug/mL Fortification Solution - Pipette 1.0 mL of the 100 pg/inL stock solution into a 100 mL' volumetric flask. Bring up to. volume with- methanol. . . ' b. 0 .1 'ug/mL Fortification Solution -- Pipette 10.0 mL o f the 1.0 pg/ml fortification solution into a 100-mL volumetric flask and Ijring up to volume with methanol.. c. 0.01 [ig/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. C a lib ra tio n 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. ' #Centre Analytical Laboratories, Inc. Study 023-015 Page 9 Exygen Research m Page 17 of 39 Ill m m ] I : M II Centre Method No: OOM-023-015 This is a typical example; additional concentrations may be prepared as I needed. ' '* Initial Cone, (pg/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 D iagram . The flow diagram of the. method is given below, followed by a detailed description of each step. i | v Weigh 1 g of matrix (O.-lg for membrane), Fortify if needed - ;- . : , - X' . . Extract with MeOH : . .` .' i Carbon clean up (except membrane) .. i . - Filter . ' ; ''; . * ' Dilution i . . ' !: . ^ . ` LC/MS/MS analysis i! . 4 .2 . Sample Processing . ' .\ All samples are received frozen and will be kept frozen (below -10 C) until time o f the extraction. . . 4.3., S ample extraction ' -. ' ' NOTE: 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 Centre Method No: OOM-023-Ol5: 4.3.1 E g g M em bran e '' 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 0.000 lg. 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 ipm 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 g g Y o lk a n d A lb um 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 mL o f MeOH, replace lid tightly, and shake on a wrist-action shaker for 15 minutes. Centrifuge tubes at - 2,000 rpm for - 10 minutes. d. Transfer about 5 mL of extract into a 15-mL disposable polypropylene centrifuge tube, add 0.5 g carbon, replace lid tightly, and 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 6C for future re-dilution or re-injection. . Centre Analytical Laboratories, Inc. Study # 023-015 Page 11 I mi . ': Centre Method No: OOM-023-015 I ' 4.4. A n a l y sis b y LC/MS/MS ,' U 4.4.1. L C /M S /M S System a n d O p eratin g C o n d itio n s (T u rb o lo n sp ra y) ' I Mass Spec: PE SCIEX API 3000,Biomolecular Mass Analyzer Interface: SCIEX Turboion Spray Liquid Introduction Interface Harvard infusion pump ; m Computer. ' Power Macintosh G3 Software: . . , PE SciexAnalyst 1.1 h WindowsNT . HPLC: Hewlett Packard (HP) Series 1100 h HP Quat Pump. ' HP Vacuum Degasser HP Autosampler - il HP Column Oven ' . HPLC Column: Genesis Cs (Jones Chromatography), 2.1 mm x 50 mm, 4p, 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 mUmin. ' Time %A %B 0 60 - 1 40 1.0 0 ' 100 i 7.0 0 ' 100 is 40 60 11.0 40 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.) can be used, provided equivalent chromatography is obtained. Ions monitored: Analyte PFOS . Approximate Mode Transition Monitored Retention Time ('min') 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. . '" . - Centre Analytical Laboratories, Inc. Study # 023-015 Page 12 * Exygen Research m Page 20 of 39 Centre Method No: OOM-023-015 4.4.2. E xa m p le T une F ile P aram eters ' 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. The mass spectrometer is tuned using a 0.5|ig/mL PFOS solution, prepared via dilution of the stock solution in methanol. The solution is infused (using a "T" connector) at 10 pL/min 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 file-is then used during routine analysis. Th tuning procedure may be repeated as necessary to ensure optima] sensitivity. Controls ' IS-Iospray OR-Orifice RNG-Focus Ring ' QO-Quad 0 Rod Offset IQ1- 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 ' TE3 Temperature ' . Set 12 13 .4 350( 4.4.3. C a lib ra tio n Procdures 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 Centre Analytical Laboratories, Inc. Study # 023-015 Page 13 Research Page nil Ili fl 1 , i j li : i i i 1 Centre Method No: 00M-023-015 appropriate software system. Any calibration standards falling outside 30%, based on its calculated concentration, must 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 (^>0.985). 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. *( 4.4.4. Sam ple A n a lysis : '' 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). & 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, 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 treated, samples. Centre Analytical Laboratories, Inc. Study # 023-015 Page 14 Exygen Research .Page 22 of 39 Il mu i a 11 ill il il i i il il I il 0 0 1 Centre Method 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 tKe LOQ. Failure to meet these criteria ' requires an investigation of cause and a full reanalysis of the affected samples. ^ i. Samples in which no peaks are detected at the corresponding analyte retention rimes 'will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention times but are less ^ a n the lowest standard will be reported as NQ (not quantifiable). j. background levels of analyte found in control/blank 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. P e r fo r m a n c e 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. ' - i First Criterion - Inject a. standard solution on the LC/MS/M5 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. ' . ' I 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. ' ' Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 15 Page 23 of 39 Centre Method No: O0M-023-01S 4.6. T ime Required for A nalysis . . 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 A n a l y t e F o u n d : . 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 - intercept1) . slope- 5.2 C om ponent Residue Concentration . Determine the component residue concentration using Equation 2 Equation 2: . ,,Resi.d, ue f,,ound ,(ng/1gS) = -(-a-n--a-ly--t-e-,-f-o--u-n--d--(-n;-g--/m---L--) --xD--F-x--F--V--(-m---L )--) sample weight (g) , ; . Where DF --dilution factor and FV = final volume Note: 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) , --------------n--g7/g~a7dTdeZd------.------------- -xlOO . Centre Analytical Laboratories, Inc. Study # 023-015 Page 16 Exygen Research Page 24 Centre Method No: 0OM-023-015' 6. SAFETY 1 ` 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 Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 17 Page 25 Centre Method No: 00M-023-01S I i TABLES * t. Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 18 Page 26 of 39 m ni m m . .......... Centre Method No: 00M-023-015 Table I: Summary of Recoveries for PFOS in Quail Egg Membrane Sample - ' Fort Level1 % Recovery (ppb) 0003798 Matrix Blank A 0 NA ' 0003798 Matrix Blank B 04 NA 0003798 Matrix Blank C 0 NA : 0003798 Spk A 0003798 Sp kB 10 10 88 : 95 ; 0003798 Sp kC 10 92 0003798 Spk D . 50 . 92 . 0003798 Sp kE 50 95 0003798 Spk F - 50 . 0003798 Spk G . 250 0003798 Spk H 250 96 " 93 ' 95 0003798 Spk I 250 93 . AVERAGE: 93 . STA N D A R D D EVIA TIO N : 2. R E L A T IV E STA N D A R D D EVIA TIO N : 3 ' i 1 t mI 1i * M i' i 11 - Table II: Summary of Recoveries for PFOS in Mallard Egg Membrane Sample ' Fort. Level (ppb) 0003802 Matrix Blank A 0 1 0003802 Matrix Blank B 0. 0003802 Matrix Blank C 1 0 0003802 Spk A 10 0003802 Spk B 1 10 0003802 Sp kC 10 0003802 Spk D . 50 - 0003802 Sp kE 50 1 - 0003802 Spk F 50 0003802 Spk G 250 0003802 Spk H 250 . 0003802 Spk I . 25 AVERAGE: . STA N D A R D DEVIATIO N : ._ R E L A T IV E STA N D A R D D EVIA TIO N : % 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 III Page 19 Page 27 of 39 Centre Method No: OOM-023-015 . Table. HI: Summary of Recoveries For PFOS in Quail Egg Yolk Sample 0003795 Matrix Blank A 0003795 Matrix Blank B 0003795 Matrix Blank C 0003795 Spk A 0003795 SpkB 0003795 Spk C 0003795 Spk D 0003795 Spk E 0003795 Spk F 0003795 Spk G 0003795 Spk H 0003795 Spk I Fort Level (ng/f?) 1 0 '0 0 10 ` 10 10 1 50 , . 50 , ' 250 . 250 250 . AVERAGE STA N D A R D D E V IA TIO N R E L A T IV E STA N D A RD D E V IA T IO N % Recovery ` ` ' ' 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 ' 0003799 Matrix Blank A 0003799 Matrix BiankB . 0003799 Matrix Blank C 0003799 Spk 0003799 SpkB 0003799 Spk C 0003799 Spk D 0003799 Spk E 0003799 Spk F 0003799 Spk G 0003799 Spk H 0003799 Spk I Fort Level (ngfc)____________ 0 0. 0 ' ' 10 ' 10 . 10 50 ' 50 50 250 250 250 . AVERAGE STAN DARD D E V IA TIO N R E LA T IV E STA N D A RD D EV IA T IO N % Recovery NA . N ' NA 83 82 ' 10Q '85 88- . 90 8785 88 88 5 '6 . NA = Not Applicable ' Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 20 Page li Centre Method No: 00M-023-015 Table V: Summary of Recoveries for PFOS in Quail Egg Albumen , Sample i 1 1 ' Fort Level (ppb) % Recovery 0003796 Matrix Blank A 0 NA II 0003796 Matrix Blank B 0, NA 0003796 Matrix Blank C 0 NA 0003796 Spk A 10 79 0pO3796 SpkB 10 . 93 II 0003796 Spk C 10 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 Sok I 250 102 i_ AVERAGE: 94 . STA N D A R D D E V IA T IO N : 7 R E L A T IV E STA N D A R D D E V IA T IO N : 7. ll Table VI: Summary of Recoveries for PFOS in Mallard Egg Albumen II Sample Fort Level . % Recovery - (ppb)' ' ; 0003800 Matrix Blank A .. 0' NA 0003800 Matrix Blank B .- 0. . NA il 0003800Matrix Blank C 0 . NA ' 0003800 Spk A . 10 . 93 . 0003800SpkB . 10 85 - 0003800 Spk C . 10 85 . * 0003800 Sp kD . 50 96 0003800 Spk E 50 . 100 ' 0003800 Spk F 0003800 Spk G ' ' 50 . 250 100 ' 98 ' 0003800 Spk H 250 102 ' 0003800 Snk I ' 250 101 ' .- ' .. ' AVERAGE: 96 ! STA N D A R D D E V IA T IO N : ' 7. ' R E LA T IV E STA N D A R D D E V IA T IO N : 7 NA = Not Applicable Centre Analytical Laboratories, Inc. Study ft 023-015 llll Exygen Research ill Page 21 Page 29 of 39 Centre Method No: OOM-023-O15 FIGURES * I Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 22 Page 30 of 39 Centre Method No: OOM-023-015 Figure 1: Representative Chromatogram of a Quail Egg Membrane Control Supli led: 11 StagliType: link CMCMtntlOM 1.000 Icq. Otti: 01/00/00 toi. H: 01:13:35m IH NO Dodlilid: Taf lecchici luto:: 1 lotThnihold: Mf AruThiuhoM: 0.31 l a Dm. Saootha: 1 n llcdw: DptctadIT: Stp. I14th: 10.0 LI e Hi 4 NO 0.20 f So;. Itl|ht: 0.01 1 tu Dp. hit litio: 5.00 Dp. Ad),latto: 4.00 Dp. Til. latto: 3.00 Dai latin li: . IN in lot. Typo: Duoli tiotloo'tin: 4.50 aia Am: 540.321 cuti lal|ht: 140 epa StartII; 4.41 ala Eod.Tloa: 4.50 a U tAH- u u u u U M U U U U U TJ -Tl Hu,* . Figure 2: Representative Chromatogram of a Mallard Egg Membrane Control Saapla'Icdu: 25 Saulalyp: lliak Cueuuatloc: 0.000 Aeq.Data: 1/09/00 Acq.Il: 05:10:31M 4M a IMUUd: tM lochili rutat: i. *w aIhtuhold: l.SS leuthruhold: 1.31 Od. Svolila: 1. NO XtfUdo: CapwtadH: 10.0 aie 4.50 ala. 1 S*p.lldU: tip. hl|ht: 0.20 0.01 m tap.Puhtatto: 5.00 Cip.Id). Ulto: 4.00 bp. Vii. litio: 3.00 IN Oaalatihi: lo IN lat. Typa: bauli buattaTU*: 4.50 ila Ana:' 514.32) eaoau n lilqht: Stirt II: 21) epa 4.44 U CadTU: .1.59 la* Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research -Page 23 Page 31 of 39 Centre Method No: OOM-023-015 . Figure 3: Representative Chromatogram of a Quail Egg Yolk Control Supli bdu: MMliTypc CucutnUm: icq. Oiti: ta|. m>: 19 * IU 4 9.009 01/09/90 10:27:13 M ttd lllid : Tm ludilat rietst! t sin tkiuhold: l.K tn i thniksld: 1.31 Ml bostb: l KTviadoii: ll. l i*e bpKtid R:' 7.11 U 5*p. (Utk;. 0.29 Stp. Itlgkt: 9.91 bp. itik btto: ' 1.00 bp. Kd], btto: 4.99 bp. n t. tatlo: 3.09 Mi U litln R: Ko IK . Typ: Huul M tutio. (Im : 4.51 aia Ans: . * 193.1 cou Iilq lt: 131.S cp Sutt T1m: . 1.13 u bd Tbi: 4.51 als ,.r k UUUUUUUUU TT--.HI U -- u " 7J 74 Figure 4: Representative Chromatogram of a Mallard Egg Yolk Control Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research Page 24 Page 32 of 39 Centre Method No: 00M-023-015 * Figure 5: Representative Chromatogram of a Quail Egg Albumen Control Iodixi S u p tt Type: C n cu tca tlo n i Aeq. D iu : Act). T i n : i lU ak O.OW 01/10/00 01:57: M H D dU ladi Yu OouA ltq raceo r: t lo in th ru te U : 1 . A c u n m l u M : 31.IS Da*. b o o e h a : 1 XT la d w . 10.6 soe 1 bpacead It: I.M l i a c Sop. lidtfc: lip . b iqkc: b p . Fiak b e t . : 0.20 0.01 5.00 i I 1 lap. M ). b e ta : 4.00 b p . V it. b c i o : 3.00 0 b l a e i n IT : Mo h e . Type t a e u c l w TIm : A cu: M ight; S ta rt Tlaa: Cad TIm : . M unul 0.0252 0.0 u 1 .0 2 ]] 0.0370 la . couaU ep . ala. lia . ... m M M0 UO m IM IN N 0 M l TJI tL u u .u u u u UUu Thumb M U u u 7J. 74 Figure 6: Representative Chromatogram of a Mallard Egg Albumen Control Centre Analytical Laboratories, Inc. Study # 023-015 Exygen Research p age 25 Page 33 of 39 Centre Method No: OOM-023-015 Figur 7: Representative Chromatogram of a Quail Egg Membrane Control Fortified at 10 ng/g (ppb) , , S upla M u ti M il lypii C O K U tatio a: Acq. to t : Acq. TIm : 14 UMC O l/M /M O tlS tlU M H oiltM i ta c k t. n e u n Noi4 T h r u h o ld : A m Threshold: Nm u S m o tto : B lU d w b p o e tto IT: l i . V idei: Up. b l tt: txp. N ik ta tle i b p - M j.. ta tti: Exp. V ii. to tio : 0 to lie iv t i : 1Too ` 1 l.SC a 1.31 u. to . * se 4.(9 i U 0.20 ' 0.01 3.00 4.00 to3 .0 0 no m m 4M . l i t . T ffll Ite m e lo TIm : Am: b ig h t: d e m TIm : End TIm : NUMUl (.3 0 2944.403 no 4 .(2 4.12 com es CpI BU 1 m M 14 u u . U U___U_ Th4u4,* U 4 4 u . u u tJ" Figure 8: Representative Chromatogram of a Mallard Egg Membrane Control Fortified at 10.0 ng/g (ppb) . Supl Typ CuaotntlMi Acq. tot: 0.200 01/09/00 Acq. TIm: 00:01:20 M h NO todlfUd: . Tm luddoq Putu: l NsIm Thnskold: 1.00 An Thnstold: 1.31 toa. Saoothi: 1 IT IlodM: 10.0 *c Exptctod KT: top. Vldth: (.30 bU 0.20 top. tolght: 0.01 Csp. hk totlo: 3.00 Exp. Adj. totlo: (.00 bp. n t. utioi 3.00 to tolieln KT: to nt ' 1 1 m 11 m wo 200 lt. Typ:. lu i To tos ttitlu TIm: Ae: * (.30 al m tolqht: Suet TIm : End TIm : 092 ep 1.(2 U (.01 Bl _ ATI 1 '- ( . 44 - * * J M 1. U U 14 U 14 44 4.1 U u ' * * `* U MM 1 f ? ( ; Centre Analytical Laboratories, Inc. Study # 023-015 Page 26 Exygen Research Page 34 of 39 Centre Method No: 00M-023-015 * Figure 9: Representative Chromatogram of a Quail Egg Yolk Control Fortified at 10.0 ng/g (ppb) ' tI.; & ' Figure 10: Representative Chromatogram of a Mallard Egg Yolk Control Fortified at 10.0 ng/g (ppb) ' '. ` .. ; . S u p li In d u : t a p i * Tjp*: C o actatn tlo a: Ic q . OiUi Acq. T i n : ' 29 0C MW 01/10/0 0 2 :n :il lit m tm fe d iti * fenati! u c to ts f e is t Thrtsbold: A cts T h ru h o ld i fe*. S aooths: IT Window: E aptctfe W : S tp. w idth: Up. I t l q t t : I p . N ik litio :. bp. M ) . l a t i . : b p . V ii. litio : Di i U l i t l n C : t a t . Typo: U t i a t l u Tla<: Am: fe iq b t: S t u t tIm : Cad T1m : TM 1 l.iC 1.31 t 10.0 l.n .2 0 0.01 5.00 4.00 3.00 No Murati 4.52 5373.3 1291.1 4.44 4 .(3 DM MM m MC 1 m ] m * 4M NS a ll to u ts eps ain ala tM Centre Analytical Laboratories, Inc. Study # 023-015 Page 27 Exygen Research Page 35 of 39 ' : Centre Method No: 00M-023-0151 - Figure 11: Representative Chromatogram of a Quail Egg Albumen Control Fortified at 10.0 ng/g (ppb) Figure 12: Representative Chromatogram of a Mallard Egg Albumen Control Fortified at 10.0 ng/g (ppb) . Supli iAdu: Maple Type: 30 QC coacaatracloa: * 0.109 taf. Otti: Acq. Tiaat 01/10/0 10:23:43 M Modified: Tea Ivuklat tictoti 1 Moise Tbrtaleld: 0.3$ Area Ifenatoldi 31.15 Mw. Somtha: 1 R Miadow: bptM (t: 10.0 4.52 TAla *Sep. fldtk: 0.20 \ Sep* Halqht: 0.01 ' bp. Hat Itelo: 3.00 ! bp. Ad). bua: 4.00 i bp. Val. belo: 3.00 1(Me Illative R: Me iiae 1100 MOO m c f im m sea tat. Type: beatelo* Tiaa: Area: .Ialite: Start Tiaa: tad T1m: Mimai 4.52 3010.1 1157.2 4.43 4.03 ala Meati cpi. la la M IN '_ Jl . ` A / 1 \V * li LI U __u______u__ Tfciu.H u u u u It M . Centre Analytical Laboratories, Inc. Study # 023-015 Page 28 Exygen Research Page 36 of 39 1 Lipovitellin, Phosvitin, anlikkLVVeery Low Density Lipoprotein (VLDL) Isolation from Chicken and Japanese Quail tgg toik tBflfiiaidi and Cook, DBA 96 [1960] and Stifani e ta l., JBC 265:882-888 [1990]) 11 * (May, 1994) 1) Break open egg, separate yolk, and roll yolk on a moist paper towel to remove adhering albumen. II 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 II M MgS04, 1 mM phenylmethanesulfonylfluoride (PMSF), and 2 pM leupeptin. II 4) Cover cylinder tightly with ParafilmM and mix well by gently inverting several times. 1 5) Using a 20 gauge needle and 12 cc syringe, transfer the suspension to Beckman polyallomer Quick-Seal tubes (tube size will depend on amount of yolk prepared and rotors available). III 6) Seal tubes, place into appropriate rotor, and centrifuge at 200,000 x g at 4 #C for 24 hours. il! 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. 1 - c-- 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 gently washed with, and then I resuspended in, a solution containing 0.45 M MgS04, 1 mM PMSF, IN III Exygen Research ill Page Ml! I II . 1 I il * . III m M II 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 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 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 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 nnM PMSF, and 2 pM leupeptin. Add one volume of cold "e-pure" water dropwise (thus solution is diluted to 0 .2 M MgS04) and let stand overnight (without stirring) at 4 C. On the following morning, the precipitate (phosvitin) 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 MgSC>4; see step 11) should be decanted, diluted with two volumes of cold "e-pure" water (final concentration of 0.05 M MgS04>dropwise with stirring at 4 C, and allowed to stand overnight (without stirring) at 4 C. The resulting white precipitate (lipovitellln) 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. nil mi Exygen Research Page 38 of 39 lit L iP o v \T E U _ | j, P rtO iv J in ^ ^ <V V U b L liou*tT7o/0 _________ F^-qi-v. e~& o '3 o i- c (. M .A a/ i n V ) E C rt Y o i_ * c, tM uo-ne-* 1*2. tj/o.fo7<M M ^Je)Y c e iv /r . (p . z o o x { r ^ z ih c z .^ ^ 1(11 I -- --------------------------- -- C ^YVc^Vo<V/ l \ -- P iU O T E ^ iiO T+ -U /tJ-(JZ_j ce<^r-- K i- I t t , H b p ^ f? a d v o \tj J h -f-c T )- v^/WK u /o .H S iH 4-S^ c ^ :M ~ CE 2 -o o *n > !U i; <e_t ' c _ _______ I___________ ~ < 1 er i o "pi-o/AJ B ltc?-(o. J-hnt^ i*c _ (J> UCA4J)) i --------- ^ o^ O lP A T n ^ r (OUCA^-P^ 1 \| K >FC pffO -- dEOU-ioLu^r e . i S M MjJ, ; -u-. PluTeti ut. + o o . IS'Kl HjJot -n ii.m .-'tu -.L eT JTuH-A tf.iJ. < Y*C_ C : ~ ~ 3 u ^ e* A i* rr> f7 o r^ - O l ' - uT T (1' ^ ) T O , o K H j i 0 *! ^ ^ V B ^ a K h ^ a sp/t^lu "TVt^ii UiiT^jTW^O o <.( *{_ 1 YeA-v-ov^> p m -- . J>iJjoLvje im o , f m Mj-Sy_ 6(lC , | : | j / * \ * -po 0 . 2.M. " H J r Y , ->f>- k , #-<-> U rr~ fnH O o y . e. t'C , J su e m k ifW C D V rffo J I p p r = L iftw /rngo O**-jh<2l0^ir*TW^. </W. ( U .. H " C -j A.'-Uo\v^.t-v-N j-Va^C Q. '( i* fitAJAT>t*T" 0<iCA<lO} AU. tv ffn y 0 0 * 7 ^ I ^ K P W F 2-Mm . LP 5 *77s= PYiivrT7A C c^ tc? fOi, 060 tf) ( A v - l u ^ '^ OfiCL^ |Ai N*Cll/K y * f ilt y Exygen Research ifl Page 39 of 39