Document J32RwgxJdRv3Qr3OMor8560OO

ftp. a & e - t i t # Protein Binding of Perfluorohexane Sulfonate, Perfluorooctane Sulfonate and Perfluorooctanoate to Plasma (Human, R at, and Monkey), and Various Human-Derived Plasma Protein Fractions STUDY ID: 9921.7 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 000090 Study 9921.7 Page 2 of7 SUMMARY The percent protein binding of four perfluoronated compounds in seven different human-derived plasma protein fractions was determined. Additionally, protein binding for the same four compounds was measured in rat, monkey and human plasma at five different concentrations. Each test article was incubated for 15 minutes at 37 C in each plasma protein fraction or plasma sample. The protein bound test article was separated from the non-bound test article by ultrafiltration using Centrifree filtration devices with a nominal molecular weight (NMW) cutoff o f 30,000 daltons. The eluent from the ultrafiltration was analyzed for test article concentration relative to a solvent standard. 000091. Corenna Kerstner-Wood, BS Research Assistant in Bioanalytical Chemistry Group Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Greg Gorman, Ph.D. Manager Bioanalytical Chemistry Group KEY PERSONNEL Study 9921.7 Page 3 of7 000092 Study 9921.7 Page 4 o f7 1. OBJECTIVE The objective of this study was to determine the percent of protein binding for each test article in human, rat, and monkey plasma as well as various human-derived plasma protein fractions. 2. SAFETY All necessary procedures to ensure the safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by the sponsor for the test articles used in this study. Procedures outlined in SOP 2-29-3 for handling human and primate plasma as well as the human-derived plasma protein fractions were also followed. 3. COMPLIANCE This work was performed using various previously validated analytical methods ( 3548, 3606, 3607). While this work was not audited in compliance with GLP regulations, it was performed in the spirit of the regulations using calibrated and validated instrumentation. 4. EXPERIMENTAL 4.1 Analytical Procedures Individual aqueous solutions of each human derived plasma protein fraction were prepared at two concentrations as indicated in the table below: Plasma Protein Fraction Albumin Gamma-Globulin Alpha-Globulin Fibrinogen Alpha-2-Macroglobulin Transferrin Beta-Lipoproteins Physiological Plasma Protein Concentraiionfll 3500-5500 mg/100 mL 965-2515 mg/100 mL 507-1037 mg/100 mL 200-600 mg/100 mL 150-420 mg/100 mL 200-320 mg/100 mL 280-440 mg/100 mL Prepared Plasma Protein Concentration f--10 % Physiological! 443 mg/100 mL 196 mg/100 mL 136 mg/100 mL 38 mg/100 mL 67 mg/100 mL 42 mg/100 mL 20 mg/100 mL Prepared Plasma Protein Concentration (100% Physiological! 4600 mg/100 mL 1768 mg/100 mL 1212 mg/100 mL 408 mg/100 mL 200 mg/100 mL 297 mg/100 mL 200 mg/100 mL Stock solutions of each test article were separately spiked into the plasma protein fractions or a control plasma matrix to produce a final concentration of 10 pg/mL. The resulting mixture was vortexed for 1 minute and incubated at 37 C for 15 minutes. Upon completion of the incubation, each sample was again vortexed for 1 minute and placed into a Centrifree ultrafiltration device (NMW cutoff = 30,000 daltons) and centrifuged at 2000 x g for 30 minutes. The ultrafiltrate was analyzed to determine the amount of unbound test article. 000093 Study 9921.7 Page 5 of7 4.2 Results The results o f all of the analyses are presented as percent bound test article in Table I-IH at the end of the report. 5.0 Conclusion The protein binding of four perfluoronated compounds was evaluated in seven separate humanderived plasma protein fractions at two different protein fraction concentrations (Table I and II). Additionally, protein binding for the same four compounds was evaluated in rat, human, and monkey plasma at 5 different concentrations (Table IQ). The data in Table I and II shows that albumin is by far the largest single protein binder for three o f the four compounds tested at both concentrations. The fourth compound, PFOS, was found to be highly bound by both albumin and beta-lipoproteins. The protein binding experiments for the rat, human, and monkey plasma show very high levels o f binding at all concentrations tested and no evidence of saturation. 000094 Study 9921.7 Page 6 o f 7 Table I Percent Binding to Human Plasma Protein Fractions (-10% Physiological Cone.) PFHS PFOS PFOC Albumin Gamma- Alpha- Fibrinogen Alpha-2- Transferrin Beta- Globulin Globulin Macroglobulin Lipoproteins >99.5 15.0 33.6 99.0 6.3 49.9 96.4 3.5 28.5 15.6 <0.1 5.4 9.0 12.5 7.9 8.0 25.8 7.2 90.1 1.0 19.6 Table D Percent Binding to Human Plasma Protein Fractions (100% Physiological Cone) PFHS PFOS PFOC Albumin Gamma- Alpha- Fibrinogen Alpha-2- Transferrin Beta- Globulin Globulin Macroglobulin Lipoproteins >99.9 26.1 13.7 <0.1 <0.1 6.4 64.1 99.8 24.1 59.4 <0.1 <0.1 <0.1 95.6 99.7 3.0 11.0 <0.1 <0.1 2.1 39.6 Table IE % Protein Binding to Rat, Human and Monkey Plasma N om inal Concentration (ppm ) 1 10 100 250 500 Perfluorohexanesulfonate Perfluorooctanesulfonate Perfluorooctanecarboxalate Rat Monkey Human Rat Monkey Human Rat Monkey Human ioo- -100 -100 -100 -100 99.4 -100 -100 -100 -1 0 0 99.8 99.9 99.9 99.5 99.9 99.9 100 99.7 99.9 99.9 98.6 99.1 99.8 99.9 99.5 99.9 99.9 97.6 98.2 99.5 99.4 99.0 99.9 99.9 97.3 -100 99.8 99.8 99.8 99.5 -100 99.9 99.9 99.6 99.4 % Binding Values reported as "~100" reflect a nonquantifiable amount of test article in the plasma water BQL < 6.25 ng/mL 000095 Study 9921.7 Page 7 o f7 6.0 References (1). Koplar, L., Pesce, A., "Clinical Chemistry (Theory, Analysis and Correlation) 1984, pg. 1416, C.V. Mosby Publishing Co. 7.0 Review and Approvals Corenna Kerstner-Wood, BS Research Assistant III Bioanalytical Chemistry Group Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Bioanalytical Chemistry Group 0G-03'o2> Date ^ 63 -63 Date ->. 1 - 0 3 Date 000096 Page 1 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 PRINCIPLE Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanecarboxalate (PFOC). The serum or urine (e.g., 0.5 mL) containing (PFOC) is fortified with an internal standard (IS), perfluorohexanesulfonate (PFHS). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5mM ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS). The range of reliable results extends from about 20 to 100,000 ng/mL of PFOC in serum and from 10 to 500 ng/mL in urine. Samples containing PFOC at concentrations greater than 100,000 ng/mL may be diluted with control blank matrix so that the concentration of PFOC will be within the range of reliable results prior to analysis. The mass spectrometry of PFOC and PFHS is accomplished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the formation of other potentially interfering ions extracted from the matrix. In order to maximize sensitivity, this method is based on mixed reaction monitoring of the negative fragment ion (M-COOH)' for PFOC. CAUTION: Since primates may carry a number of zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved primate tissue. 2.0 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 2.1 N eat Reagents 000097 ANALYTICAL METHOD Page 2 of 13 Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Seram and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.1 Water, deionized and organic free (from in-house purification system; e.g., Tngalls 210N) 2.1.2 Methanol, HPLC grade 2.1.3 2.1.4 Perfluorooctanecarboxalate (analyte), as provided by the client Perfluorohexanesulfonate (internalstandard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.1.6 Blank control monkey serum 2.1.7 Sodium Carbonate, Certified ACS Grade or equivalent 2.1.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 2.1.9 Ethyl Acetate, HPLC grade 2.1.10 Tetrabutylammonium Hydrogen Sulfate 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid 2.2 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water 000098 Page 3 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 2.2.2.1 For example, to prepare 25 mL, dissolve 4.24 g of tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilute solution of NaOH in water may be used to effect smaller pH adjustments. 2.2.3 Carbonate/Bicarbonate Buffer Solution 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g of sodium carbonate and approximately 2.10 g of sodium bicarbonate in 100 mL of deionized water. Mix well to ensure complete dissolution. 3.0 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. 3.1 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source of nitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus(e.g., Nylon-66, 0.20 nm) 000099 Page 4 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipets Micropipettor(s) with tips Culture tubes with teflon-lined caps Centrifuge Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1 mL Plastic syringes with 0.2 /an PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. M ain Stock Solution of PFOC ~ 1000 pg/mL Prepare an ~ 1000 pg/mL solution of PFOC in deionized organic-free water (e.g., accurately weigh about 10 mg PFOC into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound o o o io o Page 5 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 into an appropriate vessel (e.g., culture tube) and add 10 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFHS), --200 pg/mL Prepare an --200 pg/mL solution of PFHS in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organicfree water to dissolve and dilute to the mark with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PFHS), - 50pg/mL Prepare an --50 pg/mL solution of PFHS in deionized organic- free water by pipetting 2 mL of the 200 pg/mL solution into a culture tube and add 6 mL of deionized water. Mix well. Working Stock Solutions of PFOC To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volumetric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. Working Stock Level (WSL) Approximate Concentration (ng/mL) 500,000 250,000 Volume of PFOC solution 5 mL of 1000 pg/mL 5 mL of 500,000 ng/mL Final Volume in deionized organic free water (mL) 10 10 o o o io i Page 6 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 62,500 31,250 15,625 5,000 2,500 1,000 500 250 2.5 mL of 250,000 ng/mL 5 mL of 62,500 ng/mL 5 mL of 31,250 ng/ mL 50 /xL of 1000 /xg/mL stock 5 mL of 5000 ng/mL stock 4 mL o f2500 ng/mL stock 5 mL of 1000 ng/mL stock 5 mL of 500 ng/mL stock 10 10 10 10 10 10 10 10 4.4.2 Summary of concentrations of serum standards: Standard Level A Volume and Spike Cone. 50 fiLof 1000 /xg/mL Approximate Concentration of PFOC in serum (ng/mL) 100,000 B 37.5 /xL of 1000 /xg/mL C 25 nL of 1000 /xg/mL D 12.5 fiLof 1000 /xg/mL E 10 fiLof 1000 /xg/mL 75,000 50,000 25,000 20,000 000102 Page 7 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) F 10 pL of 500,000 ng/mL G 10 pL of 250,000 ng/mL H 16 pL of 62,500 ng/mL I 16 pL of 31,250 ng/mL J 16 pL of 15,625 ng/mL K 20 pL of 5,000 ng/mL L 10 pL of 5,000 ng/mL M 10 pL of 2,500 ng/mL N 10 pL of 1,000 ng/mL 0 10 pL of 500 ng/mL P 10 pL of 250 ng/mL 10,000 5,000 2,000 1,000 500 200 100 50 20 10 5 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. 5.1 Multiple (e.g., about three) sets of matrix standards and a matrix blank (blank + IS) are analyzed with each set of unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual ~ 20-mL culture tubes, pipet blank matrix (e.g., 0.5 mL ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL of organic-free water instead of the working stock solution. Add the 10 pL 000103 ANALYTICAL METHOD Page 8 of 13 Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) of internal standard stock (~ 5 0 pg/mL) to each tube except die blank-IS (pipet 10 pL of organic-free water instead) and vortex for ~ 5 seconds. 5.3 To each tube add 500 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. 5.4 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream of nitrogen and moderate heat (e.g., 50 C). 5.7 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual ~20mL culture tubes. If necessary, dilute an aliquot of any sample with blank matrix so that the expected concentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 10 pL of internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple of seconds. 6.2 To each tube add 500 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. 000104 Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 6.3 6.4 6.5 6.6 7.0 7.1 7.1.1 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., --50 minutes in the Turbo-Vap ) with a gentle stream of nitrogen and moderate heat (e.g., 50 C). Reconstitute the residue in 500 iLof 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 Hm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) Conditions are to be optimized if necessary. HPLC Conditions Analytical Column: Keystone Scientific Aquasil C18, 150 mm x 2 mm ED, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Keystone Aquasil C18 10 mm x 2 mm 400 /zL/min. 3 fxh A: 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: 0 - 0.5 min. 50%A : 50%B 0.5 - 7.5 min. 10% A : 90% B linear gradient 7.5-8 min. 10%A : 90% B step gradient 000105 Page 10 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Tide: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 7.1.2 Temperature: 8-11 min. Ambient 50%A : 50%B PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turboion Spray-Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter IS NC TEM OR RNG 00 IQ 1 ST ROl IQ 2 R02 ST3 R03 DF CBM Value -2000 0 400 -20 -120 5 6 10 6 10 30 40 32 250 1800 000106 Page 11 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NEB CUR CAD QPE POL VCM IP E Yalae 15 6 5 0 1 0 0 MS/MS Acquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFOC: Q1 M a fumili 412.9 Q3_M ass Camu) 368.9 Dwell Tim e ftnal 200 PFHS (IS) Q1 M ass Camu') 398.9 Mass famiii 3 9 8 .9 P aram eter R02 ST3 R03 Sari 50 60 52 Stop 50 60 52 D w ell T im e ftm l 200 000107 Page 12 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analyte PFOC PFHS IonProfile 412.9 to 368.9 398.9 to 398.9 8.2 Plot the peak area response of PFOC divided by the peak area response of the IS (PFHS) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used instead ofpeak areas. Obtain the best curve fit of the data (e.g., quadratic fit weighted with 1/concentration of die test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y = ax2 + bx + c where y= x= a, b, c Peak height response of PFOC divided by peak height response of the IS (PFHS) in standards, Concentration of the PFOC in standards, = Constants derived from the regression analysis. 8.3 Using the standard curve, calculate the level of PFHC in each unknown sample. Correct the results of samples for any dilutions. 000108 Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) NOTE: Due to unresolvable interferences with the test article and/or internal standard from the matrix, external standard quantitation may be used at the discretion of the supervising mass spectrometrist. 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120% of theoretical. 10.0 REPORTING 10.1 Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Authors: Gregory's. Gorman, Ph.D., Staff Chemist Bioanalytical Chemistry Group Approved by: es D. Joh: ^analytical S, MBA, Manager mistry Group / J S- ) 1- 0 -0 Date U - 2 7-0* Date 000109 Page 1 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 2.0 2.1 2.1.1 2.1.2 P R IN C IP L E Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorohexanesulfonate (PFHS). The serum or urine (e.g., 0.5 mL) containing (PFHS) is fortified with an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5mM ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS). The range of reliable results extends from about 5 to 20,000 ng/mL of PFHS in serum and from 10 to 500 ng/mL in urine. Samples containing PFHS at concentrations greater than 20,000 ng/mL may be diluted with control blank matrix so that the concentration o f PFHS will be within the range of reliable results prior to analysis. The mass spectrometry of PFHS and PFOC is accomplished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce file formation of other potentially interfering ions extracted from the matrix. CAUTION: Since primates may carry a number of zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description o f safety procedures to be used when handling unpreserved primate tissue. REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. Neat Reagents Water, deionized and organic free (from in-house purification system; e.g., Ingalls 21ON) Methanol, HPLC grade O O O llO Page 2 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesnlfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.3 Perfluorohexanesulfonate (analyte), as provided by the client 2.1.4 Perfluorooctanecarboxalate (internal standard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.1.6 Blank control monkey serum 2.1.7 Sodium Carbonate, Certified ACS Grade or equivalent 2.1.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 2.1.9 Ethyl Acetate, HPLC grade 2.1.10 Tetrabutylammonium Hydrogen Sulfate, Aldrich 97% 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid, 88% 2.2 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) t O O O lll Page 3 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.2.2.1 For example, to prepare 25 mL, dissolve approximately 4.24 g o f tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilute solution of NaOH in w ater may be used to effect sm aller pH adjustm ents. 2.2.3 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g o f sodium carbonate and approximately 2.10 g o f sodium bicarbonate in 100 mL of deionized water. Mix well to ensure complete dissolution. 2.2.4 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 2.2.4.1 For example, to prepare 100 mL, dissolve approximately 10.6 g of sodium carbonate and approximately 8.4 g of sodium bicarbonate in 100 mL o f deionized water. Mix well to ensure complete dissolution. 3.0 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. 3.1 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source of nitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66,0.20 pm) 000112 Page 4 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipettes Micropipettor(s) with tips Culture tubes with teflon-lined caps Centrifuge Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1 mL Plastic syringes with 0.2 pm PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion o f the analyst. Actual dilutions will be documented on the preparation sheets. M ain Stock Solution of PFHS ~1000 pg/mL Prepare an ~1000 pg/mL solution of PFHS in deionized organic-free water (e.g., accurately weigh about 10 mg PFHS into a 10-xnL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound 000113 Page 5 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 into an appropriate vessel (e.g., culture tube) and add 10 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFOC), ~200 pg/mL Prepare an ~200pg/mL solution ofPFOC in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic-free water to dissolve and dilute to the mark with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PFOC), ~ 50pg/mL Prepare an ~ 50 pg/mL solution o f PFOC in deionized organic- free water by pipetting 2 mL of the 200 pg/mL solution into a culture tube and add 6 mL of deionized water. Mix well. Working Stock Solutions of PFHS To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volumetric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. 000114 Page 6 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Working Stock Level (WSL) Approximate Concentration (ng/mL) Voluipe o f PFHS solution 500,000 5 mL of 1000 pg/mL 250,000 5 mL of 500,000 ng/mL 62,500 2.5 mL o f250,000 ng/mL 31,250 5 mL of 62,500 ng/mL 15,625 5 mL of 31,250 ng/mL 5,000 50 pL of 1000 pg/mL stock 2,500 5 mL of 5000 ng/mL stock 1,000 4 mL o f2500 ng/mL stock 500 5 mL of 1000 ng/mL stock 250 5 mL of 500 ng/mL stock Final Volume in deionized organic free water (mL) 10 10 10 10 10 10 10 10 10 10 s0 0 0 1 1 Page 7 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.4.2 Summary o f concentrations of serum standards: Volume and Spike Cone. Standard Level A 20 pL of 500,000 ng/mL B 10 pL o f 500,000 ng/mL C 10 pL of 250,000 ng/mL D 16 pL of 62,500 ng/mL Approximate Concentration of PFHS in serum (ng/mL) 20,000 10,000 5,000 2,000 E 16 pL o f 31,250 ng/mL F 16 pL of 15,625 ng/mL G 20 pL o f 5,000 ng/mL H 10 pL o f 5,000 ng/mL I 10 pL of 2,500 ng/mL J 10 pL of 1,000 ng/mL K 10 pL of 500 ng/mL L 10 pL o f250 ng/mL 1,000 500 200 100 50 20 10 5 000116 Page 8 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion o f the analyst. 5.1 Multiple (e.g., about three) sets of matrix standards and a matrix blank (blank + IS) are analyzed with each set ofunknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual ~20-mL culture tubes, pipet blank matrix (e.g., 0.5 mL ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL o f organic-free water instead of the working stock solution. Add the 10 pL of internal standard stock (~50 pg/mL) to each tube except the blank-IS (pipet 10 pL of organic-free water instead) and vortex for ~5 seconds. 5.3 For serum samples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL o f deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 5.4 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 *C). 000117 Page 9 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.7 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. If necessary, dilute an aliquot o f any sample with blank matrix so that the expected concentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 10 pL of internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple o f seconds. 6.2 For serum samples add the following to each tube: 500 pL of the TBA ion-pairing solution, 1 mL of 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL o f TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 6.4 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 6.5 Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream of nitrogen and moderate heat (e.g., 50 *C). 6.6 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 000118 Page 10 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Periluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 7.0 7.1 7.1.1 7.1.2 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) Conditions are to be optimized if necessary. HPLC Conditions Analytical Column: Keystone Scientific Aquasil C l8,150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Keystone Aquasil C18 10 mm x 2 mm 600 pL/min. 5 pL A: 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: Temperature: 0 - 3 min. 3 - 5min. 5 -8 min. 8-10 min. Ambient 50%A : 50%B 20% A : 80% B linear gradient 10%A : 90% B step gradient 50%A : 50%B step gradient PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turboion Snrav Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter IS Value -2000 000119 Page 11 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NC TEM OR RNG QO IQ1 ST R01 IQ2 R02 ST3 R03 DF OEM NEB CUR CAD QPE POL VCM Value 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 15 6 5 0 1 0 000120 Page 12 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter IPE Value 0 MS/MS Acquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFHS: 0 1 Mass Carnal 398.9 0 3 M ass Carnal 398.9 Dwell Time fins') 200 PFOC (IS) 0 1 Mass Carnal 412.9 P aram eter R02 ST3 R03 QO IQ1 ST ROl IQ2 0 3 Mass Carnal 368.9 S ta rt 35 45 37 18 19 23 19 20 Stop 35 45 37 18 19 23 19 20 Dwell TimeCmsl 200 000121 Page 13 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Detennination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analyte PFHS PFOC Ion Profile 398.9 to 398.9 ' 412.9 to 368.9 8.2 Plot the peak area response o fPFHS divided by the peak area response of the IS (PFOC) from all standards versus the concentration o f the test article in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit o f the data (e.g., quadratic fit weighted with 1/concentration of the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y = ax2+ bx + c where y= x= a, b, c Peak height response of PFHS divided by peak height response of the IS (PFOC) in standards, Concentration o f the PFHS in standards, = Constants derived from the regression analysis. 8.3 Using the standard curve, calculate the level of PFHS in each unknown sample. Correct the results of samples for any dilutions. NOTE: Due to unresolvable interferences with die test article and/or internal standard from the matrix, external standard quantitation may be used at the discretion o f the supervising mass spectrometrist. 000122 Page 14 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120% o f theoretical. 10.0 REPORTING 10.1 Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. A uthors): Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Approved by: Greg ddrman, Ph.D. Manager Bioanalytical Chemistry Group Date 9 /7 /0 3 Date 000123 Page 1 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 2.0 2.1 2.1.1 2.1.2 P R IN C IP L E Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanesulfonate (PFOS). The serum or urine (e.g., 0.5 mL) containing (PFOS) is fortified with an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5mM am m onium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS). The range of reliable results extends from about 20 to 10,000 ng/mL of PFOS in serum and from 10 to 500 ng/mL in urine. Samples containing PFOS at concentrations greater than 10,000 ng/mL may be diluted with control blank matrix so that the concentration o f PFOS will be within the range of reliable results prior to analysis. The mass spectrometry of PFOS and PFOC is accomplished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the formation o f other potentially interfering ions extracted from the matrix. CAUTION: Since primates may carry a number o f zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description o f safety procedures to be used when handling unpreserved primate tissue. REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. Neat Reagents Water, deionized and organic free (from in-house purification system; e.g., Ingalls 210N) Methanol, HPLC grade 000124 Page 2 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.3 Perfluorooctanesulfonate (analyte), as provided by the client 2.1.4 Perfluorooctanecarboxalate (internal standard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.1.6 Blank control monkey serum 2.1.7 Sodium Carbonate, Certified ACS Grade or equivalent 2.1.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 2.1.9 Ethyl Acetate, HPLC grade 2.1.10 Tetrabutylammonium Hydrogen Sulfate, 97% 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid, 88% 2.2 Prepared Solutions Appropriate changes in the solutions may be madeat the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 000125 Page 3 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.2.2.1 For example, to prepare 25 mL, dissolve 4.24 g of tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilate solution of NaOH in water may be used to effect smaller pH adjustments. 2.2.3 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g of sodium carbonate and approximately 2.10 g o f sodium bicarbonate in 100 mL of deionized water. Mix well to ensure complete dissolution. 2.2.4 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 2.2.4.1 For example, to prepare 100 mL, dissolve approximately 10.6 g of sodium carbonate and approximately 8.4 g o f sodium bicarbonate in 100 mL of deionized water. Mix well to ensure complete dissolution. 3.0 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. 3.1 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and DCA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source of nitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66,0.20 pm) 000126 Page 4 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipettes Micropipettor(s) with tips Culture tubes with teflon-lined caps Centrifuge / Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1 mL Plastic syringes with 0.2 pm PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations o f the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. Main Stock Solution of PFOS ~1000 pg/mL Prepare an ~1000 pg/mL solution of PFOS in deionized organic-free water (e.g., accurately weigh about 10 mg PFOS into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound 000127 Page 5 o f 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 into an appropriate vessel (e.g., culture tube) and add 10 mL o f deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFOC), ~200 pg/mL Prepare an ~200pg/mL solution ofPFOC in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic-free water to dissolve and dilute to the marie with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PFOC), ~ 50pg/mL Prepare an ~ 50 pg/mL solution o f PFOC in deionized organic- free water by pipetting 2 mL o f the 200 pg/mL solution into a culture tube and add 6 mL o f deionized water. Mix well. W orking Stock Solutions of PFOS To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volumetric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. Working Stock Level (WSL) Approximate Concentration (ng/mL) 500,000 250,000 Volume o f PFOS solution 5 mL o f 1000 pg/mL 5 mL of 500,000 ng/mL Final Volume in deionized organic free water (mL) 10 10 00012S Page 6 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 62,500 2.5 mL o f250,000 ng/mL 31,250 5 mL o f62,500 ng/mL 15,625 5 mL o f 31,250 ng/m L 5,000 50 pL of 1000 pg/mL stock 2,500 5 mL o f5000 ng/mL stock 1,000 4 mL o f2500 ng/mL stock 500 5 mL o f 1000 ng/mL stock 250 5 mL of 500 ng/mL stock 10 10 10 10 10 10 10 10 4.4.2 Summary o f concentrations o f serum standards: Standard Level Volume and Spike Cone. Approximate Concentration of PFOS in serum (ng/mL) A 10 pL of 500,000 ng/mL 10,000 B 10 pL of 250,000 ng/mL 5,000 C 16 pL of 62,500 ng/mL 2,000 D 16 pL o f 31,250 ng/mL 1,000 000129 Page 7 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) E 16 pL of 15,625 ng/mL F 20 pL of 5,000 ng/mL G 10 pL of 5,000 ng/mL H 10 pL o f2,500 ng/mL I 10 pL of 1,000 ng/mL J 10 pL of 500 ng/mL K 10 pL of 250 ng/mL 500 200 100 50 20 10 5 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions maybe made at the discretion o f the analyst. 5.1 Multiple (e.g., about three) sets o f matrix standards and a matrix blank (blank + IS) are analyzed with each set of unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual ~20-mL culture tubes, pipet blank matrix (e.g., 0.5 mL ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL o f organic-free water instead o f the working stock solution. Add the 10 pL o f internal standard stock (~50 pg/mL) to each tube except the blank-IS (pipet 10 pL of organic-free water instead) and vortex for ~5 seconds. 5.3 For serum samples add the following to each tube: 500 pL of the TBA in n -p airin g solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL o f deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 000130 Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis, by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.4 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). 5.7 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. If necessary, dilute an aliquot of any sample with blank matrix so that the expected concentration o fthe test article being analyzed will fall within the concentration range o f the standard curve. Add 10 pL of internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple of seconds. 6.2 For serum samples add the following to each tube: 500 pL of the TBA ion-pairing solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL o f TBA ion-pairing solution, 1 mL of 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 000131 Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 6.4 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 6.5 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). 6.6 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 . pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 7.0 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) 7.1 Conditions are to be optimized if necessary. 7.1.1 HPLC Conditions Analytical Column: Keystone Scientific Aquasil C18,150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Keystone Aquasil C18 10 mm x 2 mm 400 pL/min. 5 pL A: 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: Temperature: 0 - 3 min. 3 - 5min. 5 - 8 min. 8-10 min. Ambient 50%A : 50%B 20% A : 80% B linear gradient 10%A : 90% B step gradient 50%A : 50%B step gradient 7.1.2 PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan 000132 Page 10 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Turboion Snrav Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter IS NC TEM OR RNG Q0 IQ1 ST ROl IQ2 R02 ST3 R03 DF CEM Value -2000 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 000133 Page 11 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesnlfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NEB CUR CAD QPE POL VCM IPE Value 15 6 5 0 1 0 0 M S/M S A cquisition Conditions Scan type: MRM Polarity: N eg ativ e A cquisition mode: Profile Pause time: 5 m illiseconds Masses requested: PFOS: O l Mass famul 498.9 0 3 Mass famul 498.9 PFOC (IS) O l Mass farrari 412.9 0 3 Mass famu) 368.9 P aram eter R02 ST3 R03 QO IQ l ST Start 35 45 37 18 19 23 Stop 35 45 37 18 19 23 Dwell Time fmsl 200 Dwell Time (msl 200 000134 Page 12 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) R O l 19 19 IQ2 20 20 8.0 CALCULATIONS 8.1 At the end o f the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analyte PFOS PFOC Ion Profile 498.9 to 498.9 412.9 to 368.9 8.2 Plot the peak area response of PFOS divided by the peak area response of the IS (PFOC) from all standards versus the concentration o f the test article in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit o f the data (e.g., quadratic fit weighted with 1/concentration o f the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y = ax2+ bx + c where y= x= a, b, c Peak height response of PFOS divided by peak height response of the IS (PFOC) in standards, Concentration of the PFOS in standards, = Constants derived from the regression analysis. 00013S Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.3 Using the standard curve, calculate the level o f PFHC in each unknown sample. Correct the results o f samples for any dilutions. NOTE: Due to unresolvable interferences with the test article and/or internal standard from the matrix, external standard quantitation maybe used at the discretion o f the supervising mass spectrometrist. 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120% o f theoretical. 10.0 REPORTING 10.1 Results o f all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. A u th o rs): L_____ Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Approved by: reg worman, Ph.D. Manager Bioanalytical Chemistry Group Date 9/7M Date 000136