Document MM6nzByV48GNanbxmE878jnxL

Protein Binding of PerfJuorohexaneSulfonate, PerfluorooctaneSulfonate and Perfluorooctanoateto Plasma (Human,Rat, 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 I Study 992 1.7 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 fiaction or plasma sample. The protein bound test article was separated fiom the non-bound test article by ultrafiltration using Centrifra? filtrationdevices with a nominal molecular weight 0cutoff of 30,000daltons. The eluent from the ultratiltration was analyzed for test article concentration relative to a solvent standard. Corenna Kerstner-Wood, BS Research Assistant III Bioanalytical Chemistry Group Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Greg Gorman, Ph.D. Manager Bioanalytical Chemistry Group KEY PERSONNEL Study 992 1.7 Page 3 of7 Study 9921.7 Page 4 of 7 1. OBJECTLVE 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 fiactions. 2. SAFETY All necessary procedures to ensure the safety of the analystswere based on informationcontained in the Material Safety and Data Sheets (MSDS), provided by the sponsor for the test articles used in this study. Proceduresoutlined in SOP 2-29-3for 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). W e 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 Transfem'n Beta-Lipoproteins Physiological Plasma Protein Concentration(Q 3500-5500 mg/lOO mL 965-2515 mg/100 mL 507-1037 mg/lOO mL 200-600 mg/100 150-420 mg/lOO mL 200-320 mg/lOO mL 280-440 mg/100 mL Prepared Plasma Protein Concentration I-10 % Phvsiolopical) 443 mg/100 mL 196 mg/lOO rnL 136 mg/100 mL. 38 mg/lOO rnL 67 mg/100mL. 42 mg/100mL 20 mg/l 00 mL hepared Plasma Protein Concentration (100% .Phvsiolopical) 4600 mg/lOO mL 1768 mg/lOO mL 1212 mg/lOO mL 408 mg/100mL 200 mg/100 mL 297 mg/100 mL 200 mg/lOO 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 Centrifiee 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. Study 992 1.7 Page 5 of 7 4.2 Results The results of all of the analyses are presented as percent bound test article in Table I-III at the end of the report. 5.0 Conclusion The protein binding of four perfluoronated compounds was evaluated in seven separate humanderived plasma protein fiactionsat two differentprotein fraction concentrations (TableI and n). Additionally,protein binding for the same four compoundswas evaluated in rat, human,and monkey plasma at 5 different concentrations (Table IIt). The datain Table I and 11showsthat albumin is by far the largest single protein binder for three of 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 of binding at all concentrations tested and no evidence of saturation. Study 9921.7 Page 6 of 7 Table I Percent Binding to Human Plasma Protein Fractions (-1 0% Physiological Conc.) Table II Percent Bindingto Human Plasma Protein Fractions (100%Physiological Conc) PFHS PFOS , PFOC Albumin GammaGlobulin I >99.9 26.1 99.8 24.1 99.7 3.0 AlphaGlobulin 13.7 59.4 11.0 Fibrinogen Alpha-2- Transfemn Macroglobulin CO. 1 <0.1 6.4 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 2.1 BetaLipoproteins 64.1 95.6 39.6 I Nominal I Concentrationl I Table III % Protein Binding to Rat, Human and Monkey Plasma Perfluorohex8ncsulfonate I Perfluorooctam~onate~erfluorooctanecarboxalate I % Binding Values reportedas "-1 00" reflect a nonquantifiable amount of test article in the plasma water BQL < 6.25 n g / d Study 9921.7 Page 7 of 7 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 `corenua Kerstner-Wood, BS Research Assistant I11 Bioanalytical Chemistry Group, W Lon Coward,BS Sr. Research Associate Bioanalytical Chemistry Group 6 a 63 -C3 Date &@orman, Ph.D. Date Manager Bioanalytical Chemistry Group I . Page 1 of 13 ANALYTICAL METHOD Method NO.: BACG-3548 Title: Determination of Perfluorooctanecarboxalatein Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass SpectrometryMass spectrometry (WPLCfMStMS) 1.0 PRINCIPLE %nun or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanecarboxalate(PFOC).The serum or urine (e.g., 0.5 mL) coxmining (PFOC)is fortified with an internal standard (IS), perfluorohexanemlfonate(PFBS). The samples are then mixedwith 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 96 formic acid, 5 % 5mM ammonium acetate, filtered, and transfmd to autosampler vials, and analyzed by HPLC Mass SpectrometryMass Spectrometry (IPLC/MS/MS). The w e of reliable results extends from about 20 to 100,OOO n g / d of PFOC in serum and from 10to 500 ng/mL in urine. Samples containing PFOC at concentrations greater than 100,OOO 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 fiom the matrix. In order to maximize sensitivity, this me&cd is based on mixed reaction monitoring of the negative fragment ion (M-COOH)' for PFOC. CAUTION: Since primates may carry a number of uwmoses, 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 Neat Reagents Page 2 of 13 ANALYTICAL METHOD Methd No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalak in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.1 Water, deionized and organic free (fromin-house puriiication system; e.g., Ingalls 2 ION) 2.1.2 Methanol, HPLC grade 2.1.3 Perfluorooctanecarboxalate (amlyte), as provided by the client 2.1.4 Perfluorohexanesulfonate (internal standard), 97 % 2.1.5 Ammonium acetate, HPLC grade 2,1.6 Blank control monkey serum 4 .2.1.7 Sodium Carbonate,Certified ACS Grade or equivalent 2.1.8 Sodium Bkarbonate, 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, C d e d grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid 2.2 &pared 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 Page 3 of 13 ANALYTICAL METHOD Method NO.: BACG-3548 Title: Determination of Perfiuorooctanecarboxalatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCIMSIMS) 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542g) 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-PairingSolution (0.5 M tetrabutylammonium hydroxide) 2.2.2.1 For example, to prepare 25 mL, dissolve 4.24 g of tetrabutylammonim hydrogen 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 100mL, dissolve approximately 2.65 g of sodium carbonate and approximately 2.10g of sodium bicarbonate in 100mL ofdeionized 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 quadruple nuus spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and IKA-Vibi.ax @ platform mixer) 3.4 Solventancentration 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) Page 4 of 13 ANALYTICAL METHOD Method NO. BACG-3548 Title: Determinationof Perfluorooctanmboxalatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrametryMass spectrometry (HPLCIMSIMS) 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 solventancentration apparatus 1mL Plastic syringes with 0.2 pm PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKSAND WORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. Actual dilutions will be documentsd on the preparation sheets. Main Stock Solution of PFOC - 1000 crg/mL - Prepare an loo0 pgImL solution of PFOC in deionized organiefiee 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 c~mpound Page 5 of 13 ANALYTICAL METHOD Method NO.: BACG-3548 Title: Determination of Perfluorooctauecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass spectrometry (HPLC/MS/MS) - 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. 4.2 Stock solution of Internal Standard (PFHS), -200 crg/mt 4.2.1 4.3 4.3.1 Prepare an -200 pg/mL solution of PFHS in deionized organic-free water (e.g., accurately weigh about 10mg into a 50-mL volumetric flask). Add deionizedorganicfree 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 organiofree water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PFHS), - SOfig/mL - Prepare an 50 pg/mL solutionof PFHS in deionized organic- fiee water by pipe#ing 2 mL of the 200 pg/mL solution into a culture tube and add 6 mL of deionized water. Mix well. 4.4 Working Stock Solutions of PFOC 4.4.1 To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mLvolumetric flasksor 0 t h ~appropriate glassware. If desired a modified dilution scheme canbe used and documented in the study records. Approximate Concentration , Page 6 of 13 ANALYTICAL METHOD Method NO.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey S m atad Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) I 4.4.2 Summary of concentrations of serum standards: Volume and Spike Cone. I I Standard Approxima* Concatration of PFOC in serum 75,000 I 50,000 1 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) M 10 pL of 2,500 n g / d 50 N 10 pL of 1,OOO n g / d 20 0 10 pL of 500 ng/mL, 10 P 10 pL of 250 ng/mL, 5 5.0 PREPARATION OF SPIKED STANDARDSAND 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 doubleblank (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 f'oreach standad. For the blanks, pipet 10 FLof organic-free water instead of the working stock solution,Add the 10pL Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Senunand Urine: Sample Preparation and Analysis by HPLC Mass Spctrometry/MassSpectrometry (HPLC/MS/MS) of internal standard stock -( 50 pglmL) to each tube except the 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-pairiug solution, 1 mL of carbonatehicarbonatebuffer, 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 horizontalmixer for 1hour at a low speed setting. 5.5 Remove the tube from the shaker and place h a centrifuge (e.g., 2500 rpm)for about 5 minutes. 5.6 - Take off the top dryness (e.g., ethyl acetate layer and put 50 minutes in the Turbo-Vap it 0) into with a a clean gentle tube and stream of evaporate to nitrogen and moderate heat (e.g., 50 'C). 5.7 Reconstitute the residue in 500 pL of 5 %5 mh4 ammonium acetate: 95% methanol containing 1.5 96 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 of the test article being analyzed wiU 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 catbonate/bicarbonate buffer, and 1mL of deionized organic free water. Vortex each tube for about 5 seconds. Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalatein Monkey Serumand 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'horizontalmixer for 1hour 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 dryness (e.g., -top ethyl acetate layer and put it 50 minutes in the Turbo-VapaD) into with a a clean gentle tube and stream of evaporate to nitrogen and moderate heat (e.g., 50 "C). Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.596 formic acid and vortex briefly to mix. Fdter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. ANALYSIS BY HIGH PERFORMANCE LIQUID C!HROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) Conditions are to be optimized if necessary. 1,c C . I m 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/rnin. 3 PL A: 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: 0 - 0.5 min. 50%A : 5096B 0.5 - 7.5 min. 10%A ; 90% B linear gradient 7.5-8 min. 10%A: 9096 B step gradient Page 10 of 13 I Method No.: BACG-3548 ANALYTICAL METHOD Title: Determination of Perfluorooctanecarboxalatein Monkey Serumand 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 Quadruple Mass Spectrometer Conditions Software: PE Sciex TurboQuan 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 TEA4 OR RNG Qo IQ1 ST RO1 IQZ RO2 sn R03 DP CBM Yalue -2000 0 400 -20 -120 5 6 10 I 10 30 40 32 250 1800 Page 11 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determinationof Perfluorooctanecarboxalatein Monkey Serumand Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Value NEB 15 CUR 6 CAD 5 0 1 VCM 0 IPE 0 Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: fuMw4-0 412.9 Q.uhum@ 368.9 REdlmwm 200 p= (Is) QLMaSua 398.9 Per.lmetM R02 m R03 Qxus&n@ 398.9 start StDP 50 50 60 60 52 52 QNwm4nQ 200 Page 12 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determinationof Perfluorooctanecarboxalate in Monkey Serumand 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 smoothedas appropriate. Forquantitation, use the ion profiles at the following mass-to-charge ratios: Analvte PFOC PFHS linbfik 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 insteadofpeak areas. Obtainthe best m e fit of the data (e.g., quadratic fit weighted with l/concentration of the test article or a quadratic fit). Note: The best curve fit may be dependeat 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. , Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxdatein Monkey Serumand Urine: Ssrmple 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 frpm the matrix,external standard quantitationmay be used at the discretion of the supervising mass spectrometrist. 9.0 ACCEPTANCE AND REJECTION CRITERTA 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: Greg&y?k Goknan, Ph.D.,Staff Chemist Bioanalytical Chemistry Group Date k - 37-66 Date c . 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 SpectrometryMass Spectrometry (HPLc/Ms/MS) 1.0 2.0 2.1 2.1.1 2.1.2 PRINCIPLE Serum or wine samples m,obtained fhm cynomolgus monkeys treated with Perfluomhexand- (PFHS). The serum ar urine (e.g., 0.5 mL) containing (PFHS) is fortified with an internal standard (IS), Perfluomoctanecarboxalate (PFOC). The samples are then mixed with an ion-pairing reagmt, buffer and water, followed by extractionwith ethyl acetate. The ethyl acetate layer isremoved,evaporatedto dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5rnM ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/MassSpectrometry(HPLC/.MS/MS).The range ofreliableresults extends from about 5 to 20,000 n g / d of PFHS in serum and h r n 10to 500 n g / d in urine. Samples containing PFHS at concentrationsgreater than20,000 n g / d may be diluted withcontplblank matrix sothat the concentration of PFHS will be withinthe range of reliable results prior to analysis. The mass spectrometryof PFHS and PFOC is accomplished in the negative ion mode. The ion spray sourcevoltage is set at - 2000volts which is low enough to greatly reduce the fornation 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 tobe considered as biohazards andhandledwith universal precautions. Refer to SOP number SlU 2-55 for a description of safety procedures to be used when handling unpreservedprimate tissue. REAGENTS AND SOLUTIONS The listed reagents or their equivalentsmay be used. Neat Reagents Water, deionizedand organic b e(from in-housepurification system; e.g., Iagaus210") Methanol, HPLC grade Page 2 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of PerfluorohexanesuEonate in Monkey Senun and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/MaSs Spectrometry (HPLCMS/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 controlmonkey 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 . Tetrabutylarnmonium Hydrogen Sulfate, Aldrich 97% 2.1.11 SodiumHydroxide 50% solution, Certifiedgrade 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 m,ade at the discretion of the analyst 2.2.1 5 m~ ~mmoniumacetatein organic fiee water 2.2.1.1 For example, to prepare 4 liters,measure out ammoniumacetate (e.g., 1.542 g) and add in organic-fieewafer (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) x 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 SpectrornetxyMass Spectrometry (HPLCMSMS) 2.2.2.1 2.2.3 2.2.3.1 2.2.4 2.2.4.1 3.0 3.1 3.2 3.3 3.4 3.5 3.6 For example, to prepare 25 mL, dissolve approximately4.24 g of tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10yith50% NaOH solution. Note: a more dilute solution of NaOH in water may be used to effect smaller pE adjustments. CarbonateBicarbonate Buffer Solution for Serum (0.25WO.25M) For example, toprepare 100mL,.dissolveapproximately2.65 gof sodiumcarbonateand approximately2.10 g of sodium bicarbonate in 100mL of deionizedwater.Mix well to eflsu~6complete dissolution. CarbonaWBicarbonateBuffer Solution for Urine (1.OW1.OM) For example, toprepare 100mL, dissolveapproximately10.6 gof sodiumcarbonateand approximately 8.4 gof sodium bicarbonate in 100mL ofdeionized water. Mix well to ensure complete dissolution. INSTRUMENTS, MATERLALS, AND APPARATUS The following or their equivalentsmay be used. HPLC pump(s), autosampler,and triple quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax 0platform mixer) Solvmt-concentrationapparatus (e.g., ZymarkTurbo-Vap @ with source of nitrogen) HPLC mobile phase filtration apparatus Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66,0.20 pm) Page 4 of 14 Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass 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 Centrifuee Assorted glassware and syringes C u l W tubes (vials) forusewith 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 Appropriatechanges inthe concentrstionsof the solutionsmay be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. Main Stock Solution of PFHS -1000 pg/mL Prepare an -1000 clg/mL solution of PFHS in deionized organic-fire water (e.g., accurately weigh about 10 mg PFHS into a IO-mL volumetric flask). Add deionized orgdefieewater to dissolve.Dilute to the mark. Alternatively, weigh the compound Page 5 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Pduorohexanedfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 into an appropriatevessel (e.g., culturetube) and add 10mL of deionized organic-fkee water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solutionof Internal Standard (PFOC),-200 p g / d Prepare an -2OOCrg/mzI solidonof PFOC in deionizedorganic-fke water (e.g., z~ccurafely weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic-free water to dissolveand diluteto the markwithdeionized organic-he water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-fkee water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PF'OC), - 50pg/mL - Prepare an 50 crg/mL solutionof PFOC in deionized organic-fieewater 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 PF'HS To prepareworking stocksolutions, make theproper dilutionsas shownin the following table. Prepare in 10-mLvolumetric flasksor other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. Page 6 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) Working Stock Level (WSL) Approximate Concentration (@mu 1I VoIurfleofPFHS solution Final Volume in deionized organic fbewater (mL) 50 p L of 1000 pg/mL stock 10 2,500 5 mL of 5000 &mL stock 10 1,000 I 4 mL of 2500 ng/mL stock 10 500 5 mL of 1000nghnL stock 10 250 I 5 mL of 500 ng/mL stock 10 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 SpectroxnetryMass Spectrometry (HPLC/MS/MS) 4.4.2 Summary of concentrationsof swum standards: Standard Level Volume and Spike Conc. Approximate Concentration of PFHsinsenun (ng/mL) A 20 pL of 500,000 ng/& 20,000 B 10 pL of 500,000 ng/mL, 10,000 I C D I 10 5of 250,000 dmL, 16pLof62,500ng/mL I 5,000 , 2,000 I I I E 16 pL of 31,250 ng/mL 1,OOo F 16 pL of 15,625 ng/mL 500 G 20 pL of 5,WO ng/mL 200 H 10 p L of 5,000 ng/mL 100 I 10 pL of 2,500 ng/mL 50 J 10 pL of 1,000 ng/mL 20 K 10 pL of 500 ng/mL 10 L 10 pL of 250 n g / d 5 Page 8 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of PerfluorohexanesuNonate in Monkey Serum and Urine: Sample Preparation and M y i s by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) 5.0 PREPARATION OF SPIKED STANDARDSAND BLANKS Appropriate changes m 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 analyzedwith each set of unknownsamples.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-h water instead of the working stock solution. Add the 10pL. of intemal standard stock (-50 pg/mL) to each tube except the blank-IS (pipet 10 pL of orgmic-fke water instead) and vortex for -5 seconds. 5.3 For serum samples add the following to each tube: 500 pL of the TBA ion-pairing solution, 1 mL of 0.25WO.25M carbonateJbicarbonite buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds.For urine sampfes add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of l.OM/l.OM carbonatehicarbonatebuffer and 1mL ofdeionized 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 h m the shaker and place in a centxifuse (e.g., 2500 rpm) for abut 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 of nitrogenand moderate heat (e.g., 50 *C). Page 9 of 14 ANALYTlCAL METHOD Method No.: BACG-3606 Title: Determination of PduorohexanesuUonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryA4ass Spectrometry (HPLC/.MS/MS) 5.7 Reconstitute the residue in 500 p L of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pmPVDF or Nylon syringefiltersinto autosamplervials. 6.0 PREPARATIONOF SAMPLES 6.1 Mow each serum sample tothawto room temperature. Vortex each samplebriefly, but vigornusly. Pipet an aliquot of each sample(e.g., 0.5 mL) intoindividual-20-mL Culture tubes. Ifnecessary, dilute an aliquot of any sample with blank matrix so that the expectedconcentrationof the test articlebeing d y z e d w i l l fdlwithinthe concentration range of the standard curve. Add 10 pL of internalstandard stock (- 50 pg/niL) 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, of 0.25lWO.25M carbonatehicarbonate bufTer, and 1 mL of deionized organic 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 mI, of l.OM/l.OM carbonatehicarbonatebuffer and 1mL of deionizedwafer. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1hour at a low speed setting. 6.4 Remove the tube h m the shakerand 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 orNylon syringe filtersinto autosamplervials. Cap vials for analysis. Page 10 of 14 ANALYTICALMETHOD Method No.: BACG-3606 Title: Determination of Pduorohexanesulfonate in Monkey Senun and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (Hl?Le/MS/MS) ' 7.0 ANALYSISBYBIGHPERMlRMANcE LIQUIDCIEROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) 7.1 Conditions are to be optimized if necessary. ._ 7.1.1 HPLC Conditions Analytical Column: Keystone ScientificAquasil C18,150 mm x 2 mm ID, or equivdent Guard Column: Ehtion How rate: Injection volume: Mobile phase: Gradient Profile: Temperature: Keystone Aquasil C18 10 mm x 2 mm 600 pL/min. 5 CrL A. 5mM ammoniumacetate buff= B: 1.5% formic acid in methanol 0 - 3 min. 3 - 5min. 5 - 8 min. 8-10 min. Ambient 50%A :50%B 20%A :80%B linear gradient 10YA : 90% B Step gradient 50Y' :50%B step gradient 7.1.2 PE SciexAPI 3000Triple QuadrupoleMass SpectrometerConditions Software: PE Sciex TurboQuan TurboionS P ~SYource Note: Values listed under' M S M S Acquisition Conditions'' override parameters in this table. Auxiliary Gas: Parmeter Is Air (e.g., Grade 0.1) at 85 pounds per square inch - Value -2000 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 SpectrometryMass Spectrometry (Hp~~~) PZUameter NC TEM OR RNG QO IQ1 ST R01 IQ2 R02 ST3 R03 DF CEM NEB CUR CAD QPE POL VCM Value 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 15 6 5 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 (HPLCMSMS) Parameter IPE - Value 0 MS/MS AcauiSitionConditions Scantype: MRM Polarity Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFHS: 01Mass hmu) 398.9 9 3Mass hmu) 398.9 PFOC (IS) Q?a?wk& 412.9 E@?B!s& R02 sT3 wR03 IQ1 ST RO1 IQ2 Q3Jwu!a 368.9 rn SklR 35 35 45 45 37 37 18 18 19 19 23 23 19 19 20 20 Dwell Tirne (ms) 200 11 Time hml 200 Page 13 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end of the analyticalrun,review each chmatogram to ensure the retention time, peak shape, and peakheight and peak area detemhtionof the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the followingmass-to-chgeratios: Analvte PFHS PFOC Ion Profile 398.9 to 398.9 ~ 412.9 to 368.9 8.2 Plot the peak area response of PF'HS divided by the peak arearesponseofthe IS (PFOC) h m alI standards versus the conceatration of the test article in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit of the data (e.g., quadratic fit weighted with l/concentration of the test article or a quadratic fit). Note: The best curve fitmay be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration rangesusingthe following: y =ax2+bx +c where y = x = a, b, c Peakheight response of PFHS dividedby peak height response of the IS (PFOC)in standards. Concentration of the PFHS in standards. = Constants derived fkom the regression analysis. 8.3 Using the standard curve, calculatethe level of PFHSin each unknown sample. Correct the results of samples for any dilutions. NOTE: Due to unresolvable htdmmwiththe test articleand/orinternal standard fiomthe matrix,external standard quantitation may be used at the discretion of the supervising mass spectrometrist. . 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 SpectrometryMass Spectrometry (HPLC/MS/MS) 9.0 ACCEPTANCEAND REJECTION CRITERIA 9.1 Refm to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standardsis 80-120% of theoretical. 10.0 REPORTING 10.1 Results of aIl analyses are tabulated, and the raw data,original chromatograms, and reports are to be filed in the appropriate study file. Author@): Sr.R e s v h Associate Biodytical chemistryGroup &eg d h m n , Ph.D. MmagX Bioanalytical Chemistry Group /7/b 3 Date Da9te hA73 Page 1 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample . Preparation and Analysis by HPLC Mass SpectrometryMms Spectrometry @n?LC/MS/MS) 1.o PRINCIPLE Serum or Urine samples are obtained from cynomolgus monkeys treated with Pdwrooctanm& (PFOS). The s a w n or urine (e.g., 0.5 d)containing(PFOS) * is fortified with an internal standard (IS), Perfluomtauecarboxdate (PFOC). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extractionwith ethyl acetate. The ethyl acetate layer isremoved, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5 m M ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/MassSpectrometry(HPLC/MS/MS). The range of reliable results extends fkomabout 20 to 10,000ng/mL of PFOS in s'enrm and fkom 10to 500 ng/mL,in urine. Samples c0ntaE.q PFOS at concentrations greater than l0,OOOng/mLmay be diluted with controlblank mattix so thatthe concentrationofPFOSwillbe withinthe range of reliable results prior to analysis. - The mass spectrometryof PFOS and PFOC is accomplished in the negative ion mode. The ion spraysource voltage is set at 2000 volts which islow enoughto great~yreduce the formation of other potentially interfering ions extracted from the matrix. CAUTION: Sinceprimates may carry a number of mnoses, all unpreserVed tissues, includingblood, plasma and serum, are tobe consideredasbiohazardsand handedwith universal. precautions. Refer to SOP number SRI 2-55 for a description of safety procedures to be used when handling unpreswed primate tissue. 2.0 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 2.1 Neat Reagents 2.1.1 Water, deionized and organic fiee (&om in-housepurificationsystem;e.g., Jng& 21ON) 2.1.2 Methanol, HPLC grade . Page 2 of 13 ANfiYTICAL METHOD Method No.: BACG-3607 Title: Determination of PerfluorooctanesuVonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (PLC/MS/MS) 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.1.9 2.1.10 2.1.1 1 2.1.12 2.1.13 2.2 2.2.1 2.2.1.1 2.2.2 Perfluomoctandonate ( d y t e ) , as provided by the client Perfluomoctanecarboxdate (internal standard), 97% ~mmoniumacetate, HPLC grade Blank control monkey serum Sodium Carbonate, Certified ACS Grade or equivalent Sodium Bicarbonate, Certified ACS Grade or equivalent Ethyl Acetate, HPLC grade Tetrabutylammonium Hydrogen Sulfate, 97% Sodium Hydroxide 50% solution,Certified grade or equivalent Blank control monkey urine Formic Acid, 88% Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 5 mMAmmonium acetate in organic free water For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-he water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) Page 3 of 13 ANALYTICAL METHOD Method NO.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) 2.2.2.1 For example, to prepare 25d,dissolve4.24g of tetmbutylammoniumhydrogensulfate in deionizedwater and adjust the pH to 10with 50%NaOH solution. Note: a more dilnte solntion of NaOH in water may be used to effect smaller pH adjustments. 2.2.3 Carbonate/BicarbonateBuffer Solution for Serum(0.25WO.25M) 2.2.3.1 For example,to prepare 100mI, dissolveappmximately2.65g of sodium carbonateand approximately 2.10 g of sodiumbicarbonate in lo0mLofdeionizedwater. Mix well to ensure complete dissolution. 2.2.4 CarbonaWBicarbonateBuffer SolutionforUrine (1 .OW1.OM) 2.2.4.1 For example,to prepare 100mL, dissolveapproximately 10.6 g of sodiumcaibonatea d approximately8.4g of sodium bicarbonate in 100ml, of deionized water. Mix well to ensure complete dissolution. 3.0 INSTIRUMENTS, MATERIALS, AND APPARATUS The followingor their equivalentsmay 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 MA-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 p) Page 4 of 13 ANALYTICAL METHOD Method NO.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) 3.7 3.8 3.9 3.10 3.1 1 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analyticalbalance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipettes Micropipettor@)with tips Culture tubeswith teflon-lined caps Centrifuge I Assorted glassware and syringes Culture tubes (vials) for use with solvent- oncentration apparatus 1 mL Plastic Syringes with 0.2 pan PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appmpriate changes in the concentrations of the solutions maybe made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. Main Stock Solution of PWOS -1000 pg/& Prepare an -1000 pg/mL solution of PFOS in deionized organic-fke water (e.g., accurately weigh about 10 mg PFOS into a 10-mL volumetric flask). Add deionized organic-fiee water to dissolve. Dilute to the mark. Alternatively, weigh the compound Page 5 of 13 ANALYTICALMETHOD Method No.: BACG-3607 Title: Detenm' ah'on of Pduorooctanesulfonate in Monkey Senun and Urine: Sample Preparation and Analysis by HPW Mass Spectrometry/Mass Spectrometry (HPLC/MS/Ms) into an appropriate vessel (e.g.,culture tube) and add 10mL of deionized organic-fiee water. Mix well. Transferthe solutionto a clean vessel if desired. 4.2 Stock Solution of Internal Standard(PFOC), -200 p g / d 4.2.1 Prepan:an-2OOCrg/mI, solution ofPFOC indeionized organic-freewater (e.g.,accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic-fke water to dissolve and dilute to the markwithdeionizedoqpic-fiee water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-fke water. Mix well. Transferthe solution to a clean vessel if desired. 4.3 Spiking solution of Internal Standard (PFOC), - SOpg/mL - 4.3.1 Prepare an 50 pg/mL solution of PFOC indeionized organic- fke waterby pipettins 2 mL of the 200 y%/mLsolution into a culture tube and add 6 mL of deionizedwater. Mix well. 4.4 Working Stock Solutions of PFOS 4.4.1 Toprepareworkingstocksolutions,make the proper dilutionsasshownin the following table. Prepare in 10-mLvolumetric flasksor other appropriate glassware. Ifdesired a modified dilution scheme can be used and documented in the study records. Page 6 of 13 I ANALYTICALMETHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometryhlass Spectrometry (HPLc/MS/MS) 62,500 I 2.5 mL of 250,000 ng/mL I -1 10 ~ 15,625 5,000 50 CCL, of 1000 pg/mL stock 10 r 2,500 5 mLof 5000 ng/mL stock 10 1,000 4 mL of 2500 ng/mL stock 10 500 5 mL,of lo00 ng/mL stock 10 250 5 mL of 500 n g / d stock 10 - 4.4.2 Summary ofconcentrations of serum standards: Volume and Spike Cone. Standatd Level A 10 of500,000 n g / d - B 10 pL of 250,000 n g / d C 16 pL of 62,500 n g / d Appioximate Concentration of PFOS in serum (nIw4 10,Ooo 5,000 2,000 D 16 of 31,250 ng/mL Page 7 of 13 AN&Y"ICAL METHOD Method No.: BACG-3607 Title: Determination of PerfluorooctanesuEonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MS/MS) 5.0 PREPARATION OF SPIKEDSTANDARDS AND BLANKS Appropriate changes in the concentrationsof the solutionsmay 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 analyzedwith each set of unknown samples.A matrix double blank (blank-IS) may also be analyzedifdesired. 5.2 Into individual -20-mI, 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-fkee water instead of the working stock solution. Add the 10 p.L 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 of the TBA ion-pairing solution, 1mL of 0.25WO.25Mciubonate/bicarbonste butk, and 1 mL ofdeionized organic b e water. Vortex each tube for about 5 smnds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of l.OM/l:OM carbonatehicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfhorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis. by HPLC Mass SpectrometryiMass Spectrometry (HPLC/MS/MS) 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 h m the shaker and place in a centrifuge(e.g., 2500 rpm)for about 5 . minutes. - 5.6 Take offthe top ethyl acetate layer and put it int0a clean tube and evaporate to dryness (e.g., 50 minutesin the Turbo-Vap @) with a gentle stream of nitrogen and moderate heat (e.g., 50 OC). 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 pnPVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 N o w each serum sampleto thaw toroomtemperature. Vortex each sample briefly, but vigorously. Pipet an aliquotof each sample (e.g.$0.5 ml;) into individual-20-mL culture tubes. Inecessary, dilute an aliquot of any sample with blank matrix so that the expected concentrationof the test articlebeinganalyzedwill Eall withinthe 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 For serum samples add the following to each tube: 500 pL of the "'BA ion-pairing solution,1mL of 0.25W0.25Mcarbonatehicarbonatebuffer, and 1 mL of deionized organic fkee 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 l.OM/l.OM carbonatdbicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluor6octanesulate in Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLCMSMS) 6.4 Remove the tube h mthe 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 mthe Turbo-Vap@) witha gentle stream of nitrogenand modate heat (e.g., 50 'C). 6.6 Reconstitute the residue in 500 pL of 5% 5 mh4 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 filtersinto autosampler vials. Cap vials for analysis. 7.0 ANALYSISBYHIGHPERMlRMANcE LIQUIDCHROMATOGRAPHY MASS SPECTROMETRYMASS SPECTROMETRY (HPLC/MS/MS) 7.1 Conditions are to be optimized if necessary. 7.1.1 HPLC Conditions Analytical Column:Keystone ScientificAquas2 C18,150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Gradient Profile: Temperature: ' Keystone Aquasil C18 10 mm x 2 mm 400 a m i n . 5 PL A 5mM ammonium acetatebuffer B: 1.5% formic acid in methanol -03 - 3Sm&i.n. -5 8 min. 8-10 min. Ambient 50%A : SOY& 20% A : 80% B linear gradient lO%A:90%B step gradient 5 0 M :5O%B step gradient 7.1.2 PE Sciex MI 3000Triple QuadrupoleMass SpectrometerConditions Software: PE Sciex TurboQuazl Page 10 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determinatioa of Perfluorooctanesdfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPK Mass SpectrometryMass Spectrometry (EPLaMS/MS) Turboion SDIWS o m e Note: Valueslisted under"MS/MS Acquisition Conditions" ovenide parameters inthis table. AwiliaryGas: Parameter Is , NC TEM OR RNG QO IQ1 ST RO1 IQ2 R02 ST3 R03 DF CEM Air (e.g., Grade 0.1) at 85 pounds per square inch - Value -2000 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 Page 11 of 13 ANALYTICALMETHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanedfonate in Monkey Senun and Urine: Sample Preparation and Analysis by HPLC Mass SpectrornetryMass Spectrometry (HPLCIMSIMS) Parameter NEB CUR CAD - Value 15 6 5 , POL 1 VCM 0 IPE 0 M S M . .. .. Acaumtmn Con131t.1OI18 scantype: MRM Polluity: Negative Acquisitionmode: Profile Pausetime: 5millisecondS -PFOS: PFOC (IS) 412.9 - 9 498.9 L!zmahd 368.9 m I3lJumXrn R02 35 35 sT3 45 45 R03 37 37 Qo 18 18 IQ1 19 19 ST 23 23 Dwell T i m fmq) 200 Dwell Time (ms) 200 Page 12 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Senun and Urine: Sample Preparation and Analpis by HPLC Mass SpectrometryMass Spectrometry (HPLc/Ms/MS) RO1 19 19 142 20 20 8.0 CALCULATIONS 8.1 At the end of the analyticalrun,review each chromatogram to ensuretheretention time, peak shape,and peak height and peakarea determinationof the test articleand the IS are acceptable. The data may be smoothed as appropriate. For quantitatim,use the ion profiles at the followingmass-to-chargeratios: Analvte PFOS PFOC Ion Profile 498.9 to 498.9 412.9 to 368.9 8.2 Plot the peak arearesponseof PFOS dividedby the peak arearesponseof the IS OJFOC) h m all standards versus the concentration of the test article in the standards. Alternatively, thepeak heights maybe used instead ofpeak amas. Obtain the best curve .fit of the data (e.g., quadratic fit weighted with l/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 = d + bx + c where y = x = a, b, c Peak height response of PFOS divided by peak height response of the IS (PFOC) in standads. Concentration ofthe PFOS in standards. = Constantsderived from the regression d y s i s . Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) * 8.3 Using the standardcurve, calculate the level of PFHC in each unlcnown sample. Correct the r d t s of samples for any dilutions. NOTE: Due to unresolvable interferenceswith the test article and/or internal standard h m the matrix, externalstandard quantitationmaybe 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 standardsis 80-120%of theoretical. 10.0 REPORTING 10.1 Results of all dyses'aretabulated, and the raw data,originalchromatograms, and reports are to be filed in the appropriate study file. Author@): Lori Coward, BS Sr. Research Associate Biodytical ChemistryGroup U/>/a Date Approved by: Mkger Date Bioanalytical Chemistry Group