Document b5GdBBdrX7baRpmeapLREKOq6
Protein B inding o f
Perfluorohexane Sulfonate,
Perfiuorooctane Sulfonate and Perfluorooctanoate to Plasm a
(H um an, R at, and M onkey), and V arious H um an-D erived Plasm a P ro tein Fractions
STUDY ID : 9921.7
Southern R esearch Institute 2000 N inth Avenue South P.O. Box 55537
Birmingham, AL 35255-5537
Study 9921.7 Page 2 of7
SUMMARY
The percent protein binding o f four perfluoronated compounds in seven different human-derived plasm a protein fractions was determ ined. Additionally, protein binding for the same four compounds was m easured in rat, m onkey and human plasm a at five different concentrations. Each test article was incubated for 15 m inutes at 37 C in each plasm a protein fraction or plasm a sample. The protein bound test article was separated from the non-bound test article by ultrafiltration using Centrifree filtration devices w ith a nom inal m olecular weight (NM W ) cutoff o f 30,000 daltons. The eluent from the ultrafiltration was analyzed for test article concentration relative to a solvent standard.
Corenna Kerstner-W ood, BS Research A ssistant HI Bioanalytical Chem istry Group
Lori Coward, BS Sr. Research A ssociate Bioanalytical Chem istry Group
Greg Gorman, Ph.D. M anager Bioanalytical Chem istry Group
KEY PERSONNEL
Study 9921.7 Page 3 of7
Study 9921.7 Page 4 of7
1. O B JEC TIV E
The objective o f this study was to determ ine the percent o f protein binding for each test article in human, rat, and m onkey plasm a as w ell as various hum an-derived plasm a protein fractions.
2. SAFETY
A ll necessary procedures to ensure the safety o f the analysts w ere based on inform ation contained in the M aterial Safety and D ata Sheets (M SDS), provided by the sponsor for the test articles used in this study. Procedures outlined in SOP 2-29-3 for handling human and prim ate plasm a as well as the hum an-derived plasm a protein fractions w ere also followed.
3. COM PLIANCE
This work w as perform ed using various previously validated analytical m ethods ( 3548, 3606, 3607). W hile this w ork w as not audited in compliance w ith GLP regulations, it was perform ed in the spirit o f the regulations using calibrated and validated instrum entation.
4. EXPERIM ENTAL
4.1 A nalytical P rocedures
Individual aqueous solutions o f each hum an derived plasm a protein fraction w ere 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 C o n cen tratio n ^ 3500-5500 mg/100 mL 965-2515 mg/lOOmL 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 (~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 f100% 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 o f each test article w ere separately spiked into the plasm a protein fractions or a control plasm a m atrix to produce a final concentration o f 10 (ig/mL. The resulting m ixture was vortexed for 1 m inute and incubated at 37 C for 15 minutes. Upon completion o f the incubation, each sample was again vortexed for 1 m inute 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 determ ine the amount o f unbound test article.
Study 9921.7 Page 5 o f7
4.2 R esults The results o f all o f the analyses are presented as percent bound test article in Table I-III at the end o f the report.
5.0 C onclusion
The protein binding o f four perfluoronated compounds was evaluated in seven separate humanderived plasm a protein fractions at two different protein fraction concentrations (Table I and II). Additionally, protein binding for the same four compounds was evaluated in rat, hum an, and monkey plasm a at 5 different concentrations (Table HI). 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, w as found to be highly bound by both albumin and beta-lipoproteins. The protein binding experim ents for the rat, human, and m onkey plasm a show very high levels o f binding at all concentrations tested and no evidence o f saturation.
Study 9921.7 Page 6 o f 7
Table I Percent Binding to Human Plasm a Protein Fractions (-10% Physiological Cone.)
PFHS PFOS PFOC
Album in Gamma Alpha Fibrinogen
A lpha-2-
T ran sfe rrin
Beta
Globulin Globulin
M acroglobulin
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 n Percent Binding to Human Plasm a Protein Fractions (100% Physiological Cone)
PFHS PFOS PFOC
Album in Gamma Alpha Fibrinogen
A lpha-2-
T ran sfe rrin
Beta
Globulin Globulin
M acroglobulin
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 j
Table ID % Protein Binding to Rat, Human and M onkey Plasm a
Nominal
Concentration
(PPm) 1 10
100 250 500
Perfluorohexanesulfonate Perfluorooctanesulfonate Perfluorooctanecarboxalate Rat Monkey Human Rat Monkey Human Rat Monkey Human
-100 -100 -1 0 0 -100 -100 99.4
0
1
o
-100
-100 -100 -100 99.8 99.9 99.9 99.5 99.8 99.9
99.9 99.9 100 99.7 99.9 99.9 98.6 99.8 99.9
99.1 99.8 99.9 99.5 99.9 99.9 97.6 99.8 99.6
98.2 99.5 99.4 99.0 99.9 99.9 97.3 99.5 99.4
% Binding Values reported as "~100" reflect a nonquantifiable amount o f test article in the plasma water BQL <6.25 ng/mL
Study 9921.7 Page 7 o f7
6.0 R eferences (1). Koplar, L., Pesce, A., "Clinical Chemistry (Theory, Analysis and Correlation) 1984, pg. 1416, C.V. M osby Publishing Co.
7.0 Review and A pprovals
Cotenna Kerstner-W ood, BS Research A ssistant III Bioanalytical Chem istry Group.
Lori Coward, BS Sr. Research Associate Bioanalytical Chem istry Group
Bioanalytical Chem istry Group
0G-03'o2>
D ate
63 -63 D ate
& -03-03
Date
Page 1 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle :
D eterm ination o f Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
1.0 PR IN C IPL E
Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanecarboxalate (PFOC). The serum o r urine (e .g ., 0.5 m L) containing (PFOC) is fortified w ith an internal standard (IS), perfluorohexanesulfonate (PFHS). The sam ples are then m ixed w ith an ion-pairing reagent, buffer and w ater, followed by extraction w ith ethyl acetate. The ethyl acetate layer is rem oved, evaporated to dryness, reconstituted in 95% m ethanol containing 1.5 % form ic acid, 5 % 5mM am m onium acetate, filtered, and transferred to autosam pler vials, and analyzed by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/M S/M S). The range o f reliable results extends from about 20 to 100,000 ng/m L o f PFOC in serum and from 10 to 500 ng/m L in urine. Samples containing PFOC at concentrations greater than 100,000 ng/m L may be diluted w ith control blank m atrix so that the concentration o f PFOC w ill be w ithin the range o f reliable results prior to analysis.
The mass spectrometry o f PFOC and PFHS is accomplished in the negative ion mode. T he ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the form ation o f other potentially interfering ions extracted from die m atrix. In order to maximize sensitivity, this m ethod is based on m ixed reaction m onitoring o f the negative fragm ent ion (M -COOH)' for PFOC.
CA U TIO N : Since prim ates may carry a number o f zoonoses, all unpreserved tissues, including blood, plasm a and serum , are to be considered as biohazards and handled w ith universal precautions. Refer to SOP num ber SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved prim ate tissue.
2 .0 REAGENTS AND SOLUTIONS
The listed reagents or their equivalents may be used.
2.1 N eat R eagents
ANALYTICAL METHOD
Page 2 of 13
Method No.: BACG-3548
T itle:
Determ ination o f Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
2 .1 .1
W ater, deionized and organic free (from in-house purification system ; e .g ., Ingalls 2 ION)
2 .1 .2
M ethanol, HPLC grade
2 .1 .3 Perfluorooctanecarboxalate (analyte), 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
^
2 .1 .7
Sodium Carbonate, C ertified ACS G rade o r equivalent
2 .1 .8
Sodium Bicarbonate, C ertified ACS G rade or equivalent
2 .1 .9 Ethyl A cetate, HPLC grade
2 .1 .1 0 Tetrabutylam m onium H ydrogen Sulfate
2.1.11 Sodium Hydroxide 50 % solution, C ertified grade o r equivalent
2 .1 .1 2 Blank control monkey urine
2.1.13 Form ic Acid
2 .2 P rep ared Solutions
A ppropriate changes in the solutions may be made at the discretion o f the analyst
2 .2 .1
5 mM Ammonium acetate in organic free water
Page 3 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle:
Determ ination of Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
2 .2 .1 .1
For exam ple, to prepare 4 liters, m easure out ammonium acetate (e .g ., 1.542 g) and add in organic-free water (e .g ., 4 L). M ix well and filter through HPLC mobile phase filtration apparatus.
2 .2 .2 TBA Ion-Pairing Solution (0.5 M tetrabutylam m onium hydroxide)
2 .2 .2 .1
F or exam ple, to prepare 25 m L, dissolve 4.24 g o f tetrabutylam m onium hydrogen sulfate in deionized w ater and adjust the pH to 10 w ith 50% NaOH solution. Note: a m ore dilute solution o f NaOH in w ater m ay be used to effect sm aller pH adjustm ents.
2 .2 .3
Carbonate/Bicarbonate Buffer Solution
2 .2 .3 .1
For example, to prepare 100 m L, dissolve approxim ately 2.65 g o f sodium carbonate and approximately 2.10 g o f sodium bicarbonate in 100 m L o f deionized w ater. M ix w ell to ensure complete dissolution.
3.0 INSTRUM ENTS, M ATERIALS, AND APPARATUS The following o r their equivalents may be used.
3.1 HPLC pum p(s), autosam pler, and triple quadrupole mass spectrom eter
3.2 A utosam pler vials with inserts
3.3 V ortex m ixers (e.g ., touch m ixer and IKA-Vibfax platform m ixer)
3.4 Solvent-concentration apparatus (e .g ., Zym ark Turbo-V ap w ith source o f nitrogen)
3.5 HPLC m obile phase filtration apparatus
3.6 Filters for H PLC m obile phase filtration apparatus (e.g ., Nylon-66, 0.20 /m)
Page 4 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle:
D eterm ination of Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
3 .7 3 .8 3 .9 3.10 3.11 3.12 3.13 3 .1 4 3.15 3 .1 6 3.17 4 .0
4 .1 4 .1 .1
A nalytical balance
V olum etric flasks (e.g ., 10 and 25 mL) D isposable Pasteur pipets
'
M icropipettor(s) with tips
C ulture tubes w ith teflon-lined caps C en trifiig e
A ssorted glassw are and syringes
C ulture tubes (vials) for use w ith solvent-concentration apparatus 1 m L Plastic syringes with 0.2 pm PVDF syringe filters V ariable speed horizontal platform shaker pH m eter
PREPARATION OF STOCKS AND WORKING STOCKS
A ppropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst. Actual dilutions w ill be documented on the preparation sheets.
M ain Stock Solution o f PFOC ~ 1000 pg/mL
Prepare an ~ 1000 pg/m L solution o f PFOC in deionized organic-free w ater (e.g ., accurately weigh about 10 mg PFOC into a 10-mL volum etric flask). Add AeinninHi organic-free w ater to dissolve. D ilute to the mark. Alternatively, weigh the compound
Page 5 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle:
Determ ination o f Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrom etry (HPLC/M S/M S)
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 m L o f deionized organic-free w ater. M ix w ell. Transfer the solution to a clean vessel if desired.
Stock Solution o f Internal Standard (PFHS), --200 pg/m L
Prepare an ~ 2 0 0 pg/m L solution o f PFHS in deionized organic-free w ater (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organicfree w ater to dissolve and dilute to the m ark with deionized organic-free w ater. A lternatively, weigh the compound into a an appropriate vessel (e.g ., culture tube) and add 50 mL o f deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired.
Spiking solution o f Internal Standard (PFHS), ~ 50pg/m L
Preparean ~ 50 pg/m L solution o f PFHS in deionized organic- free w ater by pipetting 2 m L o f the 200 pg/m L solution into a culture tube and add 6 m L o f deionized w ater. M ix well.
W orking Stock Solutions o f PFOC
To prepare w orking stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volum etric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records.
W orking Stock Level (W SL)
Approximate Concentration (ng/m L)
500,000
250,000
Volume o f PFOC solution
5 mL o f 1000 /ig/m L 5 mL o f 500,000 ng/m L
Final Volume in deionized organic free w ater (mL)
10
10
ANALYTICAL METHOD
Page 6 of 13
Method No.: BACG-3548
T itle:
Determ ination o f Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample
Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry
(HPLC/M S/M S)
1
62,500 31,250 15,625
5,000 2,500 1,000
500 250
2.5 m L o f 250,000 ng/m L 5 m L o f 62,500 ng/m L 5 m L o f 31,250 ng/ mL
50 pL o f 1000 pg/mL stock 5 m L o f 5000 ng/m L stock 4 m L o f2 5 0 0 ng/m L stock 5 mL o f 1000 ng/m L stock 5 m L o f 500 ng/m L 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.
A pproxim ate Concentration o f PFOC in serum
(ng/m L)
A 50 p L o f 1000 jtig/mL
100,000
B 37.5 p L o f 1000 pg/m L
75,000
C 25 pL o f 1000 pg/mL
50,000
D 12.5 pL o f 1000 pg/mL
25,000
E 10 pL of 1000 pg/mL
20,000
ANALYTICAL METHOD
Page 7 of 13
Method No.: BACG-3548
T itle :
Determ ination o f Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrom etry (HPLC/M S/M S)
F 10 pL o f 500,000 ng/mL G 10 p b o f 250,000 ng/m L H 16 jtiL o f 62,500 ng/m L
I 16 p b o f 31,250 ng/m L J 16 p b o f 15,625 ng/m L K 20 pL o f 5,000 ng/mL L 10 p b o f 5,000 ng/m L M 10 p b o f 2,500 ng/m L N 10 p b o f 1,000 ng/m L 0 10 p b o f 500 ng/m L P 10 p b o f 250 ng/mL
10,000 5 ,0 0 0 2 ,0 0 0
1,000 500 200 100 50 20 10
5
5 .0 PREPARATION OF SPIKED STANDARDS AND BLANKS
A ppropriate changes in the concentrations o f the solutions m ay be matte at the discretion o f the analyst.
5.1 M ultiple (e .g ., about three) sets o f m atrix standards and a m atrix blank (blank + IS) are analyzed w ith each set o f unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired.
5.2 Into individual --20-mL culture tubes, pipet blank m atrix (e .g ., 0.5 m L ). Pipet in the appropriate volume as described in the table above fra each standard. F or the blanks, pipet 10 pL o f organic-free water instead o f the working stock solution. Add the 10 p b
Page 8 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle:
Determ ination o f Perfluorooctanecarboxalate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrom etry (HPLC/M S/M S)
o f internal standard stock ( --50 pg/m L) to each tube except die blank-IS (pipet 10 pL o f organic-free w ater instead) and vortex for --5 seconds.
5.3 To each tube add 500 pL of the TBA ion-pairing solution, 1 m L of carbonate/bicarbonate buffer, and 1 mL of deionized organic free w ater. V ortex each tube for about 5 seconds.
5.4 Add 2.5 mL o f ethyl acetate and extract on horizontal m ixer 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 m inutes.
5.6 Take o ff 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 m oderate heat (e.g ., 50 C).
5.7 R econstitute the residue in 500 pL o f 5 % 5 mM ammonium acetate: 95% m ethanol containing 1.5% form ic acid and vortex briefly to mix. F ilter die samples through 0.2 pm PVDF or N ylon syringe filters into autosam pler vials.
6.0 PR EPA RA TIO N O F SAM PLES
6.1 Allow each serum sample to thaw to room tem perature. V ortex each sam ple briefly, but vigorously. Pipet an aliquot o f each sample (e.g ., 0 .5 m L) into individual - 2 0 mL culture tubes. If necessary, dilute an aliquot o f any sam ple w ith blank m atrix so that the expected concentration o f the test article being analyzed w ill fell w ithin the concentration range o f the standard curve. Add 10 pL o f internal standard stock ( -- 50 pg/m L) to each tube and vortex for a couple o f seconds.
6.2 To each tube add 500 pL o f the TBA ion-pairing solution, 1 m L o f carbonate/bicarbonate buffer, and 1 m L o f deionized organic free w ater. V ortex each tube for about 5 seconds.
Page 9 of 13
ANALYTICAL METHOD
Method No. : BACG-3548
T itle:
Determination o f Perfluorooctanecarboxalate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
6.3 6 .4 6 .5
6 .6
7 .0 7 .1 7 .1 .1
Add 2.5 mL o f ethyl acetate and extract on horizontal m ixer 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 m inutes.
Take o ff the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g ., ~ 50 minutes in the Turbo-Vap ) w ith a gentle stream o f nitrogen and m oderate heat (e.g ., 50 C).
R econstitute the residue in 500 /L o f 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. F ilter the samples through 0.2 jum PV D F or Nylon syringe filters into autosam pler vials. Cap vials for analysis.
ANALYSIS BY H IG H PERFORM ANCE LIQ U ID CHROM ATOGRAPHY M ASS SPECTRO M ETRY /M ASS SPECTRO M ETRY (H PLC /M S/M S)
Conditions are to be optimized if necessary.
H PLC Conditions
A nalytical Column:
Keystone Scientific Aquasil C18, 150 m m x 2 mm ID , or equivalent
G uard Column: Elution Flow rate: Injection volume: M obile phase:
Keystone Aquasil C18 10 mm x 2 mm 400 /xL/min. 3/xL A: 5mM ammonium acetate buffo: B: 1.5% form ic acid in methanol
G radient Profile:
0 - 0.5 m in. 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
ANALYTICAL METHOD
Page 10 of 13
Method No.: BACG-3548
T id e:
Determ ination of Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
7 .1 .2
Tem perature:
8-11 min. A m bient
50% A : 50 %B
PE Sciex API 3000 Triple Q uadrupole M ass Spectrom eter Conditions Software: PE Sciex TurboQuan
Turboion Spray Source
N ote: Values listed under "MS/MS A cquisition Conditions" override param eters in
this table.
_
A uxiliary Gas:
A ir (e .g ., Grade 0.1) at 85 pounds per square inch
Param eter
IS NC TEM OR RNG
Q0 IQ1
ST ROl IQ2 R02 ST3 R03 DP CBM
V alu e
-2000 0 400 -20 -120 5 6 10 6 10 30 40 32 250 1800
Page 11 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle:
Determ ination o f Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
Param eter
NEB CUR CAD QPE POL VCM 1PE
Value
15
6
5
0
1
0 0
M S/M S A cquisition Conditions
Scan type:
MRM
Polarity:
N egative
A cquisition mode: Profile
Pause tim e:
5 m illiseconds
M asses requested:
PFOC: Q1 Mass famiA 412.9
Q3-Mass (amu) 368.9
Dwell T im e ftr)
200
PFHS (IS)
Q1 Mass (am) 398.9
Q3.Mass (amu) 398.9
Parameter R02 ST3
R03
Start
50 60 52
Stop SO 60 52
Dwell T im e fm al
200
Page 12 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T itle :
Determination o f Perfluorooctanecarboxalate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
8.0 CALCULATIONS
8.1 A t the end o f the analytical run, review each chrom atogram to ensure the retention tim e, peak shape, and peak height and peak area determ ination o f the test article and the IS are acceptable. The data may be smoothed as appropriate. F or quantitation, use the ion profiles at the following m ass-to-charge ratios:
A n aly te PFOC PFHS
Ion-Profile 412.9 to 368.9 398.9 to 398.9
8.2 Plot the peak area response o f PFOC divided by the peak area response o f the IS (PFHS) from all standards versus the concentration o f the test article in the standards. Alternatively, the peak heights may be used instead o fpeak areas. Obtain the best curve fit o f the data (e.g ., quadratic fit weighted w ith 1/concentration o f the test article o r a quadratic fit). Note: The best curve fit may be dependent on the range o f die standard curve and it may be necessary to have m ore than one standard curve for various concentration ranges using the following:
y = ax2 + bx + c
w here
y=
x= a, b, c
Peak height response of PFOC divided by peak height response of the IS (PFHS) in standards, Concentration o f the PFOC in standards, = Constants derived from the regression analysis.
8.3 U sing the standard curve, calculate the level o f PFHC in each unknown sample. C orrect the results o f samples for any dilutions.
Page 13 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
T id e:
Determ ination o f Perfluorooctanecarboxalate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
NOTE:
Due to unresolvable interferences w ith the test article and/or internal standard from the m atrix, external standard quantitation may be used at the discretion o f the supervising mass spectrom etrist.
9.0 ACCEPTANCE AND R EJEC TIO N 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 R EPO R TIN G
10.1 R esults o f all analyses are tabulated, and the raw data, original chrom atogram s, and reports are to be filed in the appropriate study file.
A u th o rs:
n, P h.D ., Staff Chem ist Bioanalytical Chem istry Group
/SI-O P
D ate
Approved by:
es D . Jo] banalytical
S, MBA, Manager imistry Group
D ate
Page 1 o f 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/M S/M S)
1.0
2.0 2.1 2.1.1 2.1.2
PRIN CIPLE
Serum or urine sam ples are obtained from cynomolgus monkeys treated w ith Perfluorohexanesulfonate (PFHS). The serum or urine (e.g., 0.5 mL) containing (PFHS) is fortified w ith an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The sam ples are then m ixed w ith an ion-pairing reagent, buffer and w ater, followed by extraction w ith ethyl acetate. The ethyl acetate layer is rem oved, evaporated to dryness, reconstituted in 95% m ethanol containing 1.5 % formic acid, 5 % 5mM ammonium acetate, filtered, and transferred to autosam pler vials, and analyzed by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/MS/MS). The range o f reliable results extends from about 5 to 20,000 ng/mL o f PFHS in serum and from 10 to 500 ng/m L in urine. Samples containing PFHS at concentrations greater than 20,000 ng/mL m ay be diluted w ith control blank m atrix so that the concentration o f PFHS w ill be w ithin the range o f reliable results prior to analysis.
The m ass spectrom etry o f 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 the form ation o f other potentially interfering ions extracted from the m atrix.
CA U TIO N ; Since prim ates may carry a num ber o f zoonoses, all unpreserved tissues, including blood, plasm a and serum, are to be considered as biohazards and handled w ith u n iv ersal p recautions. Refer to SOP num ber SRI 2-5-5 for a description o f safety procedures to be used when handling unpreserved prim ate tissue.
REAGENTS AND SOLUTIONS
The listed reagents or their equivalents m ay be used.
N eat R eagents
W ater, deionized and organic free (from in-house purification system; e.g., Ingalls 210N)
M ethanol, HPLC grade
Page 2 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
Title:
Determ ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
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, C ertified ACS Grade or equivalent
2.1.8
Sodium Bicarbonate, C ertified ACS Grade or equivalent
2.1.9
Ethyl A cetate, HPLC grade
2.1.10 Tetrabutylammonium Hydrogen Sulfate, Aldrich 97%
2.1.11 Sodium Hydroxide 50% solution, C ertified grade or equivalent
2.1.12 B lank control monkey urine
2.1.13 Form ic Acid, 88%
2.2 Prepared Solutions
A ppropriate changes in the solutions m ay be made at the discretion o f 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 w ater (e.g., 4 L). M ix w ell and filter through HPLC m obile phase filtration apparatus.
2.2.2
TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) K
Page 3 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
2.2.2.1
For exam ple, to prepare 25 mL, dissolve approxim ately 4.24 g o f tetrabutylammonium hydrogen sulfate in deionized w ater and adjust the pH to 10 w ith 50% NaOH solution. N ote: a m ore dilute solution o f N aO H in w ater m ay 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 o f deionized water. Mix w ell to ensure com plete dissolution.
2.2.4
Carbonate/Bicarbonate B uffer Solution for U rine (1.0M /1.0M )
2.2.4.1
For example, to prepare 100 mL, dissolve approximately 10.6 g o f sodium carbonate and approxim ately 8.4 g o f sodium bicarbonate in 100 mL o f deionized water. M ix w ell to ensure com plete dissolution.
3.0 IN STRU M EN TS, M A TER IA LS, AND APPARATUS
The following or their equivalents m ay be used.
3.1 HPLC pum p(s), autosampler, and triple quadrupole mass spectrom eter
3.2 Autosam pler vials with inserts
3.3 Vortex m ixers (e.g., touch m ixer and K A -V ibrax platform m ixer)
3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap w ith source o f nitrogen)
3.5 HPLC m obile phase filtration apparatus
3.6 Filters for HPLC m obile phase filtration apparatus (e.g., N ylon-66,0.20 pm)
Page 4 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
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
A nalytical balance
V olum etric flasks (e.g., 10 and 25 mL)
D isposable Pasteur pipettes
M icropipettor(s) w ith tips
C ulture tubes w ith teflon-lined caps
C en trifu g e
A ssorted glassw are and syringes
Culture tubes (vials) for use w ith solvent-concentration apparatus
1 m L Plastic syringes w ith 0.2 pm PVDF syringe filters
V ariable speed horizontal platform shaker
pH m eter
PREPARATION OF STOCKS AND WORKING STOCKS
Appropriate changes in the concentrations o f the solutions may be made at the discretion o f tiie analyst. Actual dilutions w ill be documented on the preparation sheets. M ain Stock Solution o f PFHS ~1000 pg/mL
Prepare an ~1000 pg/mL solution o f PFHS in deionized organic-free w ater (e.g., accurately w eigh about 10 m g PFHS into a 10-xnL volum etric flask). Add deionized organic-free w ater to dissolve. D ilute to the m aik. Alternatively, weigh the compound
Page 5 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
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 w ater. M ix w ell. Transfer the solution to a clean vessel if desired.
Stock Solution o f Internal Standard (PFOC), ~200 pg/mL
Prepare an ~200pg/mL solution o f PFOC in deionized organic-free water (e.g., accurately w eigh about 10 m g into a 50-mL volumetric flask). Add deionized organic-free water to dissolve and dilute to the mark w ith deionized organic-free water. Alternatively, weigh the com pound into a an appropriate vessel (e.g., culture tube) and add SO mL o f deionized organic-free water. M ix well. Transfer the solution to a clean vessel if desired.
Spiking solution o f Internal Standard (PFOC), ~ 50pg/mL
Prepare an ~ 50 pg/mL solution o f PFOC in deionized organic- free w ater by pipetting 2 mL o f the 200 pg/mL solution into a culture tube and add 6 m L o f deionized water. M ix well.
W orking Stock Solutions o f 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 m odified dilution scheme can be used and documented in the study records.
Page 6 o f 14
ANALYTICAL METHOD
Method No.: BACG-3606
Title:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
W orking Stock Level (W SL) Approxim ate Concentration
(ng/m L )
Voluipe o f PFHS solution
500,000
5 mL o f 1000 pg/mL
250,000 5 mL o f 500,000 ng/mL
62,500 2.5 mL o f 250,000 ng/mL
31,250 5 mL o f 62,500 ng/mL
15,625 5 mL o f 31,250 ng/ mL
5,000 50 pL o f 1000 pg/mL stock 2,500 5 mL o f 5000 ng/m L stock 1,000 4 mL o f 2500 ng/m L stock
500 5 mL o f 1000 ng/mL stock
250 5 mL o f 500 ng/m L stock
Final Volume in deionized organic free w ater (mL)
10 10 10
10
10 10 10 10 10 10
Page 7 o f 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
4.4.2 Summary o f concentrations o f serum standards:
Standard Level
Volume and Spike Cone.
Approxim ate Concentration o f PFHS in serum
(ng/m L )
A 20 pL o f 500,000 ng/mL
20,000
B 10 pL o f 500,000 ng/mL
10,000
C 10 pL o f 250,000 ng/mL
5,000
D 16 pL o f 62,500 ng/mL
2,000
E 16 pL o f 31,250 ng/mL F 16 pL o f 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 o f 2,500 ng/mL J 10 pL o f 1,000 ng/mL K 10 pL o f 500 ng/mL L 10 pL o f 250 ng/mL
1,000 500 200 100 50 20 10
5
Page 8 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
5.0 PR EPA R A TIO N O F SPIK ED STANDARDS AND BLA N K S
Appropriate changes in the concentrations o f the solutions m ay be made at the discretion o f the analyst.
5.1 M ultiple (e.g., about three) sets o f m atrix standards and a m atrix blank (blank + IS) are analyzed w ith each set o f unknown samples. A m atrix double blank (blank-IS) may also be analyzed if desired.
5.2 Into individual ~20-mL culture tubes, pipet blank m atrix (e.g., 0.5 m L ). Pipet in the appropriate volum e as described in the table above for each standard. For the blanks, pipet 10 pL o f organic-free w ater 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 o f organic-free w ater instead) and vortex for ~5 seconds.
5.3 For serum sam ples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 m L o f 0.25M /0.25M carbonate/bicarbonate buffer, and 1 mL o f deionized organic free w ater. 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 buffo: and 1 mL o f deionized water. V ortex each tube for about 5 seconds.
5.4 Add 2.5 m L o f ethyl acetate and extract on horizontal m ixer 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 m inutes.
5.6 Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness
(e.g., ~ 50 m inutes in the Turbo-Vap ) w ith a gentle stream o f nitrogen and moderate
heat (e.g., 50 *C).
.
Page 9 o f 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
5.7 Reconstitute the residue in 500 pL o f 5% 5 m M ammonium acetate: 95% methanol containing 1.5% form ic acid and vortex briefly to m ix. Filter the sam ples through 0.2 pm PVDF or N ylon syringe filters into autosam pler 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 o f each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. If necessary, dilute an aliquot o f any sample w ith blank m atrix so that the expected concentration o f the test article being analyzed w ill fall w ithin the concentration range o f the standard curve. Add 10 pL o f internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple o f seconds.
6.2 For serum sam ples 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 m L o f deionized organic free w ater. V ortex each tube for about 5 seconds. F or urine samples add the following to each tube 1 m L o f 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.
6.3 A dd 2.5 mL o f ethyl acetate and extract on horizontal m ixer 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 m inutes.
6.5 Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 m inutes in the Turbo-Vap ) w ith a gentle stream o f nitrogen and moderate heat (e.g., 50 *C).
6.6 Reconstitute the residue in 500 pL o f 5% 5 mM ammonium acetate: 95% methanol containing 1.5% form ic acid and vortex briefly to m ix. Filter the samples through 0.2 pm PVDF or N ylon syringe filters into autosam pler vials. Cap vials for analysis.
Page 10 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and A nalysis by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/M S/M S)
7.0 7.1 7.1.1
7.1.2
ANALYSIS BY H IG H PERFORM ANCE LIQ UID CHROM ATOGRAPHY MASS SPECTRO M ETRY /M A SS SPEC TR O M ETR Y (H PLC/M S/M S)
Conditions are to be optim ized if necessary.
HPLC Conditions
A nalytical Column: Keystone Scientific Aquasil C l 8,150 mm x 2 mm ID, or equivalent
Guard Column: Elution Flow rate: Injection volume: M obile phase:
Keystone A quasil C l8 10 mm x 2 mm 600 jiL/min. 5 pL A: 5mM ammonium acetate buffer B: 1.5% formic acid in m ethanol
G radient Profile: T em perature:
0 - 3 m in. 3 - 5min. 5 - 8 min. 8-10 min. A m bient
50%A : 50%B 20% A : 80% B linear gradient 10%A : 90% B step gradient 50%A : 50%B step gradient
PE Sciex A PI 3000 Triple Quadrupole M ass Spectrom eter Conditions Software: PE Sciex TurboQuan
Turboion Snrav Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table.
A uxiliary Gas:
A ir (e.g., Grade 0.1) at 85 pounds per square inch
Param eter IS
V alu e -2000
Page 11 o f 14
ANALYTICAL METHOD
Method No.: BACG-3606
Title:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (H PL C /M S/M S)
Param eter NC TEM OR RNG QO IQ1 ST ROl IQ2 R02 ST3 R03 DF CEM NEB CUR CAD QPE POL VCM
V alue 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 15 6 5 0 1 0
Page 12 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
Param eter IPE
V alue 0
M S/M S A cquisition Conditions
Scan type: M RM
P o la rity .
N egative
A cquisition mode: Profile
Pause tim e:
5 m illiseconds
M asses requested:
PFHS: O l M ass (amili 398.9
0 3 M ass (annil 398.9
Dwell Time (ms} 200
PFOC (IS) O l Mass (awni! 412.9
P aram eter R02 ST3 R03 QO IQ1 ST ROl IQ2
0 3 Mass famnl 368.9
Sfert 35 45 37 18 19 23 19 20
S2E 35 45 37 18 19 23 19 20
Dwell Time im sl 200
Page 13 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
Title:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/M S/M S)
8.0 CALCULATIONS
8.1 A t the end o f the analytical run, review each chromatogram to ensure die retention time, peak shape, and peak height and peak area determination o f the test article and the IS are
. acceptable. The data m ay be smoothed as appropriate. For quantitation, use the ion profiles at the follow ing m ass-to-charge ratios:
A n alv te PFHS PFOC
Ion Profile 398.9 to 398.9 412.9 to 368.9
8.2 Plot the peak area response o f PFHS divided by the peak area response o f the IS (PFOC) from all standards versus the concentration o f the test article in the standards. Alternatively, the peak heights m aybe used instead o f peak areas. Obtain the best curve fit o f the data (e.g., quadratic fit w eighted w ith 1/concentration o f the test article or a quadratic fit). N ote: The best curve fit m ay be dependent on the range o f the standard curve and it m ay be necessary to have m ore than one standard curve for various concentration ranges using the following:
y = ax2+ b x + c
where y = Peak height response o f PFHS divided by peak height response o f . the IS (PFOC) in standards.
x = Concentration o f the PFHS in standards. a, b, c = Constants derived from the regression analysis.
8.3 Using the standard curve, calculate the level o f PFHS in each unknown sample. Correct the results o f sam ples for any dilutions.
NOTE:
Due to unresolvable interferences w ith the test article and/or internal standard from the m atrix, external standard quantitation may be used at the discretion o f the supervising mass spectrom etrist.
Page 14 of 14
ANALYTICAL METHOD
Method No.: BACG-3606
T itle:
D eterm ination o f Perfluorohexanesulfonate in M onkey Serum and Urine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
9.0 A CCEPTA N CE AND R E JE C T IO N C R IT E R IA 9.1 R efer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for
standards is 80-120% o f theoretical.
10.0 R E PO R TIN G 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 uthors):
Sr. Research Associate Bioanalytical Chem istry Group
Approved by: Greg Gorm an, Ph.D . M anager Bioanalytical Chem istry Group
9/7/
D ate
9/7/03
D ate
Page 1 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample . Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry
(HPLC/M S/M S)
1.0
2.0 2.1 2.1.1 2.1.2
PRINCIPLE
Serum or urine sam ples are obtained from cynomolgus monkeys treated with Perfluorooctanesulfonate (PFOS). The serum or urine (e.g., 0.5 mL) containing (PFOS) ' is fortified w ith an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The sam ples are then m ixed w ith an ion-pairing reagent, buffer and w ater, followed by extraction w ith ethyl acetate. The ethyl acetate layer is rem oved, evaporated to dryness, reconstituted in 95% m ethanol containing 1.5 % fonnic acid, 5 % 5mM ammonium acetate, filtered, and transferred to autosam pler vials, and analyzed by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/MS/MS). The range o f reliable results extends from about 20 to 10,000 ng/mL o f PFOS in serum and from 10 to 500 ng/mL in urine. Samples containing PFOS at concentrations greater than 10,000 ng/m L may be diluted w ith control blank m atrix so that the concentration o f PFOS w ill be w ithin the range o f reliable results prior to analysis.
The m ass spectrom etry o f PFOS and PFOC is accom plished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the form ation o f other potentially interfering ions extracted from the m atrix.
CA U TIO N : Since prim ates m ay cany a num ber o f zoonoses, all unpreserved tissues, including blood, plasm a and serum, are to be considered as biohazards and handled with univ ersal p recautions. Refer to SOP num ber SRI 2-5-5 for a description o f safety procedures to be used w hen handling unpreserved prim ate tissue.
REAGENTS AND SOLUTIONS
The listed reagents or their equivalents m ay be used.
N eat R eagents
W ater, deionized and organic free (from in-house purification system; e.g., Ingalls 210N)
M ethanol, HPLC grade
Page 2 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
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
B lank control m onkey serum
2.1.7
Sodium Carbonate, C ertified CS Grade or equivalent
2.1.8
Sodium Bicarbonate, C ertified 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, C ertified grade or equivalent
2.1.12 B lank control m onkey urine
2.1.13 Form ic Acid, 88%
2.2 P rep ared Solutions
A ppropriate changes in the solutions m ay be made at the discretion o f the analyst
2.2.1
5 mM Ammonium acetate in organic free w ater
2.2.1.1
For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free w ater (e.g., 4 L). M ix w ell and filter through HPLC mobile phase filtration apparatus.
2.2.2
TBA Ion-Pairing Solution (0.5 M tetrabutylam m onium hydroxide)
Page 3 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
2.2.2.1
For example, to prepare 25 mL, dissolve 4.24 g o f tetrabutylammonium hydrogen sulfate in deionized w ater and adjust the pH to 10 w ith 50% NaOH solution. N ote: a m ore dilu te solution of N aO H in w ater m ay b e 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 o f deionized water. M ix well to ensure complete dissolution.
2.2.4
Carbonate/Bicarbonate Buffer Solution for U rine (1.0M /1.0M )
2.2.4.1
For example, to prepare 100 mL, dissolve approximately 10.6 g o f sodium carbonate and approxim ately 8.4 g o f sodium bicarbonate in 100 m L o f deionized w ater. M ix well to ensure com plete dissolution.
3.0 IN STR U M EN TS, M A TER IA LS, AND APPARATUS
The following or their equivalents m ay be used.
3.1 HPLC pum p(s), autosam pler, and triple quadrupole mass spectrom eter
3.2 A utosam pler vials w ith inserts
3.3 Vortex m ixers (e.g., touch m ixer and IKA-Vibrax platform m ixer)
3.4 Solvent-concentration apparatus (e.g., Zym ark Turbo-Vap w ith source o f nitrogen)
3.5 HPLC m obile phase filtration apparatus
3.6 Filters for HPLC m obile phase filtration apparatus (e.g., N ylon-66,0.20 pm)
Page 4 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
Determ ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrometry (H PL C /M S/M S)
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
Volum etric flasks (e.g., 10 and 25 mL)
D isposable Pasteur pipettes
M icropipettor(s) w ith tips
Culture tubes w ith teflon-lined caps
C en trifu g e
I
A ssorted glassware and syringes
.
Culture tubes (vials) for use w ith solvent-concentration apparatus
1 mL Plastic syringes w ith 0.2 pm PVDF syringe filters
Variable speed horizontal platform shaker
pH m eter
PREPARATION O F STOCKS AND W ORKING STOCKS
Appropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst Actual dilutions w ill be documented on die preparation sheets. M ain Stock Solution o f PFO S ~1000 pg/m L
Prepare an -10 0 0 pg/m L solution o f PFOS in deionized organic-free w ater (e.g., accurately weigh about 10 m g PFOS into a 10-mL volum etric flask). A dd deionized organic-free w ater to dissolve. D ilute to the mark. Alternatively, weigh the compound
Page 5 o f 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
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 m L o f deionized organic-free w ater. M ix w ell. Transfer the solution to a clean vessel if desired.
Stock Solution o f In tern al S tan d ard (PFO C ), ~200 pg/m L
Prepare an ~200pg/mL solution o f PFOC in deionized organic-free w ater (e.g., accurately w eigh about 10 m g into a 50-mL volum etric flask). Add deionized organic-free w ater to dissolve and dilute to the m ark w ith deionized organic-free water. Alternatively, weigh the com pound into a an appropriate vessel (e.g., culture tube) and add 50 m L o f deionized organic-free w ater. M ix w ell. Transfer the solution to a clean vessel if desired.
Spiking solution o f In te rn a l S tan d ard (PFO C ), ~ 50pg/m L
Prepare an ~ 50 pg/mL solution o f PFOC in deionized organic- free w ater by pipetting 2 mL o f the 200 pg/mL solution into a culture tube and add 6 mL o f deionized water. M ix well.
W orking Stock Solutions o f PFO S
To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volum etric flasks or other appropriate glassware. I f desired a m odified dilution scheme can be used and documented in the study records.
W orking Stock Level (W SL) Approxim ate Concentration
(ng/m L)
Volum e o f PFOS solution
500,000 250,000
5 mL o f 1000 pg/mL 5 m L o f 500,000 ng/mL
Final Volume in deionized organic free w ater (mL)
10
10
Page 6 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
62,500 2.5 mL o f 250,000 ng/mL 31,250 5 m L o f 62,500 ng/mL 15,625 5 m L o f 31,250 n g/m L
5,000 50 pL o f 1000 pg/mL stock 2,500 5 m L o f 5000 ng/mL stock 1,000 4 mL o f 2500 ng/mL stock
500 5 mL o f 1000 ng/mL stock 250 5 mL o f 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
A B C
Volume and Spike Cone.
10 pL o f 500,000 ng/mL 10 pL o f 250,000 ng/mL 16 pL o f 62,500 ng/mL
A pproxim ate Concentration o f PFOS in serum
(ng/m L )
10,000
5,000
2,000
D 16 pL o f 31,250 ng/mL
1,000
Page 7 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
E 16 pL o f 15,625 ng/mL F 20 pL o f 5,000 ng/mL G 10 pL o f 5,000 ng/mL H 10 pL o f 2,500 ng/mL I 10 pL o f 1,000 ng/mL J 10 pL o f 500 ng/mL K 10 pL o f 250 ng/m L
500 200 100 50 20 10
5
5.0 PR EPA R A T IO N O F SPIK ED STANDARDS AND BLA N K S
Appropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst.
5.1 M ultiple (e.g., about three) sets o f m atrix standards and a m atrix blank (blank + IS) are analyzed w ith each set o f unknown samples. A m atrix double blank (blank-IS) may also be analyzed if desired.
5.2 Into individual -20-m L culture tubes, pipet blank m atrix (e.g., 0.5 m L ). Pipet in the appropriate volum e as described in the table above for each standard. For the blanks, pipet 10 pL o f organic-free w ater 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 o f organic-free w ater instead) and vortex for ~5 seconds.
5.3 For serum sam ples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 m L o f 0.25M /0.25M carbonate/bicarbonate buffer, and 1 m L o f deionized organic free w ater. Vortex each tube for about 5 seconds. For urine sam ples add the follow ing to each tube 1 mL o f TBA ion-pairing solution, 1 m L o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds.
Page 8 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and Analysis, by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
5.4 A dd 2.5 m L o f ethyl acetate and extract on horizontal m ixer for 1 hour at a low speed setting.
5.5 Rem ove the tube from the shaker and place in a centrifuge (e.g., 2500 ipm ) for about 5 m inutes.
5.6 Take o ff the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 m inutes in the Turbo-Vap ) w ith a gentle stream o f nitrogen and moderate heat (e.g., 50 C).
5.7 R econstitute the residue in 500 pL o f 5% 5 mM ammonium acetate: 95% methanol containing 1.5% form ic acid and vortex briefly to m ix. Filter the samples through 0.2 pm PVDF or N ylon syringe filters into autosam pler 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 o f each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. I f necessary, dilute an aliquot o f any sam ple w ith blank m atrix so that the expected concentration o f the test article being analyzed w ill fall within the concentration range o f the standard curve. Add 10 pL o f internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple o f seconds.
6.2 For serum sam ples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 m L o f 0.25M /0.25M carbonate/bicarbonate buffer, and 1 mL o f deionized organic free w ater. V ortex each tube for about 5 seconds. For urine sam ples add the follow ing 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 o f deionized water. Vortex each tube for about 5 seconds.
6.3 Add 2.5 m L o f ethyl acetate and extract on horizontal m ixer for 1 hour at a low speed setting.
Page 9 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrom etry (HPLC/M S/M S)
6.4 Rem ove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm ) for about 5 m inutes.
6.5 Take o ff the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 m inutes in the Turbo-Vap ) w ith a gentle stream o f nitrogen and m oderate heat (e.g., 50 C).
6.6 Reconstitute the residue in 500 pL o f 5% 5 mM ammonium acetate: 95% m ethanol containing 1.5% form ic acid and vortex briefly to mix. Filter the sam ples through 0.2
. pm PVDF or N ylon syringe filters into autosam pler vials. Cap vials for analysis.
7.0 ANALYSIS BY H IG H PERFORM ANCE LIQUID CHROM ATOGRAPHY MASS SPECTRO M ETRY /M A SS SPECTRO M ETRY (H PLC/M S/M S)
7.1 Conditions are to be optim ized i f necessary.
7.1.1
HPLC Conditions
A nalytical Column: Keystone Scientific Aquasil C 1 8 ,150 mm x 2 mm ID, or equivalent
Guard Column: Elution Flow rate: Injection volume: M obile phase:
Keystone Aquasil C l8 10 mm x 2 mm 400 pL/min. 5 pL A: 5mM ammonium acetate buffer B: 1.5% formic acid in m ethanol
G radient Profile: T em perature:
0 - 3 min. 3 - 5min. 5 - 8 min. 8-10 min. A m bient
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 M ass Spectrom eter Conditions Software: PE Sciex TurboQuan
Page 10 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample Preparation and A nalysis by HPLC M ass Spectrometry/M ass Spectrometry (HPLC/M S/M S)
Turboion Snrav Source Note: V alues listed under "M S/M S Acquisition Conditions" override param eters in this table.
A uxiliary Gas:
A ir (e.g., Grade 0.1) at 85 pounds per square inch
Param eter IS NC TEM OR RNG Q0 IQ1 ST ROl IQ 2 R02 ST3 R03 DF CEM
V alu e -2000 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800
Method No.: BACG-3607
ANALYTICAL METHOD
Page 11 of 13
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and A nalysis by HPLC M ass Spectrom etry/M ass Spectrom etry (HPLC/M S/M S)
Param eter NEB CUR CAD QPE POL VCM IPE
V alue
15 6 5 0 1 0 0
MS/MS A cauisition Conditions
Scan type: MRM
Polarity:
N egative
Acquisition mode:
P ro file
Pause time: 5 milliseconds
M asses requested:
PFOS:
Q1 Mass (am ul
498.9
0 3 Mass tam ul 498.9
PFOC (IS) O l Mass famul
412.9
0 3 Mass tam ul 368.9
P aram eter R02 ST3 R03
QO IQ1 ST
Start 35 45 37 18 19 23
Slog 35 45 37 18 19 23
Dwell Time Cm}
200
Dwell Time tm sl 200
1
Page 12 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and U rine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
R O l 19 19 IQ2 20 20
8.0 CALCULATIONS
8.1 A t the end o f the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination o f the test article and the IS are acceptable. The data m ay be smoothed as appropriate. For quantitation, use the ion profiles at the follow ing m ass-to-charge ratios:
A n alv te PFOS PFOC
Ion Profile 498.9 to 498.9 412.9 to 368.9
8.2 Plot the peak area response o f PFOS divided by the peak area response o f the IS (PFOC)
from all standards versus the concentration o f the test article in the standards.
A lternatively, the peak heights m aybe used instead o f peak areas. O btain the best curve
fit o f the data (e.g., quadratic fit weighted w ith 1/concentration o f the test article o r a
quadratic fit). Note: The best curve fit may be dependent on the range o f the standard
curve and it m ay be necessary to have m ore than one standard curve for various
concentration ranges using the following:
.
y = ax2+ b x + c
w here
y=
x= a, b, c
Peak height response o f PFOS divided by peak height response o f the IS (PFOC) in standards, Concentration o f the PFOS in standards, - Constants derived from the regression analysis.
Page 13 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
T itle:
D eterm ination o f Perfluorooctanesulfonate in M onkey Serum and Urine: Sample Preparation and Analysis by HPLC M ass Spectrom etry/M ass Spectrometry (HPLC/M S/M S)
8.3 U sing the standard curve, calculate the level o f PFHC in each unknown sample. Correct the results o f sam ples for any dilutions.
NOTE:
Due to unresolvable interferences w ith the test article and/or internal standard from the m atrix, external standard quantitation m ay be used at the discretion o f the supervising mass spectrom etrist.
9.0 ACCEPTANCE AND REJECTION CRITERIA
9.1 R efer 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.
Authorise :
Lori Coward, BS Sr. Research Associate B ioanalytical C hem istry Group
Approved by: G reg G orm an, Ph.D . M anager B ioanalytical Chem istry Group
D ate
9/)/o
D ate
