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M am '*, ) BIOANALYTICAL REPORT
Title:
Date: Authors:
Quantitative Determination o f Ammonium Perfluorooctanoate (APFO), M easured as the Perfluorooctanoate Anion (PFOA), in Human Serum from Study DW04 Using Turbo Ion Spray LC/MS/MS
30 September 2003
David J. Anderson, M.S. Susannah L. Murphy, B.S. Jennie L. Romney, M.P.S.
Advion BioSdences, Inc. 15 Catherwood Road Ithaca, New York 14850
Prepared For:
DuPont Haskell Laboratory for Health and Environmental Science 1090 Elkton Road P.O. Box 50 Newark, D E 1971
Advion Report No.:
<
Advion Intanai Study ID:
02331AD JA JP U .D O C DW04
Number o f Pages:
19
Summary and Conclusions
The concentration o f ammonium perfluorooctanoate (APFO), measured as the perfluorooctanoate anion (PFOA), was determined in human serum samples collected during the Study DW04 using a selective, accurate, and reproducible analytical method developed byAdvion BioSciences, Inc., Ithaca, NY.
Human serum samples (0.025 xnL) were extracted by a liquid-liquid extraction procedure to isolate PFOA and the internal standard (9H-hexadecafluorononanoic add, 9H-HDFNA). Following reconstitution, sample extracts were analyzed by turbo ion spray liquid ctoomatography/tandem mass spectrometry (LC/MS/MS) in the negative ion mode.
A ll samples were successfully analyzed within one run. The lower limit o f quantitation was 0.05 pg/mL for PFO A The precision o f this assay (CV) as determined from the analysis o f quality control (QC) samples for PFOA was 4.11%.
om pany S aftnized. Dobs n ot co n tain TSCA CBI
00134
Title:
a d v io n b io sc b e n c e s
SIGNATURE PAGE
Quantitative Detennination o f Ammonium Perfhiorooctanoate (APPO), Measured as the Perfluoraactauoate Anion (PFOA), in Human Serum from Study DW 04 Using Turbo Ion Spray LC/MS/MS
Advion Report No. 02331ADJA DU.
Reported by:
David J. Andason, M.S. Manager o f Bioanalysis
S s e P zoS Date
Authorized for Release by:
D irector ofB io an aly sis
Date
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QAU STATEMENT Periodic inspections of the bioanalytical portion o f Study PW 04 were conducted by the
The study was inspected on die following dates:
Phase o f S tudy Inspected
D ate o f Inspection
Sam ple L o o n B inder Data R eview R eport
8 January 2003 31 D ecem ber 2002 17 Septem ber 2003
Dare R eported to M anagem ent
8 January 2003 31 D ecem ber 2002 18 Septem ber 2003
Based on the inspections and the data reviewed, this report is a complete and accurate representation o f the data.
George C. Amedro, B.S. Quality Assurance Auditor
D ate
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.' )
TABLE OF CONTENTS
SIGNATURE PA G E................................... .............:......................... -............................ ,...2
QAU STATEMENT........................... ........................................................ - ,....................... 3
TABLE OF CONTENTS................................ .......-............................................ .................4
LIST OF TABLES-- .................. ....................................................... ...................................5
LIST OF FIGURES......................................................... .................. .......................... .....-- 6
1. INTRODUCTION....... `..................................................
7
L L Study Description and Objective......... ...................... .........-- .......................--.7
2. METHODS...................................................................................................................... 7 2.1. Analytical Procedure(s) --....... -,.......- ,......................... -- ............................... -- 7 2.2. Assay S ite . .,.................,................................................... ...........................- ......... 8 2.3. D ata Processing........................................... ....................... ...................................... 8
3. R E S U L T S -................................................................................
8
3.1. Assay P erfo rm an ce...................................... ............................................ ............. 8
3.2. Analytical Results for Study DW 04....... .............. .................................................... 9
4. DATARETRIEVAL..................
9
5. REFERENCES.........................................
9
6. TABLES..... .......................
..1 0
7. FIGURES.............................
16
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LIST OF TABLES
Table I: Summary o f Sample and Assay Information for Study D W 04............... ,........10 Table 2: Precision and Accuracy for PFOA Quality Control Samples in Human Serum
for Study DW 04......... ,.................................................................................. 11 Table 3: Precision and Accuracy for PFOA Calibration Standards in Human Serum for
Study DW 04.... ...................................................................................................12 Table 4: Calibration Curve Statistics .for the Detennination o f PFOA in Human Seram
for Study DW04...............................,.......................... ......................................13 Table 5: Concentrations o f PFOA in Human Serum Samples from Study DW04..--....14
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LIST OF FIGURES
Figure 1: Selected Reaction Monitoring Chromatograms o f PFOA and trie Internal Standard in Control Blank Human Serum E xtract............................................ 16
Figure 2: Selected Reaction Monitoring Chromatograms o fPFOA and the internal Standard from a Human Saum Extract Containing Internal Standard Only (Zero Sam ple)............................................. ................... ....................................17
Figure 3: Selected Reaction Monitoring Chromatograms o f PFOA and trie M arnai Standard from a Standard 1 Human Serum Extract (0.05 ng/m L)..........
18
Figure 4: Selected Reaction Monitoring Chromatograms of PFOA and trie Internal Standard from a Standard 9 Human Serum Extract (10 pg/m L)......................19
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1. IN TRO D U CTIO N
1.1. Study D escription and Objective
The objective o f this study was to determine die concentrations o f ammonium perfluorooctanoate (APFO), measured as the perfluorooctanoate anion (PFOA), in human serum sam ples collected during Study DW04. The study ID was generated by Advion BioSciences, Inc. (Advion) for internal tracking purposes. Human serum samples were analyzed using a selective liquid-liquid extraction, turbo ion spray liquid chromatography/ tandem m ass spectrometry (LC/MS/MS) assay. The work described in this bioanalytical report was conducted in accordance with Advion standard operating procedures (SOP).
2. M ETH O D S
2.1. A nalytical P rocedure^)
PFOA concentrations were determined jh human serum according to the validated LC/MS/MS m ethod (1), Serum samples (0.025 mL) were extracted by a liquid-liquid extraction procedure to isolate the analyte and internal standard (IS) finm human serum. Following reconstitution, sample extracts were separated by reversed-phase chromatography on a 2 x 50 mm (5 pm) Betasil C column (ThermoHypersil-Keystone, Bellefonte, PA) w ith an initial mobile phase o f 45% Eluent B (90:10 methanol: 2 mM ammomum acetate) and 55% Eluent A (10:90 methanol:2 mM ammonium acetate). 9H-hexadeca0uorononanoic acid (9H-KDFNA) was used as the IS for PFOA. PFOA concentrations were determined by turbo ion spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the negative ion mode.
Following background subtraction (for calibration standards, QC samples, and zero samples), study sample concentrations were determined from a weighted (1/x2), quadratic regression o f peak area ratios (peak area o f PPQA/peak area o f 9H-HDFNA) versus nominal concentration ofnine calibration standards. The nominal concentrations o f the calibration standards were 0.05,0.1, 0.25, 0 .5 ,1 ,2 ,5 ,7 .5 and 10 fig/mL PFO A
The lqw er liirnt o f quantitation (LLQ) for this assay was 0.05 pg/mLPFOA. Quality control (QC) serum samples at three different concentrations (0.15,3, and 8 jig/m L PFOA) w ere analyzed in replicates o f three.
The acceptance criteria for calibration standards stipulated that at least three-fourths o f the calibration standards must remain on the calibration curve for each analytical run. Calibration standards may be rejected if they show deviations >15% from th en nominal concentration (20% at the LLQ).' The coefficient o f determination (r2) must be >0.98.
The acceptance criteria for the quality control samples stipulated that two-thirds o f the individual QC samples must be within 15% o fthe nominal concm tiation, and at least
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one-half o f the replicates at each QC concentration must be w ithin 15% o fthe nominal concentration.
A summary o f the sample and assay information for Study DW 04 is given in Table 1. Trip blanks were prepared at Advion in advance o f the collection o f serum samples in this study. The trip blanks were prepared by adding control hum an serum to collection tubes. The purpose o f the trip blanks was as a precautionary means to m onitor fin potential contamination o f study samples during shipm ent
2.2. Assay Site
All samples were analyzed at Advion BioSciences, Inc., Ithaca, NY,
2.3. D ata Processing
The data were collected using turbo ion spray LC/MS/MS in the negative ion mode. Peak areas were integrated by the Applied BioSystems program MacQuan, version 1.6, residing on a Macintosh computer. Following peak area integration, the results tables to m MacQuan were saved as text files, which were used to perform background subtraction, using Excel 98, to correct for the presence o f PFOA in the control serum lots. Background-subtracted data were saved as MacQuan text files and uploaded to the Advion file server where a weighted (1/xz) quadratic regression was performed using the software package W atson (v 6.1.0.03, InnaPhase Corp., Philadelphia, PA). Concentration data were downloaded to m Watson with three significant figures!. Statistics were downloaded from Watson with three decimal places and rounded to three significant figures prior to reporting.
3. RESULTS
3.1. Assay Perform ance
The performance of the assay for PFOA as determined from the analysis o f QC samples is documented in Table 2. The intra-assay precision (CV) o f QC samples ranged from 3.14 to 4.11% for PFOA. There was no marked inaccuracy in the results from these Q samples; mean accuracy (RE) ranged from -10.7 to -6.33%.
The performance of daily calibration curves is documented in Table 3. There was no marked inaccuracy in the results from these standards; m ean accuracy ranged from -2.60 to 5.00%.
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The slope, y-mtercept, and coefficient o f determination (r2) for the analytical run are presented in Table 4. The r2 value was 0.9958 for PFOA in human serum.
AD Advion personnel assigned to analyze samples for this project were required to successfully extract QC samples and calibration standards. Results o f the extraction were reviewed by a trained analyst against the pre-defined acceptance criteria for this assay. Results are maintained in the study records o f Advion.
Representative chromatograms from a PFOA control blank and zero sample are presented in Figures 1 and 2, respectively. Representative chromatograms from PFOA standards 1 and 9 are presented in Figures 3 and 4, respectively.
3.2. A nalytical R esults for Study DW04
PFOA concentrations in human serum from Study DW04 are presented in ta b le 5. A ll data were rounded to no less than three significant figures before reporting in Table 5. The two trip blanks are identified as subject numbers 98009 and 98010.
4. DATA RETRIEVAL
The calculated concentration data from the analysis o fhuman serum samples for Study DW04 are maintained on file in the archives o fAdvion BioSciences, Inc., Ithaca, NY in Advion Notebook 02331.
5. REFERENCES
1. Advion BioSciences Validation Report 02120VDJA_DU.DOC. Method Validation for the Quantitation o fAmmonium Perfluorooctanoate (APFO), Measured as the Perfluorooctanoate Anion (PFOA, 0.05 to 10 pg/mL Concentration Range), in Human Serum by Turbo Ion Spray LC/MS/MS. 12 July 2002.
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6, TABLES
T able 1: Sum m ary of Sam ple an d Assay Inform ation fo r S tudy B'W04
Species/M atrix:
H um aa/Seniip
Sam ple Collection and Storage Inform ation:
Anticoagulant/Stabilizer: Date Received at Advion: Storage Temperature at Advion: Number o f Study Samples Received: Number o f Study Samples Analyzed:
Not applicable 10 October 2002 -20 C 62 62
Assay Inform ation: Assay Period:
29 October 2002
A nalyte: Analytical Standard:
Lot No: Source:
PFOA PFOA (supplied as ammonium perfluarooctanoate, APFO) 421207/1 Sigma-AIdrich
internal Standard: Lot No: Source:
-9H-HDFNA None available
S igm a-A ldrich
Calibration Range: Regression Method; Weighting Factor: Lower Limit of Quantitation: Upper Limit of Quantitation;
Quadratic i/x 2 0.05 pg/mL lOpg/m L
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U
Table 3; Precision and Accuracy for PFOA C alibration S tandards in Hum an Serum for Study DW04
Run Number STD1
STD2
0.05 0.1
1 0.0444 0.0984
0.0534 0.111
Mean
0.0489
0.105
C V (% ) RE (%)
NA -2.20
NA 5.00
CV * (SD/Mean)*lOO
RE = [(Mean-Nominal)/Nominal}* 100
NA = N ot applicable
STD3 0.25 0.232 0.262
0.247 NA -1.20
PFOA Concentration (pg/mL)
STD4 0.5
STD5 1
S D 6 2
0.495 0.512
0.948 1.00
1.93 2.11
0.504
0.974
2.02
NA 0.800
NA -2.60
NA 1.00
STD7 5
. 4.92 4.85
4.89 NA -2.20
STD8 7.5 7.68 7.53
7.61 NA 1.47
STD9 10 10.2 9.86
10.0 NA 0.00
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Table 4:
C alibration Curve Statistics fo r the D eterm ination o f PFOA in H um an Serum for Study DW04
Run Number Quadratic
Linear
Intercept (Q
Coefficient (A) Coefficient (B)
1
-0.004962
0.357472
-0.001110
y ~ A ? + Bx + C, weighted 1/x', after background subtraction
Coefficient o f Determination (i2)
0.9958
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TafaleS:
C oncentrations of PFOA in Hum an Serum Sam ples from Study DWQ4 .
Subject Number
57178 57179 57180 57181 57182 57183 57184 57185 57186 57187 57188 57189 57190 57191 57192 57193 57194 57195 57196 57197
57198 57199 57200 57201 57202 57203 57204 57205 57206 57207 57208 57209 57210 57211 57212 57213 57214
PFOA Concentration (pg/mL)
0.168 0.175 0.0788 1.65 <LLQ <LLQ <LLQ 0.0561 <LLQ
1.13 1.11 ' <LLQ <LLQ <LLQ <LLQ <LLQ <LLQ <LLQ 0.0543 <LLQ 0.0978 0.347 0.127 0.0834 0.277, . 0.0506 0.297 0.233 0.0527 0.174 0.125 <LLQ 0.0565 0.210 <LLQ <LLQ 0-658
.. .continued
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Table 5:
(Continued) C oncentrations of PFOA in Human Serum Samples from Study DW04
Subject Number
PFOA Concentration Gig/mL)
57215 57216 98009 98010 98039 98040 98041 98047 98048 98049 98055 98056 98133 98134 98135 98136 98137 98138 98139 98140 98141 98142
0.0910 0.0750 <LLQ <LLQ <LLQ <LLQ 0.244 0.0598 0.137 <LLQ 0.106 0.199 <LLQ 0.0885 0.242 0.226 0.359 0.122 0.0629 <LLQ 0.127 0.264
98143 98144 98145 98146 98147
0.0807 <LLQ <LLQ 0.0576 0.313
<LLQ: Less than the Lower Limit of
Quantitation (0.05 pg/inL)
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7. FIGURES
Figure 1: Selected Reaction M onitoring Chrom atogram s of PFOA and the In tern al S tandard in C ontrol B lank H um an Ser am E xtract
tniz = 413.0
=* 169.0, PFOA
1.45e2 cps
C ontrol B lank, R an 1, Injection 7
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Figure 2:
Selected Reaction M onitoring C hrom atogram s o f PFOA and the In tern al S tandard from a H um an Serum E xtract C ontaining Internal S tandard Only (Zero Sample)
m /z = 413.0 -> m /z = 169.0, PFOA
2.05c2 cps
[,, ^ 0.9
i
1.8
Time, min
-- --------- r" 2.7
Zero Sam ple, R un 1, Injection 93
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Figure 3:
Selected R eaction M onitoring C hrom atogram s o f PFOA and th e In tern al S tandard from a S tandard 1 H um an Serum E xtract (0.05 jxg/mL)
m /z = 413-0 -> m /z - 169.0, PFOA
9.10e2 cps
~ r--'--:-- -----~t~-- :------ ---*"-- !--r
0.9 1.8
2.7
Tim e, m in
Standard 1, R un 1, Injection 8
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Figure 4:
Selected Reaction M onitoring Chrom atogram s of PFO A an d the In tern al S tandard from a S tan d ard 9 Hainan Serum E x tract (10 jig/mL)
w/z = 413.0 -* m/z = 169.0, PFDA
1.68e5 cps
Relative Ion A bundance (% )
8060-
40-
20-
------ --------- !----- j------ --------" ' i ------ '-- r-
0.9 1.8 2.7 Tim e, m in
S tandard 9 , R on 1, Injection 16
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