Document jBq5XzbObDvNQq9emvqZwOEJk

M a n . 5l/J>0 000046 Afi.a&6 - dK-00 National M edical Services November 9 ,2 0 0 1 Mary Kaiser, Ph.D. DuPont Centrai Research & Development /ilmington, DE 19880-0269 j r ) Dear Dr. Kaiser: We have completed work on the development and validation o f a new procedure for the analysis o f PFOA in blood. Please find enclosed an updated Test information Summary Sheet and a summary o f our findings. As always, we look forward to and welcome discuss on concerning the particulars o f this analysis. Specifically we greatly anticipate your comments concerning the proposed methodology change. Sincerely, j. Does not contain tSCA CBl 000047 PFOA Method Change Proposal November 4, 2001 Background: In an attempt to better certain aspects o f the method currently in place to test for the presence and amount o f perfluorooctanoic acid (PFA) a new sample preparation procedure was developed and validated. In brief, an internal standard (perfluorodecanoic acid) is added to the sample and the acids derivatized to their methyl esters utilizing methanohc HC1. AU derivatives are then extracted into hexane prior to analysis by GC-ECD. The main advantages o f using this new procedure in conjunction with the results of the validation for it are outlined and discussed below. Reporting Limit: The reporting limit for this analysis has historically been lOppb and in this regard it is of importance that this value be consistently achievable. However, during the course o f analysis we observed interferences that did not allow us to reprodueibly detect this critical lOppb point. In essence, this point was "buried" in the baseline and as a consequence the reporting limit had to be increased to a greater value. Investigation revealed that this problem was in fact due to one of the reagents that used during the sample preparation part o f the procedure. Because purchasing fresh material did not alleviate this problem, it was determined that a new method be developed. By using the proposed method the interferences that caused the problem detailed above are no longer present. A reporting limit of lOppb is consistently obtained (See Figure i). Patient Results: To test the validity o f the method 33 patient samples, along with die appropriate quality controls, were analyzed using both types ofsample preparation procedures and their results compared, With the exception o f four patient samples, all results are comparable with values not differing by more than 30%. The patient samples where this criterion did not hold true were samples where the described interferences were present. Overall ConclusionJ: 1. A reporting limit o f 10 ppb is consistently Obtained. 2. Interferences that were observed and, in some samples, affected accuracy f results are no longer present. 3. Patient results are comparable when analyzed using both methods. 4. The greater ease o f sample preparation allows us to handle a higher volume o f samples at one time. I 00048 NAME OF TEST: SYNONYMS: ANALYTE GLASSIFICATION: METHOD PRINCIPLE: ALTERNATIVE METHODS: SPECIMEN REQUIREMENTS: PERFLUOROOCTANOIC ACID NMS # 3425 PFOA, FC-143 Component. Perfluorocaprytic acid Fluorinated Organic Acid This lest is a quantitative analysis of perfluorooctanoic acid in whole blood and serum specimens. This is achieved by adding an internal standard (perfluorodecanoic acid). The acids are then derivatized to their methyl esters with methanolic HCI, which are then extracted into hexane. Analysis is by gas chromatography with an electron capture detector on a QB1701 capillary column. None available SPECIAL HANDLING: REPORTING LIMIT: TAT: PHARMACOTOXICOLOGIC DATA: LIMITATIONS OF METHOD: REFERENCES: I Store refrigerated. 10 ng/mL Set-up on a by-arrangement basis. Little toxicological data is available on perfluorooctanoic acid. It is known to have a very long elimination half-life. 1. Quantitative results cannot exceed the highest working standard or be less lhan the lowest working standard for that run. 2. There are no known interferences for this analysis. 1. Belisle, J, and Hagen. D.F.. A Method for Determination of Perfluorooctanoic Acid in Blood and Other Biological Samples. Analytical Biochemistry, 101.369-376(1380). 2. Ubel, F A .. Sorenson, S.D., and Roach, D.E., Health Status of Plant Workers Exposed to Fluorochemicals - A Preliminary Report, American Industrial Hygiene Association Journal, 41, p. 584 (i960). 1 F ig u re 1: Low point (I Oppb) on calibration curve using liquid extraction procedure. Software Varsion : 6.1.2.0.1:D19 O perator : lamadrtd Sampla Number : 053 AutoSampMr : NONE Instrum ent Name : E03 Interface Serial H : 3730910097 D eiaytim e : 0.00nrtt Sampling Rate : 7 1420pts/s Volume Injected : t.OOOOOOut Sampla Amount : 1.0000 Internal Standard Amount : 1.0000 Data Acquisition Time 4/12/01 11:42:30 AM Date : 4/10/01 248:53 PM Sample Name : Low Curve: 10 ng/mL s tu d y : Homemade MeOH:HCt RactdViat : 0/0 Channel :A A/D m V Range : tOOO EndTim e : 21.00 min Area Reject : 100.000000 Dilution Factor : 1.00 Cycle : 33 Raw Data F o e : V\P42001\MainUti\Biv\E0-31DataV041101V>FOAtpft>a4J41101hgM3.raw <Modified Result F ile : ttpa42W)1VI4ainUbUErw10-3\Dala\t>41101\PFOA\pfoaO411Q1hgf-038.rst Inst M ethod: \\pe42001\MafnLatAenvteO-3Wata\040301\pl<0-1000mewfmm Ytpe42OO1VMalntab\Env1EO-3\DataWH11O1tPFQAtpfoo4M11Q1hgf*030-rst Proc Method :V\prs42001\MaW-ab\ErMEO-3\DataW1101\PFOA\PFOA 10-2000441101.m tfi Calib M ethod:\1pe420O11MatnLebfmAE(KFOata\0411Q1VPFOA1PFOA10-2000-041101.mth Sequence F ie : (tpe42001\PenEite\TcCSW er6.t2\Tom p\pba4341l01hgf-2012610903-20010116-t44723 idx Pi Peek tim e R et.H T * tm inj | Time{min] PFOA by GC-ECD (E03) Area Height Area/Height ISTDResp Blank Column Component Name Concentration itiV -s ] jjlV J JsJ R atio ng/mL 3 3.008 0.733 40421 14846 7 4.1.05 1.000 636461 23S754 3.262 2.688 0.6627 18000 PFOA IS T D -P F O A 684882.251600 1.0627 9 3 ooooso Figure 2: Freeze Drying Method (Low Q Q Software Version : 6.1X0.1:019 O perator : hagaraa Sample Number : 025 A utoSam pler : NONE Instrum entName : EOS Interface Serial * : 7230910097 Delay lim e : 0.00 min Sam pong Rata : MUJOOOpts/s Volum e Infected : 1.000000 m L Sample Amount : 1.0000 Internal Standard Am ount : 1.0000 Dota Acquisition tim e : 4/7/016:23:55 PM Date : 4/15/01 4:15:44 PM SampfeName : Low QC S tudy : WUB11904 Rack/Vial : 0/0 Channel :A NO mV Range ; 10000 End ta n e : 21.00 min Area Refect : 100 000000 Dilution Factor : 1.00 Cycle : 25 Raw Data File :pe4200i\MalnUbtBnAEO-3tOata\04070IVPFOA4M0701HGF4K5uaw <ModiRed> Result File : pe4200HMeinLaW EnvMVData\040701\PFOAt>4070IHGF4)25.ret In st Medtod :ttpe4200HMaInLabtEnVC0-3tMethads\PFOA 0-1000 euhre-121600ftom \\p42001tMainLabtEnrv1E0-3t0at3t040701VPFOA-0407O1HGF-025.r5t Prue Method : Vtpe42001tMalflLebtemAeOStdahA040T01tproa 2004000S40701.m tb PFOA by GC-ECD (E03) Peak Time R el.R T Area Height Area/Hslght STD Reap Blank Column Component Name Concentration 0 fnunj IpVaJ (pVf [r] R atio rtq/m l 2 2.871 0.652 45541 1861* 4 4.401 1.000 909923 298336 955*6* 316950 2.447 3.050 0.062* 1.0000 1.0624 PFOA I S PFOA 3* ----- ,-- 34 4 000051 P F O A Method Comparison F re e ze Dried Method versus Liquid Extraction Melhod of Perfluoroaotanooto ^methyl ester from Blood ample `.u m b e r C on trol Number . 1 Low QC 2 3 4 5 6 7 Mid-QC 8 Low QC 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mid-QC 23 24 25 26 27 a 28 y 29 LowQC 30 Mid QC 1^ 32 33 34 35 36 37 38 39 AVG SD RD (%) FreezeDrying PFOA ISTD ISTD Ht. Ht. Resp. ng/mL 17222 243073 8053 247169 0.071 0.033 48 13 21927 252004 12329 208416 8168 212227 21538 274340 0.087 0.059 0.038 0.079 63 37 18 55 50612 183210 17312 251466 76733 195603 71339 253949 71436 226788 60243 256633 116082 194232 34108 184427 39543 247914 4C585 238558 56678 233943 45512 255324 63205 218966 2178 216499 49680 220045 68453 223282 40573 249224 3309 236671 47659 248200 10975 239652 0.276 0.069 0.392 0.281 0.315 0.235 0.598 0.185 0.160 0.170 0.242 0.178 0.289 0.010 0.226 0.307 0.163 0.014 0.192 0.046 256 46 410 2700 310 2100 810 160 538 140 220 1500 2800 0 2000 298 140 0 673 25 12113 249430 0.049 28 78299 229317 10037 167416 52963 191978 139762 3518 198673 153525 149912 7358 166129 79791 264217 19411 137065 117280 263930 59401 272670 0.341 0.060 0.276 0.000 0.018 0.000 0.000 0.044 0.302 0.142 0.444 0.218 340 38 261 0 0 0 0 23 1169 117 2438 784 Liquid Extraction PFOA Ht. ISTD ISTD ng/mL Ht. Rasp. 22049 256135 20971 281763 0.086 0.074 49 38 27751 291816 24076 297100 15910 285704 21041 303446 0.095 0.081 0.056 0.072 57 44 22 36 79640 243951 24268) 273951 130769 307048 85973 281786 101613 300800 68749 293512 207364 325202 60516 340561 28829 291402 46740 302389 68624 303009 26280 266231 73665 258508 5060 310214 55696 263645 86938 295411 58153 331323 7399 321543 228640 323630 12966 303100 0.326 0.089 0.426 0.305 0.338 0.234 0.638 0.178 0.099 0.155 0.226 0.099 0.285 0.016 0.211 0.294 0.176 0.023 0.706 0.043 299 53 420 2800 310 2000 730 140 627 120 190 1249 2543 0 1752 265 140 0 860 10 15902 314680 0051 17 100095 302138 24353 304819 85457 274960 7902 298280 9273 281886 10052 303660 7813 326413 21948 343206 55987 318673 57208 329714 90354 323824 42979 336052 0.331 0.080 0.311 0.026 0.033 0.033 0.024 0.064 0.176 0.174 0.279 0.128 310 44 233 0 0 0 0 29 1386 136 2477 909 % diff > + /-3 0 /J 2 90 * * 10 17 20 42** 15 14 2 4 0 5 10 13 15 15 15 18 10 N/A 13 12 0 N /A 24 86 * * 49 * * 9 15 a N/A N/A N/A N/A 23 17 15 2 15 . 220457 37941 17 300294 24257 8 00052 PFOA 30% Error Bars {0-4000 ppb) PFOA 30% E rro r B ars (0-500 ppb) j Frecaa Dtying o Simple grtractfoo oooosa PF 0 A P * D,VlnO (PPW PFOA Mathods Comparison O 500 1000 1500 2000 - 2500 3000 PFOA S im pl E xtraction (ppb) | PFOA Data Une y x --------- Regression (PFO A Data) ! Pfoa Melhod Comparison PFOA F r Drylna (ppb) 0 SO 100 150 200 250 300 350 ito o 450 500 PFOASimpl* Extraction (ppb) | PFO A P a ta ........... U ne y c x -------- -U n g a r (PFOA D a la jl 000054