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p. 1 ~R 0 aG .~ 339 7 1 (30001 7 p. 2 ~R .2~6 - 339 ~ ~. . . ~~~ E . I . du Pont de Nemours and Company, Inc . Polymer ~oducts Deve..-tment . . B 465 .9000 Code OrN igia nal 3 Apr 81 Page--]. of 2 2 BLOOD c Determination of Pea].uorooctanoic Acid Gas Ciromatogravhic Method I . Scone and Aavlicationa This is a method for the determination of perfinorooctanoic acid (C8) and its salts In blood . Approximately 1 g of sawgle is required for measurement of concentrations down to 0 .01 Dg/g . The method has been evaluated for analysis of human and rat srho?e blood, but pre?iminsrr experiments indicate that it could also be for serum . A similar procedure can be used for the analysis of aqueous solutions (as from air impingers), and a modified metho d used for other solutions and solid samples . used II . P.inciule (See Note l . ) The water is removed from sample a.Lqucts by freeze-drying (lyoph'_ization) to ge_-mit derivatization . Addition of inethanolic HC1 to the dried residue, along with pe_rYluor.odecenaic acid (C10) iaternal standard, converts the acids, which are not amenable to GC analysis, to mor e i volatile methyl esters . These are extre.cted from the reaction mixtur e ~ into hexane solution for CC ana:,ysis . An electron capture detector (ECD) Is used because of its sensiti~ity and selectivity, with either a packed or a capillary col iac . 3N C1 a> C7F15C0+0 M + ~s30g C.~F1,CJCCg3 + HOE IN+ = NH4+, Na , fi + TIT, Interference s As in aqy GC analysis, comDotmds with the same retention time as the compcund of in-terest will not be distinguished from it . These can include =mpuritiek present in the reagents or other comnonentsin the samoles themselves . ' Interfering aee'xs from solvents, de :ivatizing reagent, etc ., are not generally observed in this laboratory with the reagents specified below (See Note 2), but each aecr batch shot :7.fl be checked end a blank include d in every analysis . A small neeit at the C4 ester gosition Is found in reagent blanks containing peralueradeceno=c acid internal steaua_3, orobaoly from Ca uresent as an imuurity in that material . This cr.! .1 give an intercept slightly greater than zero in the ca .Lib:'ati*Cn olot , ; but will be a significant contribution to the C8 teak area coly at the lovesL concentratiors (about 1 1 3 of the total for 0 .0_ ~g/g C8, :rhen about ' ~g/6 C10 is added) . l,'~ ~ Company Sanitized . Does not contain TSCA CBi OQ0 0 1.8 p. 3 I ' Page 2 of 22 . A very small peak at that position has also been found in some "blank blood" sas:oles, as shovn in the exasmle in Figure 2a . Whether this represents a trace o* C8 (about 0 .001 - 0 .005 {ig/g) or some other component of the blood would be difficult to determine at this low concentration and with the limited number of samples examined to date . IV . Sensitivity, Precision, and Accuracy The method has been vsed over the concentration range 0 .01-150 Ug/g 08 . Above 10-20 p g/g, however, interferences with the C10 internal standard are general.~y encountered so that some sa,dification must be made for analyses at higher concentrations (See t(ote 15) . Due to the relatively narrov linear range of the ECD, the calibration curve must always cover the region of interest _*or quantification . From the data available at this time, precision is estimated . to be 10% relative . Becovery of spikes is quantitative within this uncertainty 'when calculeted relative to the C10 ta.ternal standard., although absolut e k~ recovery is affected by the presence of dried blood solids (See Note 3) . Accuracy will also be affected by the choice of c8 standard (particularly when the packed colasn analysis is used.), :mich should correspond t o the -luorosu_r:aetant composition of the samples as nearly as possible (See lfote i~) . V . Avoaratus Instruments and equipment used, in this laboratory are sneci*ied here ; equivalent apoa.-atus can be substituted . 1 . Gas Chraato ranh and 8uovlies - For packed column analysIs . See Appendix 1 for capillary colnnn analysis . ) qevlett-Packard 5830 GC with F.P 18803 Electron Capture Detiector, equipped for on-colvIDn injection with g?ass-packed column s 10 ft x 2 mm id glass commas (HP configuration 5 with CD adapter), packed sr3.th lOx OV-210 on 70/80 mesh Chr'omosorb W .AW .DAdCS . and con 'c_itioned at 200 C Heailton 7011 10 uL syringe $ , % Company $aniHzed Does not contain TSCA CBI 000019 p .4 t ~ y ~~~~k Code No . B k65 .5000 Original 3 Apr 81 . . Page 3 of 22 2 . I o phi2izer ~ Labconco No . 75352 bench-too freeze dryer (12 port, dry ice cooled) Lsoconco No . 751 06 ( 150 mL) or No . 75108 (300 mL) Fast-Freeze flasks, vith No . 75 1+T6 stainless steel adapter s Vacuum ~anso With avniliaxy cold trap Mc:,eo3 gage or electronic vacuum gage reading in the 5-0 .005 torr range 3 . Thermastn.ted Reaction Block Pierce No . 18800 Reacti-Therm Heating Module, with. Reacti-Block to bold 2-dr am vials end thermometer ( Block C, No . 18804, has 12 holes, but they must be enlarged to about 19 ~ to accommodat e the 2- 'c-.-em vials . Block B, No . 18802, can be used as is, but v111 hold only 9 vials an.d Provide s poorer thermal contact .) Lab-;sine blu7.ti-Blok Heaters can be used With the same blocks, and will hold 2-6 in a single uai .t . b . Vials end. Septum Cac s Wheaton No . 22k881: or Pierce No . 13028 2-dram ( e:oout 7 mL) screyr cap septum vials (borosii .icate glass ) Pierce No . 12713 TEFLON-silicone septa and No . 13216 open-top screw caps 5 . P=ets and Disvense_s Gilson P200 Pipetman variable volume micropipet for quantitative delive ry of 20-200 l!L aliquot s 1, 2, and. 5 mL volumetric pipets for measuring sample and reagent aliquots . (Brinkmen Dispensette bottle-top dispensers are a great convenience for reaetitive delivery of solvents . ) 1 6 . Deposable l yoode reic syringes , 1-3 3L, if blood sales are to be taken from closed "9acutefners" . 7 . Vol~etrj c il asks, 10, 25, end 50 m:, 8 . Brans on 3-220 ultrasonic cleaner (See Note 5 .) 9 . tinaj.ytical balance 10 . Coon laboratory e?u;pmect (including Z refrigerator for storage ~ s amcls ) Company Sanitized . Does not contain TSCA CBI 000020 p. 5 ~ I Page 1~ of 22 VI . Reagent s 1 . C8 standards : Pe rtluoro-n-octanoic Acid ( PCR Research Chemicals), PC-1b3 ammomium perfluorooctanoaate ( 3K), or other material in use in the area to be moaitored ( See Note Ii . ) 2 . Per_luorodecanoic acid ( PCR Research Chemicals ) 3 . Methanol (Fisher ~I.C (A-452) or Fisher Certified ACS (A-bl2)) (See Note 2 . ) 4 . Derivatization re agent, 3~ HC1 in methanol, prepared from Applied Science No . 18053 Instant Methanolic HC1 Kit, but substituting Fisher RPI.C or ACS methanol for the Lipopure methanol provided (See Note 2) . The reagent should be stored in the refrigerator, end can be kept for about 1 month . 5 . Hexane ., Pliillips Spectrograde or Applied Science Lipopure (See Note 2 . ) 6 . Water, di stilled or deionize d ~ ; 7 . "Bleak" Blood, to spike for standards . Heparinized "Yacutainer" blood collection tubes ( 9-D No . 6 1 80) have generally been used for this, as well as for samples . Yacutafners containing liqui d EDTA anticoagulant can also be used . NO'1'J : All blood salesust be stored under refrigeration . 8 . Perflvwrodecanoic acid ( C10) standard solution in methanol (See Note 6 . ) Prepare a stock solution and sequential dilutions as fc,Uovs Solution Preoaration [ C_0], ug /mL (F] Cg/mi, I Weigh (to 0 .1 mg) about 25 mg perfluorodecanoic acid into a vial . Add ~ 10 nL methanol and shake to dissolve . ( Solvent volt can be a&justed as needed to corresaond to the actual sa~ie wt .) 2500 175 5 II Dilute 2 mL I to 50 mL 100 70 .2 in methanol . III Dilute 2 mL _TI to 10 mI ., in methanol 20 .0 1L .0 ~ompany Sanitized. Does not contain TSCA CBI 000021 p. 6 ~~ . . . ,' i ~ Original 3 Code So . 3 1;65 .5000 Apr 81 Page 5 of 22 ' 9 . Pe=fliorooctanoic acid (C8) standard . -solution in water (See Note 6 .) Perfluorooctanoic acid is only sparingly soluble in aat er, and. dissolves very slowly . At least 1-2 hr shaking is required at this concentration ( about 2 mg/mL), and it is best to prepare the stock solution one day in advance of the dilutions . Acid con- centration can be checiced by titration . Concentrations will vary, but solutions are needed from wbich at least four spiked blood standards can be prepa :ed to cover the range of interest . For the e xamqle shown in the diromatograms and calculations below ( 0 .01-0 .9 1,tg/mL), a stock solution end sequen tial dilutions wouil.d be prepared as foll.oas : Solution - Prevaration (C8)-, ug/mL [F], ug/mL I Weigh ( to 0 .1 mg) about 45 mg perflu,orooctanoic acid into s vial . Add 20 mI, distilled water and shake to dissolve . (Solvent volume r.en be adjusted as needed . ) 2200 1511; ( for k kmg samnle ) ~ II Dilute 3 ml. I to 50 ml. ~ in ft20 . 132 90 .8 III Dilute 5 ml. _TI to 26 .1; 18 .2 25 m.lin E20 . IV Dilute 2 mL II to 5 .28 50 m.l in K20 . 3 .63 V Dilute 3 ml. IV to 0 .63; 1 25 mL in H2O . 0 .1L36 VII . V==I . Doerating Conditions Safety and Eeath Precautions Human blood should be considered potentially infectIous, and the Laboratory 3uideljies ( See Reference 1) for handling, storage, end disposal of sammles should be followed . The 3M ConDany has found in preliminary studies that C8 caused birth defects srhen fed to rats in a laboratory ezperiffint . Female emnloyees of childbearing capability should not engage in any portion of this procedure which offers significant chance for exoosure . Although no health problems are kno wn for workers exposed to C8, it has been found in blood s2~les and may be very slowly eliminated frcm the body (Se e ~ Reference 2) . Standard laboratory safety practices for handling toxic, embryotoxic, etc ., materials and for corrosives should be used in working with these perfluorinated acids and their solutions . Company_ Sanitized. Does not contain TSCA CBI 000022 p. 7 age 6 of 22 VII! . Safety aad Health PecaaxUons 'd) I The electron capture detector contains a radioactive source (63Ni), and the manufacturer's instructions for safe operation must be observed . Care should be taken to avoid contact with the low-temperature baths used for freeze-drying . ~ IC . Procedure A . Preparation of Blood Sam lea end Spiked Standards for Anai,ysis See Note 7 . A13. blood samples should be well shaken before taking aliquots to insure a unit ore distribution of cells and . plasma . l . Spiked standards For each standard to be prepared, and for one or more blaaks, pipet a 1 at aliqnot of "blank" blood into a. 2-d_'-am vial . Spike each Srith Cg standard soltttion to give the range of ccncentratfons required, as shown in the following example : S~ike [C8], ug/mL blood [F], ug/mL b]ood 110 33 1 L 26 .4 jrg /mL C8 in water 0 .873 0 .600 i.iL 5 .28 }ig/mL C8 in water 0 .581 0 .400 69 " 0 .36L 0 .25]. kl " 0 .216 0 .114 9 110 ILL 0 .634 ~tg/mL C8 in water 0 .0691 0 .01480 16 " 0 .010 0 .0070 Blank (no spike) 0 0 Mix gently, keeping the solution in the bottom of the vial, and sonicate for about 2 mia for additional asf .xcng (See Note i) . ~ Stare in the refrigerator until needed (See Note 8) . 2 . Sales If the sample vclvae is sufficient, prepare in &xp1inate . For each sample, pipet a 1 mL a"g,not into a tared 2-drain vial and weigh to 0 .1 :ng . (Aiiquats can also be taken with a B_spas able sjr'_r..ge, if it is felt preferable not to open the containers ) . Company Sanitized Does not contain TSCA CBI U00lJ23 p. 8 ~ ~ B . Freeze-Drying - Code No . B 465 .5000 Original 3 Apr 81 Page 7 o. 22 1 . A schematic diagram of the freeze-drying apparatus is show n in Figure 1 . Bach o~ the 12 ports has a separate valve so that flasks can be attached or removed while the system is under vacuum . The epparatus should, be pumped down to an acceptable vacuum (See Note 9) and the cold traps filled before the drying I flasks are attached . 2 . Freeze the samples before placing them under vacuum to prevent bioing . To reduce the chance of contamination or sample loss, cove: each vial with a light filter beforehand ; a small piece o~ "Kimtrl.pe" held in place with a rubber bend rorks rreLt . Freeze the solution in each (See Rote lo) and keep the vials at dry ice temperature until all of them are ready . 3 . P'I.a .ne up to four vials in each drying flask and attach to the drying chamber . Th.e pressure should, drop below 0 .5 torr again within a few minutes if no leaks are present, and. the seamles snould remain frozen as sublimation takes place . Allow them to dry for about iC hr (See Note U) . C . Derivatization ) 1 . To the dried residue in each vial, add 2 mi, methano3ic HC1 and ~ 50 uL C10 standard solution (about 20 Ug/m:, in methanol) . Prepare a reagent blank similarly, using an empty vial . Close with a septum screw cax~ and shake well . Sonicate for about 2 slin (See Note 5) and shake again . 2 . Thermostat for 1 hr at 65C, then allow the vials to cool to room temperature and stand overnight (See Note 12) . 3 . Add 2 mm each of hexane and distilled water and shake for about 2 mm . 4 . After the phases separate, the upper hexane layer can be sampled directly for CC analysis (See Note 13) . The solutions ~ are stable in this form for at least several days, although hexane will gradnaZ].y evaporate after the septum has been pierced . D . CC Ana:ys_s - Packed Column . See Aonendix = fo- caoil.I.ary column analysis . 1 . GC Conditions - Instrument and column as shown in V , Apparatus . Temueratures : Injection oort 200 C .)~ Detectcr 325C Column oven 100C, isothermal company Sanitized. Does not contain TSCA CBi 000024 p. 9 ~ n n Page 8 of 22 ~ Carrier gas : 90% argon/lOw methane (or nitrogen if recommended for the instrwnent) flow about 30 mt,/min Injection Voinme : 2 y L Sensitivity : Attenuation as needed to keep peak height measurable ., 25-213 on HP 5830 . Run time : 11-25 min, depending on sensitivity (See Note ] .b .) ; After installing the column and establishing the conditions above, check the ECD base frequency and noise leoel according to the manufacturer's instructions, to insvre that they are stable and within acceptable limits . 2 . Run each solution twice (or r~m duplicates once each), bracketing samples with standards of similar concentration in the second series of injections . Renresentative chromatog:ans are shown in Figure 2 for blanks, stendars and samples . Measure either peak height or peak area . As noted above, the calibration curve must cover the concentra . If some samples are found to fal l -tionragefhsmpl above the range of prenarefl standards, the hexane solution can be diluted, with that frosl a blank ( e .g ., 100 UL + 400 UL for 5X dilution) to reduce the C8 concentration without changing that of the internal standard . This type of dilution can also be used if peeks interfering with the internal standard are observed at high C$ concentrations in either sample or standard preparations (See Mote 15) . I . Calculations (See Note 16 . ) 1 . Normalized C8 Peak Value s Alabu3ation of peak heights for analysis of a series of standar s (prepared as shove) and samples as shown in Figure 3m . To correct for any variations in injection vole, reagent volumes, etc ., the raw C8 peak heights (or areas) are first normalized relative to the internal standard average : i C10 (average) C8 (corrected) = C8 X. 10 Calculation of the C10 average also provides a aeasl : :e of the arecis :on in sample preparation and GC analysis . ' ~O"'~' 5~~ 0oos ~ot coneaM rsca csr O00025 p .10 ~ i 1 Original 3 i _ ~2 . Calibration Plot Code No . B 165 .5o00 Apr 8]. Page 9 o* 2 2 A calibration curve obtained by plotting corrected Cg peak height vs concentration of the spiked standards is shown in Figure 3b . In this case, straight lines can be fitted to sets of points at higher (0 .61-0 .15 pg/mL) and lover (0 .15-0 pg/mY.) concentrations, and the slopes and intercepts calculated by linear least squares analysis . In some plots no really linear region may be found, and . a saooth curve shonld be drawn through the data points . 3 . Calculation of Sa~le Concentration s Using the corrected Cg peek heights (or areas) for the samples, corresponding values of pg F/sample can be calculated from the LIS equations (as in Figure 3) or can be read directly fron the calibration curve . Divide each value by the sample weight (in g=ams) to obtain g F/g blood, and average the results for duplica;a rims or duplicate preparations . Report as ug F/g blood (See ~Tote 6) . ).Y } XI . Calibration (See Under X, Calculations .) ]QI . Notes 1 . Initial method development was based on a procedure for determinatIon of pe : fluorooctzaoic acid in serum or plasma recently ublished by 3M (See Reference 3) . In that method, C8 is extracted from acidified serum into hexane/ether, the solution concentrated by eva~oration, and diazoanethane added for dervatination . With the lyophilization procedure described here, sample hand'!ing, time, and material requi=^ed are reduced, and the use of diazomethene is eliminated . Eovrever, concentrs.tion of Cg from a large volume of sample as in an extraction, is not possible . A sample preparation procedure similar to that in the 3M method has been used for aqueous solutions in tthis laboratory and can be followed by the sane metaenolic PCl de-lvatisatioc and CC analysis as lyophilization . The extraction has not been tested with whole blood in either method, however . GC analysis was initially done with a packed column (as in :.be 3iK method but with a different stationery phase) . A capillary column has also been used recently for better resolution of the branched C8 isomers, and modifications for that analysis are given in Appendix I . ( ~ Inpany SanlNzed. Does not contain TSCA CBI 000026 p .11 I i Page 10 of 22 ~ 2 . The solvents specified in Section PI were found preie _-able to several ~ tried for this analysis, but others may also be satisfactory . ~re n with the same reagents, the appearance of the blank may depend on the condition of the detector . With the i.Cl) used here, contamination results in a significant decrease in selectivity and, increased reagent interference . 3 . Recovery was determined by spiking blood, water, and methanol I solutions at equivalent concentrations, obtaining the response {actor for Cg relative to C~0 in GC ana?ysis of the water and methanol spikes, and using his factor to calculate the C8 concentration in the blood spikes . Average recovery (Cg found/added) was 100 # 5s for 18 samples of hmuan blood spiked with 0 .0ih-46 ug F/mL (FC 1k3 or perfluoro-n-octanoic acid) end for 7 samples of rat blood spiked with 0 .007-67 ug F/mL . Absolute C8 and C3 peek heights were about 1 5-205 lover in the presence of dried solids from 1 g blood, however, so that a corresponding error tmuld result from exterae3. steadard. calibratioa against aquecus solutions . ? ~ ! The questicn of whether recovery of spikes provides en accurate measure of recovery of the same material from samples should also be considered, particalarly with a complicated, substrate such as blood . Agreement between the results of this dna2,ysis and those of Wickbold Torch analysis for organic fluorine in a series of test samples seems to indicate that the spikes are a reasqcsb?e model for samples . k . The FC-1L3 s amples examined to date all. appear to be a mixture of about 70-75w straight-chain material with several branched isomers r :esent at about 1-10X ( de termined by F-19 NI4Et, capillary GC, end GC/) . Ln contrast, comnercial oe=fluoro-n-octanoic acid (?CR) contains about 95' : straight chain C8 with about (Cf3)2CF(CF2)kC00H end traces of the other isomers . As shown in the ch_cmatograms in Figures 2c and 2d, in the parked coluffi OC analysis, only one of . these is really segarated from the main peak ( small peak at 3 .7 sin) 'while others are visible as a shoulder folloving it - Chromatograms of samp les and standards obtained vsiag the capillary caluna analysis are shown in Figure IL (Appendix I) ; under those condit_or.s, at 'east Dartial resolution of foti+ isomers from the main peek can be ~btajned . Since EC7 response appears to be higher for the isomers than for the straight-chain ester (by a facto : of 2 or more), the total. area/ concentration ratio also becomes higher as their percentage increases . In the packed column analysis, the response factor calculated for Ca relative to C10 is about 90-kOx higher for ?C-lk3 standares than for perrl:ioro-n-octeaoic acid standards, so that it becomes very important to use the one most similar to the aaaples to be analyzed . Although s:a-'-'_ variations In isomer distribution can be seen in human blood sarmles, a1I of those examined to data resenbie pera'1uo=o-n-octznoic ac :d much more than FC-143, and t has been an appropriate standar d Company Sanitized. Does not contain TSCA CBI U0pp2~? p .12 ~ . 4 ItI . Robes ( Cont' d) Code No . B 465 .5000 Original 3 Apr 81 ' Page 11 of 22 h . ( Con.t' d ) for avantification within the uncertainty c_* the method . In a series of rat blood samples, however, considerable variation in distribixtien was observed . As seen from the chromatograms in Figure L, neither standard would have been a good armroxiamation to some samples, and analyses were done with the capillary cclum _ i ( of spikes 5 . Gentle sonication (with the vials immersed about half-way in the water bath of a. sonic cleaner) has been used to aid in mf zin g and in promoting contact of derivatizing reagent with the dried blood solids . Additional experiments are planned to determine whether this step is necessary or can be eliminated . 6 . Since results that are to be coxpared with nonspecific analyses for total organic fluorine must be expressed as fluorine rather than as C8, both concentrations are given for the standard solutions . Values for vercent fluorine used in the conversion are : perfluorooctanoic acid, 68 .82%, am'non+_um pe-"flaorooctanoate, 66 .10e, perflvorodecanoic acid, 70 .22% . 7 . An aliquot volume of 1 mL is generally used, for both sasmles and m~ standards, but it cube reduced to 0 .5 uL when concentrations are expected to be relatively high . Coagulation is generally a problem with rat blood samples kept for more than a week or two before analysis . When {t is no longer a homogeneous liquid, the entire satrole must be lyophilized., mined veil, and portions of the dried powder weighed out for analysis . The exact ratio of dry to liquid weight must be determined for each sample, but is about 0 .2, so that 0 .2 g powder Will be comparable to 3 mL liquid . This procedure greatly increases the time required for sample handling and the probability for error in the weight, and it should be avcided if possible . 8 . Spf1kes prepared for method development have been allowed to stand at 1 east overnight before analysis, but a period of a_esr hours appears to give eqt:ivalent results . 9 For this freeze-drying application, pressures less than 0 .5 torr seem to be satisfactory, although 0 .025-0 .05 torr may be achieved . Readings will also depend on the type of gage used, since the Mcl,eod gage does not read the pressure of water vapor as electronic gages do . company Sanitized. Does not contain TSCACI 000028 p .13 Page 12 of 2 2 ~ 10 : A sma'_l dry ice/acetone bath or the center cold veil of the drying chamber can be used for rapid freezing of the solutions in the bottom of the vials . As nosed in Section V=I, care mast be taken to avoid contact with the cold bath . 11 . A. h-h_Y drying time has been adopted for preparation of blood samples by comparison with 1 mL water samples, where drying can be monitored by d3sapnearance of the ice . Experiments with spiked blood samples indicate that longer drying times (8-15 hr) can also be used without loss of C8 . 12 . Derivat_zation mixtures from aqueous samples can be partitioned with haxane%ater immediately after cooling . In the presence of dried blood solids, however, additional time for equilibration of th e CS end C10 esters aeems to be necessary . After standing overnight, peek areas for both Cg and Clp are lover than in reaction mixtures prepared from aqueous or methanolic standards, but the relative resvonse factors are equivalent (See Note 3) . Preliminary experiments indicate that it might be possible to eliminate the overnight equilibration by spiking with aqueous C10 before lyophilizstion, rather than with methanolic C10 after . ~ ,,v to 13 . Although a clear hexane layer is obtained after standing for about 1/4-1/2 h_, the solution has e relatively dark rust color and undoubtedly contains nonvolatile or high-boiling impurities . In the packed column analysis, these are injected onto the colum n head ; and at the end of the day, the oven should be programmed 180C and held for several hours to reduce build-up . No deterioration of performance was observed, in a column used for several weeks of analyses, but it may orove preferable to adopt flash venorization with a glass injection port line= which can be changed daily, as in the capillary coiumQ analysis . 14 . As seen in the chromatograms in Figure 2, some small, broad peaks can anoear late in the run . These appear to come from the C,0 standard and might be eliminated in purified material ; others seem to be characteristic of older blood sa=les . At the high sensitivi ty used for low Cg concentrations, these can interfere with the following rand must be allowed to elute before the neft injection . 15 . Several minor comoonents are present in both the perfluoro-n-octancic acid end FC-1h3 standards which elu`.e near the C internla standard under these GC conditions and can interfere with quantification . Similar peaks are also often observed in samples at higher concentrations . With the C70 concentration generally added (about 1 pg/g), this seems to occur above about 20 pom C8 in the 2api_13-p column, analysis end about 10 :eg/g with the pac ked _o'_umc . In some cases, an obvious shoulder c an be observed on the C , p peek , varying in sise with the C8 concentration . In others, an un reso'_ :ed peak may be indicated by an unusual change in C 1 0 ?e ak size, but is only seen if a sanole is prepax-e without added internal standard . Deoenc3ing on the type of analysis to be done, rarious techniques can be used for cuant_fication under these coud..t tons . Thp5fl! Sanhized Does nof confain TSCA CB1 -- ------- GU002 ) p .14 I I ~ i , ~ Code No . B 11 65 .5000 Original . 3 Apr 8Z Page 13 of 22 ~ (a) Standards can be prepared onl,y up to about 10 ug/g, and ' any samples found to be above th is range dilated and rua again, as described in IX, Procedure, Part D . (b) If only relatively high concentrations are of interest, the Clp concentration can be increased and/or the sample size decreased so that the interference becomes negligible . (c) With the increased resolution of the capillary coltaon, components of the pe r1noro-n-octanoic acid standard can be resolved from the 010 and the roblen eliminated for standards . Samples must still be examined for interference, however . (d) The calibration curve can be used for an e=ternal standard calculation, xith uncorrected. 08 peak areas or heights . While good reproducibility is possible with care in measuring the 'injection end reagent volnees, it is obviously more critica], here than in an internal standard calca.lation and must be checked regularly at each point . lo' . From the calibration plot in Figure 3, it can be seen that the ROD response is not linear trith concentration over the entire range . Linear plots may be obtained in some cases, but they still have concentration-dependent response factors . The calculations describe d ' here thus make use of a calibration curve, in *rhich the internal i ) standard is used to no* mp + ze raw peek values before plotting . For en ECD which shows a more linear response and. constant relative response factors, calculations could be done by a general internal standard procedur e instead. . (A ca:ibration plot of this sort has been observed with a C'ax^_an 3700 G^, under similar condit ions) . XIII . Reference s 1 . ^ R cperj.mental Station Rules and Procedures for 3i man and Other Primate Blood and Blood Products", 3/80, Safety Office, Experimentai. Station . 2 . F . A . tToel, S . D . Sorenson, and D . S . Roach, "Fee"th Status of Plant Workers Exposed to F1;ioroczemicels - A Preliminary Renorr" M. Ind . HyR! Assoc . .T . 4l , S$k ( 1980) . 3 . J . Belisle and. D . F . Eagen, "A Method tor Detern;aat i on o f ?er:].uorooct an oic Acid in Blood and. other Biological Samples", Anal . 3iochem . 101,369 ( 1980) . ~. ~ Origin : r xperimeatal Station Prepared by : S . S . Stafford 4nvro ved bp : L . J . 2apa , Anproved by Laborstor; Methods Coamit t ee, 9 ?iov 87. ~ ES-56T ~F< .. '. t%ompany SsMBz~L Does not contain TSCA CBI 000030 p .15 ~ . 1 bo m~ E ~ UmUaO . ~ C 7 0 ~ ~Q > H I g~Y ~ H + 1 \ fr ~d M. . +~ t. - Code No . B 465 .5000 Original 3 Apr 81 Page 11 of 22 . . W I N ~~ p ~ ~ o a" w- .. ae~ ~ O O O . " i~ W rs ~~ ) ~ z ~ m ~ ~t-~ 1 P E .-! r1 d .. ~ .r+ C `!s N ~ ~ C \ \ \ \ ~ \ ~ ~ ~ O . ~ > c ~ > n m M ~ti o ~o a m ' r. :. d 7 9 ~ ? O . E U G H ~ Qm M ~ Q 'I N C, .. A ~ V - -- company S 5flBtZed Does not contain TSCp CBI 0 0-(1031 p .16 " - - -Y '^ Page 15 Code oS No . B 465 .5000 Original 3 Apr 81 22 FIGURE 2- CffitOMATOGE#A26 FOR CP DEZ'ERMINAI01P PACIG~D COLOt .QJ ANALYSIS GC conditions as on p . 8 . Attenuation as noted for 0-6 mm, 27 after 6 minutes . j Chart speed 1 cm/min . Retention time (m3nutesI indicated by each peak . (a) Blank blood, no C8 spike or C`0 (b) Standard, blood spiked with standard edded 0 .01 uglmL pu~ _ uoro-n-octanoic (97I+/8o) " acid (0 .007F/mL ) - (9/k/80) II ~ ~ : C10 ester :eek at CS position, attn 25 C8 ester ~ ~ ,. r i :~ . at y 25 ~ A r .. . y ~ M A,y = ~ = R ~ f s y w. ' n w n . ~ ~ (c) Standard, blood spiked with 0 .9 u8/mL (d) Sample F.2291h-2?-1 per'luoro-n-octanoic acid (0 .6 pg F/-L) (9/10/80 ) (9/10/80) ; - 'I 0~ CS ester, attn 27 ~ attn 27 ~ i nv C_0 ester C1C este- I ~ ~ . ~ ~ ' ~ ~ ~ ~ w ~, ~ ~ i ~ I = I CbIflpany Sanitized. Does not contain TSCA CBI G00032 p .17' I 1 ~; :y~ .,'~ f ( ~ 1= E -={~ -- code No . s 465 .So00 t O :: :_r _' ig : .-E : : :i :- . . n - a.l 3 Apr 8Z ' Page 16 of 2 2 -.3 M ,,~~, U ~t .~i . :.:: .::t.:. ::;: : ~~..~,~. L - - :f f: : :f : L :f: . . - . . . . . :- : a .. .. : . . _ f: ~f : :f~: . :i -:i : . :: _i=e~cf .[. . . . :~;r.: : F~ . C a ~ _ :~: - ;~' ;: :f : :~ : .' :f ~ : :: e jOb U m " :i: :i ::fc i: : i : :_:_f : : :i: :. ~ Tr % I . g I , Z~ L 17_ ~ ~ d - f=. ~:: :~: :E:~:-.. ~ .. '.~_.~.M : ..::.'- l L .l' -L. . :~' :S : :i:: _ _ =i ; -ci' :ii' :: :: : . . : : :.L ~ m :f : c[f~ :._ _ ~ C ~;fL :~c: - :t: .~ : :`': ~ .;-; ; =f :_ ~('.:.~ : :_ - 1 :f: 'i- ~~i: - :~ - =_ f = - L = _ ~: 1~ : ._f~=-- _:~ ~ ~ ,~ ai G ~ o ~ ~_ . .. . C r := ~_:t- - _i: ; --4 cf= : fc _:r :?: _' ..:;f :f: :z: fr: _:r:: -L: .-- =..4-_~ ~ :f:: 41--E4-= - - ::f : i=f--L' 4 ' -- - I :i ,,!' ..f _w_: .. -' -_t _ 1 . . i~ ! - _'f . = _.._~L:I :: - =~f :~ a' .. : f: -_ __ - _ __ _ ~ .i' L- _:f . . : _.: : :- :f: - :i- :_ -.i:: _ -_ '[: . .c-- - . . .. . ....... _r_..E .... :=.:::.i:.....__...= . .... .......1 . .. .....= ....... :..::1= :_..-:..r.:_f:=..===._ ::: )C .-4 C a N 0 uCi 0 iti N C-i .e ~ `p2FaH c C CO o ~i ~. Na N eVi ~ O .i C .. L N N l'- O 0 > 7 O O 6: C L A,1 ~ V o C o .O.i c ~. .H +~ 'O O " C : o: r r v a m r. ~n r a ~ tn 1-1 o .n oi F . .. vs F. rr m ~n ei ~O o O: mm rI N i . 3 2 wf . 4) t t c0 ' e ~. C 4^'1 Irf f O~ .1 m ~N .4 y 0 v. a. N 4- , . m 'n ao v . .-t p 11 ' CL A 1 O 'O m t- O~ d .~ O ~a O m N O Vs ~ C= ^ ~ r r r r r- ao m r- m e- m e- r ~ - ^ 43 G F. _G6 O a J m~ c i r ~ '0 U .. W +' ~"~ vl p U C T L_ N T O C - ~-~i m .M~ ~ 4 io r . 4 ; '0 a~ c~ o v' .a ~n Y+ . i ~ : i ~ e~- s ri~ ~ ~f .-4 o ti ~ ~ vs 4 .i +~ ~ O a rs L .4'0 Q CJ y ~ 0 U rl O y O m : c L so o .y LV .+ P1 2 [ PIV , as !+ vs .r O U rr W C '0 U .b'C= .1 V,'C. v y .: 9 n u -+ ,.0i '0 1. ti v ~-~ U ~ S0 ~ ~ a m ^ V I4, ;. a Em V ~ a O ~o O 4; O 01 .. o 0 ~ O O U m u u O 201 s o ~n Q W OC~ ~ P .-1 . . O O O O J y1 P . . 4 ' o LO. Y r-~ f . O ~ Y i1 ~ 1 1!1 so r-1 f5 y + a! V: O O O # Company Sanitized. Does not contain TSCA CBI 000033 p .18 ~?:. ,. ~ ) Code No . E 1i65 . 5000 Origina13 Apr 81 Page 17 of 22 APPENDIX t Modifications for Caa Uary GC Analysi s As discnssed in the ae+, .hod, a capillarJ column can be substituted for the original packed column in analyses where separation of the C8 isomers is necessary for quantification, or where determination of the isomer distribution is of interest . Chromatograms obtained with the column and conditions currently in use are shown in Figure 4 . A wide-bore WCOT is used to permit large injections (1-3 UL, as Tr3.th the packed column) for detection of low concentrations e.lthough the efficiency is probably less than that of a lower capacity column . The major isomer peaks are generall.y resolved well enough for quantification, hoverer, or at least to indicate whether a significant peak is present (as in Figures 4c and d) . Comparison of these chromatograms with those in Figure 2 shows that the analysis time is presently longer than with the packed column ; this may be reduced by further colwan modifications . Preparation of semples end standards is the same for both analyses . Modifications needed in apparatus, GC conditions, end, calculations for the capillary analysis are as follows : i . AnDaretus (V ) HP5830 GC with 3P18803 ELectron Capture Detector, equipped for spiitles s injection, with glass capillary columns . A flame ionization detector should also be available on this or another capillary instrument . Glass injection port liners (inserts) for use with the capillary system in solitless mode . Silanized glass wool for packing injectcn port liners . Glass WCOT column, 50 m x 0 .5 mm, OV-210 (high load) . (The column currently in use is a custom-coated column from iitech Associates . It can also be specially ordered from Quadrex or Chrompack, but is not presently available as a stock column . ) 2 . GC Aaa:ys s (ji%, Procedure, Part D ) Instrument and column as above ; straighten the coium ends, recoat them with ?FAP in Cfi2C?Z, and install the column according to the manu:actt :.re_'s instructions . Before inserting the glass injection port liner, pack it loosely with about 1 cm silanized glass wool . "~' ._-'s should be positioned just below the point of injection, to prevent nonvolatile sample components from reaching the cclunLn . { -Company Sanitized. Does not contain TSCA CBI - 00003-4 - - p .19 Page 28 of 22 GC Conditions : , Temperatures : Injection port 200C Detector 325 C Colt~ oven programs (a) 15 min 5 5 C, 80/min to 130C, 5 min 130 C (b) 10 in 6oc, 2/min to 76C then 6/min to 120c, 6 min 120 C ~ Carrier and make-up gas : 90% argou/10% methane colt~ pressure 3 psig, about 2 .7 mL/min flov at 100 C meke-u about 26 mI,/ain (total flow about 30 m ;,/min) splitter about 100 mL/min Injection volume : 2~tL, splltless injection vith 60 sec hold before opening splitter vent Sensitivity : Attenuation as needed to keep peek height measurable 25_213 on R'5830 Run time : About 30 min (depending on program) plus cool-down . Temperature program and. column flow rate s+ill vary with the condition of the ~ co1wsn, instrument coat'ig.tration, carrier gas, etc ., and must be chosen and modified as needed to maintain adeq,uate resolution . Col*nffi oven program (a) was used for the chromatograms in Figure 4 ; program (b) and sim:,la: ones have also been used . After installing the column and establishing the conditions above, cheek the ECD base frequency mfl noise level according to the manufacturer's instructions to insure that they are stable and within acceptable limits . Base frequency should also 'be checked before each day's operation . Maintenance of a clean system is especial .ay important for temperatureprogrammed EC,'D analyses at high sensitivity . A clean injection port liner and new septum (low-bleed) should be installed at the end of each day, and the column ovpn teaeperat :ie raised to 130C (or the maximum program tesoerature) overnight and when the instrument is not in use . 3 . Calculations (X ) The calculations are done in two varts, first determining the concentration of the main component, then adding a percentage correction for the secondary isocers . (e) Concentration of eerfluoro-n-octanoat e Calculation of noroalized C8 peak values, preparation of the calibration curve, and use of it and the samle veihts to calcu~ate ug/g for each Company SaniHzed. Does not contain 7SCA CBt 6000j5 p .20 Code Be . B 465 .5oDD Original 3 Apr 81 pSBe =9 of 22 (' sample are done as in the original method. Since oa]y the area of the main peak is used,-however , the concentrations of the standards mast first be co--ected to correspond to it rather than to the total Cg concentration . For the u erfluoro-n-octsnoic aci d standard used, in this laboratory, the concentration of straight-chain acid is taken to be 95% of the total ( See Note k above . For some samples, such as the one shovn in Figure kd, the isome r peak j ust ahead of the main peak may nat be large enough to be resolved but still makes a significant contribution to the total . Peak height rather than area, can be used to mirimize the error in a_uantification of the main peek . (b) Correction for secondary comoonents i . \ { ( 1) Relative re3ponse factors .. As discussed in Note 4 shove, ECD response for the branched isomers is higher than that for the straight-chain ester, so that relative response factors must be determined in order to calculate their concentrations . Since F~.--~i response appears to be more nearly equal for all of the isomers, the factors can be estimated by comoaring peak area percent values for standards analyzed on the same column by the two different detectors . Differences in carrier gas, reagent interference, and relative peak sizes make it difficult to reproduce the chromatography exactly, but conditions should be chosen to make the analy ses as similar as possible . Calculate average area percent values by both detectors for the groups of five C8 isomer peaks, then use the ratios to determine ECD response factors relative to the main peak, as in the _ollosring example for an FC-143 st andesrd: Peak Number, 1k .95-17 .22 aia in Figure 4b #1 #2 N3 (main peak) AFL #g LCD area percent (ave rage of several runs ) 7 .38 11 .94 5= .76 10 .02 18 .91 FID area percent 3 .55 p+ .38 72 .88 8 .bL5 10 .73 (average of several rims ) ~ A '' 1l x/( ftn ir i ~ ;M CD \A i ~ FID 2 .93 3 .81i 1 .00 1 .67 2 .48 _ .{i . C~ttfpa`ny Sanitized. Does not contain TSCA CBI 000036 p .21 Page 20 of 22 For pe_fluoro-n-oetanoic acid standards (Figure ka) the only measurable isomer peak is # 5 . The factor calculated for it generally differs scevhat from that for FC-lk3 standards, as might be ex.xcted from the incomplete resolution and . different contributions from neighboring peeks . For samples ranging in type between the two standards, the average factor is usually taken . (2) Percent correction for total concentratio n For each sample run, use the peek areas and response factors to calculate the concentration of isomer peaks relative to the main I peak as in the following example (peak numbering as in the factor calculation above) : Figure l+e ; Sample 80-686o9 . Run #21, 12/30/80 Main Component Concentration 7 .63 ug/g F Peak Fi Peak Area w of main peek <Aiy(2) (iaterataits ~ 2 3 h .93 83 560 L }I2 8o 3 .81 70 460 3 .09e 1 .00 59 1000 (100 ) (main pealc) 7-63 Total p correction = 21 . 1 x 1 .2b1 .= 1 .67 32 130 3 .27*.r 9 .5 ?pm F total. 5 2 .73 20 9300 12 .91 *Peak 12 is freaueatly visible as a shoulder, but not resolved sufficiently for quantitative measurement . Depending on the size, the correction may be eonsidered insignificant or can be estimated by comparison vr_th the other isomers . e*'_his value is orobab1y too high, by compe .rison with peaks fl and #3 in the chromatogram in Figure he . Since the correction is only a few percent, however, the effect on the total concentration will not be appreciable . These calculations clearly involve many approximations, especially or samples unlike either of the standards in isomer distribution . Determination of the isomer concentrations is difficult in those cases, since the "response factors" for a group of poorly resolved peaks 'will be influenced by the relat :.ve sizes and contributions from each other as well as by detector response . For peeks 12 and gk in particular, the inaividual values must be cor_sidered estimates only . Since the contribution cf each isomer to th e . i ~otnpany Santtized. Does not contain TSCA CBI 00003~? ~ I i p .22 -. ~ Page - Code No . B L65 .5000 Orl8ina7. 3 Apr 8l 21 ai 22 ~ total concentration is usual?y small, however, the overall uncertainty will probab]y sti1l be less than if the composite peak were used . an.d no correction is made tar differences in composition . In most of the blood samples examined to date, the composition has appeared similar to that of pex3lnpro-n-octanoic acid ( See Note k), so that the isomer correction is small and calculated for the veil-resolved, peaks 1 an.d Ii . For those samples, the pe,cke3 colmin analysis should give equivalent results ('within the uncertainty of the method), but the canillesy analysis has the advantage of verifying that a sigaificant error has not been introduced, by undetected isomer peaks . I }; - Company Sanitized . Does not contain TSCA CBI ~_ ... OQa038 p .23 ~ '"3@~~ "~ `~~~ ~ .. .. . .- ..--._-- . --'- -o __---_~ -'. - '-- -- -- - -- Ori~inel 3 Apr 8i Page 22 of 22 /30/80 conditions as on p .18 seeracure program= ~ 35'C, 8'/min + 130'C, ( a) Standard, rat blood spiked with ".12 ppm perflnoro-n-octanoicx C8 esters , 130C _ acid .; C1p ester (ti15 ppm) iart speed 1 cm/min = ` w R w w ~ w = ~~ +. _^ ~_t',~,, ~w ~" tennation 210 for'20 min, - Q ''.- ~ ' ~ t w+~; " - ~ ~_~.. w~ Zafter " ww~A : S I (b) Standard, rat blood ~ spiked with 'ti16 ppm ; echyl perfluoro-n-octanaate FC-14 3 ,pears at 16 .6 min, branched Li .~w*~ at 16 .9 .16 .3, 17 .0, and C ester s 8 (14 .95-17 .22 riLe) ' .2 minutes . ~ Jr w -~ - C ~ i " s' ^. . M .~-.~ 'r+ti'~~ . - ~ '~^ s - w ~ - ~ -~ ~ .'~fi~ h Y-~~~ .. M ~ i i r cooparison with the (c) Hat blood sanple e piatograms in =igure 2 80-68629 : ~ .( ad column analysis) : ~e peak ac 14 .9 cain corresponds ~i ~ chat at 3 .i min i ie peak at 16 .3 fl in Iies under ~~ ~ le nain peak ~ e i ' : ~ ~ - :~'M t r.~,s :~'~-~"` i ~ ~ ~ ~ ~4 ii w ~ .^i ~ ~u ze peaks at 17 and 17 .2 min as a shoulder on the 3ck of the main peak I - ` : ,pear (d) Rat ' 7blood sampZe 80- 860 6 O i w w w : _ ~ ~:~ = ~ w~ ~~wr,, I ~ i ! J~ 'ii w 1Ce) Hat blood sampl e 80-68609 ~ : - .. ~ - ` 05 ~ : ~ s~`f"i' n T Company Sanitized. Does not contain TSCA CBI 000039 BEST COPY AVAILABLE