British·American~obacco C~rmpanpltd FUNDAMENTAL RESEARCH CENTRE P.O. Box 90, C~dr Pu~ Rwd, SwmMgWn, 202 7FS Engrd 'I~kphonn swlhMlppn (0703) 722111 Tekxe QIZb9 Fu: (07113) 779715 RRBlBTM 6 June 1991 See Distribution ReDodNP,r~228 The Measurement of Sub Nanoaram Ddiv~ienf 2naPhthYlamine and eaminobUenvl in htei~tt~am Smoke This Report describes the development of the ana~ methods for hvo substances on the SRG "other noxae' list, The developed method indicates that the levels in modem, norwentilated cigaret am at the subnanogram level. Earlier values reported by Mi~uda and Hofhnann for plain cigarettes, and quoted in reports such as that of the US Surgeon General, appear to be too high. R~J~~ $~~ R~R~~ar Distribution Mr. A.I. Hard, MAlba~e Dr. PJ. Dunn, m, canada Dr. S.R. Massey, m, Canada Ptof. E. Ritlershaw, BA'TCF, Germany Mr. I,C, Brwvn, BTC, Southamp~on Dr, J,S, Wigand, B&W, USA Dr. E, Kausch, BATCF, Germany Dr, CJ.P, de Siqueira, Souza Cruz Mr, T,I, Wilson, Australia Mr, H,V, Thomsen, ~nma~e MI, E, Grant, Arger~na ~ Melnber of the B~k~ Indurtricl Group Regd, Offte: r,O, Box II Wenmimter Ho~ne 7 Millblnk, London SWlr 312, Intorpontcd in London No, 139762, N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REPORT TYPE (RD w T): FI1E NO: (Dc#nrined by Is~c~ PROJECTNO: Yil CtCOC7 REPORT NO: SKllrilY CIPll~rKlliOn: @aerminedbIssocr) REPORT 1IT1E: Ill~k~tt ~FIE ynt~P~u e~E~l~v~i ai(SJ~cnogmHn D~utRICS 41 ~·rapt~r~aMlntlAM Fid(;Rplg· NO: ~·lcvu\~·o GIPb~L in ~od~~ FIITE~ I DIOWIRB Ofiiec) AVIBOR: VL""'An~ C~]4pl~I FglCnp~P·No: ~CI·A.k~J (CcmpotaDrmn) CO.WORKERS: ~kJ 1.R ~-pJg. PbolopsC·Ho: SEC~~ON 1EAI)EIUREVIEWER: ISSUER: R.~· BU'ce~ owRIBvnoN: RB(I):A~H(1): MIJ( (2): PID(1): SRM(1): TIW CZ): ER(1): EK(1): CmdeS(1): HYI(I): RPO(1): Ilkuy(2): Filt DISK REF15RENCE: IYPPIG MECIWG SipmuclDPeSgt Ike ~e Alo~bo~l DDe L~Pa Dot R#eivedJRmwAdl Reviram fo~ Ik·4~1~1 r14- Irrlan4t I· 2-5-51 rll clrbfC f,5 ?gYil?S SI 'D~LLfC~D ) j~l 13sij/ l'd·SPli- B~ ~L~G Comctiow E~ ~s~q~ Rep~e Ih~e BwPd CoW lssoc YY ~ ~, I I k rb ~91 · Thir prdw~M is i~drm~d ~faci8ote and daoil rk ~ otlu~vnz R~D CU~L Rlpmfmn ~ t ryplspipl st~b~ uorilrlv npon irF~a~ ~pp~pyl~l ond isrud~ The pq'o~M s~ho~d k co~Pnmecd or k n rims ~rhl ininbl IYPil oFd occo~npnn~ 1K IYPCKI~P~ (rmd n~srcr ~fn pnPorprion) or oil svbsopvmr L ~u. In~bcs~.roC~RD~Rl~o~drl$l(~navhnthr~ponCi~ld. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 THE MEASUREMENT OF SUBNANOGRAM DE~VERIES OF 2·NAP~rmVLIIMINE AND CAMINOBIPHENVL IN MAINSTREAM SMOKE REPORT NO:T~228 6 June 1991 DISTRIBUTION: Mr. Al. Heard Dr, R,R, Baker 2 Mr. I.C, Brown 3 Dr, J~S ~gand 4 Dr, J,S, Y~Y~F~gand 5 Dr, PJ. Dunn 6 Dr, S.A, Massey 7 Prof, ERItterabaua 8 Dr, E. Kausch 9 Dr, C,J,F, de Sigueira tO Mr, E, Grant 11 Mr, H,V, Thomsen 12 Mr, T,l, Wilson 13 Library 14 library 15 ,,, MCP~I~.DMlmPYOllhD~.launulhP~~plDnr ~p Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 fundamental Resea~eh Centre, Bli~shdme~iean Tcbac~ Co, Ud, SO~TTt~AMPTON~ ~t~E MEASUREHEHT Of SUB·NANOGRAM DEUVERES OF BNAP~HYLI~INE AND eAMINOBIPHENYL IN MAINSTREAM SMOKE REPORT NO:T~I 6 June 1991 AUTHOR: Gn Few ePWORKER: LR, fade PROJECT MANAGER: R~A. Crellin ISSUED BY: R,R, Baker COPY NO, O 1991 BAt O Ud, Do not wW o show to urw~tho~od pe~ws C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Centre, British·American TdMeeo C~~ ~M,, SOCrmAMPTON, GGFIBTM 6 June 1491 THE M~ASUREIIIEKT OF SUB·NANOGRAk~ DELIVERIES OF SNAPHTHVLAMINE AND 6AMINOBIPHENYL IN MAINSTREAM SMOKE REPORT NO.t.228 SUMMARY: InvesS4afon of the levee of ~naphttrr~amine and deminabiphenyl in mainstream smoke is being undertaken in FRC because may are of po$ntial interest to regulatory authorities, Reports in the sdentfie literab~re give their levels in mainstream smoke trom plain cigarettes as a few nanograms but there are discrepancies in the reported values which may be due to problems 1 the anal~dcal methods used. This Report describes a new a~ctical method developed for their determination in mainstream smoke. Alter trigping in phosphoric add, the aminea are exbacted into hexane. In the hexane they are then derivabied by heptaRwro~u~rie anhydride and ana~e~ed by gas chromatography with a mass selec~ve detector in the single ion mode. Apolieation to a standard, fluecured, king shed filter cigs~ene has indicated the presenceof the amines atthe subnanag~am level. These results imply thatthe earlier work of Patrianakos and Hoffmann in 19'29, who measured the deliveries of the dnes from pla~ US and French cigarettes, were of the right order, i.e. ~ ·5 ng, andthattheresults ol MasudaandHafhmann in 1 969far2aapMhylamine, who quoted 22 ng for a plain US e~s~e~te, were incorrect. Q 1891 BAT Q Ud. Do 1 coW orsno~r pp unaulholised pe~pla C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 INDM 'TER~S: WJaphthylamine ~C·Aminobiphenyl Mairstream Smoke GCMS ventilated ciaarettes F~lter Cigarettes e 1991 B~T Q. Ud. Do not ooW or s~w to unw~orbad perg#rs~ N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 1, INTRODUCTION In 1997 the BAf Scientific Research Group (SRG) considered those substances regarded by external aganirah~onslregubtory authorities which have specific toxicological pmperties and have been reported in cigarette smoke. G priwi~ list of substances for further investigation was produced which included 2·naphthylamine and eamincbiphenyl (1). The levels of the ~s~o substances reported in cigarette smoke have been reviewed previously (2). YRh regard to 2naphtylamine, reported levels in smoke seem to be confused In 1967, Muller and Stedman (3) failed to identify conclusively 2napWiamine in the mainstream from commercial cigarettes. Allowing fordetee~on limits, they concluded that the delivery must be less than 40 ng. They noted that Austian workers (4) did not dated Pnaphthylamine from Mended cigarettes of undescribed origin. In 1969, Masuda and Hdffmann found 22 ng from US plain cigarettes(5). This was refuted in 1979 when Patrianakos and Hoffmann (6) found 1 ng and 1.7 ng of Snap~llamine in plain 85 mm US and plain 70 mm French cigarettes raspecblely. Equivalent levels of Caminobiphenyl were quoted as 2.4 ng and 4,6 ng respectively, The Hoffnann school atbibutes the reduction in2·naphthylaminehom 22 ng to 1 ng for a plain US cigaretteto product changes, at least partially. However, bey note that they cannot exclude the effects of differences be~Neen the analytical techniques used, It seems unlikely bat produd changes would explain a 95'/0 redudjon for plain cigarettes which suggests a difference arising from the ana~ses. If Patrianakos and Hoff mann's 1 and 2,4 ng delivery results for Snaphthy~amine and 4·aminobiphenyi respectively from plain cigarettes are coned, modern filterlventilated products would be expeded to deliver subnanogram quantities of the two amines, this presents a considerable anal~cal challenge. The objective of the present stu~ was to develop such anal~cal methoddogy, 2 METHOD DEVELOPMENT The deriva~don of fairly involatile amines to vola~le amides using Lorinated acid anhydrides followed by gas chromatography with an electron capture detector (ECD) is a well established mute to their quantitation. A number af fluorinating agents are available wib, perhaps, the two most popular being pentafluoropropionic an~dride (PFPA) and heptafluombuybic an~dride (HFBA). Given that the smoke levels of the amines to be quantitated were expected to be at the low nanogram level (no more than 1 or 2 ng) and h~at the sensitivity of be ECD increases with be number of fluodne atoms in the molecule, HFBA was chosen for this wok Method development soon exposed the pitfalls of using an ECD. Itr ert~eme sensit~i~j meant that before the wmplexi~ of the O 1991 BAt Co. Ud. Do not ccW or show to unaubassed persons. N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 sample matrix was taken into amount censideraMe care had to be taken to minimise #mtaminsli6n entering the system, The following preeautiom had to be taken: (i) The hexane sdvent had to be purified so as to be acceptable fdr ECO work T~ was provided by Rathbum Cherrjcals Limited at the suitable level of puri~, n awas found that twas impcrtantto use an inert J8i~m or one wilh a PTFE mating fadng the eokmn. ~ prevented any interaction between the HFBA and the septum and eradicated sepblm bleed. Fl HFBA is very la~ile, behg hldolysed readily by wa$rto give HfB add. Inthis wcrkitwasstored under an atmosphereof dry ni~ogen. Having been satisfied that ro contaminants interfered with the elution of the amides, the comp~xity of the sample matrb: tan exbaeted smo~e solution) was insatiqated, Alhough the ehromatogram was found acceptable far one of the amides, itwas totally unaecsptable forthe o~er, multiple cpelulion a high ievei occurringaround its retenliontime. Giventhatawnsiderableamountof method developmentwould have had to have been invested in elea~ing up the sample mahix, Lwasdeddedtochan3eto amass selec~vede$ctor(MSD), this a~pwing single ion monitoring for each of the rideJ. Single ion monitoring had tee adv~tages war the ECD: ($ The chmmatcgram obtained would befor one iononly, equUnt to the mdecular mass, and hence definitive for an amide. (ii) Single ion monitoring would be aquivel8rtl to several deari~ up stages, 2~ Present Methodoiogy 11I1 Reaeente Hexane and water solvents were obtained hMn Ra~um C~mica~ Limited The 2·napMhylamine and baminobiphenyl were synlhe~ised by Salford UltraCne Chemieak Limited, Their punty wls unspeeif~d but OGMSD sb~dies 'u~di~ated a high level of purS, HFBA was obtained fmm the Pierce Chemical Company as 1 cm~ a~npaules, Sodium hydroxide pellets were ob~ined from BDH limited, O 1991 BCT Co. Ltd Do not CC9Y Or ShOW1P Inaump6wd persons. N ·P Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 21,2 Preparation of the Sample Using one port of a Flbona 302 8·pon smoking engine or a Bcrg~dt s~gle polt smddng engine, 20 cigarettes are smoked to CORESTA standardsthrough a double bubble trap each containing 20 #nl of 0.1M phosphoric add. The smoking engines are protected by a Camkidge fiLr pad afterthe bubble traps. After smoking, the contents of the Ir~ps are fitered through a No, 1 Wha~an titter paper. Rw oon$nts of the finttrap are filts~ad into a 50 eml volumet~ flask,the contents of the seoond trap are then poured into the First trap and then also hltered into the lask. The second trap is then washed 13 eml 9qud of the phosphoric add soiu6an which is then used to wash the first trap before fiCaticn, Thewashlng and liltrafon a~e then repeated at filbatas being collected into the 50 an' flask Some more ph39phoTic acid is then added, 1 necessary, to bring the level of the acid up to the neck of the flask (this tadiitates ease of removalof thectganie phase after hexaneextradion). The contents of theflask am then made alkaline by the addSon of mugh~ 1 g of sodnum hydroxae pellets to the sol~bn, the contents of the flask being vortex mixed until the pellets dissolve. This ensures that the andnes are in b~efree state, i.e, not protonated, In the free state they can be extracted by a na~polar solvent. Having allowed the Aadtlo coal, the a]kaline solution is then exb~eted manually by a4 em3 a~udof hexane for 15 minutes using w~t action shaking. After 15 minutes, 3 an' of the hexane layer is removed to a graduated 10 mm x 75 mm, disposable, Pyrex culture tube and EoncenM8d down te~old by a gentle stream ct rdtrogen. At a volume of300 ul the culture tube is mad with a ground glass stopper to await b~e reagent for the formation of the amides. 21,3 Formation of the AmMes Addon of HFBA to Zaapt~llamine and Q·aminabiphenyl will form the HFB amides as shown on the following page. a 1991 BAT Q, Ud Do not capy or show to unauthorised penons~ N b Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 H.M (H·~ 010 ~pli~niw II K(2~pm~l) I~ H.M I H·u I H·u H~ cinQphwrll I H·fCb~ II~ 3 ~ ef HF~ it added te he Plrex witure tube ~bh it hen r~sto~~ ad piard iA a 'Reactivial' heating mantle at SO'C fer or hcur. 300yl ef w;der it ha added b the tube which it ,sSoppered and vertex mixed br 6 minutr te remwe ·ao HEBA ad HW rid. ~, ueel hexane layer it new rea(fj br analysil, Scme auhors adviJa he uae ct a weak Ylno riden te remeve he exlls H~ e.O. 53b ammodur hydrexide, it hr been feund inthiswcrkthat aJtrong Yins riden will aw hydrciyait d he amidr ad in fat rp bm erur even ton weak 1Yi it od, Hence, it it raemmaded hat water alone he wed k remeve he excess H~ ad HFB dd~ O 111 ~ Q. lfd nO rt eDW P tnOW 1 UPIUlmOl(Wd q01. ClibPDF - v~~fastio.soni 21.4 GC·MSD Analysis 2 Irl of the hexane layer are injedad into a Pe~kin·Elmer Sigma 3 gas chromaiograph interfac~ed to a Hewletl Packard 5970 MSD, fhe interface was held at 240'G and the gas dhromatographie co~ions were as follows: Column: Sepulco Inc., fused siEca capillafl SP&5, 60 m, 0.32 mm 1D, 1,0 pm Nm thickness. injection port temperi~ure 250'0, Temprature program: 180'C to 220'0 at rChninute, then hold for 20 minutes~ Helium carrier gas column p~essun: 10 p~si. Gas k VIrIuBh splittef: 5 cm'lm~ute. The #SD was adjusted lor the singh ion monitoring of the mdecuiar ion of N-pnap~i~yl)·HFB add amide, r, 339, The e~cb~n mulSplier voltage was 2400V and the speetrometer wasnot switched on until after the solvent passed though the system. Under Vlese chromatographic conditions the retention time of the nap~t~ylamaewas arwnd 25 minutes, Its mass spechurn is shown in Rgule 1 and the single ion c~uomatograms from a 25 pgl~ standard and the analysis of a standard, Ruecured, finer king sire cigarette are st~awn h Figures 2 and 3 respecb~ely. Following the ronitocing of the m, 339 ion a further 2 id of the hexane layer is injected with the MSD setto monitor solely the mdecul ion of the N~4·biphsnyl~HFB acid amide, m,365, whose retention time was amund 37 mi~nes. Its mass spedrum is shown in Rgure ~ and, as forlhe naphthykmide, the single ion ehromatograms hom a 25 pg~ standard andthe ana~sis of a standard, fluecured, filter king sire cigarette are shown in F~ures S and 6 respectively. 215 RepraBon of Standards 2·NAPHTHYLAMNE AND CAWNOBIPHENY1 ARE KNOWN HUMAN CARCINOGENS, WORK wrm me souos rO MAKE UP STOCK SOLUTIONS MUST BE UNDERT,AKEN WITH PARTICULAR CARE to REDUCE ME CHANCE OF SPIUAGE OR HUMAN CONTACT. HUMAN CONTACT YAY BE AVOIOED By WORKING IN A FUME CUPBOARD IN AN ISOLATED LABORATORY WEARING DISPOSABLE LABORATORY COATS AND GLOVES AND SAFETY SPECTACLES, AMINES CAN BE NEUTRALISED BY CONYERSION '10 THEIR HYDROCH1ORIDES. O 1991 BAT Co. Ub Do not eoW or snow to unaloia~d pcow O C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 his advirahle, becauseof theca~drogenic nature of these substances, to reduce stock solutions to the minimum. In this wo~e 20 mg of each amine are weighed intothe same 20 ad Mlume~ieRask and hexane added En give a concentration of l~gl~lforeaeh. DluiionsafWssMde solution aretheo Mdetogive solutions con$iningl and tOrgi~of each amine. TwoSO an'volume~ieffa~kruetaken and 0.1M ph06ph0lC add added to heel unSI the me~scur of the pid is at the neck of the masks, into the phosphoricacid of one of these flasks ~ pi of the 5 naj~ solution is injected wib~ immediatevorlex mixing. Into~eadd of the ether, 1 ~ofthelOng~lrlsolubbn is injected, again, withimmedhlswr$x mixing. The fonner flask provides a 125 pgllJ standard, ~ut~Etthe latter pcwides a 25 pgCI standard. Afterafewrrjnutss o~vMtexmi~ing, roughly 1 gof sodium hydraxae pellets areadded to each mask, again wt vortex mixing. Whenthe pellets have dissolved and the masks have cooled,4 cm3 of hexane is added to each flask. Each flash is then manual~ shaken using a wrist action for 15 minutes. The flasks me then allowed to stand for awhile ta allcwthe pharestP separate. For each ol the flasks, 3 crn' of Ihe hexane phase is removed and concentrated tedoM by blowing down using a genbe sb9arn of ni~ogen. Theln standards are now ready to be taken through the deivadisaticn to give heane layers con~d~ing 125 and 25 pgbl of the amjdes. Thess soYons are bjeded into the GC and monitored by single ion monitoring of the m, 339 and m,365 ions, 1 the MSD chanaing its Fmgram at 30 minutes into the run to aeecmmodate single ion monitoring of both ions in one run, Only two standards are prepared and run for each sample since the response is linear in his region and this is the region of interest with regatd to modem dgarettes. As the standards become monger, a calibri~ien curve is produeed which deviates mom and more pos&ely from Inearity~ It would appear from the anaiysis of second extradions of the standards with fresh hexane that recovery at th~ level is ahnost 100%. AU soiledplassware is ento soakinll hydrochb~add. Oispasal isbydiiut~n to waste. 2.1.6 Calculstlon of Dellvwles let Se " integllf8d abundance of Ihe 339 ion in the sample let SR tintegrated abundance of the 365 ion in the sample let 4 :: integrated abundance ol the 339 ion in the 125 pallJ standard Let~Lintegated abundance of the 365 ion in the 12.5 P4~J standard lel yp' integrated abundance of the 339 ion in the 25 pgl~ standard let H~·integaled abundance of the 365 ion in the 25 WhCI standard O 1991 BAT O. Ud. Do h31 copy or show to Lnaulho~d p~wrau. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill I~ll~ldli~fi~·4 ~~y~~)6~·BI~P·4 ~,~,~~aph~mi·l~l~' ~~t· b Condrs· dki·lr91~ bl~ 1C ~'~·t ~ad2~ap~lyl·l··G r ~~r erad~b~~·G I~~~ r pa~aie rROI ·DWIDB~OY~ WYaln1~~ d ~pmF~l~i'd~~ $~'~ ~Y~ md,m ~ dgarsttes, P, ·~ b ~ Y~ ~~I L Y~ ~ 4~'' ~J bdl~~~ h nL ~ gk~ ~ rd ~~ ~ tdg d P~~I ad HBP b 119 ~ ~b ~'d dlY* dl ~ p q d ~~~b~yla~nbs adP~S~J~inc~iphe~CIYI" US d ~ mt ~m ~arattes· ~~uette 2·NpMlt~~ia OP 01 01\P6 O 111 ~ O. U. Do dla ~ ~ uM~tm0rmd pmo~J. O ClibPDF - v~~fastio.soni REFERENCES i. SRG btber noxae' lid Odober 1967. 2, BATUKE Report No, AD2146, 13,7,1999, 3, Millerp.L, Stedman,R.L, Tobacco, 165, 32 (1 967). 4. Pailer,M,, Hub~e~WJ., KSnP., Fach. MW~ Ostene~h, Tabalueg,, 7, 1(196r)~ 5, MasudaY., HolhnannD, Anal, Chem., 41, 650·652 (1969). 6. Padriarakcs~C. HoRmaM,D., J. Anal. To~deol., 9, 15015( (1979). 01991 BA~ ~. U DorotmWorshcwmurJlmorksdpfna· C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 RG, 1 T, 220 MASS SPECTRUM OF N·I2·NAPHTHYU·HFB ACIO AHIDE ~8401 nit 339 Ilr no .c~~ol i· P~u R12 ~m Jt UZ s/ ~rN 1- olt ns ~BUNDAN[E ns ll.OE5 6,OE5 a9 339 ~ as no ZOt h' / zn nz a C 100 200 300 nASSII:HAR[iE c\ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fit. 2 T. 221 SINGLE ION [HROMATOGRAM FOR q 339-2L INJECTION IK A 25 pglpl STANOARO ABUNDANCE 1600 1100 1200 1000 15 20 25 30 TPIE Imin,) Flli. ~ SINGLE ION [HRQMATdORAM FOR ml319·2dlNJ[tfN1N AOM A ~9uwoc~la STANDARO. FLUT~CURED. KING SIZE FILTER [IljllPE~~E ANALYSIS H· 12·nlphth~·Hf8 Ibd id~ 6 20 25 30 !S to a TIHE Inin,l ' O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Rh, L I nl ~n~ss sPrrraul M~c-aPHEnu-HFa ~oo ~no~ P, ~t 36 Ilr 1)1 be \~ P 1, ~ ~ t-- ~lr ns Ilt 151 U H h31 ~r 152 ~uolw~ lil 1.OES 1~5 (! . I · ~' 2.OES ns :C~ a 2(7 311 o 10~5 I . i I~ I 181 21 310 o MASSIMAR5iE N ClibPDF - v~~fastio.soni F& S f. 221 IHGLE HIN WIORIlO[iRAH iOR m~di-lylHII[TION Of A ii pglpl STINOBO. ABUNDMIIE 1800 1600 1500 noo looo 1~ io is so 35 40 TI~ Inin,) nt, 6 SIN[ilf Ilul MROnlm[iRAM FOR in, 1IZ·?ui IYIE[~KIH fROn I STAHOIRO RUE-[U(m. m(E SeI FLTER [IEbREITI INMYSIS ~euwD1~N[E 1000 H· Il·biplMyll -~B rid wi~l 2000 1000 O if ~o is co ^4 ~IHE (Rin.). i ru Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~I I yll BRmsH AMERICAN TOBACCO CoWRlgu~EtForm PPUdNun~be~l6 Organization: Dept, of hstice - Legal Health, Canada r Request Details )~·i -'i5 Request Date: ~...~~.~.~..~~...~....I~~~.~~.~~..~..~.~·~·····~·~~···~·~····~~···~···~~··~······r File Hum~~ FIW Ijl~~ta3,.,.....,.,Bor Number: ~P OMxl*.t.*ll. 111111111 111)11))111 Page Range: First Fag Last Page rlo lalrlolllhlYIZI· 14 10 lal~ lo aplaa B~WnN~nll····l?~n~~hi ···~j~ermnr~~~~~ ~~~~~~ Details below will be filled in by Depository Staff Only U: CopyDehils Copied By: ~o, LL 1111((1111111111 )111111111111)11)1 111111111111111 11(((111 (IlIIIIIIIIII(I Dlh: ...I~.:~;·~~:~~..·..·I······(···tl~···T(~ (·a·~~G~··ehl. Copy Checked By: 11(111111111.d:..~a~K~ 111111111111111~1111)1)IIIII(III)))YI Date: ....~~.,~.~~.~~~~.· .I.IIII. I~I)~()I))111111I)I IIIIIITime: 1(I(IIIl 111)(1111111111)1~1~1)11 III Delivery Details Checked $: .··.···.~~~~~··.·..·~·.1·11..1(1111··..1111 Date: I·(I11I)III1II(1I1I1·11)(1111·11 Sent By:IIIIIIIIIII,,I, II1,(1(11I 11111111111111111111 1·11 Date;IIlII( 111111111( )1))111111111)11( C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 British~Ameriran~obarro~ompanpltd FUNDAMENTAL RESEARCH CENTRE pc. sox 99 ~kpmt~s Puk Rosd, Swlhsmpl~, 909 7FS Ef4ud Tekohpne: Soulhunplon (9792) 7112111 Teox: 61264 Fu:(OtW) Tf9715 RRB~BTM 6June1991 See Distribution Reaort No, T,228 nle Measurement of Sub Nanooram Deliverles_of 2naPMhvlamine and ~amlnoMDhenvl in Mainstream Smoke this Report describes the development of the analy"doal methods for Bva substances on the SRG 'other noxae' lid fhe developed method indicates that the levels in madam, ro~ventilated cigarettes are at the subnanogram level, Earlier values reported by Masuda and Hoffmann for pbjn cigarettes, and quoted in reports such as that of the US Surgeon General, appear to be too high RI1SnJ$IJ R,R, Baker Distribution Mr, A,L, Heard, Millbank Dr, P,J, Dunn, m, Canada Dr, S,A, Massey, m, Canada Prof. E. Rittenhaus, BATCF, Germany Mr, I,C, Brown, ETC, Southampton Dr, J,S, Wyland, B&W, USA Dr, E, Kausch, BAfCF, Germany Dr, CJ.P, de Siqueira, Saura Crur Mr, T,I, Wilson, Australia Mr, H,V, Thomsen, Denma~ MI, E, Grant, Argentina A Memher of the BA~I. Indur~i* Group Regd. office: P.O. Box 4112 Wmmimts Hou!, 7 Millbonk, London ZtP ~E~ IncarpPntcd in CMldon No~ 239712, N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Il~nnrle,·n: f~ Tilt MO: (D~mirdbY rran~: 91 I·C) ~C lg~~ MO: ~dt Mhe @aeanined CIISOCI) Im,~~B: ~E ~~~n~v~45~ ~bl05m~ ~,~ler 8 I.mpS~,~ ra ~h~·ME C·~O l~lOP: V~n,Mco t~J 4 rC ~ I F~Cn~ · n: ~~·l.k~ (CaplPW ~~o~n: gdCn~ 1 1.1.~1 Pklgn~·MI: ~~10R ~EWREIIO~: b~~~R: IF): mOk mg,: wb): ~~: k DISK REFE~A~ MPG I CHBC~PIG ti~m~e I~~ a Alhc"l I~JR~ L Reetived I Re~Emad I Revimer ~t I I b~f *I~p~ Ic~lLnlt I 2-51 t 1 I - 1 17 gq( (1~ fi ~a ) i~l 111~1 Papre 1~·541-~ j ~ Concctio~ ~i Cc·9l~i; E~ r~3~( Repre ~ BOIIPd ~ rd·gl~ Irdwmo i LMJII ~focili~rJY~ pro~ns~~dATLI~Er ~1C~ L C ~ni~ ~ ~ ~ C iW Ippmcd~ ~B p~jwn~l~~pld~~ h k ~~ ~ ~ ~ ~GO~b~n)~ rlPaaipl ~3C pllj~PljOllJ I~IYbS1PWnl CU~PI Rccwds ~Y ~ Ma~a ~ ~ ~ C ~ ClibPDF - v~~fastio.soni fHE YEASUREMEM OF SUBNANOGRAM DUIVEAIES OF 2·NAPMHYLIIMINE AND CAMINOBIPHENVL IN RIAINS'TREAM SMOKE REPORT NO:T228 6 June 1991 DISTAIBI~ON: MI. AL Heard Dr, R,R, Ba~cer 2 MI. I,C, Brown 3 Dr, J~S. Wigand 4 Dr. J,S, ~gand 5 Dr, PJ. Dunn 6 Dr, S,R, Massey 7 Prof, E, Aittershaus 8 Dr, E. Kausch 9 Dr, C.J,P, de Siqueira 10 Mr, E, Grant 11 Mr, H,V, Thomsen 12 Mr, 7.I, Wilson 13 library 14 Libmtl 15 01991 BIT CD, Ud mlY~mPy ol~harm ms~ho~ p~l~onr C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundmnental Research ~~I ~~Ck~a fcbaceo ~~ Ud, ~lrFHAM~~~ ME MEASUREMENl OF U&~IQYY DEUYERIB Of 8r~gtWllr AMD ~YYOBIPH~L lr ~YW~Y ID(t REFORT n0:T~2#I 6 June 199~ AUMOR: G~ Few eO·WORKER: 1R. Fade FROJECr IMYW: Rk Clln lSSUFO Br: R,R~ Raker eOPV NO, elrl BATalld oc~4'~lun~ho~d pwprP. ClibPDF - v~~fastio.soni Fundamental Research Centre, British·American Tobacco Co, 13d,, SO~T~HAMPTON, GAFIBTM 6 June 1991 THE ME4SUREMENT OF SUB·KIINOGRAM DELIVERIES OF BNAPHTHY1AMINE AND eAMINOBIPHENYL 1N MAINmREAM SMOKE REPORT NO,r,229 SUMMARV: InvesSgation of the levels of Baap~tylamine and daminabiphenyl in mainstream smoke is being undertaken in FRC because they are of po$rdial interest to regul~P~ wthoriti~s, Repa~tJ in the sc~ntifie lilerah~re give their levels in mainstream smoke from plain cigarettes as afew nanograms but there are discrepancies in the reported values which may be due to problemswth the analytical methods used. T~ Report desaibes a new anllyrcal mel~d deve~ped for their determination in mainstream smoke. After tapping in phosphoric acid, the amines are extracted into hexane, In the hexane they are then dei~vab~ed by heF~aRwrobu~6e anhyddde and a~sed by gas duomatography with a mass selective detector in the siqle ion mode, Application to a standard, Ruocured, king sized filter cigarette has indimted the presence of the amines a(the sub·nanagram level, These results imply b~af the earlier woh of PatriaMkos and Hdfmann in 1979, who measured the deliveries of the amines from plain US and French cigarettes, were of the ~M wder, is. 1·5 ng,andthattheresulof MosuCPndHdihnannin 1969for2aapM~mine, who quoted 22 yl for a plain US ciaamtte, were incomci, ~1991 BAT Co. Ud. Do rot coW orsnow lp wulholi~ed pppls. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 INDEX TERLIS: 2NapMhllamine ~Aminobiphenyl Mainstream Smoke GC~S Ventilated Ci~arettes Filter Cigarettes a 1991 BAT Ca, Ud. Do not eoW oc show to onollth#tsod p#BwQ· N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 1, INTRODUCTION In 1987 the BAT Scientific Research Group (SRG) considered those substances regarded by external orgnira~:nhJregulatory authorities which have spedic toxicokgical properties and have been reported in dgarette smoke, G priority list of substances for further investigation was produced which included 2·napM~lamine and 4aminobiphenyl (1), the levels of the two substances reported in cigarette smoke have been reviewed previaus~ (2), Wlh regard to kap~yh~nine, reported levels in smoke seem to be confused. In 1967, Muller and Stedman (3) failed to aerldy conclusively 2naph~lamine in b~e mainstream from commercial cigare#es, Allowing fordeteetion limits, they concluded that the delivery must be less hclan 40 ng, They noted that Austrian workers (4) did not dated kaphthylamine from Mended cigarettes of undesnibed origin. In 1969, Masuda and Hoffmann found 22 ng from VS plain cigarettes(51. This was refuted in 1979 when PatriaMkos and Hoffmann (6) found 1 ng and 1.'I ng of PnapMhylamine in plain 85 mm US and plain 70 mm French cigarettes respedblely. Eoulveierrt levels of Q·aminobiphenyl were quoted as 24 ng and 4,6 ng respectively. Tne Hoffmann school atbibutes the re$uct~n in Pnaphthyla~nine from 22 ng to 1 rgfora plain US cigaretleto product changes, at least parb~ally. However, they note that they cannot exclude the effects of dfferences between the analytical techniques used, It seems uolbly that pcoduel changes would eqlain a 95% redudjon lor plain cigarettes which suggests a difference arisiq from the analyses, n Palrianakos and Hoffmann's 1 and 2,4 ng delivery results for anap~rmylamine and baminobiphenyl respectively from plain cigarettes are coned, modern filterlvenblated products would be expected to deliver subaaragram quanlities of h~e ~Na amines, This presents a conside~4le ana~wl challenge, The objedjve of the present study was to develop such anal)"6cal methodology, 2 METHOD DEVELOpMENT The derlvatisation of fairly involbdle amines to vola~le amides using Ruorinated add anhydrides followed by gas chlomalography with an elecbon Fapb~re detector (ECD) is a well established route to their quantitation. A number of luorinating agents are available wYh, perhaps, the two most popular being pentafluoropropionic anhydride (PfPA) and heptafluarabu~ic anhydride (HFBA), Given that the smoke levels of me amines to be quantitaled were expeded to be at the low nanogram level (no more than 1 or 2 ng) and that the sensitivit~of the ECD inereaseswit~ the number offluodne atoms in he molecule, HF:BA was chosen for this work, Method development soon exposed the pitfalls of using an ECD, Its extreme sensitk~i~j meant thz~ before the complexity of the ~ fssl BAf Co. Ud Do not ceW M show to unauthorised PBIJOnS. N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill sample matrix was taken into arx~o~vn considerable care had to be taken to minimise contamination entering the system, The following precautions had to be taken: (i) The hexane solvent had to be purified soas to be acceptable for ECD work This was provided by Rathbum Chemicals limited at the suitable level of puri~, (ii) Ilwas found that it was importantto use an inen wplm or one with a P~FE coating facing the column, T~ prevented any interaction between the HFBA and the sepb~m and eradicated septurn bleed. (ii) HF84 is vey labiie, being hydolysed readily by water to give HFB acid. Inthiswo~ itwasstored under an atmosphere of d~ ni~ogen. Having been satisfied that no contaminants irtedered with the ei9on of the arnides, the mmplexit~ of the sample ma~e tan extracted smoke solution) was inveslgated. Although the chromatogram was found acteprab~ for one of the amides, Swas totally unacceptable forthe ether, multiple cpelu2on at high level ocouning around its retention time. Given that a wnsiderable amount of method deve~pmentwould have had to have been invested in dea~irg up the sample matrix, twasdecidedto ehangeto amass selective detector (MSD),this a~wing single ion monitoring for each of the sass. Single ion mcainodng had ~no advantages over the ECD; The chromatogram abtained would be for one ion only, equUnt to the molecular mass, and hence defiritive for an amide. iii) Single ion monitoring would he equivderrllo ~everal c~aning q stages~ 21 Present Methodology 2,111 Reagents Hexane and water solvents were obtained horn RaVlbum Chemicals limited. The Pnapht~llamine and esmbabiphenyl were syntheeU by Salford U~trafine chemicals limited~ Theirpuiny was unspeei~d but GC~SD studies indicated a high level of puritj, HFBA was obtained trom the Pierce Chemical Company as 1 cm" ampouies. Sodium ~dmxide pellets were oMahsd from BDH limited. g lggl SAT Co. Ud. Do not coWPr show to wlaumdi80d ps~o~ ru P Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 21,2 Preparation of the Sample Using one port of a fin~na 302 kpn smddng engine or a Boga~t single port smoking engine, X) dgarettes ;Ue smolced to CORESTA standards through a double bubble bap each containing 20 wn5 of O.tM phosphoric add, The smdd~ enghes are pmtected by a Cambridge filter pad afterthe bubMe traps. After smoking, the contents of the traps are filtered though a No. 1 Wha~nan alter paper~ The contents of the first trap are filtered into a 50 ~n' volumetric lash, the contents of the second trap are then poured into the fnst trap and then a~so AUered into the lask, The second tap is then washed with 3 cml aliquot of the phosphoric aca wiulan which is then used to wash the first trap before fbidion, The wa~hingandrltrat~n are then repeated, a]lfiitrales beirg ooileoted into the 50 cm~Rad~ Some mom phosphoric acid is then added, I necessary, to big Iha level of the acid up to the nede of the Rask Pis lacililales ease of removal of thecrganiephase afterheuaneeutradion). The contents of thelabk are then made akaline bythe addiEon of roughly 1 9 of sodium hydroxae pellets to the solution, the contents of the flask being vortex mixed udl the pellets dissolve, This ensures that the aminas are in thefree state, is, not protonated, In the free state they can be extracted by a nonpdar solvent Havingallowed the Radrto cool, the alkaline sdut~n is Ran extracted manually by a4 cm5 a~uoldhexane for 15 minutes usingmaacCon shakirg~ After 15 minutes, 3 cn' of the hexane layer is removed to a graduated 10 mm x 75 mm, disposable, Pyrex culture tube and Concentrated down tercfold by a gentle stream olni~ogen, At a volume of 300 1J the culture tube is mad with a ground glass stopper to awa~ the reagent for the fonaCon of the amides, 2.1,3 Formation of the AmMes AddiTon of HFBA to 2dapMtJhmine and daminobiphenyl ~O form the HFB amides as shown on the fdlowing page. O 1991 SAT Co, Ud Do not copy or show to unaulho~iJed penons~ N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 H·~ 010 l~w~h~lmiw II Kn~qm~l) I~ ~·M I H·~ Iol i H·~ H~ I·min~bphnll I N~ldjJwyq II~ 9 ~ of H~ b d~ ~ be qrex abl ~ ~bh b ~n ~b·s~opp~ nd ~~ i~ a'~d~~' ho~g A~ ~ 50'C ~r on hour 8~ d r+r C ~~ d~ ~ b~ t~ ~~ i ,stopps~sd ~~ #~ ahd ~r 5 Rh~ ~ ~~ r~ H~ d H~ ~, ~I r~r hlm~ I~r e o~ reao~ ~r nS~it, ~n, ~oa ~~ ~ u# d ~ wePk Yine al~n ~ ~cs La sx~ H~ e~O~ 5K n~~um ~IRYI, ~ hO ~nku~ in hint~ I Jtrong ~in, ~ldon wl ~r ~n~ d ~ ~i~ md In ~ ~m ~w ~r nan ~en weak JMI 1~ u~. Hno, ~ is ~mm~d ~ rlr 8n ~ ~ b ~~ ~ r~ H~ nd HW dd~ O 191 ~ Q. ~d ~ ct ~ r ~ ~ ~nwYI#lsod ~o·, ClibPDF - v~~fastio.soni 2~~4 EGMSD Analysis of the hexane layer are iriected into a Perkin·Elmer Sigma 3 gas ehromatagraFh interfaced to a Hewlett Pactard 5970 MSD, The interface was held at 240'C and the gas chmmaographic #Indi~ons were as follows: Column: Sepulco Inc,, fused s2ca capillary SP&5, MI m, 09 mm ID, 1,0 pm film thickness. Injdw port temperature 251]'C. Temperature program: 150'0 to 220'0 at 2'Chninute, then hold for 20 minutes. Helium carrier gas column pressure: 10 p.sj. Gas Row through splitter: 5 #n"lmLrute, The #SD was adjusted for the singh ion monitoring of the mdecuiar ion of N·pnap~yl)·HFB ae~ amide, m, 339, the elec$on mulS~ier voltage was 2400V and the specbPmeter was ~ switched on until after the solvent passed though the system. Under Ihese chromatagraphie conditions the retention time of b~e naphthylamide was arwnd 25 minutes. Its mass spec~um is shown in Agure 1 and the single ion chrornatograms fmm a 25 pg(lJ standard and the ana~ysisofastandard,flue~·cured,filler king size cigarette are shown in Figures 2 and 3 respectively, Fdlavirg the monitoring at the m,339 ion a further 2 pi of the hexene layer is injected ,iVI the anso set to monitor solely the molecular ion of the N~dbipherrjl)·HFB acid amide, m, 365, whose retention tim, was around 37 mirwtes, Its mass specbum ~ shown in Figure 4 and, as for the naph~ylamide, tne single idn chmmatograms from a 25 p9ilJ standard andthe aaaiysis of a standard, lue~ured, Lr king site c~aretle are shown in Figures 5 and 6 respectively. 21.5 Pr~pratlan of Standards 2·NAPHTHYLAMINE AND 4·ALUNOBIPHENYL ARE KNOWN HUMAN CARCINOGENb WORK wrm me souos To MAKE UF STOCK SOLUTIONS MUSt BE UNDERTAKEN WITH PARTICULAR CARE TO REDUCE THE CHANCE OF SPILlAGEOR HUMAN CONTACT. HUMAN CONTACT MAY BE AVOIDED BYWORKINGINAFUMECUPBOAROIN AN ISOLATED LABORATORY WEARING WSPOSABLE LABORATORY COATS AND GLOVES AND SAFETY SPECT~C1ES· AMINES CAN BE NEUTRAUSED BY CONYEASION To THBR HYDROCHLORIDES. O 1991 BAT Co. Ud Do nor mW olshav Lo uns~wised P~P~, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 his advisa~e, because of the cardnogenic nabJre of these substances,to reduce stook solutions to the minimum. In this work 20 mg ct each amine are weighed into the same 20 cmJ volume~ie flask and hexane added to give a concerdra~on dl CBCI foreach, Dlutionsof VSstocksolution arethen Mdetogivesolutions containing 5 and 10 ng~~ of ea~eh amine. ~wo 50 em'volumebicflasks aretaken and 0,1M phosphoricadd added to bolh until the meniscus of the act is a the neck of the tasks. Into the phosphoric add of one of these flasks 1 ~ of the 5 ngilJ sdut~n is irjededw~ immediatevortew m~ing~ Intotheadd of the other, 1 ~ of the l0ng~~solution is h~jected, again, with immedhlevonex mixk~. The fonner flask provides a 125 pgllJ standard, u~i~t the latter pmvides a 25 pg'ut standard. After afaw minutes of vortex mixing, roughly 1 g of sob~um hydmxide pellets are added to each flask, again wt Mrtexmixing, Whenthe pellets have dissolved and be flasks have cooled, 4 cm3 of hexane is added to each flask. Each flask is b~en manually shaken using a wrist action for 15 minutes, The tasks are then aliowed to stand for a while to allow the phases to separate. For each of the masks, 3 cm' of the hexane phase is removed and concentnted ten·fPld by blowing dorm using a genb~ stream of nloogch Thehn standards ,,ready to be taken through the delvatieaZwllo gLe hexane layers containing1L5 and 25 pg~l of the amidas, These solutions are injected into the GC and monitored by single ion monitoring of the m, 339 and m,365 ions, 1 the MSD eha~ing its pmgam at 30 minutes into the run to accommodate single ion monbring cf both ions in one run, Only ~n standards are prepa~ed and run for each sample since the response is linear in this region and this is the region of intere~et w~i~ regard to modem cigarettes. As the Standards become stmrger, a cal~raSM curve is produced which devisdes more and more positively from lineari~j. n would appear tron the ana~sil of second ert~ons of the Mnda~ds wl fresh hexane the recovery at this level is almost 1009b, AU soiiedpiarswue is lefflo soakin~h~ hydlae~o~add. Oi~pos~ isbydilution to waste, 21,6 Calculation of Deliveries .s, .integrated abunlance of Ihe 339 ion in the samp~ Let S~Jli · integrated abundance of the 365 ion in & sample Let I~~integrated $w~dance of the 339 ion inthe125 pgilJ standard let b' integrated abundance of the 365 ion in the 12.5 pg~l standard Lel H,·inlegraled abundant of the 339 ion in the 25 pq~J standard let ~E~~integratedlundance of the 365 ion in the 25 pqhC] standard 01991 BAi Ca, ud. Do not wW or Jhow to una~ho~sd pwws, ft C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill I~D~LlOn·~f,~·~ .,~~scl;.-j4S~b·~ Y00flp01#Bt~P1J~ UU II~ II' ,~,,~,~ilrrm·t· CwleKnn~oa dC·blr~ ~ ~' ~' ~ Lt·C arad2~rup~rln·G ' ~~r o~~adc~Sbsl.G ' ~~~ 0UIIB FBOI YODII on aeu~ ~ ymlr mdm dg dgarettep, ~ Idr I~~lluecund, C~~ 4~ " ~~ h dl~l~. ~ r~L a O~D ~ d ~1 ~ td~~ d PYanakos ~ Hrl ~1PI R ~~ ~1(,~ dl ~ 2 ~ d ~qMhllarnPs ·d 2 ~ 5 ~ d ~~ph~ ~I Y I " ~ d m m F,rh e$arettes· ~qatats ~sp~Ynb, 08 01 0~ 0' elH pau ~~~'C~LN~b~~ ClibPDF - v~~fastio.soni REFERENCES 1, SRG 'dher nowae' lid Odober 1987. 2. BATUKE Repon No. Rb2146, 13,7.1999. 3. Millerp.L, Stedman,RL, fobacm, 165, 32 (1961), 4. Pailw,M., Huhsch,WJ., Kuhn$l, Fach, MY~ Osteneich. Td384reg,, 7, 1(1967). 5. Masud4Y., Holhann~D, Anal Chem., 4~,650652(1969). 6. PalriKakcff C. HoRmann,D., J· Anal. TFaiml., 3, 15blw (1979). B 1991 BAT Co. ua Do rot coW wshww Io u~umai~ed prso~~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 HG, 1 T, 220 MASS SPECTRUM OF N·(2·NAPHf~fi)·HFB AOD AHIOE -y,l O~lp' nit 339 Ilr no ~~~ i· Rll~m nlr UZ H 1· nit 215 ABUNDANCE 115 a,oES 6,0ES a9 339 as 120 207 n~ / zn 212 O C 100 200 300 nASSIIHAR[iE C\ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FE 2 T, 221 SINIjlE ION CHROMAtOGRAM FOR nt 339-2L INJECTION DF A 25 pglpl STANDARO ABUNOAN[E 1000 1100 1200 1000 15 20 25 30 TIME IninJ Ai3 SINGLE ION CHROMATO[IRAM FDR m2 33992u\ INJICfWN FROM A Aam~oAn VWDARD. FLUE-tVREO. KLNG_SltE RLTER .[l[illPLltEI~ALY~~LS 7000 6000 N· I2·nlpCth~O·HFB Kid ~pid~ 15 20 25 30 !5 CO a TIME Imin,l ' O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Rti, I T. 21 'MASS SPELTRUM H-(4-BIPHEMYL)-HFB A~Z QO·Y~1t~·' t LF./ nIr 315 11I11 nIz 63 111 115 nit 1H _L ABUNOANCE 111 1.DES 3.OES (( · I ~15 21ES ns 2)1 311 O 1015 N a a 100 zn 3)0 o M~SIIHARBE ClibPDF - v~~fastio.soni FIG. 5 f, 221 IXl[iLE 108 [WIORI;TO[IR~UI iOR ra1315-Zpl INJI[TI)H OF A ZI ppipl STUIOARO. ABUNDANCE 1100 1600 fSDO 1200 1000 Ij it zs ~o ~s co fmE Illin,) Fl[i, 6 SINELE ION [HROnAmLiRAM rOR m,1613ei IIIE[~KIN FROn A STAWOIRO RUE-[UIED, m(E SIZE FILTER [IDIRDTI ANALYSIS ABUWIAN[E 300( #· Il·biphM~I -HFB Idd ~i~ 1000 O -ir - io i is io · TIME (Rin.). , ru Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~·I II BRITlsn A~rH~a TolAcco C~A hllat ~tn Lql~ Hllla: 1( Op~lh~ol: D~ of Ya - LCI Bc~lL, Cllldl I Bcq~nt Ddl% Beqant Dltr: )7-5'$5 II*(U~lnll*CIIIHI*nlnU~~l~~ln~HHI~IIIIIIHHIUI rPe Mllbre A~,1~1S,..,,.ea BpCec aCP 0~r**1111 nnni nl~(~l~ll Plfl hale: Fi~ Pile Lrt Pip Ylo la lrlo111614 IZ i' 1~ 10 lal~ ~ll*sr~c.~JL~-~ IJ er~ A ~ H~I*IIIIHIIIIII**lnn** Ddls ~1~ war ~ ~ned a ~ D~i~o s~ only n c~r D+~ C~picd $:Cu L~ ~~~iuci ·rH~~~rnn,*r~ncHrrrrUnlnlHw* *. Ji:~~9-----h.P_~S~ c,m Cbelhd BI:,,,,, nlHC(IYIII*llnYHHC)II DI~: HHIHll~~~nl~enllnnnYII**I**WTiC: nlCLeYYYI*~IIWnnll m Ddbc~ Dt~ib CLChl $: Y**UYUIHH IIH..-IIUI**· D1~: rlnr*·ull*rlrlrnl QePf b:~~H~(C~IILnllnll w**rrurrHw~ DlfCtr~~uHI*Nlnll IIIHn~ ClibPDF - v~~fastio.soni British·i~merieanTob~ao CompimgUd FUNDAMENTAL RESEARCH CENTRE "o. Box PQ, Regulrs Pull Road, soumunpton, SCTJ IFS Enptvd Tekphone: So~hunplon (0703) tBZ111 RRB/BTM Tdu:dT1284 Fu:(0700)779715 6 June 1991 See Distribution ReP~NaT~rm The Measurement or Subdann~Pram Dslivadss ol kanMhvlamine and 49~noMDhendin Mainstream Smoke fhis Report descrbeJ the development of the anal~al methods for hM wbstances on the SAG "ob~er noxae' 1~1, The developed method indicates that the levels in modem, nonventilated cigarettes are at the sub·nantlgram Gel. Earlier values reported by Masuda and Hoffmann for plain dgare#es, and qwted in reports such as that of the US Surgeon General, appear to be too high ~ni$w R,A. Baker Distribution Mr. A.L Heard, Mllba~e Dr, P,J, Dunn, ~L, Canada Dr. S.R, Massey, m, Canada Pmt. E. Rittershaus, BATCF, Germany Mr. I.C, Brown, ETC, Swthamp~on Dr. J.S. Wigand 8&W, USA Dr. E. Kauech, BATCF, Germany Dr. CJ.P, de Siqueira, Souza Cruz Mr. T.I, Wilson, Australia Mr, H,V, Thomsen, L~nma~ MI, E, Grant, Argentina ,1; A Member of the BAT. Indunrin troup l~gd, Mfitr: P.O, BPlld2Wmminn(ll~uu, t MillbmL, London IW1P 31E. IncPrpontrd in London NP. /3376L f~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REPORT TYPE (RD w T)~ FI1E NO: (De#r~inedbylseoa) PROIECINI): Ylj C~ COL? REPORT NO: SLeurily CIPrdTw(ion: @aermined by IsPw) REPORT TITlE: Chntkljst ~t~HE y~64sje~mtvl; ofi S11~5 ~a~n~ogm~k~t Dk\vhC1Gf Pj ~'m~hjlA~hly IAM FibyCnphr· No: in·~PJ~ 4-·1~Ullh0 bipktn3i i, pmodt~ F~IM I (Toh"xh~O&e~) ~mHOR: Vt""'A~t~ c'JLtrr~k~ FiglCnph~·No: ~CI·A.(C~~ (CompPta~mn~ CO.WORKERS: ~n,J le C-~s WO~OvgphS·No: SECI~ION IEADEWREVIEWER: Irsal: IZ.A. O"~GJ olSIRIslmon: Re (1): ~ (1)1 MU((2): PJD(I): SRM(I]: 'IIW[2): ER (1): EK(1): CmdeS(1): HVr(1): RPG(1): Ilkuy(l): Re DISK REFERENCE: TIPPIG eHEQWG Sig~nP~JDucSmt Dme Dm Amb~c~ Dot 1~ D+ Reeeivcd I Rebuntd I Reviewer fw ~p~g b~tf (2' II Itrlanp a W,~,a,, 2-5(1 211 ~o~C I ~ 5,5 c(t, I alslt' 4 1 r I I IpderTcl~D I i~l Il·(';jj ii Plep~e~E~e 11'6·5~1 Co~tiow Es )rC~~fi Repere One BOOpd CDW Lrae I ~k·d·91~ Dir prq'ormo it irtlrrd~d o~f~cili~ofl Md daoil th plogmr o/BI~UXE R~D Curat Rqomfmn ~he t rlptrnipt sra~ rd L npot isFMUy ~Wmvalond iuucd. TA( p~on~ shPIJd br cannmllad ot L n k 4ftEA initiol typing Md ofconpMy L rlp~saip~ (md nurstrr ~r prepqr~ion)m 1 subrrqurm ·C~ S1OBCS, it mwt C srntro CulaalRrcwds wiJI kMPd~t whn&npnirisnld. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 THE YEASUREMEHF OF SUBNANOGAA~ DUIVEAIES OF BNAPHTHYL9MINE AND eAMINOBIPHENYL IN ~AINS'TREAM SMOKE REPORT NO:T~228 6 June 1991 DISTRIBUTION: Mr. AL Heard Dr, R,R, Baker 2 Mr. I,C. Brown 3 Dr, J~S, Wlgand 4 Dr, J.S, ~gand 5 Dr, PJ, Dunn 6 Dr, S,R, Massey 7 Prof, E, Rittershaus 8 Dr, E, Kauseh 9 Dr, CJ,P, de Siqueira 10 Mr, E, Grant 11 Mr, H,V, ~homsen 12 Mr, T,I, Wilson 13 Library 14 library 15 01991BaCP.Ud DDMte~W~~On~vP~hP~dp~nt~.~ ~p C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Contra, BritishdmeriEan Tabaceo Co, Ud, SOU~MPfON, THE MEASUREMENT OF SUB·NANOGRAM DEUVERIES OF BNAP~HY1AMINE AND CAINOBIPHENYL (N MAINSTREAM SMOKE REPORT NO;T~228 6 June 1991 AUTHOR: GA Few eQJNORKEA: LR, fade PROJECT MANAGER: RA, Clellin ISSUED BV: R,R, Baker COPY NO, a 1991 BA'I e4, Ub Do not mW arhcur lo Inwlhwbsd pncna O 1 hi Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Centre, Bri~sh·Ameriean Tobacco Q, Ltd~, SO~TrYQMPTON. GARBTM 6 June 1991 THE ME4SUREMENT Of SUB·NANOGRAM DELIVERIES OF 2·NAPHTHVLAMINE AND eAMINOBIPHENYL IN MAlNSTREAM SMOKE REPORT NO,T.228 SUMMARY: Investigation of the levels of Znaphtt~ylamine and daminobiphenyl in mai~stream smoke is being underta~cen in FRC because they are of potential interest to regulatory aub~wities, Reports in the scientific literature give their levels in mainstream smoke from plain cigarettes as a few nanograms but there are discrepancies in the reported values which may be due to problems with the analyb~cal methods used. This Report describes a new analyh~ca~ method developed for b"leir determination in mainstream smoke. After trapping in phosphoric add, the amines are extracted into hexane, In the hexane they are then derivathed by heptafluorobu~rie anhydride and analysed by gas chromatography with a mass selective detector in the single ion mode, Application to a standard, fluecured, king sized filter dgarette has indicated the presence of the amines at the sub·nanogram level, These results imply that the earlier wo~ of Patrianakos and Hdhnann in 1919, who measured b1e deliveries of the amines from plain US and French dgarettes, were of the rght order, i.e, 16 ng, and thatthe results of Masuda and Hoffmann in 1969 for 2·napHbrllamine, who quoted 22 ng for a plain US cigarette, were inconect. O 1991 BAT Co. 1W~ Do not copy of Shnr lo wulhor$d persons, N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 INDU( TER~S: 2·NapMhylamine ~dAmirobiphenyl Mainsb~eam Smoke ~JC·MS ventilated qarettes Filter Ciga~ettes O 1991 BC;I Q. Uh, Do not wW or show to uHulh#lssd pqo~, N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 1, IN~RODU~OH In 1 BQbo 8A'T8cientific Research 0Rp (SRG) considered has suhstenoes regarded by external ogn~~~~y authoritlee which have speci~e toxicoiogloei properties ad ha ban reportd In cjOerefie smoke, A ~o~ list of substances for tmsr invesl;gation was produced which included P·nqhbylnino and 4dminobipheoyi ~~ ~ levels of he two substenca reoortd in eiga~ette smoke ha dan reviewed prevlouaiy (2). m reOerd to kqht~tamln,, reported levels In amd(e seem to he confused In 1967, Muller ad Stedmen (2) filed to identify conclusively 2~q~~ir in he mdnstream tom commercial ciOaretfee, Allowing brdetee~on limits, they concluded tW the delivery must he las ha 46 ng, They noted hi Austria workers (4) did not detect 2nephthyiemine tom blended cigarettes of undescribed origin. In 1969, Made and Hofiman found 22 ng tom US plain cigarettes (6). This was refuted in 1979 when Petrienekos ad Hofmnn (6) found 1 ng ad 1.7 no of Od~~~ino in plain 66 mm U8 ad pYn 70 mm French cigarettes respectively. Eouivaient levels of Cpminobiphen~ were quoted as 2,4 ng ad 4,8 no Mpk~, 'The Hofiman school rb~ the reduction in 2nephthyiemine from 22 no h 1 1 for a pin vs cigarette to product changes, at least ~~C. However, mi ah that they eannot exclude he effects of differences ~~wn me eniyticai technioues used. h mts uniikeiy hat product ohenees would explin a 969b reduction for plain dgarettes which suggests a difference arising from he n~~. I Petriaakos ad Hdm#nk 1 ad 2.4 no delivery results for PaqY~mins ad eaminobiohenyi npdvely tom plain cigarettes a correct, modem l~o~sn~d products would be expected to del~ suk~a qu·LI of he two aina. This presentJ a considerable anslytid chdnga. 'The objective of he presat study was to develop such enei)"dcai methodology, 2 YaHOD UFVELOPiFHl ~e derivetisetion of u4 invoU~ emines to ~l~ls aides using fiuorinated aid ahydrides foiiowed bl gr Qpp~~l In a electroR capture detedor (ECU) is a rl estebiished mute b ~t quntitation, A number of fiuorinating agents a available wt, perhaps, he ~ most popular Yng pentafiuoropropionic on~dYe (PFPA) end heptefiuorohuytrie ahydride Given hi he smd(e levels of the emines h he puanb~tatod were expected to he i ms low nanogrem level (no more ha 1 or 2 no) ad hi he sensitivity of he ECD increases with he number dluodn atoms in he molecule, HWA was chosen forthis rt Method development soon exposed mo pitfalls of using a FCU. its extreme tsns~ meat that before he ~npledf of he O 111 I~ Ca Ltd no not coW a Jhow to mauthorlJed pHsons. N ClibPDF - v~~fastio.soni sample mabix was taken into account considerable care had to be taken to minimise contamirntion entering the system, The following precautions had to be taken: (i) The hexane solvent had to be pured so as to be acceptable for ECD work T~ was provided by Rathbum Cherrjcals limited at the suitable level of puri~, Fi) It was found that itwas importantto use an inert Jsplm or one wi~ a mA coating facing the column. fhis prevented any interact~n be~ueen the HFBA and h~esepb~m and eradicated sepb~m Meed. (ii) HFBA is very labiie, being hydolysed readily by water to give HFB acid, IntMsworkitwass~oredunde~an atmosphere of dry nitrogen, Having been satisfied that no eontaminants intarfe~ed with the elution of the gmides, the complerj~ of the sample m~EI tan extracted smoke solution) was investigated, AIVlough the chmmal#lnmwas found seteFlahle forone d the amidss, Iwas totally unacceptable forthe o~r, multiple cpelulion a high level ocwrring around its retentiontime. Giventhat a considerable amauntaf method development would have had to have been invested in E~a~nl upthe aampie mahix, itwasdecidedto changeto a mass selective detector (MSD), this a~owing single ion mooitoing for each af the amides. Single ion asdoring ~d ~n advantages over the ECD: The duomatogram obtained would be for one ion only, equUnt to the molecular mass, and hence defirlive for an amida. Single ion monito~ing would be equivalent to several dewi~ up stages~ 21 Present MethodologY 21,1 Reagents Hexane and water solvents were obtained from Relhbum Chemica~ Limited, The 2·naphthylamine and 4.amino~iphenyl were synlhes~ed by Sa]ford Ulbafine Chemicak Limited, Their purity was unapec~d but OCICSD sRldies indicated a high level ofputty. HFBA was obtained horn the Pierce Cherical Company as 1 on' a~npbuies. Sodium hydmxide pw were obt~d Irnn BDH limited. e 1Dol B~T Ca. Lid. Oc roll Ec9Y or s"" U"BL~PW persons, N Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 21.2 Preparation of the Sample Using one port of a ~ilb9na 302 B·port smoking engine or a Borgwaldt single port smoking engine, M cigarettes are smo~ed to CORESTA standards through a double bubble bap each containing 20 wnl of 0.1M p~phorie add, The smoking engines are protected by a Cambridge filter pad ~ter the bubble traps. After smoking, the contents of the b~s are filtered through a Na. 1 Whatman filter paper, 'The contents of the first trap are filtered into a 50 anl volumetric flask, bcle contents of the second trap are then poured into the ~rst tap and then also filtered into the lask. The second trap is then washed with 3 cm' ali~u0t of the phosphoric acid solution which is then used to wash the first trap before filtration. The wa~~ing and filtration are then repeated allS~batas being collected into h~e 50 em3 flask Same more phosphoric acid is then added, if necessary, to bring Re level of the acid up to the neck of the lask (this facilitates ease of removalaf thecrganie phase after hexane ewtradion). The contents oltheflask arethen made aa(a]lne bythe adLn of mug~Jy 1 g of sodum hydmxae pellets to the solution, the contents of the flask being vortex mixed until the pellets dissolve, this ensures that the amines are in bL~e free state, i.e, rot pmtonated, In the free state they can be extracted by a nan·pda~ solvent, Having allowed thelasklo cool, the alkaline solution is then extracted manuaily by a 4 cm' al~ual of hexane far 15 minutes using mist ae6on shaking. After 15 minutes, 3 cmJ cf the hexane layer is removed to a graduated 10 mm x 75 mm, disposabeq pymx ~bxe tube and concentrated down tsdold by a gentle stream of nitrogen. At a volume of 300 IJ the culture tube is Mted with a ground glass stopper to await Re reagent for the formaEon of Re amides. 2,1,3 Formation of the AmMes Addition of HFBA to 2mqhthylamine and Caminobiphenyl will farm the HfB amides as shown on the following page. e 1991 BA'T Co, Ud Do not copy orshow k wadhwiJed persons~ N C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~·u ,H.M ~nqm)lr~w II H(2·npm~f) I~ U.M ~H·u I ng Crixbph3 ~I H·t(J~wn~ II~ 1 ~ d H~ C added ~ n( ~ a~R ~ ~bh D ~n ~PP~ d ~~ I+ ~'Rdd~ bLg ~de ~ SO'C ~r oa bl ~ d ~r C ~ id~ ~ OI C~ ~~ C ,skppsrsd J ~~ ~~d ~ 6 ~b~ ~ ~~ aor H~ d HW dd, h oRr ~~ lyr C nr rea~y Ir n~it, &a, Jon ~~ ~ u# d ~ weak Ylne J~n ~ ~move ~, Ix~ Hr ,q, SX~dm hydrl~, ~ hn~~d hLLatntl~ #In ~dJon ~I r~~ 4dmCli d ~ m~ ad h ~ b~ br ~r even ~on nl JYI b u~. ~a, ~ is #m~~d Y ~lr ~on ~ r~ ~ ~a ~ Ix~ H~ nd HW ~~ O 111 ~ Q. ~d ~ rt ~ r ~ ~ ~wuYlor~d ~or. ClibPDF - v~~fastio.soni 21.4 EC·MSD Analysis 2 id of the hexane layer are injected into a Pe~i~·Elmer Sigma 3 gas chrwnatograFh interfaced to a Hewlett Paekard 5970 MSD, The interface was held at 240'C and the gas ehmmediographic conditions were as follows: Column: Sepulco Ine,, fused silica capillary SP&5, 60 m, 0.32 mm ID. 1,0 pm film thiduleas, Injdwl port temperature WI'C. Tempeature program: 150'0 to 220'0 at 2'Clminute, then hold for 20 minutes, Helium caniel gas Edumn pressure: 10 p,sj, Gas daw through splitter: 5 cm4mhute. The MSD was adjusted for the single ion monitoring of the mdecular ion of N·P·nap~yl)·HFB sea amide, m, 339, The elec$on muCplie~ voltage was 2400V and the spedpometerwas not mitehed on until after the solvent passed though the system. Under these chmmatograpHe conditions the retention time of the nap~~wnide was around 25 mirwtes, Its mass spee~um is shown in F~ure 1 and b~e single ion chmmalcgams from a 25 pg'yl standard and & analysis of a standard, fiuetured, flte~ king site cigarette are shown in Rgures 2 and 3 respedively, Following the mwlitorhg of the m, 339 ion a further 2 id of the heuane layeris injected wBthe MSD setto monitor solely the mo~cular ion of the N~dbiphenyl)SiFB add amide, m,365, whose retent~n time was around 37 minutes, Its mass spee~um Is shown in F~ure 1 and, as for the naphthylamide, the single ion chromatograms from a 25 pg~J standard andthe ana~sis of a stanja~d, luecursd, filter king she dgaretle are shown in ~us 5 and 6 respeeb~vely. 215 Preparation af Standards 2HAPHTHYLAMNE AND eAUINaBIPHENn ARE KNOWN HUMAN CARCINOGU(S, WORK wrm TnE 301105 TO MAKE UP STOCK SOLUTIONS MUST BE UNDERT~AKEN WITH PARTICULAR CARE TO REDUCE ME CHANCE OF SPILLAGE OR HUMAN CONTACT, HUMAN OONTAOT MAY BE AVOIDED BY WDRKING IN A FUME CUPBOARD IN AN ISOLATED LABORATORY WEARING DISPOSABLE LABORATORY COATS AND O1OVb AND SAFETY SPECTACLES. THESE AMINES CAN BE NEUTRALISED BY CONVERSION TO THEIR HYDAOeH1ORIDES. O 1991 BAT Cc. Ub Do not coW orshow to una~lhc~ed pwsons. 0~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Itisadvisable, becauseof the eareinogenic na~reaf these substances,to reduce ,toJe solutionsto the mininuun. Inthis work 20 mg of each anine are weighed into the same 20 #n3 volumebiclask and hexane added to give a mncenb`alicn of 1 ~gl~lforeach. Dilutions nfl~ stock solution are then made logive solut~ns containing5andt0~i~aleachamine. Two50cmlv6lumebicflarJ~Earetaken and 0,1M phosphoricadd added to Wh uahlthe men$us of the aca is athe nede of the Rasks~ Into thep~K#phPriC acid of one of these fiasks 1 IJ of the 5 naklsolution is injected wah immediate wrtex mixing, Intatheadd olt~ other, 1 id of the 1 O ng~~ solubbn is injected, again, with immedbtewr$x mixing. The farmer flask p~a~ides a 125 pgild standard, wMlst the tatter pmvides a 25 pgllJ standard. Afte~afewminulesofvo~ex mUng, roughly 1 g of sobum nydmxide pellets are added to each Rask, again with vortexmixing, Wnenthe pellets have dissolved and Ihelasks have coded O #n) of hexane is added to each ffask Each flask is then manual~ shaken using a wrist aet~n for 15 minutes. The flasks are then allowed to aan31oralletoallawthephabestosepa~ate. For each of b~e flasks, 3 cm' of b~e hexane phase is removed and coneenlnted 18~1old by blowing dw~nuling 1 ge~ stream of nbg,n, mehw standards are now ready to be taken through the derivatisalion to give hexane layers containing 125 and 25 pg~ of the ami~les, These solutions are injected into the GC and monitored by single ion monitoring of the m, ~39 and m,365 ions, with the MSD changing its program at 30 minutes into the run to accommodate single ion monitoring of hath ions in one run, Onlyt~ standards are prepared and run for each sample since the response is linear in this reaion andthk is the region of interest with regard to mojem cigarettes. Ar 1~ standards become stronger, a ealbraSon curve is produced which devii~es mom and mom positively from lineari~~ It would appear trom the analysis of second exbadiom of the standards with fresh hexane tha recovery at this level$ a~nost 1MPlo. AIIJoiied~iasnva~eblefltos0linlMhydmchloric8dd OisFas~is~diiln to waste. 2d.6 ealcuiatlon of Deliveries LetSS3o·mtegrated abundance of the 339 ion in the sar~e LetS~t~ntegrated abundance of the 365 ion in the sample Letb,integra~ed abundance of Me 339 iolin the 11-5 pd~ standard Letlwointegated abundance of Ma 365 ion in Be 125 pgCI standard let HSIO-integaled ~undance of the 09 ion in the 25 Wild standard lel Hur I integralsd ~xmdance of the 365 ion in the 25 P9~ Standard O 1941 BaC~, lid. Ooratapyccrhnvla ma~hei~dPf~ons, L~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill Meanres~oMshclwlolhe339ion·ji~i~iRF. Meanrespraslacla~lolhe365ion.[~i~;EL~, Cone~e~a~n of 8neplhylamino in hewanelayer (pgl~l)·~' C, Concentdon of 4amirobiphenyl in h~me layer (P91~1) .~' Cuc oel~Je~uof2~apht~m'"'C~B X i~~lCjB Oeliveryof eaminabiphenyl. Ca I ~~cig 3, DEUYERIES FROM MODERN DAY #GARE~ES Todeterminethe levels ol2·napM~an~ine and4~uninobip~nyl deberadtmm modem day dgarettes, the srnd(e rem a standard, fuecured, Br king she oigsratts was andysed in duplicrde, The results us given bebn and reinlo~ce the frdngs of paaianakos and Holhnann in 1979 (6) who lound del~e~aof 1 to 2 ng ol 2napMhylamine and2w5ngp14dmmbipheyIfmmpla~85mm US and 70 mm French eigarettes· nqidqarette 2·naphb~ylamine 02 0.4 baminobiphenyl 01 0.6 O 1991 B~f Co, lid. Daml coWM~haulpu~dho~dp~om. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REFERENCES 1~ SRG "ather noxae' lid Odober 1967. 2 BATUKE Repo~ No. RDd46, 13,7.1989. 3. Msle~R.L, Stedman,R.L, tdwcco, 165, 32 (1967). 4. PailerM, Hubsc~WJ., Kuhn)l,, F~t~, ~MI~, Os$nsich, Tab~kree,, 5. MasudqY., HoMnann,D, Anal. Chm., 41, 650652 (1969). ,~s, HoffmaMv(D., J· Anal. Toaiml., 3, 159-131 (1979), elosla~co.ua oomte~,MI""U"Yd"e~~pIW~MO Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 H[j, 1 T. 221 MASS SPELTRUH OF N-(2-NAPHTHW·HFB AOO AHIOE ~040, -t~~tl nlr 339 Jr no ·riP( i· GIT ~n7 air 162 s/ ~rN 1- nlt nl ABUNDANCE 115 6.0E5 69 1 Ijl ( 339 3 O5 170 207 h` zn zn O c 100 200 300 nASSltHARIEE c\ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIG. 2 f. 221 SINGLE ION [HROMATOGRAM FOR s2 339-2(11 UE[TION OF A ZS pQICIIVANIIABII ABUNDAW[E 1600 1100 1200 1000 B 20 25 30 'TPIE (min,) Fit. 3 SINGLE IOH CHROMATbLIRAM FOR m2 339-2uliNJ[[fn]N FROH A ABIPIDAHB STANDARO. KVE-EURED. KING SblE FILTER [6AREIfE ANALYSIS 7000 6800 N· I2·nlphth~ll·HFB Kid 1Djd~ 13 20 23 30 33 lo TlnE Imin,l ` o Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Rti. C I PI ~MASS SPECTRUM M·II-BIPHEMIL)-HFB AO~ 0 O4191 ~2 3LS nit 110 i;"" \~ P~ ~ 00; nit 133 nit 115 II~ 1SI ~I 132 ABUNOANEE 11I 4.OES 10fS (1 I . 21ES ns :~P a 2)1 311 O 1IES I I , 1 I 100 210 300 o M~Y[HARBE N ClibPDF - v~~fastio.soni flG, 5 T, 221 nNIilF NIN [HROFIATOGRAM ~OR I,111-2IIHIE[TION OF A Ii pgl~ STAHOIA[I. ABUNDANCE 1800 1600 1100 not 1000 15 20 25 30 35 CO tmE Init,) RE i SINELE Kn [HROMATOGRAII FOR ~ 365·2111 HIE[TION FROH A S~MWIRO, FM]E-[UREO, KIHE SIIE FILTER OGIR~ITE ANALYSIS. ABUHOANCE 3001 N· Il·biphcnyl) ·HFB dd ui~e 2000 1000 o u a 15 20 25 30 35 TIME Inin,l, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 a O a c ru rr O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 t 126 Fg~l .NAPH~HYU·HFB A[IO AMIOE MASS SPECTRUM OF N·I2- 'ioI .I ~Nn/uFpp, nh 339 lit 179 nit 121 nit 112 Y ~rn I- air 16 ABUNDANa H5 ~nES ~a 339 69 O IS 110 Iv 201 2n 2'12 9 aa ~oo r~ 100 0\ nASSI[HAR[j~ o\ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fl(i, 2 T, 221 SINGLE ION CHROMATOtRAM FOR q 339-2L INJECTION OF A 25 pplyl STANDARD ABUNDANCE 1600 W00 1200 1000 15 20 25 30 Tmf Imin.l Fni,3 SINGLE ION CttROMAtOtRAM FOR_ma 339-Zul~NJE[TION FROH A ~suwo~ncE STANDARD, RVE-[uRED, KING SIZE FILTER UliARLfTE ANALYSIS 7000 0000 H· 12·aphthN-HFB Icid Imidt 15 20 15 30 3r 40 nnE Imin, I c C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fl[i. I f, 221 MASS SPECTRUM N-(l·BIPHEHYLI·HFB A[IO AMIDE BC~iOt~u:,, s 08? n~h 365 mh tli 1~· ~~i~· i· ~Q nit 153 R12 161 i., 0~1 Q~ Ce ~4-0 MI[H dz 115 nit W1 as nit 152 ABUNOMI[E 1611 s,OEs 3,0fS 365 00 13 2.OES 115 l~'gz 201 311 p ~,DES I o o N 1DD ZW ]00 0\ E1ASSI[HARGE Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 AS~ 5 T, 221 9a · olarn RL ~l~i UBllr 16 d~ ~·II ~auno~r II~ 16)0 1C00 1200 1000 13 ic a ]I ~s ~o me C~I F~1 91LE DI aDIBE*l FOR L I~tlUB11 RI I PYIIL RL~IB. 16 a ant oumt wwnsCIGARE~TE AHAIYS6. ~~O~~f I~n I· R·$~l~l -HfO 1Q ~I 1000 -a ~t I ~d u nMt l~ihl ClibPDF - v~~fastio.soni