I Britishdmerican Tobacco Compan~ Limited FUNDAMENTAL RESEARCH CENTRE PO Box 90 Regents Park Road Southampton 909 tTS England Telephone: Southampton 10703) 782111 f~kx: 477269 Fax: 167031779715 7 April 1992 ARBIBTMIRepons See Distribution Reoort No,Z2;16 TheDeterrnination of A~tamid~in Mainstream .C)oareae_Smake This Report describes the development for a method for the analysis of acetamide in smoke, a substance of relevance to ROOT Technology, The development was undertaken because of discrepancies which existed in the literature between deliveries determined by two earlier methods, ~e;J~d &kr R,R, flaker Distribution Dr. P,J, Dunn, In, Canada Prof, E, Riaershaus, BAtCF, Germany Mr, E, Grant, Argentina Or, C,J,P, de Siqueira, Soura Cna Dr, E, Kausch, BATCF, Germany Mr, A,G~ Stephenson, 91"C, Southampton Dr. S,A. Massay, Itl, Canada Mr, H,V, Thomsen, Denmark MI, G.A, Read, Millbank Dr, J,S, Wigand, 9&W, USA Mr, T,I, Wilson, Australia End, I~cDOma nwnK KnomtGnra St~rm mi9 IOY ii: A Menu or rwl BAT Iwwnn~e~ Gnwr ~.vrmolhW a l~ws ~io 1~91~ 17\ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REPORT TYPE (RD or T): ?-23j FI1E NI): (Dctermined by Issuer) PKOJEIT NO: REPORT NO: Security ClassiFicaliun: (Deam;btdbytsuu) REPORT TITLE: ~k Dlenr~~h~ Etikeiawidr It B~PU Mu~L;hi~ ~ahi~ 5vs~ ~C~bf~ nnor 4 ~ Fe~,FigslCraprs· No: CO;WORKERS: L·cu4RK Phulo~apb· No; SECTION 1EADEWREVIEWER: ISSUER: ~ Rb omRrsOTloPI: Re (1): ALH(1): M131(2): PJD(I): SRM(1): TI~(2): ER(I): nr (1): CJPdeS(1); HYT(I): RFG(I): Library(2): R DISK REFERENCE: TYPING CMQSffi SigPa~Pre I Date Smt Date Date Autbod Date Issuer Date Received I Returued I Revic~wer .I Draft for Typ~g C-ih' Iluljl J'i~e ,If, Sir Indcx'lams Is~. Rel~~Il;caJw I qLW 2·lt-i2 ~ly~'' Comaioar Prepare C~e BormdCoW Irme rB~'r ~.rin C TLit prafor~M is buended to focilitdre nod daPil Ihr progress $ BATU~ RltD Cenae Repotrsfrorn ~3 ~Y ~Y~CSMiP' IIPQL until the report is fnnlly opprovrd md issued. The profonno ~i~o~d be commmd p L h linu of the iniridi typing and oecompany the ?Ip~snipt (ond rMstu ofter prepmorionl or all subsrquc~u o stage. it mlln be sent fo CaholRecordrwCh the Moner whm the report is issYd. L; C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 THE DETERMINATION OF AC~AMIDE IN MAINSfREAM CIGARE~E SMOKE ~PORT NO: T,236 fApril 1942 DISTRlBUTION: Mr, GA~ Read Dr. RR Baker 2 Mr, kG. Stephenson 3 Dr, J,S, Wigand 4 Dr, J.S, Wigand 5 Dr, P,J. Dunn 6 Dr, S.R Massey 7 Prof, E, Ri~ershaus 8 Dr. E, Kauseh 9 01. C.J,P, de Siqueira ~O MI, E. Grant Mr, H,V, Thamsen 12 Mr, f, I. Wilson 13 library 14 library 15 5 1992 B~T Co, ltd. Do nol copy or shea ro uruuthorised persojj. L: C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Cenb~e, British-American Tobam, Co, ltd,, SOUMAMPTON GAF~BTM THE DF~ERMINAfION OF ACEfAMIDE IN MAINSTREAM CIGARE~TFE SMOKE REPORT NO: T,236 7 April 1992 AUHOR: e,A, Few CP WORKER: i. clark PROJECT MANAGER: Rk Crellin ISSUED BY: RR Baker COPY NO, h* log: 9~T Co, Ird Do not copy or shoa~ to utl3uthorisod p#sons, L" C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Centre, British American Tobacco Co, ltd,, SOUHAAMTON GAFIBfM t April 1992 ME DETERMINA'TION OF ACEfAMIDE IN MAINSTREAM 8GARETTE SMOKE REFORT NO, T,23B SUMMARY: Recent studies have shown that ROOT technology will increase the levels of acetamide found in cigarette smoke. However, this observation was made when the analytical method at that lime was essentially semi~uaditative. Since then, the method has bean improved and made quanttative. This report desen'bes the method and presents results Irom applying it to various versions of Marlboro lights and to a standard, Rue~eured, king site filler cigarette, The analysis extracts a Cambridge tilter pad with methanol and then injects the methanolie solution into a gas chrornatograph fitted with a mass selective deteetor~ Quantitation is by single ion monitoring of the 59 mass, INDE( TERMS: Aeetamide Mainstream Smoke GC·MS Ventilated Cigarettes Filter Cigarettes C 19P2 B~T Co. Ltd. Do not copy or rhoH to unnuthonred persons. Z1~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 INTROOUCnON During the course of recent studies involving ROOT Technology the question was asked as to whether it enhanced the levels of acetamide in cigarette smoke, The rationale behind this was that it was known tom the literature that acetamide existed in smoke and the origin was unkown, ROOT technology, providing the possibility of reactions bCeen sugars and ammonia or ammonia compounds, was seen as a possible contributor to acetamide levels, Previous workers had in fact quoted conflicting deliveries with recent work by R J, Reynclds quoting 4 to 5 pg for the Kentucky 1R4F reference cigarette (1) and Japanese workers quoting 20 limes higher values for plain, unblended cigarettes (2). Even given the level olventilation and filtration efficiency of the 1R4F reference cigarette, there does appear to be a diserepancy~ To help to clarify the level of aeetamide delivery and discover whether this was influenced by ROOT Technology, a simple anal~lieal method far acetamide was developed, This merely smoked a small number of cigarettes onto a Cambridge filter pad which was subsequently extracted with a few mi of methanal. ARs concentrating a mi of the methanal 2 fold, 2 pi of the methanolic solution was injected into a gas chromatograph fitted with a mass selective detector. Quantitation was performed by a single ion monitoring af the 59 mass. This method produced results which were similar to those found by R, J~ Reynolds; however, the ehromatography was unsatisfactory with considerable tailing of the acetamide peak furthermore, the results indicate Ulat, indeed, inclusion of tobacco which had been treated by ROOT technology did increase aeetamae deliveries (3). To eradicate the large tailing and make the method quantitative rather than semiquanlitative, the method underwent further improvement and was then applied to various versions of Manbam Lights and a standard, Rue~Eured, king site filler cigarette, 2, METHOD DEVELOPME~SI The initial improvement work used a 50 m polymethykilaxane (SE54152), fused silim, capillary column of internal diameter 0,32 mm and S Clm film thickness. This was housed in a Hewlett·Pad~ard 5890 Series II gas chmmatograph with a Hewlett· Packaid 5971 Series mass selective deteetor~ Injections were made in splilless mode with a 50:1 split purge being activated 45 sees afferthe injection to sweep cut the injection port The carrier gas was helium with a ~ mUmin fiow rate through the column. The gas chromatographic conditions were 65'C for 10 min followed by a ramp of 4'Clmin until the aeetamide eluted, The oven temperature was then raised to 240'C to clean off the column, The mass selective detector IMSD) was operated in single ion monitoring mode wlh a dwell time of 1001 set for the mother ion mass of acetamide, mZ 5Q~ The MSD was not turned on until 8 minutes into the run in order to avoid GCI excess pressure ham the methanol solvent and it was operated with the electron multiplier at around 2200V, G f 199? BIZ~ Co, Ltd, Do not eopv or show to uFarhorised praons. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill In the earlier work it had been concluded that acetamide leveV unit of tar was dependent on the number of cigarettes smoked, since results seemed to decrease as the TPM on the Cambridge filter pad [44 mm) increased. This was interpreted as arising from a pH change. Initially, the pad in water gave a pH of 9, This reduced to pH 6,5 with 45 mg of tar on the pad. Hence, with new chmmatography, this earlier observation had to be veined or eliminated. this was achieved by smoking standard, Ruecured, king sire cigarettes, varying in number from between 5 and 20, on a Borgwaldt 20 pen smoking engine and on a Bcrgwaldt single port smoking engine. Each pad after smoking was placed in a 50 mi eoniealOask, 1D mi of HPLC grade melhanol was added, the Oask was stoppered and the pad was then edraefed by gently swirling MI a HARVARWLTE multi·shaker for 15 minutes, At the end of this time the pad was pale grey in colour and 2 pi of solution was injected into the gas chromatograph lot analysis. A typical chromatogram b shown in Figure 1 where the acetamide peak is highlighted, the following conclusions were drawn: 1) The acetamide peak was much more intense than that which was previously observed and this meant that it was no longer necessary to concentrate the smoke solution before analysis. The inwease in intensity was the result of focussing the acetarnide on the column at 65' C, 2) The acetamide peak was still, however, unacceptably broad, being almost 1,5 minutes in width, 3) It made no difference to the results which smoking engine was used. 4) i3ver the range of cigarettes smoked, i,e. 5 to 20, no bs of acetamide was observed. 5) Replicate smokings showed good reproducibility~ Although a tremendous improvement in chmmatography had been achieved, when the quantitative results tom the calibration curve using peak area were compared with those obtained tom using peak height it became clear that tailing was still affecting the quantftatlon, The peak height results were sligMly less than those obtained from Be peak area. Hence, I was decided to swth to a more polar column with Vie intention of narrowing the peak width. under the conditions used, adetamide had eiuted at around 21 minuter Earlier work had suggested that acetarnide would exhibit much the same retention lime on a shorter BP20 column (e~uivalenl to a CW·20M or a Supelcowax 10 column) with better peak shape, On substituting a 25 m BP20, fused silica, capillary column of internal diameter 0.22 mm and a 0.25 pm film thickness and using the same ~o chramatographic conditions as before, acetamide was found to elute at G around 22.5 minutes with a peak width of only 30 seconds, a deat 1992 Bi\T Co, ltd. Do nor copy or showlo ualurhori~ed ptrranr. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill improvement on the earlier result. A typical chramatogram for a standard, fiuecured, king size cigarette is shown in Figure 2. Furthermore, when quantitative results from the calibration curve using peak area were compared with those from using peak height, there was complete agreement. Typical calibration curves using peak area and peak height are shown in Figure 3~ Preoarat~an of standards Standards must be prepared from crlslalline acetamide, not Fcwdered. Powdered acetamide degrades over a period of time, One or two gr;tals weighing 20 mg were placed in a 20 mi volumetric flask and HPLC grade methanoladded up to the mark Once the acetamide had dissolved and the flask been well shaken, a 10 fold dilution of 1 mi of this stock was made to give a 100 nglpl standard. 5 mi of this standard then underuent a 2 fold dilution to give a 50 ngl pi solution. This standard was then subsequen~ diluted to give a 25 ngl ui solution which undenvent the final dilution to give the lowest standard, 12.5 ngl yl.'lhe calibration cu~e was drawn through the peak areas of the 50, 25 and 12.5 ngl pi standards and through the origin. The volume injected into the gas chromatograph was 2 ~1, It is advisable that standards be made up freshly each day since the lower standards do degrade during the course of a day. The stock solution may be retained for a couple of tap but it is prudent to monitor its peak area as a sentinel to as and when degradation takes plaee~ Calculatiaoof results After reading from the calibration curve the amount of acetamide within the 2 . pi injection, if 10 cigarettes are smoked this should be halved to give the delivery per cigarette in ug, since..,, Y ng12 pt is equivalent to Y12 Clglml therefore in ~O mlthere is 10Y12 Clg from 10 cigarettes hence, aeetamide deivery is Y12 Clgldelivery 3, RESULTS AND DISWSSION The acetamide deliveries for 8 variants of Marlboro lights from different markets and a standard, Ruecured, king s~c~e cigarette are given in Table 1, From this table it may be seen that the reproducibility of the method is generally good, given that no internal standard is used. Furthermore, the consistency of the aeetamideltar ratios (especially acetamideiPMWNFi impbs stability since I is believed that same of the markets ate in fact marketing the same product, i.e. US (expod versioni. a The contrast between the aeetarnideltar ratios for Marlboro Lights versus a N standard, fiuecu~ed, king size cigarette is of particular interest. The n t 1992 BAl Co. ltd, Do not cop~ or show to unauthorised penofs~ · U; Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill acetamaelPMWNF ratio for Marlbwa lights at 26 pglmg is 3 limes larger than that far the RuPcured cigarette at 0.6 ~glmg. Returning to the work of R.J Reynolds (~j and the Japanese who have smoked Ruceued, Burley, Turkish and Malsukawa j2) cigarettes suggests that a US blended cigarette should produce a lower ace~amideltar ratio than a flue~cured cigarette. Hence, since 1 is known that Philip Mcri6, the manufacturer of Marlboro, uses ROOT Technology, it implies that ROOT Technology does in fact increase aeetamae in smoke, This finding agrees with the conclusion from the earlier work (3) which showed that the introduction of ROOT Technologies developed by BAT companies increased acetamide delive~s by up to 50%. Finally,it must be said that the results from this method certainly Can toward those of R J, Reynclds Bather than the findings of the Japanese. However, this may not be surprising since Reynolds used an eleetroslatic predpitalor to collect the smoke and this work has used a Cambridge filter pad. The Japanese had a cold trap behind their glass fibre SUer pad and hence would have collected any acetamide in the vapour phase. The results suggest that there may be a significant con$ibutidn to acetamide from the vapour phase. A lypiwl chrcmatogram from Marlboro ~hls is shown in Figure 4 for comparison wlh that tom the nuecured cigarette which was shown in Figure REARENCES i. White, E, Uhrig,M,, Johnson,t., Gordon,B,, Hicks,R, Borgerding,M,, Coleman, W,, Elder,J~ J, Chrom~ Sc~~281 393·399 (1990) 2, Sakuma, H,,KwsgaaM., Yamaguchi,K and Sugawa,S,Beitr, Tabakforseh., 12, 5, 251·258 (1984) 3. BAT Report No. R02203, 26.11,91 I' 19E B~T Co. Ltd. Do not copy or show to unauthorised persons, iT~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~B1E ~ AC~AMIDE DEUVERIES FROM MARLBORO UGHTS AND A STANDARD, fLUE·CURED IONG SIZE QEARE~E A a c O$in Aeeta~d~ OCery TPM PMWNF IVB PJC Australia 23, 25 14,6 12.0 1.64 2,00 France 18, 19 10,8 9.3 1,71 1,99 USA 2325 13.7 1.75 2.11 Japan 23, 21, 22 14,5 12.1 1.51 1·82 Hong Kong 2223 13.4 11.1 1.6s 203 Germany g~ll 6.a 1 5.8 ldg '·r2 Saudi 13, 15 9.1 7,7 1,5d 1.82 Tai~Jlan 22, 22 13.3 11.1 1.65 1.98 FlubCured 9 15.4 14.5 0·45 0·62 e 199? BAT Co, ltd. Do not copy or show to unauthorised person, in Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 BGB i, SlvGlE DH CHRO~OC~I ~ 19, Or S#OH FROM I ~~~, ntiErCL;RED, MNG SIPE aC~RE~I~E ON I ~~W COIUMN ;lbundancc AF~E U"i 1200 1100 1000 900 100 700 600 100 400 300 A~l 100l O I~ a 00 16 00 18 00 20 00 22.00 24.00 · Q ClibPDF - v~~fastio.soni FIGURE 2, SINGLE ION CHROSldTOG1~UI, m,9, OF SblOIIE FROM ~ STAND~WI, nUECURED, MNC SIZE QGWTIE ON ~ WthX COLUMN i""'"'i 11Pd i r300 22,70 1200 ~~TAMIDE li001 1 ~26135 29, 1000 900 ,52 800 900 600 500 16.60 26, 25, 400 16,69 (17j~91 J00 t22J~'~,OP;"b 11, loof~g ul~e~w,~3 looi 0' ru 10:00 1s.0(1 Zd.00 25.00 h Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 nGlrRE 3. Z,) PWII IIIW CbWBIL~TIO~ CI11YE FOR .4 POWI COll;hR', B) PE~ ~mQfl c~wsRVlOa CLrRYE FOR h POUR COLUMN 16- 14 i 4 12 4 '" j Se d 'i d 20 10 60 80 10d ng bCET~I~IIDE ,, i B) 18 ' * 12 i lo : 1 6- 4- P 2- /r N O O ao 40 as 8o loo ng AC~A~IIDE Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ncfiRE 4, SINGLE ION CMIOM~IOGR~M, mZ 55, Of PtOM FROM ~URIBORO UCIFI~S ON A ~OIAR COlli~i ~unCznce jjOO · ;'LA~I~DE II · 5000· 4500- 4000 : 3500· 3000· j i 23127 25001 1000: 1500? loaa; o.~~ ·\ i :11 a 500- ...~ .....,,, 1~ ~n II, nn tiso 1;.60 23,50 24,00 24 50 TiX -)20.00 211.)0 Il.uu ~~.~u r..*...··- --·-- h Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111