,·B~T· Britishdmerifan Tobacco Company Limited TECHNOLOGY CENTRE PO Box 90 Aegenrs Perk Road SourhamDton 909 KS Englano Telephone: Southompton 101031 782111 telex: 477269 Pax: 101031779715 26 May 1992 RRBIBTMIReports See Distribution Report No, T,242 The Determinafion of the Urea Content of Tobacco the method developed in this study is relatively simple and does not use the expensive radio·labelled compound used in the B&W method. The study has shown that urea is naturally present in tobacco. psbA~$r R,R$aker Distribution Dr. P.J, Dunn, ITL, Canada Prof. E. Rittershaus, BATCF, Germany Mr, E, Got, Argentina Dr, C,J.P, de Siquaira, Souza CIU2 Dr. E. Kauseh, BATCF, Germany Mr, A,G, Stephenson, ETC, Southampton Or. S.R, Massey, Itl, Canada Mr, H,V, Thomsen, Oenmark Mr. G.A, Read, Millbank Dr, J,S. Wigand, BLW, USA Mr, T,I, Wilson, Australia Encl, Rico Om NILCwu C~OFIE Gib Sru~ZI ~U;j IDr 0~ A ~nnr, os rnr BIT IrPums CI~·r Iscon~olms I\ 1;lWI No:~O:: C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REPORT TYPE (RD or T): 7242 FI1E ~O: (Delumimd by issuer) PROJEI1' NO: REPORT ND:T Security Qassilicacion: (Dctumirad by Issuer) RrwRTmLE: I ~J,z ~C~k M~ LL~ G~~t 4 Trku F~;nphs· No: rTo Dnwine Office) AUTHOR: GA ~CU F~E~l;np~· No: I E*C (ro~pputuDnwn) ~VJCYI· CO·WORKERS: PbolugraphJ· No: SE~ION 1EADEWREVIEWER: I~wm: RR~J·L~ DISTRIBUTION: RE (1): ALH(I): MW1(2): PJD(1): SRM(1): TIW(2): ER(1): m(l): CJPdeS(I): HYT(1): WG(I): library(2): me DISK REFEREI~C~: TIPING CIICCENG Si~arme I Date Sent Date Dare APthorl Date Issuer Date Re#ived I RcauPed I Rnriewa Daitfor wing IPdn Terms 4i· Cweaions I I I ~--tzO·s·Y1 1~E~ iiS Prepye One CoW 211\> ~his proforma is intended to facilitate and Llail the progress of BATUKE Rd~D Gnlr~ Reports from the ~plsnip~ stage wuil the report is Fnolly approved p~ isr~rd. h ptojwmo should he con~nmccd at the G rime of the initial typing and occ~mpany the typescript (and uJler ~a preparation) at ail subsequent srages. it RUL~t be sent 10 Central Records With the Master When the report is issued C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 THE DETERMINATION OF THE UREA CONTENT OF tOB1ICCO REPORT NO: T,242 18 May 1992 DISTRIBOTION: Mr, G,A, Read 1 Dr, R,R, Baker 2 Mr, A,G, Stephenson 3 Dr, J,S, Wigand 4 Dr, J,S, Wigand 5 Dr, P,J~ Dunn 6 Dr, S,R, Massey 7 Prof, E, Rittershaus 8 Dr, E, Kauseh 9 Dr, C,J,P~ de Siqueira 10 Mr, E, Cirant 11 Mr, H,V, thamsen 12 Mr, T,I, Wilson 13 library 14 Library 15 i 199: BAT Co. ltd Do nor cop;v or show to upnuthorised persons. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Centre, British·American Tobacco Co, Ird., SOUTHAMPTON GAFIBfM THE DETERMINATION OF ~HE UREA CONTENT OF TOBACCO REPORT NO: T,242 18 May 1992 A~rmoR: G~A. Few ISSUED BY: R,A, Baker COPV No, E 199? B~T Co, Ltd. Do not cop? or show to unauthorised pcnoar. 0~ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Fundamental Research Centre, British American Tobacco Co, Ltd., SOUTHAMPTON GAFIBTM 18 May 1992 THE DETERMINATION OF THE UREA CONTENT OF TOBACCO REPORT NO. T,242 SUMMARY: A method is described for the measurement of urea on tobacco. It entails derivatisation by heptafluorobutyric anhydride followed by gas chromatographic analysis with a mass seledive detector used in the single ion monitoring mode. When applied to fiuecured tobacco, a natural level of some 200 ppm was found. INDE( TERMS: Urea Tobacco Chemical Properties GC·MS " 19?3 B~T Co, Ird. Do not sopy or show to un3uthorisd persons, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 1, INTROOUCflON It is commoniy aeeecte~ ttat the nitrogenous content ct tebaeen accounts for a considerable fraction cithe taste and flavour of cigarette smoke, Hence, the addition of nitrogenous e3mpounds to tobacco can en0anee the taste and fiavour of the product, One compound of particular interest is urea and this has been applied to tobacco and tobacco sheet and the treated tobacco incorporated into cigarettes To establish the urea loading on the tobacco an analytical method is iequked and this report describes one such method, Prior to this development, a literature search revealed that B&W had analysed for urea on tobacco by siiylation followed by mass spectmme~y with ~adio·labelied urea as an internal standard (1), This method, although perfectly satisfactory, has Ule drawbacb that 1 uses an expensive radio· lauled compound and a silylaling agent which will rapidly di~ the source of the mass spectrcmeter, The work presented in this report has endeavoured to develop a more simplistic method, This has been applied to tobacco with the discovery that urea is a tobacco natural, occuning at the ppm level, The method uses acylation via a Ruorinated acylating agent followed by mass spectrometry in the fashion of the BBW work, 2, Method Development Acylation, using fluorinated ajlating agents to form volatile derivatives which may be analysed by gas chromatography is a common route to the analysis of amines, Application to urea on the ether hand is more complex, owing to the two possible sites of attack, HeptaRuarobu~yTie anhydride (HFBA) was chosen as the acylatjng agent with dimethyl formamide (DMF) as the inert solvent, The reaction is given below. The choice of dimethyl formamide was driven by its solvating power, CF3CF2CFTC:O HZN CF3CfZCFTC:O CF3eF1CfrC:O O C~O HN HO CF3CFZCFTCSO HZN CtO HFBA H2N The problem in this reaction is whether to add either a large excess of HFBA in an effort to acylate both sites or just enough to acylate one site only~ To determine if the latter was possible sc saving on the amount of reagent used and minimising exposure of the gas eh~cmatography column to a highly acidic material, 1 ~gl~l, 500 nglpl and 100 nglul standards made up in DMF were reacted separately with 3, 6, 10 and 20 ul of HFBA, 300 pi of each standard was taken and placed in a Reaeti·vial, the relevant amount of HFBA was added and, following vortex mixing and capping, placed in a Reaeti·vial heating mantle for one hour at 70'C. At the end of this line, 1 uI of the e 5 199! B~; Co. Itb I)o not copy or sbor to unwurhorised peacms, in Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill reaction mixtures were injected into the gas chromatograph which was fitted with a mass selective detector, 2,1 Chmmatanraahv and masssPectmmetru Chramatogtaphy was achieved wing a 25 m BP20 fused BEa capillary column with a 0.22 mm internal diameter and a 0.25 pm film thicloless housed in a Hewlett·Packard 5890 series gas chromatograph, The column was initially held at BO'C for 5 minutes and then ramped to 180'C at 5'C per minute. The flow through Vie column was ImVminute with a 10:1 split Tne chromatcgraph was f~ed with a Hewiett·Paeka~ 5971 series mass seleeb~ve detector, The mass selective detector was used initially in the scan mode with a mass range of 33 · 550 units and with an electron multiplier voitage of 1870 volts. In this mode only 3 major peaks make up the chromatogram, as shown in Figure 1. From the mass speetra, the first or these is a reaction product from urea and HFBA, and its mass spectrum is discussed in Appendix ~~ The second is HFBA itself and the third heptafluorobutyric acid. The reaction product is thought to be the one site moiety because of the presence of an ion of mass 166, No evidence was found for the existence of the two site moiety, even taking the column up to 230'C in temperature, Quantitative measurements based on the peak at 10.6 minutes were taken using the mass selective detector in single ion mode and allowing it to spend all its time monitoring only the 166 ion, The results of varying the HFBA level for the three standards are given in Table i, Table 1 HFBA levels for the Three Standards Urea Standards 100 500 1000 HFBA 166abundaneex 3 0.75 3.1 7.0 6 1.82 10.3 18,9 10 1,06 17,5 22.7 20 0.16 6.7 8,1 HFBA = heptanuorobutyric anhydride, Table 1 clearly indicates that the addition of 20 Cll of HFBA destroys the single site moiety and, in fact, some loss occurs with the addition of 10 CII, The n addition of 6 ~1 of reagent produced the linear Calibration curve shown in G Figure 2. ~ 1992 B~I Co. Ltd. Do not copy or show 16 unauthorised peaops, ir; C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 2.2 Tobacco e~aetiao Owing to the strong solvation power of DMF, 0.1 g of finely powdered Rue· cured tobacco was e~xtraeted with 5 mi of solvent for only one hour in an ulb~a· sonic bath, Iml of the slurry was then filtered through a 0.2 Clm, 13 mm nylon filter disc, From the filtrate, 300 Cll was taken for reaction with 6 ul of HFBA, The resulting single ion chromatogram is shown in figure 3, where it may be seen that the region of interest is absolutely clear of interference, This is because of the rarity of the 166 ion, The area of the peak shown in Figure 3 is 0.08 x 104 units and comparing this with the calibration curve given in Figure 2 suggests a natural urea level in fluecured tobacco of over 200 ppm, The calculation is given below: The curve is essentially linear and 100 nq of urea standard gave an area of 1.82 x 104 units Hence, b,08 x 104 is equivalent to 4.4 ng which was contained in a 1 ~I injection, This means that 5 mi of DMF would have contained 22.2 ug of urea which was extracted from 0.1 g of tobacco. Hence, 1 g of tobacco would have contained 222 ug, a concentration of 222 ppm, 23 f~a~ It was noticed earlier in the work that a blank (300 Cll DMF, 6 Cll HBA @ 70'C for 1 hr) appeared to contain a tiny amount of urea. At the time this was believed to derive from solvent contaminaticn. In latter wch the blank showed an insignificant amount present as shown in Figure 4, However, it is recommended that a blank is run, 2.4 Materials 'The urea used was of Anaiar quality (99.5K) from BDH, as was the dimethyl formamide, The HFBA was obtained from Aldrich with a puri~ of 98%, 3, Further work Further work needs to determine the reprodueibil~ of the method, If results are too variable an internal standard may need to be incorporated, This must be totally inert with regard to the reagent, It was discovered in this work that even an aldehyde such as veratraldehyde (which has a large mi! 166 abundance) will react with HFBA There are, however, no current plans to do this further work in Southampton, due to changing work priorities, C 199? B~T Co, ltd. Do not copy or show to uF3uthorised persons, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~FE~HCE 1, BB,W Report No. PGS·803689, 20,6.1989. O 1992 B~T Co, ltd, Do not copy or show to unauthorised persons, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 APPENDIX ~ 'The mass spectra of heptafiuorobutyric anhydride and heptanuorobut~ic acid am dominated by ions of mass 69, 100, 119, 150 and 169 (see Figure 1) because of the fragmentation patterns producing ions CFg C2Fql C2Fg, C3Fg and C3FI respectively, Subseguent~, any derivative of HFBA will also have speetra dominated by these ions. In cases where the background ionisaticn is in low abundance, identification of the derivative may be impossible, This is not the ease for the reaction between urea and HFBA. The mass spectrum from the moiet~ eluting at 10.6 minutes contains two singular ions of mass 166 and 146, and a large mass 44 abundance. It is the authors belief that this reaction product is the result of a 1:1 reaction between urea and HFBA; the tentative mechanism is presented below, CF3CFZeFZ~~O IJH ·--. nl~L~ ' alcha~c;0 1,44 CsO 'NH m, 168 CFtCFZ CFTCF2 CFTCFZ ·CO H' CFZ-NHZ (I-- CF2 CtO CF2 C:O N~ mt 1IW N H2 H I·HF CFTCFZ CF~NH mx 146 6 1992 B1\T Co. ltd. Do not Fop? or show to unauthorised persons. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 T,2~2 FIGURE ~~ CHROMATOGRAM FRQM UREAIHFBA REACTION MIXTURE WITH MASS SPECTRA OF ~9JOR PEAKS Abundana TIC 300000· 280000· ,, 1 9 ~u :I 2600001 ii : ··I·· II II, 240000· i 220000 7 . / ' I I! j ill 200000 I ) I I I leooooi I I j ~ io lio l~o rOa JD 100 ~p 16100Pj ;1 P1 Y(i IH' / IP 100 !50 200 i~ooooli 1 I ; I ' 120000 1 i I I looooo i aooool i I ii d 1 60018j jl!I It / 40000l 15k~jli~: 20a00- Time -~ IO,OD 15,00 20, Oa 25,00 30.00 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIGURE 2, UREA CALIBRATION CURVE · 6 ul OF HFBA mt 166 ABUNDANCE x 10E4 I np3~8 800 11CO 1,2W 1,242 FIGURE 3. SINGLE ION CHROMAIOGRAM ,mz 166, FROM A TOBACCO EXTRGCTIHFBA REACTION MIXTURE Abundance TIG: TOBACCO.D 10.65 92· 90- BB· 86- 84- e2.; i i i 80- 16 I i /6 'I 74 72· 70- 68; 66- 64· 62 Time ->9.00 9,50 10,00 10, JO !1,00 11,50 4 0 N a ;3 S 0\ [I` C Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 T,?B2 FIGURE 4, SINGLE ION CHROMATOGRAM, mt ~66, FROM A DMFI~FBA BLANK A~undanee TTC: BLANK~D 86· 84- 82~ 80· ,,i i i I I ; i; 1 111 1 II i 'f 4 !(1) I 1 1111111111 1 i 1(11 1 II IIIYIIIIII(HI at lla1 a~lepl i IpCll6ll I1I Iplo Iq I I TP1 1 811111r iii CCI I : 12 I I ii ' I I I~I i ! ' ' 70· bB; 661 64; 62~ So56 j 5ZJ 48 ~i~e ->9,00 9,50 10,00 !0,50 11,00 11.50 IV Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111