51 ~cc~ Iltt IEPDm lo, ~t ~o REPm TIT1E security Mllslflcatlan: ngrlenphl WD: ~I~~~LldCid A~~'% 4 ~~sbn~e ~rmhb Iho~pClb: I I File Ya: I~ aUThOR: p, ~.~o~ZkLi~ CO·POIEIS: SfQIOY LEaglp: R~~wlu:,e ~~ mu~l: R.R.dGkr OISrPIBUTlaY: pi (1) : KH ( I: EEK (2) : pJD (1) : S~ (1) : RI (2) : ER (1) : EK (I) : WP de S (1) : HYT (1) : RFG (1) : library (2) :Flle OPI t~ ~qPA~~I nPI16 CHE~IYG 1gnl- ( Date I Oate 1 01~ trre gent I Icc'd Returned I ~Se~tloR Date Issuer I gate leader ~"C *r yjt ';? D~a di~ j i~l ~ Zdre ~ R JI ,tr~l Irqrl ~r ~k I(I bC)Iu~l Bo Master: ~k~paet~d Corrcetlon~ prepare 1 I~~ I ih ?~ lli~F 4 IIIIE IJl?kk I &~1 Sb nL Thll pro fon~ II 1 to facilitate and detail the progress of B~TUKE R1D Centre Reports frP the typescri pt stage until the report Is flaalll approved and Issued, The pro fon should be e~aced at the tlw of the Initial typing and aecMnplny be typeserlpt (and easter after prrpantlanj at all rubsequent stages, It lust be sent to Central Fflfng with the easter when the report is Issued. TOE: 1~ OlWTS for typing fHOOLD IE CLBR11 lE6~11~, I,e, original typescript, coeouter printouts, ete, (not earbons or photofopfcs) or hanbrlttcn in Ink, 2~ after typi no, the typescript should loRtll be returned to be author, who will subseguently obtain the Scetlon leader's and Issuer's approval, 3, Clteulatlon should be deterlrined by the Issuer, a, Tke tyoeserfpt eust not be presented for printing until the Issuer has Initialled the pro forea against "Prepare Iaster", 5, after printing, one bound eoff eust be returned to be Issuer lot final n epurevel era the Isruer u~l enlure bit one eom ii rcen by the Mlnagcr, O ClibPDF - v~~fastio.soni eAJ (U~K, Rlreh d ~lopment C~,I ~~AMPTON. MWP YODP ~DIE9 ~ ~C QEYI~I gy R~ROSAIME F6R~OM RBO~ WO:~2M1 12 Aogr~ 1~ AmHOR: D~ hu\noM C~OR~g: C.F. HEWETT R~I~B BY: P,C. 8~# ·UB 81: R.R, ~KER DI~RIBU~O#: Dr R. Binn YL A.L Ho~d 2 Yr Y.1 R~pdb 3 YL Y,L R~Y 4 Dr PJ. Dunn 5 Dr S~R, ~ry 6 Yr TI m~ 7 Y1 TI m~~ a Hs~ E, kahrl g Dr E, lul~ 10 O~ C.J,P, d~Siquein 11 Yr H.V~ ~l,n tt Yr R.~ G1~1 13 COM ~. olll# ~~llrlr~uuuhwtid~ ClibPDF - v~~fastio.soni e,A,r (U~K. 8 Export) ltd,, Researct~ & Development Centre, SOI~T~AMmON. DAVWPH20 12 August 1988 MODEL SfllMES ON THE CHEMISTRY OF NITROSAMINE FORMATION REPORT NO, T200 SUMMARY: Chemical model experiments have been ear~d out to gain a more thorough understanding of the chemical mechanisms involved in the formation of tobacco·spec~ie nibosamines~ T~i~ involved nbsating aHtaloid precursors in aqueous solution, This inlormation may assist understanding some of the reasons for observed variations in nitrosamine levels behueen tobacws and brands, FCMte was confirmed as the agent responsible lor the nitmsation of alkaloids to nitrosamines. Nitrate was round nottobe reactive under the conditions studied. Both nomicntine and anatabine were mnvened in good yield to nitrosonomicotin (NNN) and nitmwanatabine (NAtB) respectively. The nitrosatbn was mae efficient at low pH in the pH range studied (2.5 · 8.0), indicatingvlatnitmusacidishenihasatinga9~ Astudyo~lheeneclolreadion time showed thatanatabine is more raptly nihasated than nomicaline. A mechanism to explain this has been proposed Nitrosation of nicotine gave two nihosamines, NNN and d(Kmelhyl·N·nibosamina)-l ·(3·W~1)1·butarone (NNK), anhwgh the yields are much less than those from nomicotine Or anatabine. In this ease there appeared to be a pH opSmum of ca. 4, Additionally, at higherpH, pmportionally mere NNK was produced than a lower pH, e lass BATUKE Do not mW or show to mumerlJed PF9MI. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 The remits indicate fhst $ mkh~sm of ri~ols~an rd Ihe Iw~l amina nicotine, is different trom 'hal of k pemds~y mines, ranimCne and anatabin. A mechanism t, the nilrastdion of nicotine to NNN and NNK is proposed, INOU( ~RB: Chemical Model System Readdon Mechanism NibPsamines Tobaceo Speeifie Nitrosamines NitrosoMlmieotiM, Ntrosoanata~irm NNK NLate Nitrite FFieoSne Nomicotine Anatabine pH Measurecnent Reaction Products E1~8 BATUKE Do MI mW or~no~ to unsuhPllsod pe~c~· I Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 INTRODUCTION Tcbaee~specific nitrosamines (tSN) are not present in freshly harvested (green) tobacco but;ue pmdud during curing (~). The amounts of nihPaamineli produced during the curing stage are dependent on three factors: (i) Method of curing (e,g, flue- or aiKuring); (ii) Tobacco nitrate and aata~oid levels; (iii) Tobacco type, It has been found that the method of curing has b~e major influence on resultant TSN levels j2 19). Ruwuring produces more nilrosamines than aircuring for a given tobacco type with similar nitrate and nicotine alkaloid levels. Thus in the unusual event of fiuezuring Bu~y tobacco, high levels of TSN are obtained. while ajrzuring V~rginia tobacco gives very low levels of nitrosamines (3), Under mnventional circumstances, aircured Burley lxKmelly has higher levels olTSN than nubcured Virginia lobaao (3 a 4)· The fonalian of nitmsamirss during curing is thought to be due to La build up of nitrite levelsin tobacco, There are b enzyme systems involved in Re tobacco plant's assimilation of ni~ate: a nitrate reductase system which reduces nitrate to nitrite, and a nitrite reductase system which reduces nitrite fuRher for ineorpodPn of nitrogen in amino acid pplheJis (61. When tobacco is cured a is thought that the m6re fragile nitrite reductase system becomes inoperative o, loses its effeetiveness~ This leads to an accumulation of nitrite within the curing leaf, Nicotine a]kaloids then read with nitrite to produce tobacco specific nitrosamines, if is also known that different me~ds of curing gie rbe to different proportions of individual nlpsamines, in particule NNK(6), little information is available on the detailed chemical trawformations which produce nitrosamines in leaf, The present study has been cart~d out to gain further information on the mechanisms involved. Due to the inherently chemically complex situation in tobaew leaf, a model chemical system has been used rather than tobacco leaf itself, There is an appreciable munt of literature avajlahle an the reeeSans of nitrosating agents with secondary and tertiary amines (7 6 8), However, there is much less information available ,, the chemistry of the formation of tabacc~specific nitrosamines, There are certain structural differences (i.e, Wridyl ring) which could influence the nitrosation mechanism and subtly alter rates of nitrosation, It was decided to make the model as simple as possible and to carry out the niksation reacCans in aqueous so~ron, c 19BB 8ASUKf. Do not copy or show to unsuthreised peo#n C~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill BPERIYWTAl DEBGH ~ YY amnenb ~nd 1 labacea · ~ldnr InIfdioo Rd niootine · whioh n known ~ ~ preourfal b ~lonaYn (Hm~ Ibtwlttabl~ (NAt8) d yk~~13·niro~M1~44·(3·p~61F l~a~s(MH~, 110 ohooon for Cg, 11001500 roIofionI Ilo mrrid out wn dbo nn h qlal dll b+~ b I 1111 pH within 1 q, d~~7aL 711 ~~~a ~~ nomiootino and anlpbin, were ~~ ~ ~ mao mmed to 16 r ab ono aduot 111 koned d 1~1 Ydb Cd~ nicotino) cwld bs eY ~~t tno fm" 1~ Ydd o~hd. On~~S, ~ of fhb Y doIonZIod in Le ~ roilltes to L, nlmJa d nanioofinI and rYh, Irmoosh Yd dO from nYal roIofias IIo I]oo gkn· Aooaxlo(O.l 1) Ya del h~~ C vIiuos d 85 ~110 4A~ 5J, 7.0 and 8.0 were obtained bY the1Y~ pe(hodb dodbd in k IL~n pH 25 Id 125 C1 1 ~uau h~~bde dd wIs dY 1 al 1 q#ul ~~m hydro9In pr~+ udl n, d,ld oH ~~ r rkd on h, pH nJr pH IQ IT ~ 5~ P1 Y Aqueous die bldldd~ ·blo I Jd,d C P1 1 glee p~~p hydmoon pY11. pH T.D Id 5,0 11 1 ~ab IdlD hYaOwido 1I1 1 lddod 1 P1 1 qll rJun d~PI," pblphl~ pgCDUAE AMD AMAUIS BuLr Ioiufion (5 Mn') re p~d h I 100011 ~~51~ ·d nY b 1#,) Iddon~ ~) rll d~~ b~iJ~ llioId B h ddr of alkaloid solution (1 em4~ MI orocoduroI ~dh du~~ ondtho raUHjnO IYrr 1IR n· Y 1 fho dlk for I Ipdo Odd d Imc. h ~uaul o~l·~ were pourod let in Yur tubo' ~r 1 1~ d~lahand o~bn of ICYLS· in ~ to JRL movthss of ~grhn r ~bt ~b oYnl I mdgla tkl to d~ ~'6' ~Cr Cb·l manulleRnsd lnd ·~ld by h Mink, Olr West Gs·any. c 1I1 WKE Do id 4 a sho" I'~~ P"O~' ClibPDF - v~~fastio.soni T~e nitrosamines were removed by washing the contents of the tube wi~ dichloromethane (200 #n3 into ,glass column pad(ed with anhydrous sodium sulphate (100 g) to further dry the eluent Tne eluent was concentrated (KudemeDanish)$ approxma$ly 3 ern~ and the nitrasamine content measured by injection of a sample onto the GCrTEA system. In this way awes po~e $ idenly L nbsdmine fumed P My), the total yield and the percentage yield (based an starb'ng akaloid). Nrm6SATION OF NORNICDTINE AND ANATABINF To confirmthat nitrate could not direct~ nitmsate alka~ids, buffered ~H 25) aqueous mixtures ol nwrieotine or anatabine and sodium nitrate wars allowed to stand ovemight a room temperature. Fommation of ni(msamines (NNN or NAtB) was not detected in measurable amounts. In initial experiments the effect of pH was studied. h was thought appropriate to work at a tenth cf the mdar concentration of the buffer solutions; consequently 0.01 M (final wmentnion) nomicotine, an~sbi~e and 0.01 M sodium nitine solutions were,~Bd~ After mixing the substrates as desabed previously, the mixtures wsre left far 2( hours at ambient temperature prior to work·up and nitrosamine measurement, The results obtained far namicotine and anatabine are given inTables 1 and 2 and plotted in Figures t and 2. Nitrosamine #mcsntraion decreases as pH k~creasm inthe range 2.5 · 5.5. This is especially so for nitrosoanatabine but is less clear for nitroso· nomicoline. I~tB was not produced in any significant amounts at pH 5.5 or higher, while scme NNN (471 W cnr) was produced at pH 7.0. Neurath at al. have rep6ned that the optimum pH for nlmsabon of ncmimtine was 3,3 (~0), This seems to be co~ary to the findings or this study where there would appear to be no pH optimum around this region for either the nitrosation of nomicatine or anatabine, EFFECT OF REACTlaN Ll$E AND SOLUTION pH Tables 1 and 2 elea~ demonstrate that the yields of NNN and NAtB, especially at pH 2d - bO, are guile high. In subsequent experiments. the concentration of alkaloids ,, reduced to reduce the level of nitrosamines formed. The effect of reaction time and Jcl~on pH fur dmsln of 0.0002 M namicotine and anatabine by 0.01 M nade was studied and the results are shown in Tables 3 and 4 and plotted in Figures 3 and 4, These results "8aMnomiwtine and analabine were purchased Irwn ~casler Synthesis Ud., U,K. c 1988 BATUKE Do rot copy or shwlm unaulhwisd pooora~ C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill confirmed that higher yields of both nibasamines were found at the lower pH range, As anticipated, nitrosamine formation increased with increasing reaction time such that at pH 2.5 quantitat~e yield of NAtB is achieved by 4 hours (98010). For NNN the formation rate is less mold for the initial 4 hours. After 4 hows at pH 2,5 the yield is 63.4b, after which no further NNN is formed. It would appear that, for pHs 25 and 3,25, there is a slight drop in yields of NNN for periods longer than 4 hours. this is proba~ly not a real enact and it must be assumed that maximum formation of NNN has occurred for pHs 2.5 and 3·25 by 4 hours. Even after 11 days the yields of NNN at the various pHs are not ~uanftative (ca 55·MIZ). The reasons why quan61at~e yields of NAtB can be achieved but not for NNN are not understood. Perhaps the nitrosat~n of nomicoline is reversible and an equilibrium concentration of NNN is attained, Clearly, anatabine is nitrosated at a faster rate than nomicotine (Figures 3 and 4), After 1 hour at pH 2,5 the yield of NA1B is 91"/0 compared with a yield of 32% for NNN under the same conditions, This finding is surprising as both nomicotine and anatabine are secondary amines and have similar chemical structures, The only difference k that anatabine has a C·membered ring containing a carbon carbon double bond, THE EFFECT OF DIFFEAENt CONCENTRA'TIONS OF AIKALOID AND NITRITE ON NlTROSAMINE FORP~1ATION It is appreciated that tobacco leaf may vary in the relative levels of alkaloid and nitrite during curing; therefore, the effect of different concentrations of nomieotine and nitrite were studied, Three eoneentrafians of nomieatine and nitrite were examined (0.01, D,M)1 and O,M)01 M), The different concentrations were mixed as desc~bed in Table 5 and kept at pH 4.0 for 1 hour. As anticipated, reaction of the highest concentration of nwnicotine (0.01 M) with the highest concentration of nitirte (0.01 M) gave the highest yield of nitroscnomicotine (40.3 pg emJ, 2,3"10), Reaction of the lowest concentration of nomirotine(0,0001 M) with the lowest concentration of nitrite (O.MIO1M) did not produce NNN in any measurable amounts, whilst intermediate concentrations of nomicotine and nitrite gave intermediate amounts of NNN. Using the Table 5 data far one hour of reaction to obtain initial rates of reaction gives first order kinetics in narite and an order of 0,7 in nornicctine, The va~id5 of this treatment is supported by the data for 1 and 4 hours of reaction at pH 4.0 in Table 3. c 1988 BATUKE Do not copy or show to unavb~odsed persons, IV C: I i I; PDF -!::!!::!!::!.f3 StlC.i: Dill in parenthesis~ (t is clear for bath the 24 hours and a days data that the relat~e amounts of NNN and NNK fanned are dependent on solution pH. There is a steady decrease inNNN relat~e amounts on increasing pH (944b, pH 25 ~ 69%, pH7,0, a days data) Throughwtthis experimentthereisalwals an excess of nicotine and nitriQ. Therefore, b~e differences be~ettn the relative amounts of NNN and NNK cannot be viewed in terms of cwnpeliljva format~n of either ,itmsamine from nicotine, and the data is only a rsAecSwl of the dmsrent rates of tonnab~n. The existence of a pH optimum for the nibosation of nicotine wd the absence of one for the nitrosation of nornicotine and anatahine suggests that the mechanisms of nibosation are different, MECHANISTIC INTERPRETATION Nornicotine and Anatabins Farmatlon Nrwus acid (HNOJ or its anhydrlde (1S~03) are the nitmsating species and not nY$ ion. Nibite icnreacts withpmlonsin aquecs solvt~nwpmducenitmus acid and N,4 z~o;tw'= UQIO, =N,4fH,O Thus the reaction is PH depended: the greaterthe conc~mraSon of proton the more nitmus act is produced as the ecluilibnum 4 pushed to the ipl of the stoichiomsltic aqua6on. t is assumed IY the direc~ niVcaating spede~ is the nitramium ion(NO') produced by icnic dissodaion of nilrws acid or N,4(H). HNO,f ~OI+OH' N,O, ~NO'+NO; This species can react wah s8ronda~y a~rin~s expelling a pmton to p~o~e a ni~osamine. R H a' N-H R F 1988 BATUKE. Do rot mW wsho~tPurPumarissdp~ncM· C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Pusiby the dmerence in react~n rates between anatabine and ncmicatine can be explained in terms of the presence of the urban urban double bond in anatabirm~ Perhaps, initially, a charge~t~pfer cornplex is formed with the NO· ion, N N~ H H and then bond formation with the basic nibagen oewrs, eliminating a proton, regenerationof the double bond, and N418 Iwmsfah The boat mntorm~8ion of the sb~membered ring would place these gmups within the required interatom distances, H NrcO 1988 B4TUKE. no not copy or shw,to un~nwls~d psmw· C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 for b~ansfer of the NOI ion, This mel elcp~n the laster rate of nitmsln of .atabiMI. Three~dimensional molecular models Indicate that the proposed mecha~ism is po~sble, A simpler rplanaCwl is Rat basidty dffletemeJ between nomicotine and anatab~e could explain he rlffarence in readdPn ratea. Haveve~, the douMe bond in anatlb~o i, most likely to reduce b basiei~ nd possi~ reduce the nib~a~ion rs$~ Ntcotine When nicotine is ni~·osated bL~ereb a requiremen~ for an oxidetive step as wdl as ~it~osation, For example, in t~B famrsdon of NNN trorn nicotine the N·melhyl group is replaced by a ~ilroso group. Yi ,I 5 r N R HecM at id. (12) have suggested that namsation occurs Fast, which, bner eliminat~n of nitroxyl (HNO), leads t,the produc~on of two possible imminium ions. 'i v B .HNO [H, [H, lip `"; -- NNK N yj Iln so, NNNC~j~l N CHI c 1988 BFltllR Oo not coW or snow to uMlhc~ed psoKYa· C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Hydro~sis of the in$rmediale imminium ions is proposed, folloned by nibosaticn (HNO,J to either NNN or, altemativeiy, direct reaction of nibite (F10$ with the imminium ions, I is suggested by the aub~w that the oxaaCon step may occur first to produce the ~Na possible imminiurn Ions, the oxidanl being nibaus acid, 'i P~t/\N/~ HNO, N' [H, N' [H, Reaction of the imminium ions will lead to the eventual formation of NNN or NNK, In the case of the formbdon of NNN, fwmaldehyde is eliminated. For NNK pmdue~on no such eCminabbn occurs and a ca~onyl group is generated and the N·methyl grwp retained. IHP F,, [Hi O N=O (yCO ,N~O CH~ CI''"'"" N N c 1988 BAfllK~ Do R# mW or shoe # uuamP~d pwUrg. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 REFERENCES 1. Heeht, 6S·; Chen, Ct~CHong 8,; Daug, M.; Omaf, A,M·I Halfmann, D~; Tso, 'T,C, Beitr. Tabakfarsch, (1977)9(1), lb. 2 BATR&D Repd~lNo. R01943·R, 13.10·83. 3~ BAtLIKE RLD Report No. RD2062d, 6.11.88. 4, Andenen, RA; Kemp,tR cancer Res. (1S85145 (11 pt 1), 5287·5293. 5, Ibraheem, S,M. SETTA Ann, Tab, Section 2 (1966) (20), 557, 6. BATUKE RBDReportNo.RD2096R, 5.10.87. 7. "OpenChejn NRrogeo Campounds',~ls I 8 II. Author: PA.S. Smih~l PubTffhed by WA. Benjamin, Int. (New Ywk) 1966, 8. Smi~, P.A.S,; loeppky, R.N. J, Am~ Chem~ Sac, 89, 5 (196fJ, 1141·1157. 9, "Handbaok of Chemistry and Physi#", Slsled., 187DH, PuYhed by The Chemieal Rubber Co,, page 104~ 10, Neuralh, G.B.; Ounger, M~; Pain, F.G, "Environmental N·Nitrcso Compounds Ana~sis and Fwmln", IA.RC. (lyon), 1976, 227·236. Il,'lnarganic Chemistry', pumll and Kotr, 19TI, Published by W.8. Saunders eo,l p,OlB. 12. ~l~c~, S.S.; Cheh Chi~ang a.: Omal, R.M.: Jaccts, E; Adams, J.D.; Hoffmann, D· J. Org. Chem, 43, 1 11978), 72·76. 198B B~IVKE. Do 1 copy or show to ~n~auno~s~d P~W· h) C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE~ NltroJetlan o10~1 NI Nornieotine with R01 Y Nltrite 24 hours Solufion p~ Nitmeonomicotine (NNN) Pe~c~ntage Y~ Concenh~ation (~~ emJ) (O~) 250 1305 74 3~25 1550 88 4.00 1175 66 4,75 368 21 5.50 15.6 0,9 7,00 47.1 2,7 TABLER N~oaUon o10,01 M An8tabinewlth 0.01 M Nlhite 24 hours Solution pH Nihot~anatabine (NA1B) Percentage Yield Concentration (R anJ) (O 2.50 1590 84 3.25 1100 58 4,00 1150 61 4.75 298 16 5,50 o.n a~o4 7.00 725 0.38 8,00 0.37 0.02 e 1988 BA~UKE. Do Mt mW w shor lo una~lhPrked pcKwra, IV Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 fABLE 3 Nlllooatlon of 60002 M NomkoHne 1 am M Nllde al Dmerant pH Deer Oiffsrent nme RriPt 1HOUR 4 HOURS I HOURS 11 D~vs Sdulkn NNN Pwe~r~ NHN Pag~ HNN Psre~ NNN Psreent· pH Cona~ 806 I COMIF r 8ge 119400 Ybld IPlbn Yk~ ll96MI T#ld balion Yt~d (Ileolr) (~k) Ika~ll (1C) (rcJcn~ %) liWa"J)I ~k! 25 11.3 32 223 63 201 n Is.t 56 325 8,3 24 18.0 51 17.0 (5 20.3 n 4,0 24 6.8 92 26 18.6 53 2R8 5Q 0.9 15 12 3.4 26 7.3 17,4 49 65 0,5 1.4 0.9 25 1·5 4.2 93 TABLE4 Nn~ooation of O.DM12 hi Ans~ine 1 0.01 51 Nllrils at Dmtnnt pH ovar Dmerenlflme Periods 1HOUR 4nWRS IHDURS IDAYS NAtB~IPereent·l NAtB IPeresB Sdutkn NAIBIPereem·l NAIB IPMeent· C~Mn~ ape pH Cc~ln· spa 180 Ca"c~~ 8BB 118560 Yrld 1~MI YWd IrslPn b~a~ian YleM (re ern~ I (11) Ik~) (Wc"r)l (sc) 21 34.1 P1 16.9 98 121 1W 38.0 loo 34 28.8 76 35.P 95 98.3 100 1[LI 100 4.0 127 9( 265 70 381 1W 3f.2 98 4.75 5·1 11 50 16 21.0 55 313 86 6.594 5.1 11 18.T 1 26.5 fO c 1488 BATUKE Do nd coW MshPn 10 u~a~horbed PIOMLS. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 fABLE5 B Beet of DH~eront Cpncantrations ol Ncm)eot[na and NIMle on Nitroronwnlcatine Formation NomieoEne Nihite Pereentaq% Concent~aUon Concentralian ConcanbaSwl Y"M (MOL9R) (MO1AR) (141#"1) (01~ 0.01 0.01 403 2.3 0.01 0,301 3.48 0,20 0.01 0,0301 0·39 0.022 0.001 0,01 10.92 0,001 0.001 0.90 051 O,M11 0.0001 ND 0,0001 0,01 1.M) Q,O 0,0001 0.001 0.14 O,Bt 0,0001 o,ooot ND ND · Not Detected Solution buffered at pH 4,0 Reaction ~me 1 hour TABLE 6 Nltmsslion or 0·D1 61 NieobiM wfth 031 M Nible 24 hours Nitrosamine C~Mtentration Percentage Yield Sdution pH (A em') (4/0) NNN NNK NNN NNK 2.5 32 0·2 0.18 0.01 3.25 41 0,3 023 0,04 4.0 5.6 2,0 0.32 0.10 4,75 1.2 0·3 0,07 0.01 5.5 0,3 0.05 0.02 0,002 7.0 0,55 0·3 0.03 0.01 B.O 0.6 0.4 OR3 0,02 e 1988 BA~VKC Do nol ODW Pr IhOW a, ula36o~d penons, h, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE 7 M~m~llon pl0·001 H Nleotlm wllh 0,01 M Nnm at DiffsPnt pH OYH Dms~ent nme Periods ,WUR i HOURS 21 MOORS BD~VS So~n NNN NNK NNN NNK NH( NNK NI NNK Concen·IC#K~n· Concsb I C~n~n·l Comrm· pH ~ce~ 929911 (rs8kn ba~kn 92902 tratbn tratkn Irat~n 82992 (Ilg anJ)( lup~rl'l (I~,~l~w~w~ (Wan~)(lrllmrl(lRolr] 25 0.1 ND 1.M ND tn 0.15 tie 0,20 pr) (9) 144 (6) 38 1 635 ND aec No Ist ale 051 (91) (9) (89) (11) 4.0 1 0.19 ND 316 ND 1.53 0.11 638 213 p) (21) (75) (25) 175 n22 ND asl No 1~25 0.35 7.12 297 118) (22) (il) (29) 15 021 ND O.11 ND 0,13 020 6.9b 2rj (78) (22) (72) (28) 1.0 HD ND NO ND ND ND ND · Not Detected c 1288 BATUKE Do nol coW or~o~ to u2gtl8nbod pwswu~ h) C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 T, 200 Fi[i, 1 NRROZOIIORII[OTN [DN[IUTRITOII YIRIV~ IOUIIIOHP NHN ~1l0 Ir~ ~o"i 1600 O 1400 O 1200 O 1000 800 600 600 200 O i i 1 i iii' pH n, a o o N o Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Pit. 2 T. 200 WITROS)NITABINE [ON[ENTRATION VERSUS SOLUflOU pH NA,B IEUI I~ mP 16001 O llDO 1200 t000 O 3 4 5 L 1 I OO PH Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 T. Z00 PIG. 3 NIIROS~IION OF 8000RI NORNI[OTIHI: WI'IH O.lfl HITRITt ~T DliiUitnT DH OMR ~RIAIHI nnt ~tAloo~ ~-O pH 2.5 ~--w PH 3.25 O-el pH C,O c~ pH I,fS PERaNTA[iE YELD NNH ,,~ pH 5.5 60~ o ? b a 16 18 21 11 GAYS REAC'IK~ LlnE IHRSJ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIG, L t. ZDO NITROSATION OF 0,0002M ANA'TABINE \JITH 0.01M NITRITE AT OIFFERENT pH OVER OIFFERENT'IIME PER1O05 PER[EN'TADE YIELD NAtB 13cl 100~ d O ~Q pH 1,5 n --~ pH 3,25 P-C1 pH, k0 PH 1,75 t-+ pH 5.5 a ~ a 12 16 20 SC 8 N DAYS 5, REA[tlON TIME 0105,1 O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIG, 5 t. 200 WITROSONORWIOTM~ ~NO WM( [ON[~ITAATMN VERSUS SDLUTION pH NI'TRI)SAHINE ~NCWTRATION Icld g'l 6,0 ~d NN11 Icr NNK NNN NNK O o D i j 6 ; 1 II a PH Q Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 T, 200 FHi.b NIIROIII Oi IPPW Il[OnL ~TH OAll liRT~ i\T IIIIRAtWT PH iOR (OLYI NITROSAF1IIE COIIENtRAtlON IW 'm'l 0,00 7,00 b,DO 5.00 4,00 3.00 2,00 1,00 I 3 " 5 1 1 0 "o pH N o o N o Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111