826 



GUUCURONIOeS OF NCOTING AND (TS METASOUTeS 



193 



nitCT cenlnfiigc nib« (Hiir.iii Imtrumfni Co, Wofaum, MA) ««d fofo*- 

 riKoTxl by ccairi/\icui<iT< uadcroit urae coaoiilani. Tea ralcrotlicn or 

 '.hi5 rillritc were lnj<ctcd In duplkaie ooto (he LC/MS lyitos. 

 i.C7A/i'. Th« tlxrmosnmy LC/MS method used ii i iBndific«tioo ol 



M rcpnrUd m«tii<kl, w4w3ra tirin* il i(\)«cl*d dif^cliy onto • poifencr 



lulrtini odunn for sqjintiaa of the mcuboUta (I >). In the modin- 

 cailon, tlK wjtcr/metlunol gridlefU bejltu »iih lOOX wiie/ tnd loa to 

 3(Wl metiunol a 3.1 rain. 4S% methanol it J min. ir.d firt« tt>«th«««il 

 jt i miA. PocioalUfnn Addition of 0.1 M Aaun.>niufn acciAle bufTec m 

 •cc<nI^p;uhc<i ••in) ■ wato'/mcDvuiot "rcrwwd-giaOlcni" lu milch tnn 

 iir the anilytlcil colunui. Tlli pnovidc] I coniifleot compoiiiion of 

 water, mcthiooi. UKl amittonium tocuu (a Uie Ihemiaipray iotafUE. 

 Th£ fevenad-<iraili«nt tcchni^ua produooa a mor* coa«i*(«nt bajdioc 

 aad invirarcd icjTrodiKalnlity oTropoiue (br (h: varioai tatsy\t% (i). 

 ions monltorct] ror quantlUUcia nere m/t 163 (nicotlat iiid deitiethyl- 

 CJOtiiiiBt), 177 (oodtaae). 179 (ruoitincl'.W-oiiiS!), ItO (i<,<otii\ln« 

 iat^rnal ctaadard), a»d 1 91 (ootinJno-^-oKHla and (rciiu-3 '-hydraxyoQtin- 

 inc), with a dwell time cfO.U HC nir actl lOIL 



Detmnination of Conjitsatu. Ift order to aaifltify elocuronide con- 

 jiUUlei in tmokcn' orinc urine mmpta were mxtci with ^^uouronl- 

 daao ( 1 4). ITrina tampkt were Tnt adjuttai tc pH 7.0 by addldOQ of 4 

 K NjOH nod exTf«t«d twict wWi eqatl volurocj of cnethyleoc cfaloridf 

 (o remove at mudi of th£ free metzbolhes xs ooiabte. AiDRuuunm 

 tixtats buflber (0 J M) w« addad ta • Sral eoooentraLoa of OOi M 

 aoeui«. Tbe luiiptc wai adjostcd to pH 3,0, fdlil 4 N HQ. Fof cacll 

 umpic, trlpticalc 3 mi eliquou were lEmovcd tnd 3.i50 U fi-ilucurooi- 

 dax were idded (o eich. Somole* «<re iocuhued Car 24 hr ii 37'C, A 

 cemplc of th« 4xmoud uricc wm4 incubated under idcntjcai COOidilloai 

 without tlw addMoa of eniyme co rertiy ttui relezx oribe nciabolites 

 wu dtM to tbe eiuytnc Tlu imounl of free cietabolitea in liir cazymc* 

 lieattd taispla wu avengad for (ha tripUsalai. TIm amount of free 

 maabolilaa ia tbo control aanple tm isbiracicd ftom tills iven^ (0 

 dctcnnloe uie unaant of eaeb metiboUte that exUted u • gltJcuionide 

 coajuftUc. 



Sakjactt. Elavao hMliliy laaU voluntaen, i<«d 29 to 47 )rcan and 

 waehios ISO z 24 (t> (mam ± iX>) wtio tiul vnokrd 12 to (0 Ctteroi 

 ctginnet per cax for it leaa 4 ytxa. were fluilied Vidds for ilieir unul 

 dffuette brxixl] ivcngcd \.CLi ±i i.Q (m«an i: SO) mg of *t«r' aod 0.6 

 ± OJ m^ of oicouaa par cj^sireac aa detcnnic^ by ttzndanf FTC^ 

 metixMologles (15). Smokett vrtrc uksd to tmote tbdr usual brmdi u 

 they would aarmilly 4o ortr a 24-hr peiiod. Thsy were ufccd to reacird 

 the exact nnmbae ot oifareftM Kookad duria|t this period. Tbe 24-br 

 uiiiw aunptea i«m cnUoelcd In Bertie IxsoKs «ad nored »t 0'-5X: 

 t)c<wtta cooeolon. Total 244r urioc voiome *ad urine pH woe meu- 



'Aocmumno toM in: FTC, Fcderd Trada Ccnvxlntxx NlST, Nadonii 

 iniatua of 3tanaam and Tacrrolaay. 



orcd immcdiaicj) aftor collccuoo. Crciilnlne was detcraUnod (roceyth 

 Laboratory Medicine, Wiaiion-Sakm, NCX and all nluei TeQ wtthia tbe 

 oormil nivee of 0.6 to 2.8 t/24 hr. The amount of nia'wiac cadi amotar 

 itMort>«d wai ratimatAd by multiplytnj the number of d^xrctta tmokod 

 tiT 0>a 7i<ld or ntoottnc (mlllienmt (icr cl(arctlr), .u nxctdJy dcxcnntned 

 (Of out C^arUtc iKJnd hy the FTC m"hod (1 S I The atnouol of atnortjod 

 nteotiac exarted in urine ai sKtalulit^ ww cilcuUiMl by multipljHaa 

 t^ total Aoi^wnt mea«ar«d Tor a^oh metabolite by the ratio of (he 

 molccvdar weigtro Of olcoUnc to the irtutxxiie. 



vtndatioa (or CMlahw. llic method wa^ nUditui durint this jutty 

 foe COlinijte Oliltt NlST Reference Mufrvil «444, "CoOnine in Fraaaa- 

 Dried Urma' (16). Itefactooe Material 8444 aa^Xnra cotimoc m udae 

 at tw^ l<rrct>; appioxlmucly X tod 300 Oj/mL The Ueh itandixd 1:11 

 in the lower part of our calibrated nut Triolioite moasu/emenu inade 

 on thu (tiatcTiil cave a value of 445 ± 10 nrjvA, whicti i< -tfitittn IOCS of 

 Oi« NlfT noammtediai -rauc of 4!l] t*/- 10 ng/inl. 



lUnItt 



Trcxtment of urine umplee. which had been extncted with 

 mdhyknc cblorkk, wttb ;>-glucuranidase tnt^rcAied th; coacen- 

 iration df nicotine, cotinine. tnd .•rjTuJ'.liydimyeotinioe (fig. 

 I). Tbe combined selected ion chromslosram in lig. \A b thai 

 Of unirtaied uiliK tnnected from an individual smokEr. Tbe 



(raiu-} '.^ydroxycotioine, ootinine, and Dicolinc peaks ore dearlj' 

 diaocmiWe. The ctiromatogram in Og. ifl b ttie amc urine 

 nmpl; foUowing duplicate eztractiona at pH 7 aith metbyleiu 

 chlorid*. Mote than 90% of the ccttninc aod tiJootllK were 

 removed, but lesj than half of the £ra«-3'-hydroxycatintnc vrai 

 eztncted bee«ii«« of iU hj^lrophDicity. Tbe efaiomatogriin in (ig. 

 1 CshoiKi the same exoacted urioc utQple following ^-£luctiroa- 

 idue tieattoeat tx Dteviouily dociibed. Cooueatnlion of firee 

 nicodns, ootimiie, arid tnni-'i '-hydnxtycQtininc in die ezoaoed 

 nrine ioaeaied b? ^-dtictiroaidaM tresUnenc witli cotimce 

 tJiavring tbe gnateit {xtcmw. ITemsthylootiiiiae and ntcotino- 

 ;v -oxide, as wen as ottier nicotine metabolite) wtndi were aol 

 detected in stnoloerc' uriae. did ixit v?^tu at glocuroaide oon- 

 jugatea within tfac detection limits of the metiiod (20 to 200 i]g/ 

 ml). lactibatioiu of smolcers' urine uoder idcnticel conditk»u 

 vuith i2eat4ikActivat«d 9-sloe<jronidiac or ^-elucaimidax in tbe 

 preseDce of laccforolactoik: yieided no additionai nicouae, co- 

 tinine. Of (nntT-J'-hytlrozycotiiune. 



Attcmpta to ideatify the gluaironlde (»nju^te$ la the mas 

 r^ocea that wonJd be eitsected were uniuoceofuL Analyiia of a 

 model gluouronide, «}-aaphthy{$iucui<otude, by thennoipny VC/ 



L ««KIJ.I^WE 



S ii:f*CT1U^fie*, 



C Ftttrt^^.tf^iiounYtf 



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Ca^i^i^^J fciocicd Ion ihnjmnrurrrjfnr 0/ ntcoflne (NfCj. ctXM/flc (t.(/T;. drtrf (rais-S' hy(lia,:ycain:rc i]HO for (4) */hDle unrv. (B) 

 'Txiramd U'inr ami (C) i*):(rnctfri unnc (olki^^irjf crni'^c -ruaimfltti 



