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"•flolc^iT Aad £K4>«<<iito«lAt T1><rapw«Mai 



Vol. 30, No 3 

 rrmrd«il.i,S.J, 



EVIDENCE FOR URINARY EXCRETION OF GLUCURONIDE CONJUGATES OF NICOTINE, 

 COTININE, AND r/WWS-3'-HYDROXYC0TININE IN SMOKERS 



GARY O. BYRD. KUO-MEl CHAN3. JACKIE M. OneeNE, AND J. COM OeflETWIZY 



PtiarwacoKxiy OMikir. A X KoynMt foOtcco Contfitny 



(fleceivcri JUr tS. 19911 ocrapMid OctoOar 22.1991) 



ABSTflftCT: 



Urtnarr nlo«*n» mf6«lte aulpul »<•• pn>fl«0 (or 11 Mnokara Mho 

 •(n*4<*4 ttt«b r*g«lar eljarvM* bnn^a *^ MJrum. Th<rm**^«y Hquld 

 c>v<Mn*ts9r«phy/ma«< «p»c1ro<n«bv "«« u**^ *" in«jii»»f Hle«iln» 

 and aiflM RMtabolltca. hdutfng oluevnxiitlA r.anJuoi«*< al nlc<]4in«, 

 CQtlnln*, and Crant-S'-hyctvxycotinlx* Itat wme dalamilned Indu 

 focty uiing ainrnM li)rdro*r<<L TTist* imuKi ««.•• ut*d la ostimata 

 an ittrag*, ilMdy-fltats ooneentritlon In a 24-tir urine •amota 

 during «d Wftuni amoklng am) io iiteia Vittrtndirklval variaWMy in 

 ttM ticrtHon of thaaa mataboWw Tha varfeUMy In ab>«Ju(« amouM 

 among tti» nh« antlylM rangMl fmn 3S to 70% tor thsie invokore. 

 Tlw glucurwilde con^ugcMa constitutad an anrag* o( 29% ot aX 



urinary nmaoouM* monitored In Mi atudr. b'jnt-.J'-rtydroiycDiMna 

 In ttn ti«a (o«m oomoonca tne liro«<i aIngN mttiborita In anvotcat*' 

 uiina, witti an anrag* ol 33% or tne (oUL Tn* tixnt at nkotVia 

 m«UboltM detenninad hare mn nqr cKns la Ui« r^eoaral Trade 

 Commlnton jrMda c* Naa^nt <or iha favil numtnr o( cigarsnet 

 tmo4ad by «M4* «w> ) a«t« 4u><f«3 lh» utlrx eoltoatfvti Inlwntitf. TfWM 

 r«euHt tndlceaa dMi ■ imrgt ptopartisn ol «»• niaadrw abaorbod wM« 

 tmoklns can b« accouaed lor ■■ urinary melifcott** af n4o««lne, 

 including |>ucuronld« conjuotiea o4 r.loatlne, eaUnkxt, and (nne-3'. 

 hrdroiyco«n«n«. 



Alifaough oicotiac dispasitioa in smokefi t)u be«n studied for 

 deoides with a aamber of mctaboUto reported ( I X t^e atnlity to 

 account for all of ifaa nicotine ibmrbocl by smokcis hai been 

 diiliaih becauce of a iack of undcratnttdins of what Ibc major 

 mctabolitei ace and how they vxry from one individual to the 

 next. SlJCh an utKterstandiag ic nf particular iroporttso* for 

 bioasnys (hat atiantify exDOfuic to xcnobtoiics like cicotine <2, 

 3). Pivgres i In anahflical inetbodolojy hai made it patsible to 

 dctennine in biological fluids metabolites of nicotine tiiat have 

 a wide range of polarity. The nelativdy nwcm detentiination of 

 a major tnetabolite, fta«>3'-h>ilroxycotinir>e (4, 5), aixJ die 

 reports of glueuiooide coojupita of oicotioe, cstioiae, and iraru- 

 3'-hydroxycotinine (6-9) have ag lificamly improved the poten- 

 tial (be quantlfjicg nicotine absorptioa in Sfflokert. 



Many analytical taettods JUCB as GC (10), GC/MS (11), 

 HPLC (I), and cnzyrae-linlted IraniunosortJeot aissyj (12) havt: 

 bead utititod to nooitor the distrlbutloa of tiicotJne and its 

 metaboUtea in biological fluids. Hf LC with U V and ndlomctiic 

 deteetiOD (!) U putioolariy usofol for d c l cf mimng mosi bxlaiice 

 from administered csdioiabeled nicolioe. However, :t ia not 

 practical to introdtiee radiolabeled nicotine into unoken wtio 

 are absortang nicotine u'bile tmolang their regular brand* ad 

 tibitiwi. 



We have dcmonstrited the uulilv of a thermoa>niy LC/MS 

 method for determining nicotine and many cf it'; metabolitcs-in 

 the nrine of human smokers, including the major metabolites 

 »ra«-3'-hydroxyootinine and cotinine ( 1 3) and glucuronide con- 

 jtigate* of tilootiac, tnBrLf-3'-hydroxycodnine, and cotinine (8). 

 The major advaniagca of ihermospray LC/MS are its ability to 

 determine all major iud several iiiinoi nicolioe mctabolilcs in a 

 aogl* chfomatogrAphic run Without dtrivariiatifm techniques. 



S«fKj rWxVil rmtia»ml» u, Or dtrj n 9,™ rnainnooiu.)y otrlaon, R J 

 •lorroui Torvaoco CotKDanv. Bu»ono WO-} wi"tv»> .Sjii.il kg ?"oe 



This study examines the amount and distiibutioa of urinary 

 nlooiinc mKaboUUa among i J smokers wbo imoked their rsj- 

 ular cigarette hrands j4 liiltum. hficoxioe and eight metabolites 

 are moflitoicd using thennospray LC/MS, including traiU'V- 

 hydrorycDtiniix; eotJoiat, alcotlxie-/V.oxVle, cotiniiie-^'-ojcide, 

 demethylcatiAiac, and the iliree gluuiironlde conjtigstes de- 

 torib«d previotoiy. By lookjoc it these mqjor metabOll(£S in 24- 

 hr urine nmplw of 1 1 anioken, a survey of nicotine (lispositlon 

 um be coottructad to define an average, rteady-itste comxncra- 

 tion doting ad libitam nnoking and aeoeM iotcrindividual vati- 

 atality in the excrritinn of thece metaboUtec 



Materials tad MellMdK 



Mafnialt a;-)N!cotiBe and (-)eatinioe were purchased conu^e^ 

 ciiUy (Slfeu Chemical Cs, St. Louii MO). Donetiiylootiinae <ta( 

 Jrnthaiird Ox our tjbontory b> I3f. George Neuralh (Inrtit^t fiir 

 Bloph«rtBai«nti*sh« MikroaAalfUk, Himtruig, PR.O). (mu-J'-Hydltrt- 

 ytotimae, nieatirK-r~V-euda, xai wenuc-V-owdc were ayntfaesUed 

 for our laboratory by Dr. Peter Crooks (UivivutUy oflCcoiuefcy, L«iae- 

 ton). AB samptet vrejt a.wsjed for parity hy GC/MS. t«ceot for niootine. 

 //'•cxide (whicti is thenruuiy casiaMe) ud fouad to be at least 99J% 

 ciirc ort)ettor based on wtil ion oitrtoL Thcnioo(iot-l '•A'.oiicle purity 

 wu iiaKsxd br LC/MS aod Ibund lo be 97.4%. Caiuune-meth/Wi was 

 purcluaarl avmaioidtlly (CMsbndec l«an>f>e LabontDry, Camht1<]|e, 

 MA) ind determiood lo be 98.4 jiom« (l«ii<ritia> by GC/MS. 0- 

 Glucuronidase from bovine liver (424.000 Fishimui uniu/t (olid) wu 

 purchased comniercjllr (Sijnu). HrtX>^r«dc methanol, inelhyfcne 

 chloride (Amencsui Buriick & Jsdaoo. Miulccioo, Ml), and hith-()urity 

 water (Mtni Q Flltrjnoa SjrjKn, MiUipoie Corponitioo, IledfaroL, M\) 

 «wc uKd *> lolTcnu. Rcogcm tf*^^ ttnmoniu.'n iccaic (Mallindcrodl, 

 Puif, KY) w«i uwJ to pnpnrc the b^iircr loluiJons. 



Methods Sample P'tfiata/ion. S^mrtle pcepanAttoa wu by s sinipic 

 metluxl tint bis been described prrvioujlr (S, I H Urloe umcilai <••«> 

 ihawrd just prot lu use. Or/e-raUliliia iligucu wttt Irsnsfctrod to a 

 plajlc iiibe lo *hich <^u kl.VsI ir.tmal flundsrd (</|-cotinine in mctli- 

 aiiol) 11 appruilnijifir 2,000 t\fjn\ fli: simple wis Jcrilnfugw; it 

 l6.5fA-ro<-)f"in(Ep;)cn«iori'MM3;i.ls 3. eprrtxlorf CirnhK. HimhurR. 

 GtrtranvX :qO '"» .-nl of i»»e 5\ipcrr.«'.*ni .vis .-rrriyvcd tr> 1 I? *iM 



19? 



