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1 A Novel Method for the Isolation and ~. . a 1 ) ! '.Quantitative Analysis of Nicotine and Cotinine in Biological Fluids/* ~' Laboratory, Oak Ridge, Tennessee 37830 \ NOTICE . This m=t:r.al may bs Ixotcatad Ly e.plri=ht law (TiiIP 17 U.S. Code):_ ~~ M.P. Maskarinec, R.W. Harvey, and J.E. Caton,Analytical Chemistry Division, Oak Ridgt: National Abstract A rapid quantttatlve method has be.n developed for the esllmatton of nicotine and eottntn. In blood and urine. Isots- tton Is aeeom;ltshed by adsorptlon of the alkaloids on Amber- tite XAO.2 ristn and subsequent Nutlon with chtorotorm/ methanol. No sotvent extraction or further purUieaUoa Is requtred. The flnal determinatton Is mad. by high perform• ance liquid chromatography using UV drateetlon. Absolute recovery of nicottne was at least tlOX In aU samples. The accuracy of the method Is-ostlmated to be = 5% on standard addition measurements. Dal.ctton limits of 2 ng/ml urine may beTouttnely obtatned. _ Introduction The adsorptior•.; distribution, and metabolism of nicotine have been investigated in a variety of animal species (I-3), as have the pharmxological_a_ctions of this tobacco alkaloid (4). However, in spite of the widespread use of tobacco products, there exists no simrle quantitative method for the detcrmina- tien of nicotine and cotinine, its major metabolite, in body fluids. Previous methods for nicotitie and cotinine in urine and su am ttave relied mainly on solvent partition schemes for isolation and purifiatioit, while the final determination is made by gas chromatography, using conventional ilame iotti- zation detection (S-7), nitrogen-sclectire detection (8,9) or. deetron capture detection (10). While the sensitivity of these methods has been entirely;sufficient, the isolation schemes have been generally non-reproducible and tedious. In addition; total analysis times have been rather long. The advent of High-Performance Liquid Chromatography (HPLC) as a rapid, reliabie analytical tool has led to the development of a method for the estimation of nicotine and cotinine by HPLC using UV detection (11). This method also employed solvent partition for isolation. Earlic* work on the screening of urine for basic Q'rugs of abuse by adsorption onto Amberlite XAD-2 (12) led us to `.nvestigate the practicality of •Rescarch sponsored by the Natinnat t'ancer lustitute under lnter. agency A=reemenCNtfl :NC1) 40-+tLS-74 under l7ninn Carbide Cnrp- oration contraa W-740S•:zg-2b with the L'.=. Departmennot Energy. Send reprint requests to: M.P. Maskarinec. Aralyticat Chemistry Division. Oak Ridge National Laboratory. P.O. boa ::. Oak Ridge. TN 37830. Z - 124•JULY/AUGUST 1975 this approach to the rapid quantitative isolation of nicotine and its metabolites from body fluids, while relying on HPLC for the final determination. . Materials and Methods . ~ Amberlite XAD-2 was obtained in prefillcd; precleaned columns (Isolab, Inc., Akron, OH). "C-nicotinc was obtained frorn Amersham Ine.. Arlington faeights, IL- "C-cotinine (13) and nictxine-I-N.oxide (14) were synthesized according to established procedures. Nornicotinc was obtained from Pfeliz & Bauer. Flushing, NY. A Packard Tri-Carb Model C2425 Liquid Scintillation Counter was used for recovery experiments. Liquid chromatography was done using a\Vaters Model ALC-202 isoeratic liquid " chromatograph equipped with a Rhcodyne sample injector and a UV absorbance detector (254 nm). Columns were 25 cm by 4.6 mm i4 Zorbax-Sil (Dupont). All solvents were spectro-quaGty or better. Urine (20 ml) was adjusted to pH 9 with saturated NH,Ci buffer (pH 10). Plasma (1-5 ml) was diluted to )0 ml using a )0°fs saturated buffer. "C-nicotinc (0.27 pCi) was added to each sample to measure recovery. Ten ml of the 101re buffer solution was allowed to flow through the XAD-2 column for equilibration. The sample was then passed through the column at a flow rate of approximately 2.5 mI/min. After a second wash with 10S's NH4C1 buffer (15 ml), the alkaloids were eluted with I ml acetone, foilowed by 20 ml methanol/chloro- form (I:3). The eluate was biphasic. The upper (methanol) layer was discarded, and the lower (ch'oroform) layer gently evaporated to dryness after removal of a SO-Nt aliquot for liquid scintillation counting. The residue was redissolved in 200 pl dioxanersopropanol/NI+M'40Ii (50:3:0.4), x"hich was alxo used as the mobile phase. A 20-p1 aliquot was applied to the liquid chromatographic system and cluted at a Iflow rate of 1.0 ml/mia. Results and Discussion .. Six replicate analyses of urine samples indieatcd a mean recovery (after evaporation) of 8S.7r,'s for nicotine and 86.4% for cotinine, with standard deviations of 4.6?'# and 0.93°.r., respectiveiy,u shown in Table 1. Adsor;.tion of both nicotine JOURNAL OF ANALYTICAL TOXICOLOGY-VOL. 2

o-•.-a•c n• 1ofo Table I. Recovery ot "C-Nicotln• and "C-Cotintne from ' . Urine g.l r.l . . Nicotine Cotinine ® I Detet7ninations 6 6 / 7. Unretained 03.69 = 5.10 01.14 10.28 Y I i , 1 % In Methanol 04.20 s 0.47 03.87 t 0.29 9. Chloroform 86.50 s 0.48 86.80 s 0.49 . !. Recovered After 85.70 = 4.80 86.40 s 0.98 1 ) ) ? ) Evaporation • Units are 9r =1 Standard Deviation. and cotinine on the XAD-2 was virtually quantitative. While the alkaloids partitioned between chloroform and methanol approximately 95:5, the urinary pigments (which would be expected to interfere in the final determination) were parti- tioned into the methanol phase. The accuracy or the method was determined by standard addition of nicotine to a urine sample in which no nicotine was detected (<2 ng/mI). •The results are shown in Figure 1. Over the range 10 ng/ml - 10 jtg(ata all determinations were within SRo or the correct value. .Evpt at a level of 10 pg/mb. no significant breakthrough of nicotine on the XAD-2 columns was observed. STANDRRD EIDDITIDN OF NICOTINE TO URINE Figure 1. Results of standard addition of nicotine to urine. A comparison of the liquid chromatograms of the urinary extracts of a smoker and non-smoker is shown inFigure 2. In addition to the.nicotine and cotinine peaks. nornicotine was also tentatively identified (by retention time comparison with an authentic standard) in the smoker's urine. The levels in this individual (a heavy smoker of non-filter cigarettes) were 247 ng/ml nicotine and 521 nj/mI cotinine (after recovcry eorrea tion). The nicotine level in the non-smoker was 7 ng/ml while 0 r 7 iT''-1' T l"! I t t• i~ t- t T t • t-......... o a . i 6 •0 oa N $60 2 4 . 0 to .2 0. 14 Iw(1r1 TT~Z'Try-TTT1 T`1 t-TT:Z.'• . . t . . . . . . 0 2 6 . 6 1 40 Q 44 06 0 2 4 6 • a •0 ot 04 K tlYr10M. WlNK M) Figure 2. Comparison of liquid chromalograms of the alkaloids from the urine of a smoker (a) and a non-smoker(b). Conditions: column-Zorbax-Sil (Dupont) 25-cm r 4.6-mm i.d.;' flow rat. • 1.0 mi/min; detector - UV (254 •nm); sensitivity - .16 AUFS.. the cotinine level was below the detection limit of the method (2 ng/ml). The extracts were further analyzed by glass capillary gas • chromatography on a 50-m x 0.26-mm i.d. OV-10] column using simultaneous non-selective (FID) and nilrogen-selcctive (NPFID) detection (Perkin-Elmer Model 3920 Gas Chroma- tograph). The results were within ST's of those obtained by HPLC. To further test the validity of the method, a series or urine samples from smoking baboons were analyzed (blind) for nicotine and cotinine,and'the results were in close agreement (p <.05) with those obtained in a separate laboratory by gas chromatography-mass spectrometry (1 S). While urinary levels of nicotine and its metabolites are important in screening for cigarette smoking and in determin- ing total smoke dose, it was of interest to examine the possibility of determining plasma nicotine levels by this method. Recovery of plasma nicotine was found to be similar to the urinary recovery. A typical liquid chroinatogram ob- tained from a S-rni plasma sample of a dog cxposed to cigarette smoke is shown in Figure 3. The recovery corrected nicotine level in this case was found to be 112 ng/ml. .~- twcie~t e ~ r ar.. . ' ddertn ~ti ~i~~t~ Conclusions A method has been developed which allows tht rapid, quantitative determination of nicotine and its metabolites in physiological fluids. The method has been applied to the. measurement of urinary levels of nicotine and its metabolites in humans and smoking baboons and also to the study of plasma nicotine levels in dogs exposed to cigarette smoke. The: simplicity and rapidity of the method allows up to 40 samples/day to be conveniently run by one technician. Therefore, the method should find wide applicability in I kbo- ratories involved in nicotine screening on a routine basi:. In ,oases where radioactive tracers are not readily available, a suitable internal standard may be added for recovery measure. ment: One such compound is desmcthylimipraminc (11). ~ 1 JCURNALOF ANALYTICAL TOXICOLOGY-VOL. 2 JULY/AUGUST 1975-125

: ~ • • .... t a ) I . 0 2 4 6 6 10 12 /4 .1i TtME to" 0 2 4 6• e /0 12 14 16 ELuTION vOLUME ta.t1 Ftgure 3. LlQuid chromatogram of nicotine Isolated from 5 ml dog plasma. Conditions: as In Figure 2. Sensitivity •.08 AUFS. Acknowledgment The authors wish to thank Dr. Patricia Buhl (Borriiton Research Lab, Temple Hills, MD) for supplying the dog plas- ma samples and Dr. Walter Rodgers (Southwest Research Institute, San Antonio, TX) for the baboon urines. Manusaipcreceived June 16, )978 1.P. Jcnner. J.W. Gorrod. and A.H. Ilcckett. Factors affecting the in vitro metabr of R-(i•)- and S-(+nicotine by 6uinca•piY . liver preparation. .,'ennbiaice. 3 (9): 563•S72 (1973). 2. P. Jenner, J.W. Gorrod. and A.H. Ueckett. Species variation in the metabofismorR-(+)-and S•H- nieotine bya-C- and N-oaida- tion in vitro. Xenobiotica 3(9): S73-580 (1973). 3. P. Jcnner and J.W. Gorrod. Comparative in vitro liepatic metabo- lism of some tertiary N•methyl tobacco alkaloids in various species. Res. Coerar. CFere. Path. and PhereraroL 6 (3): E29-b43 (1973). 4. M.S.G. Clark. M.J. Rand. and S. Vanov. Comparison of • pharmacological activity of nicotine and related alkaloids occur• _ ing in cigarette smoke.Arrlk. lnt. Pbsrnrocady>,.aTAerap. 156 (2): 363-379 (1965). S. A.H. Beckett and EJ. Triggs. Determination of nicotine and its- . mctaboiite. cotinine. ia nrine by gas chromatography. Nat,.re. 211: 14t5•1417 f 1966). 6. H. Schievelbein and K. Grundke. Gas-chromatotraphische methode zur bestimmung von nicotine in blut and gea•eben. Z Awal. Clatm. 237:1-b(196a). - 7. !.E Burro.s. PJ. Corp. G.C. Jackson. and B.FJ. Page. The determination of nicotine in human blood by gas-liquid chroma• tography. Awofyst 96: 81-8/(1971). g. P.F. Isaac and MJ. Rand. Cigarette smoking and•plasma levels of nicntine. Natyrc 736: 306-310 (1972). 9. C. Dumas, R. Badre. A. Viala. J: P.Cano and R. Guillerm. Alicro- methode de determination de la nicotine et de Is continine dans Ie sang et 1'urine par chromato`nphie en phase gazeuse. Resultats obteaus ehez divers fumeurs. Ear. J. TozieoL 8(5): 280•286 (1975). 10. L Nedakantan and H.B. Kostenbauder. Electron capture derivative for determination of nicotine in sub•picomole quan- tities. AnoL Chem. 46 (3): 452-454 (1974). 11. lan D. Wat.son. Rapid analysis of nicotine and cotinine in the.• urine of smokers by high-performance liquid chromatography. l. Chroareropr. 143:203-206 (1977). 12. M.P. Kullberg and C.W. Gorodctsky. Studies on the uie of XAD-2 resin for detection of abused drugs in urine. CGn. CAe,n. 20 (2): 177.183 (1974). 13. E.C. Taylor and N.E. Borcr. Pyridine-l•oxides. IV. Nicotine•1- oside. Nlcotine•1'oside. and N'rcotine•1.1'•dioaide.J. Org. t'ieni. 24: 275•277 (1959). • 14. E.R. Bowman arid H. McKennis. Jr. (-)•Cotinine. Biacheer. PreP. 10: 36•39 (1959). 15. W. Rogers: Personal Communication (1978). . 128•JULY/AUGUST 1978 JOURNAL OF ANALYTICAL TOXICOLOGY•YOL. 2