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Journal of Chromatograph/¢ S~ience. Vot 28, Aprit 1990 The Determination of Nicotine and Cotinine by Ion Pair Reversed-Phase Chromatography Laura J. Lewis', John D. Lamb, Delbert J. Eatough, Lee D. Hansen, and Edwin A. Lewis Department ot Chemist~, Brigham Young University, Provo, Utah 84602 An ion chromatographic method is described for the determination of nicotine and ¢otlnins in aqueous solutions. This mthod Is humid on a type of reversed-phase ¢h~mtatography involving ion pair formation of protonated nicotine, ¢otlnine, pyddine, and pyrtdine derivatives. Oetactlon i~ sccompli~hed by measuring the UV absorption ~t 262 rim, Det~tio~ limits for nicotine and ¢etlnine are 8 ng/mL and := ng/mL, respectively. Analyses of environmental sample~ m~d spiked anvironments| samples by both this ion chromatographic method and a previously reported gas ~:ttcomstogrephi¢ method have been used to demonstrate the accuracy and predslon of this technique. The results ~ the Srmlyses ol both sots of samples by the two methods Me in excellent agreement with a linear correlation eoeflleiont of 0.97. Introduction Current rescerch into the effcc= of passive exposure to tobac- co smoke has led to an increased interest in methods for the determination of nicotine and nicotine metabolitcs in both biological and environmental samples. Sensitive methods for the determination of nicotine (I-methyl-2-(3-pyridyl)pyrrolidine) and ¢otinine (l-methyt-$-~3-pyridyI)-Z-pyrro[idone) in biological fluids and environmental sample extracts have been reported which employ a variety of techniques including gas chromatog- raphy (GC) 0,2), radioimmunoassay (RIA) (3), and high-per- formance liquid chromatography (HPLC) (4). Methods based on ~as chromatography sccm to be the most widely used at present. Samples in which nicotine is to be determined are often most easily obtained as aqueous solutions. For example, in studies where nicotine is used as a tracer of environmental tobacco smoke (ET$), the samples are sometimes water extracts of acid- coated filters or dcnuders that have been used to sample indoor air (5). Nicotine and its principal metabolite, cotinine (6), are considered reasonable biological markers for ETS, and in this regard, both nicotine and cotinine have been determined in blood (7), urine (8), and saliva (9). In each of these biological samples, the alkaloids are present in an aqueous solution. The use of GC techniques must necessarily involve steps in which the nicotine or cotinine is extracted into an organic solvent with the possibility of some loss. For this reason, ion chromatographic (IC) analysis offers an auractive alternative for a more direct determination of these compounds in aqueous solution. The aqueous sample need merely be filtered or centrifuged in some cases, and inject~ directly into the IC for simultaneous detection of nicotine and cotinine. Unlike the familiar reversed-phase HPLC, in this method, nicotine and its analogs are protonated in the eluent stream and separated as ion pairs on a neutral column in a reversed-phase mode. Sensitivity of any method for the determination of these com- pounds in environmental samples is crucial. The expected con- centrations of nicotine and cotinine in the urine of smokers are at or below 100 ng/mL { 10). Plasma, again in smokers, usually contains nicotine at or below 50 ng/mL and cotinine at or below 16 ng/mL (!1). For nonsmokers exposed td ETS, nicotine concentrations in saliva are at or below i[00 ng/mL white cotinine in urine is at or below 10 ng/mL (12). Nicotine and cotinine are present in filter extracts of ETS samples at levels of approximately 1000 ng/mL (5). The ion chromatographic method described here has adequate sensitivity to accurately analyze aqueous solutions for both nicotine and cotinine at most of these concentration levels with a required sample volume of only 0.10 mL. Experimental Reagents. ( - )-Nicotine was purchased from Eastman Kodak Chemicals. Hexancsulfoni¢ acid was obtained from Dioncx Corp. ( - )-Cotinine, 4-ethcnylpyridine, and pyridine were ob- tained from Aldrich Chemicals, and HPLC-grade acetonitril¢ was purchased from J.T. Baker Chemicals. Deionizcd water was obtained by passing distilled water through a Milli-Q water purification system (Millipore Corp.). Apparatus. The analyses were performed on a Dionex Model 2000i/SP ion chromatograph equipped with an isocratic pump. The detector was either a Shimadzu SPD-6AV UV-Vis detector or a Dionex Detect/Ion UV-Vis detector. The recorder used was a Cole-Parmer model 8373-20 with the attenuation set at I V 200 Re~1~(ph~-~/i~)~it~a~nten~thi~rna~i~hib~i~m~.G~6~G9G w~ oi th th sli th~ 606000696 PRODUCED FROM B&W WEB SITE full scale and the chart speed set at 0.~ cm/min. The separa- tion was performed on two Dionex Mobile Phase Ion Chro- matngraphy guard columns in series (6 cm x 0.5 cm). The eluent consisted of 5 mM hexanesulfonic acid in 5% (v/v) acetonitrile-water. The flow rate was 1.0 mL/min. The detector wavelength was set at 262 nm (a 254-nm filter was used in the case of the Dionex detector). All analyses were performed at room temperature. Procedure. Calibration standards for both nicotine and cotinine were prepared at different concentrations { 162 ng/mL- 162,000 ng/mL) by dilution of the respective 162,000-ng/mL concentrated stock solutions into a total volume of 1130 mL The concentrated stock solutions were prepared by dissolving the appropriate weighed amounts of nicotine or cotinine into a total volume of 500 mL. All of the solutions were prepared with deionized water. Two types of environmental tobacco smoke samples were analyzed. The first were ~.0-mL aqueous extracts of benzene* sulfonic acid coated denuders that had been used to collect sampl~ of the basic gas phase compounds produced by smoking cigarettes in an environmental chamber at Yale University (5,13). The second were extracts of benzenestdfonic acid coated dcnuders used to collect samples of nicotine and cotinine from a 30-m~ Teflon bag environmental chamber at Brigham Young University ($). The BYU denuder extracts were spiked by adding small volumes of standard solutions. For comparison purposes, the Yale samples were also analyzed by a GC technique that involved reextraction of 2.0 mL of the aqueous solution into 4.0 mL of methylene chloride (14). Results and Discussion Originally, a conductivity cell in conjunction with a membrane suppressor was used as the detection system. However, excessive noise, unexplained interferences, and non-Gaussian peaks led to the change to the UV-Vis detector. The use of a UV-Vis detector employing a monochromator (Shimadzu 6AV) rather than a filter wheel allowed better detection of both nicotine and cotinine, as the UV absorbance of the eluting compounds could be monitored at a wavelength at the absorption maximum (262 nm) for both compounds. Also, a chart recorder was used rather than an integrator since peak height was adequate for deter- mination of the concentration in the desired concentration range. The separation, i.e., baseline resolution, as well as peak symmetry is illustrated by a chromatogram for a mixture con- mining 1.0 mM concentrations of cotinine, pyridine, 4-ethenyl- pyridine, and nicotine, shown in Figure 1. Calibration curves for nicotine and cotinine are remarkably similar. The equations relating peak height to concentration of nicotine and cotinine are given by equations I and 2, respectively. Peak Hr. = 0.0442 + 17.731 (nic] Eq I Peak Ht. = -0.0223 + 18.168 [cotl Eq 2 where the peak height is in cm (for a detector sensitivity setting of 2.0 absorption units) and the concentration is in raM. Note that for both compounds the calibration curves do not pass through the origin but instead intercept the subordinate axis slightly below the origin. This is most probably an artifact of the fitting and results in a negligible error in the concentration determined for any sample containing nicotine or ¢otinine at concentrations near zero. Figure 2 is a UV abs~rptlon spectrum of nicotine in the IC eluent. Nicotine has a strong absorption band at 262 nm and cotinine has a strong band at 264 rim. Setting the detector at 262 nm re~dt~ in a loss of peak height for cotinine of only 0.3%. Under the chromatographic conditions, the retention times are 2 rain for cotinine and 11 min for nicotine. Since pyridine ab- sorbs strongly at 254 nm (1% peak height loss at 262 nm) and 4-ethenyipyridine at 266 nm (5% peak height loss at 262 nml, the optimum detection limits for these two compounds would not be achieved at this particular wavelength. The minimum detectable levels of nicotine and cotinine were found to be 8 ng/mL and 2 ng/mL at a signal-to-noise ratio of 2 with an in- jection volume of 0.10 mL. The reproducibility of concentration measurements using the IC technique was determined by making ten replicate measure- ments on two standard solutions, one with a concentration of 162 ng/mL and a second with a concentration of 1620 ng/mL. A RETENTION TIME (rnin) Figure 1, Chromatogram for a mixture containing (A) 1.0 mM cotinine, (g) 1,0 mM pyridine, (C) 1.0 mM 4-ethenylpyridine, and (O) 1.0 mM nicotine. 60600069? 201 606000697 PRODUCED FROM B&W WEB SITE 4 In both instances, the standard deviation of the measurement was less than 1%. TEe recovery of nicotine from spiked environmental denuder samples is illustrated in Table 1. Recoveries in excess of 100% nicotine may result from either the presence o1" an interfering contaminant or the uncertainty in preparing the lowest concen- tration standards with Eppendorf pipettes (ca. ± 2%). However, the results show that nicotine can be reliably determined in the desired concentration range. The IC method was also used for the determination of nicotine in environmental samples collected earlier in a labora- tory intercomparison study (l l). A typical chromatogram for an environmen~l sample is shown in Figure 3. Table li illustrates the correlation between [C analyses and GC analyses for seven of these samples. These data show that the IC and GC results are generally in good agreement, with the IC concentrations tending to be slightly higher than the GC concentrations. This is not an indication that either technique is more accurate than 0.4"1 ,., o.3~. o.ol- , 1~ ~. ~ ~ 0 0 0 0 0 0 ~AVELENOTH ~ ~ ~-~m ~m ~m for ~ ~) 8~.~mL n~ne ~ ~ (B) ~.0 ~mL ~ni~ ~ • ~ ¢ et~t. The ~ m~ ~ ~ ~ co#~ are ~I ~ nm ~d ~ ~cm-+m~-~ ~ mm of cotmme m B.+8 x +~ ~L~-~m~-+. Table I. Analyses of Nicotine-Spiked Environmental Denuder Solutions Mlol/hll Arna~ Tnhd n/co~ne ¢oncm~rllion added found % (ngmL) (no~nL) (.~mLi nesovo~ Amb/ent samp/e" 2430 22 23 107 852 40 42 105 8906 81 77 95 783 162 158 97 10430 1620 1609 99 Benzenesulionic aci@ 0 16 17 105 0 162 163 100 0 1620 1618 99 0 3240 3240 100 B A . 1, FIEurl 3. Chromatogram of an environmental samde collected inane 01 BYU's trailer, (A) 4-ethenylpyr~dine, (B) nicotine. 202 fiUEOOOG S 606000698 PRODUCED FROM B&W WEB SITE the other but may simply reflect differences in the calibration of the two techniques or differences in the recovery of the anuiyte in sample workup procedures. In any case, the IC and OC data are highly Linearly correlated, with a correlation fident of O.~7. Conclusions The ion pair chromatography technique described here has been used for the analysis of air samples collected in both en- v/romnental chsmhers (BYU's Teflon bag and Yaie's stainl~ steel room) and from a number of other indoor environmenu such as restaurant, bar~, and hom~ in which smoking occurr~i. None of the ~amples showed peaks that eneluted with nicotine or ¢otinine, indicating that the method is sufficiently selective sendtive fo¢ the study of environmental samples and may be useful for the analysis of biological fluids obtained from smoke¢/. However, in the case of nonsmokers passively exposed to ETS, the alkaloid concentrations in blood and urine may be too low for d/rect analysis and a concentration step would need to be employed. Acknowledgments The authors acknowledge the technical assistance provided by Brenda Sedar in making many of the IC determinations. This research was funded by the Center for Indoor Air Research through a grant to Hart Scientific Inc., Provo, Utah. Table II. Determination o! Nicotine in Environmental Samples using Ion Chromatography and Gas Chrommogmphy" 87122 ~30.0 + 2.0 259.8 :1:24,0 67123 12.5 d: 1.1 8.7 d: 1.0 87124 233.8 ,.1- 2.1 244.9:1:21.2 67125 6.8 ::t: 0,6 8.2 + 0.9 67126 814.8 ::1: 5.0 742.0 + 56.1 97127 56.6 + 4.8 42.5 :i:2.2 87128 49.6 :t: 3.5 42.1 + 2.6 References 1. S.K. I-lammotK:l, B.R Leederer, A.C. Roche, and M. Sche~ker. lectfofl and analysis of nicotine as a marke~ for anvironmentaJ ~oacco smoke. A~mo¢ Er~iron. 21:457-62 {1987). 2. G.B. OMakar and F.C Conrad. Estimation of effect of environmen- ~I ~tmocO ~noke on air qu~dy within l:mseenge~ csbin8 of com. metclad aimralt. Environ. Sc/. ~Ich. 21:994-99 (1987). 3. F.W. Henderson, R. Meeds, A.F. Reid, RC, Hu, J.L. Mumford, L Forehand, R. Burlon, J. Lewta¢ and S.K. Hammond. Serum and urine ¢oltnine as quantitative mnasums d passh~e tobacco smok~. Indoor Air '87 2:18-21 0987). 4. R.D. Badow, P.A. Thompson0 and R.B. Stone. Simultaneous re|halloo o( nfo~irm, cot~nine and five additional nicotine metab- otitne in the udne of smokers using precofumn dedvati~tion and high perkxmanca liquid chromatography. J. Chromatogr. 41g: 5. DJ. F.~, C.L. Banner, J.M. Bayona, FM. Caka, H. Tang, LJ. Lewi¢ J.O. Lamb, E.A. Lewis, ~nd L.D. Ha~se~. Sampling for gas- phase nfoo(ine in environmental Iobecco smoke with a diffusion denude¢ and n paseive sampler. In Proceedings of ~he 1987 EPA/APCA Symposium on Measurement o~ Toxic a~d Rel~ed A/r Po/~,nts. 198Z pp. 132-39. 6. M.A.H. Rumll. Estimation of smoke dosage and m(xlaltty of nonlmokem from environmental tobacco smoke. Tox. Le£ e--is (1~'). 7, N, Hong~n and M. Hongan. Gas-liquid chron~raphic data'- ruination of nicotine and cotinine in plasma. Clin. ~. 24: 5O-52 8, R.G. Wilcox, J. Hughes, and J. Roland. Verification of smoking history in patients after infarclio~ using urinary nicotine and cotinine measurements. BriL Mad. J. 2:1026-28 (1~T/9). 9. MJ. Jlu~is, M.A.H. Russell, C. Feyerabend, J.R.M Morgan, R Gammage, and E.M. Gray. Passrve exposure Io tobacco smoke-- Saliva cotinine concenlrations in a representative population-- (lSaS). 10. A. Bibet, G. Schare¢, I, Hoepfner, F. Adlkofer, W.D. Hailer, J.E. Haddow, and GJ. Knight. Determination of nicotine and cotinine in human serum and urine--An intedaboratory study. Tox. Let 35:45-52 11. O.A. Machacek and N. Jiang. Quantification of cotinine in plasma and saliva by liquid chromatography. Clin. Chem. 32:979-82 (1~6). 12. O. Hoffman, K.D. B~unnemann, N J, Haisy, O.W. Sepovic, and ing under controlled conditions and the etiminafion of colinine. indoor Air '87 2:13-17 (1987), 13. F.M. CaP, a, DJ. Eatough, E,A. Lewis, H. Tang, and J. Crawford. An intercompartson of sampling techniques for nicotine in indoor environments. Environ. Sci. Tech., submitted for publioalion. 14. O.J. ~h, C.L Banner, J.M. Bayona, F.M. Caka, G. Richecds, J.D. lamb, E.A. Lewis, and L.D. Hansen. The chemical compo~ lion of environmental tobacco smoke I. Gas phase acids and bases. Environ. Sci. Tech. 23:679-87 (1989). Manuscript received April 3, 1989; revision received July 25. 1989, 606000699 203 606000699 PRODUCED FROM B&W WEB SITE