Anal. Chem. 1084, 56, 1947-1950
LITERATURE CITED Kbnig, W. J . Prakt. Chem. 1904, 89, 105-137. Epsteln, J. Anal. Chem. 1947, 19, 272-274. Asmus, E.; Qarschagen, H. Fresenius’ Z . Anal. Chem. 1953, 138, 414-422. (4) Nagashima, S.; Ozawa, T. Int. J . Environ. Anal. Chem. 1981, 10, 99-106. (5) Nagashlma, S. Anal. Chem. 1983, 55, 2088-2089. (6) Nagashlma, S. Water Res. 1983, 17, 833-834.
1947
(7) “Standard Methods for the Examlnatlon of Water and Wastewater”. 15th ed.;American Water Works Association: Washington, DC, 1980; Part 412. (8) “Standard Methods for the Examination of Water and Wastewater”, 13th ed.; American Water Works Assoclatlon: New York, 1971; Part 207.
RECEIVED for review February 21,1984. Accepted April 23, 1984.
Determination of Nicotine in Tobacco by Circular Dichroism Spectropolarimetry W.Marc Atkinson, Soon M. Han, and Neil Purdie* Chemistry Department, Oklahoma State University, Stillwater, Oklahoma 74078
A circular dlchrolsm (CD) spectropolarimeter has been used as the detector for the determination of (S )-(-)-nlcotlne In chopped tobacco leaves, after a straightforward single extraction of the analyte Into methanollc KOH. Other compounds extracted from the leaf absorb In the UV range, but none is CD actlve, eilminatlng all possible Interferences. Results are reported lor the nicotine contents in a few smokeless tobaccos and clgareltes.
Circular dichroism (CD) spectropolarimetry has been successfully applied to the identification and determination of alkaloids in a variety of complex matrices (1-8). Since two prerequisites must be satisfied for CD activity, namely, the analyte must simultaneously absorb electromagneticradiation and be optically active, determinations can be made quickly and directly, after simple solvent extraction and without separation from the other components in the mixture. Previous work was devoted to the determination of opiates, Lcocaine, and D-LSD in confiscated specimens and of tetracycline in human urine (9). In d of these instances the analyte was either the only CD active or the predominant CD active component in the mixture. In this work we have characterized the CD spectrum of @)-(-)-nicotine in aqueous, 2-propanol, and methanolic KOH solutions and have determined the total nicotine alkaloid content of several cigarette brands and smokeless tobaccos. These experiments are preliminary to later studies of the nicotine contents in tobacco smoke, in pesticides, and in biological fluids and to a full analogous study of the cannabinols in marijuana. Nicotine determinations are of particular importance to the tobacco industry and in the area of toxicology. As expected, the analytical techniques applied to this purpose are many and varied. A fairly thorough review of these methods (10) pointa up the significant contributions from chromatographic procedures: thin-layer, liquid, and gas chromatography, often times coupled with mass spectrometry. Other techniques include potentiometry (1I), absorption spectrophotometry (121,and polarimetry (13). Many of the methods involve a prolonged extraction and work-up procedure prior to determination, but the comparisons among the resulta for nicotine in leaf, in smoke, and in biological fluids are excellent. We have found no reference in the literature to the determination of nicotine in smokeless tobaccos. 0003-2700/84/0356-1947$01.50/0
The gas chromatographic study of Severson et al. (14)describes a simple extraction procedure followed by a rapid determination of the relative distribution of the nicotine alkaloids in cured tobacco leaves. This is a significant contribution in that in most other methods the analogues were determined as total nicotine, The important minor nicotine-type alkaloids constitute about 5% of the total alkaloid fraction. A knowledge of their distribution is important to product development, but not necessarily to quality control. An earlier CD study of nicotine-type alkaloids was done for the purposes of a preferred conformation study (15). Spectra were not included for reference, but molecular ellipticity data are included for nicotine, nornicotine, anabasine, and methylanabasine, the last three being minor constituents of tobacco. The evidence suggested that the analogues are indistinguishable from nicotine by CD. This was confirmed in our laboratory for anabasine. Results are reported, therefore, as total nicotine. EXPERIMENTAL SECTION (SI-(-)-Nicotine, as the liquid free base, was obtained from Eastman Kodak and used without further purification. CD spectra were measured for the analyte dissolved in aqueous HCl, aqueous NaOH, and in a variety of buffers over the pH range 6.2-8.6. Extractions from tobacco leaves were made using either analytical grade 2-propanol or 0.05 M methanolic KOH (Baker Chemical Co.). Samples of smokeless tobaccos and cigarettes were chosen at random from local stores. UV absorption measurements were made on a Perkin-Elmer Model 552 spectrophotometer. CD measurements were made on a JASCO-5OOAautomatic recording spectropolarimeter fitted with a DP-500N data processor. Daily calibration of the ellipticity scale was made against a standard solution of androsterone in dioxane as recommended. Measurements were made over the wavelength range 230-320 nm, with base line correction made for the blank by spectral subtraction on the data processor. Sensitivity,scan rate, and repeat functions were selected which optimized the signal to noise ratio. Sample weights were chosen both for nicotine and for the tobacco extractions such that the nicotine concentration was in the range of lo4 to M. Cell sizes were either 1 mm or 1 cm path length. For the extraction experiments, the sample weights for the smokeless tobaccos were on the order of 200 mg, while individual, randomly chosen, cigarettes were used with average net weights (filters removed where appropriate) in the range of 550-850 mg. All determinations were done in duplicate. For 2-propanol extractions, the samples were agitated with either 5-mL or 10-mL aliquots of solvent for 1h on a mechanical shaker. For methanolic KOH, extractions were made during a 1 h ultrasonication pro@ 1984 American Chemical Society
1948
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
1. 4 0
I 10
20
30
40
50
60
Figure 2. Extraction of nicotlne from Skoal smokeless tobacco expressed as nicotine by welght of sample vs. time in mlnutes. The broken line corresponds to % nicotine by weight extracted by Soxhlet.
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The CD spectrum of (S)-(-)-nlcotlne in methanollc KOH, normalized to molar concentration. The units on [e] are mdeg/(M cm). Figure 1.
Table I. CD Spectral Characteristics for Nicotine
2-propanol 7.2 buffer 8.2 buffer
methanolic KOH
222 220 220 229
236 236 240 243
[41.0] [34.2] [25.0] [37.0]
244 247 249 255
255 [-77.81 264 [-63.31 264 [-64.41 265 [-91.51
261 [-87.61 267 [-62.51 268 [-60.01 271.5 [-92.21
cedure (14). The extraction efficiency was compared against the results obtained from a 15-h Soxhlet extraction. Any suspended particulate matter was removed by rapid centrifugation. An aliquot of the supernatant solution was added directly to the sample cell and the complete spectrum was recorded. The whole spectrum was recorded as a precautionary measure to safeguard against the appearance of any unexpected, interfering analyte. Once it is confirmed that no interfering compound is present, subsequent determinationscan be made from data taken at a few selected wavelengths. To assure a more uniform moisture content which would make comparisons among the various produch more valid, the leaves were first allowed to equilibrate for 48 h in a desiccator prior to the extraction step. When Pz06was used as the desiccant, the extraction efficiency was about 10% of that accomplished by Soxhlet extraction. An excellent correspondencewas achieved, however, after the leaves were allowed to equilibrate in an environment of 81% relative humidity maintained by a saturated solution of ammonium sulfate.
RESULT AND DISCUSSION The CD spectrum for @)-(-)-nicotine in methanolic KOH is shown in Figure 1. The spectral parameters are listed (Table I) for a variety of solvent systems. The wavelengths of the negative maxima k-mm correspond reasonably well to the anticipated absorption maxima for a substituted aromatic ring chromophore. The molar ellipticity coefficients [e] were calculated from the slopes of the corresponding correlation curves of the experimentally observed ellipticities, plotted against the molar concentration of nicotine (cf. Beer’s law), according to the equation
where q, and t R are the molar absorption coefficients for the left and right circularly polarized components of the incident beam, respectively, and b is the pathlength in cm. The molar ellipticity [e] is defined in this way to conform with the analogous molar absorbance coefficient, since CD is correctly identified as a modification of absorption spectrophotometry. This definition is not to be confused with molecular ellipticity for which values of [O] are one hundred times greater. Correlation coefficients for the data for the three maxima were
all 0.999 and standard deviations were on the order of 1.3 x lo4 deg/(M cm). The same general spectral characteristics were observed in aqueous media. Maximum wavelengths were red shifted between 4 and 8 nm. As the pH of the aqueous medium is systematically increased, using a series of buffers, a gradual change in the relative magnitudes of the principal negative bands is observed with inversion having occurred at pH 8.2, Le., = -60 and [e],, = -64.4 deg/(M cm). This change is correctly associated with the changing proportion of the monocation to the neutral molecule on titration of the alkaloid (15). In an earlier CD study of nicotine and its analogues (16), a single unresolved negative band was reported for each analogue whose wavelength maximum occurred in the narrow range of 263-266 nm. Only a slight increase in [e] was observed on changing to basic conditions. The resolution of this Cotton band into two in this study is attributed to instrument improvements over the intervening years. Cross-over points AD and the positive Cotton band characteristics are in good agreement between both studies. The instrumental limitations of the technique set the linear dynamic range for nicotine measured in a 1-cm sample cell at an upper concentration limit of M and a minimum detectable quantity around lo4 M, dependent upon the wavelength chosen for quantitation. There being no restrictions on sample availability, a minimum limit of 0.16 pg/mL is achieved with a 10-cm cell. This figure is well within the predictable range for the determination of nicotine either in tobacco smoke or in biological specimens. The limit could be further improved by expanding the sensitivity scale of the instrument. The present figures were calculated from data measured at a sensitivity of 2 mdeg/cm ellipticity. An additional 10-fold extrapolation is easily accessible. A number of solvents could have been chosen for the extraction of nicotine from the chopped tobacco leaves. 2Propanol is commonly recommended for alkaloids (17). However for tobacco, extraction with a strong basic solution, even an alcoholic KOH solution is recommended (14, 18). After equilibrating at 81% relative humidity, the moist pliable leaf was suitable for efficient extraction. As seen in Figure 2, a 30-min extraction time under mild conditions would suffice as an alternative to the prolonged Soxhlet procedure. Multiple extractions, performed simultaneously, effectively reduce the time for determinations to less than 10 min per sample. An advantage, inherent to the CD technique over UV spectrophotometry, is illustrated in Figures 3 and 4. Superimposed on the UV spectrum for nicotine is the UV spectrum for the methanolic KOH extract of Skoal smokeless tobacco (Figure 3). The spectrum of nicotine is masked by the cumulative spectra of other compounds extracted, and nicotine determination would require a spectral deconvolution strategy. In contrast, in the CD spectral comparison made for the identical solutions (Figure 4),nicotine appears as the
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
1949
Table 11. Nicotine Content of Commercial Tobacco Formsa brand
descriptionC
MeOH-KOH (81% RH)
MeOH-KOH (P20,)
2-propanol (81% RH)
Now Barclay Camel Marlboro Ultralight
F, S, 85 mm F, S,M, 85 mm R,NF, S, 70 mm F,S,85 mm F,S,85 mm
1.49 1.53 1.55 1.42 1.30
1.66 (0.024)* 1.69 (0.017)b 1.72 (0.150)b 1.58 (0.135)b 1.49 (-)
0.29 0.38 0.43 0.53
Skoal Hawken Kodiak Silvercreek
WG WG WG WG
1.28 0.36 1.01 1.73
2.24 0.47 1.95 2.56
1.02 0.06 0.49 1.10
Results expressed as % nicotine (w/w). *Smokenicotine analyses data taken from ref 19. F, filter; S, softpack; M, menthol; R, regular; NF, no filter; WG,wintergreen.
/
350
-L Flgure 8. Comparative absorption spectra of (a)nlcotlne and (b) Skoal extract. Absorption is plotted In arbitrary unlts.
only CD active component in the matrix and its determination can be made directly. Data at the positive maximum were not used for analysis, because of the obvious deterioration in the signal to noise ratio. Results for the nicotine determinations in cigarettes and smokeless tobaccos are shown in Table I1 for both extraction solvents. Values are averages calculated from data taken a t the wavelengths of the negative maxima, and are reported as percent by weight. Only the 81% RH values were determined by CD detection. Results reported for the dry specimens were calculated after measuring the moisture lost on standing over Pz05until constant weight was obtained. To our knowledge the results for smokeless tobaccos are the f i s t to be reported. Results are averages of at least five independent determinations, Considering the inhomogeneousdistribution of nicotine within the chopped leaf blends, the reproducibility is excellent. Most determinations of nicotine content in the many forms of tobacco reported in the literature and in annual reports by the Federal Trade Commission (19)deal with the content in smoke. Only a few references to the determination of nicotine contents in chopped tobacco leaf are available (18, 20, 21). Direct comparisons among the many methods are difficult because of the variety of blends and smoking conditions. The present results obtained after either a 1-h extraction procedure under mild conditions or a prolonged Soxhlet extraction are in excellent agreement, as was previously reported in a study which used GC detection (14). For cigarettes, good agreement was obtained with earlier literature results for chopped leaves,
Figure 4. Comparative CD spectra of (a)nicotine and (b) Skoal extract. Ellipticity is plotted in arbltrary unlts.
where values on the order of 2% (w/w) were reported (1420, 21). No earlier information is available for smokeless forms for comparison with the present data. Neither flavors nor sweeteners, currently added to products to make them more palatable, interfere with the direct CD detection, although these may have been extracted. For example, the compounds responsible for the very dominant band from 290 to 325 nm in the absorption spectrum (Figure 3) were CD transparent and noninterfering. In summary, it has been demonstrated that CD spectropolarimetry can be used very effectively for the determination of nicotine contents in chopped tobacco leaves, after a simple and direct single extraction into methanolic KOH. The method is both economical and time effective, because internal standards are not required, detection is free from interferences, and instrument calibration is necessary only on a daily basis, at most. Detection limits can be extrapolated to achieve a level of tens of nanograms per milliliter. However, the method does not distinguish among nicotine and its structural analogues and results must be reported as total nicotine. The analogues generally account for less than 5% of the total nicotine fraction, and the lack of discrimination is therefore of little consequence, except when the analogues are detrimental to the quality of the product. Registry No. Nicotine, 54-11-5.
Anal. Chem. 1984, 56, 1950-1953
1950
LITERATURE CITED (1) Bowen, J. M.; Purdie, N. Anal. Chem. 1980, 52, 573. (2) Bowen, J. M.; Crone, T. A.; Hermann, A. 0.;Purdle, N. Anal. Chem. 1980, 52. 2436. (3) Crone, T. A.; Purdie, N. Anal. Chem. 1981, 53, 17. (4) Bowan, J. M.; Crone, T. A.; Head, V. L.; McMorrow, H. A.: Kennedy, R. K.; Purdie, N. J . Forensic Scl. 1981, 26, 664. (5) Bowen, J. M.; Purdie, N. Anal. Chem. 1981. 53, 2237. (6) Bowen, J. M.; Purdie, N. Anal. Chem. 1981, 53, 2239. (7) Bowen, J. M.; Crone, T. A.; Kennedy, R. K.; Purdle, N. Anal. Chem. 1982, 54, 66. (8) Bowen, J. M.; McMorrow, H. A.; Purdie, N. J . Forensic Sci. 1982, 27, 822. (9) Bowen, J. M.; Purdie, N. J . Pharm. Sci. 1982, 71, 836. (10) Green, C. R.; Colby, D. A.; Cooper, P. J. Recent A&. Tob. Sci. 1980, 6, 123. (11) Efstathion, C. E.; Diamandis, E. P.; Hadjiioannon, R. A. Anal. Chlm. Acta 1981, 127, 173. (12) Harvey, W. R.; Handy, B. M. Tob. Int. 1981, 183, 137.
(13) Klus, H.; Kuhn, H. Fachliche M/tt. Oesterr. Tabakregie 1977, 17, 331. (14) Severson, R. F.; McDuffle, K. L.; Arrendale, R. F.; Gwynn, G. R.; Chaplln, J. F.; Johnson, A. W. J . Chromatogr. 1981, 217, 111. (15) vonEuler, U. S.,Ed. "Tobacco Alkaloids and Related Compounds"; MacMllian: New York, 1965; p 308. (16) Testa, B.; Jenner, P. Mol. Pharmacoi. 1972, 9 , 10. (17) Merck Index, 9th ed.;1976; p 5069. (18) Bush, L. P.; Grunwald, C.; Davls, D. L. J . Agric. Food Chem. 1972, 20, 676. (19) Federal Trade Commisslon Report, Washington, DC, March 1983. (20) Harlan, W. R.; Moseley, J. M. Encycl. Chem. Techno/. 1955. (21) Schmeltz, I.; Hoffman, D. Chem. Rev. 1977, 77, 295.
RECEIVED for review July 5, 1983. Resubmitted April 2,1984. Accepted April 30, 1984. We wish to thank the National Science Foundation for the support of this work under Grant NO. NSF CHE-7909388.
Determination of Ethylene Oxide in Air by Gas Chromatography George G. Esposito,* Kenneth Williams, and Rodolfo Bongiovanni
US.Army Environmental Hygiene Agency, Aberdeen Proving Ground, Maryland
A method for the collection and analysis of airborne ethylene oxide (ETO) has been developed. Thls procedure Is based on the entrapment of ET0 on a chemically Impregnated air sampilng tube where It Is converted to 2-bromoethanol. The 2-bromoethanol Is subsequently analyzed by gas-llquld chromatography using an electron capture detector. The method was tested In the 0.5-20 ppm concentratlon range and was shown to provide recoveries of 92-112%. Statlstlcai treatment of laboratory and field data Is presented.
Ethylene oxide (ETO) is an important chemical having wide industrial application for the synthesis of polymers, surfactants, antifreeze, etc. Within health care facilities, E T 0 provides the only practical means of sterilizing heat-sensitive surgical tools and equipment. The effectiveness of E T 0 as a sterilant residues in its inherent toxicity; consequently, there is an acute concern regarding its toxic effects on exposed workers. Furthermore, at concentrations below the current limit of 50 ppm as an 8-h time weighted average (TWA),E T 0 is suspected of being a carcinogen. Recently, the U.S. district court ordered the Occupational Safety and Health Administration (OSHA) to issue an emergency health standard of 1 ppm. If the proposed standard of 1ppm is adopted, current ET0 monitoring techniques will be severely stressed to accurately measure down to that level. The National Institute for Safety and Health (NIOSH) sampling and analysis method for E T 0 (1) uses activated charcoal and carbon disulfide to collect and desorb ETO. Analysis of the desorbed material is accomplished using gas chromatography (GC) with a flame ionization detector. Other charcoal tube methods have dealt with various parameters such as different charcoal tubes (2) and different equipment (3). Coyne and Pilny (4)conducted a validation study of the E T 0 charcoal tube procedure; they studied storage stability, air flow rates, and the effect of relative humidity. The generally accepted method utilizes the large charcoal tube (390/700 mg) based on the procedure recommended by Qazi and Ketcham ( 2 ) .
21010
Even though the charcoal tube method has found wide acceptance for monitoring ETO, this technique has certain shortcomings. Some of the problems associated with the E T 0 charcoal tube method are interferences from other organic compounds, limited sample size because of breakthrough, the negative effect of high relative humidity, the need for refrigeration to avoid losses, and the overall complexity of sampling and analysis. In addition to charcoal tubes, methods using passive monitors (5,6) have been used for the determination of ETO. These devices offer convenience and ease of operation; however, the cost per sampler is significantly greater than that of a charcoal tube. This cost is offset, to a great extent, by savings in time and labor. Recently, a new method (7) for E T 0 was developed by OSHA at their Salt Lake City Laboratory. Samples are collected by using two charcoal tubes connected in series and desorbed with 1% CS2 in benzene. The desorbed E T 0 is derivatized with HBr and analyzed by GC with an electron capture detector. This method provides good data; however, the limited capacity of the charcoal tubes requires sequential samples be taken to monitor long term exposures. In addition, tube storage is limited to approximately 3 weeks. The object of this study was e0 circumvent the shortcomings of the OSHA method by simultaneous adsorption and derivatization of E T 0 in a collection tube. During the initial stages of the study, several sorbents were tested for their suitability as substrates for the derivatization reagent, HBr. Of the materials evaluated, the one that provided the best results was Ambersorb 347, a spherical carbonaceous material prepared by the controlled pyrolysis of polymeric beads. It is sufficiently retentive to entrain E T 0 long enough for derivatization to occur; with a mixture of toluene and acetonitrile, the derivative is quantitatively desorbed within 30 min. In the proposed method, Ambersorb 347 is treated with HBr and packed into glass tubes. Air is drawn through the tubes where E T 0 is converted to 2-bromoethanol. The tubes are desorbed with a mixture of acetonitrile and toluene and subsequently analyzed by GC using an electron capture de-
This article not subject to US. Copyrlght. Published 1984 by the American Chemical Soclety
