Anal. Chem. 1986, 58,565-568 (3) Sullivan, J. J.; Iwaoka, W. T.; Liston, J. Biochem. Biophys. Res. Commun. 1983, 114,465-472. (4) Sommer, H.; Whedon, W. F.; Kofoid, C. A,; Stohler, R. Arch. Pathol. 1937, 2 4 , 537-559. (5) Taylor, D. L.; Selinger, H. H. "Toxic Dinoflagellate Blooms. Developments In Marine Biology 1"; Elsevier/North Holland: New York, 1979; p 505. (6) Prakash, A.; Medcof, J. C.; Tennant, A. D. Bull.-Fish. Res. Board Can. 1971, No. 177,87 pp. (7) Quayle, D. B. Bull.-Fish. Res. Board Can. 1969, No. 168, 68 pp. ( 8 ) Permewan, W. Lancef 1888, 2, 568. (9) Evans, M. H. Br. J. Exp. Pathol. 1965, 4 6 , 245-253. (10) Wong, J. L.; Oesterline, R.; Rapoport, H. J . Am. Chem. Soc. 1971, 93,7344-7345. (11) Barber, M.; Bordoli, R. S.; Elliot, G. J.; Sedgewick, R. D.; Tyler, A. N. Anal. Chem. 1962, 54, 645A-657A. (12) Williams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.;Taylor, L. C. E. J . Am. Chem. SOC. 1981, 103,5704-5706. (13) Nakamura. M.; Oshima, Y.; Yasumoto, T. Toxicon 1984, 22,361-385.
565
(14) Maruyama, J.; Noguchi. T.; Matsunaga, S.; Hashimoto, K. Agric. Biol. Chem. 1984, 4 8 , 2783-2788. (15 ) Hall, S.;Relchardt, P. 8.; Neve, R. A. Biochem . Biophys . Res. Com mun. 1980, 97,649-653. (16) McNeal, C. J. Anal. Chem. 1982, 54,43A-50A. (17) Heller, D. N.; Yergey, J.; Cotter, R. J. Anal. Chem. 1983, 55, 1310-1 313. (18) Ligon, W. V., Jr. Int. J. Mass Specfrom. Ion Phys. 1983, 52, 169-193. (19) Rinehart, K. L., Jr. Science 1982, 218, 254-260. (20) Puzo, G.; Prome, J. C. Org. Mass Spectrom. 1984, f 9 , 448-451. (21) . . Shimlzu. Y.; Hsu, C. P.; Genanah, A. J. Am. Chem. SOC.1981, 103, 605-609. (22) Rogers, R . S.; Rapoport, H. J. Am. Chem. Soc. 1980, 102, 7335-7339.
RECEIVED for review June 4,1985. Accepted October 10,1985.
Determination of Tobacco-Specific N-Nitrosamines by Capillary Gas Chromatography/Selected Ion Monitoring Mass Spectrometry R. F. Arrendale,* W. J. Chamberlain, 0. T. Chortyk, and J. L. Baker Tobacco Safety Research Unit, Agricultural Research Service, United States Department of Agriculture, P.O. Box 5677, Athens, Georgia 30613
M. G . Stephenson Crops Research, Agricultural Research Service, United States Department of Agriculture, Coastal Plains Experiment Station, Tifton, Georgia 31 793
laboratory falls into this latter category. Consequently, we have developed a nitrosamine analysis method, using our GC/MS system in the selected ion monitoring (SIM) mode. In this technique, the mass spectrometer is adjusted to selected masses for defined time periods, during which time the signal response is recorded (26-19). This technique has two major advantages; (1)it provides detection limits that are 2 or 3 orders of magnitude lower than are possible in the normal scannling mode, and (2) by choosing ions that are characteristic of the compound of interest, the mass spectrometer in the SIM mode functions as a selective detector. Integration of the recorded signal response can then be used for the determination of component concentrations. We have also utilized the many advantages of capillary GC in the development of this method, especially the use of cold on-column injection. This technique has several advantages over the now conventional split/ splitless techniques (20-28). Some of these advantages are (1) elimination of discrimination between low and high boiling components; (2) reproducibility and linear response; (3) excellent determination of concenThe determination of concentrations of the two major totration of individual sample components; (4) elimination of bacco-specific N-nitrosamines, N-nitrosonornicotine (NNN) thermal or catalytic decomposition of sample components due and 4-(N-methyl-N-nitrosamino)-l-(3-pyridyl)-l-butanone to rapid heating; and (5) analysis of sample components at (NNK),is very important, as both compounds are carcinogens ng/pL concentrations using a flame ionization detector. We and are found in tobacco and tobacco smoke (1-4). A variety also compared our recently developed cold on-column inlet of methods for the determination of these important comand a commercial inlet, operating in the splitless mode, for pounds have been developed (5-14). Separation of N the determination of concentrations of NNN (29). nitrosamines by GC or high-pressure liquid chromatography An analytical capillary GC/MS technique must also include (HPLC) and detection with a thermal energy analyzer (TEA) a capillary GC/MS interface, that provides reproducible, have long been the methods of choice (11,13,15).However, linear, and quantitative transfer of the components eluting many laboratories do not possess a TEA, mainly due to its from the capillary GC column to the MS source for detection. expense and limited application to other areas of research. We have designed and constructed an open split interface But, most laboratories have access to a GC/MS system. Our (OSI), which satisfied these requirements (30). These imMethodology was developed to determine the concentrations of the tobacco-speclflc N-nltrosamlnes, N-nitrosonornlcotlne (NNN) and 4-(Nmethyl-N -n~rosamlno)-I-(3-pyrldyl)-l-butanone (NNK), In tobacco and tobacco smoke, based upon gas chromatography/mas spectrometry (GC/MS) analyses In the selected Ion monltorlng (SIM) mode. The N-nltrosamlnes were separated by caplllary GC on a fused silica Superox-4 caplllary column. Both splitless and cold on-column Injection modes were evaluated, with the latter produclng the better resutts. Our recent advances In analytical methodology, such as a laboratory-constructed cold on-column capillary GC inlet and an open-spllt interface for caplllary GWMS application, were Instrumental In the successful analyses of these tobacco-speciflc N-nltrosamlnes. The development of this GC/ SIM-MS method and its appllcatlon to analyses of N-nltrosamines from tobacco and tobacco smoke are presented and discussed.
This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
provements in methodology were successfully combined in the development of this analytical method for N N N and NNK in tobacco and tobacco smoke. E X P E R I M E N T A L SECTION Materials. The NNN, used as a standard, was prepared from nornicotine by the method of Chamberlain et al. (14). Standard NNK was obtained from D. Hoffmann and S. Hecht of the Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY. The internal standard (ISTD), 2,4’-bipyridyl, was obtained from Aldrich (Milwaukee, WI) and was used as received. Dichloromethane (methylene chloride) and methyl alcohol were “distilled-in-glass” grade (Burdick and Jackson, Richmond, CA) and were used as received. Tobacco Sample Preparation. Flur-cured tobacco (3 g) was ground (20 mesh) and extracted ultrasonically for 1 h with 150 mL of aqueous citrate buffer (pH 4.5) (I4,31). The citrate buffer, adjusted to pH 5.0, was extracted with dichloromethane (3 x 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, concentrated to 5 mL, and chromatographed on 30 g of Activity I11 basic alumina, packed in a glass column, with a bed length of 150 X 15 mm (31,32). The N-nitrosamines were eluted with dichloromethane (250 mL), and the solvent was removed on a rotary evaporator. One milliliter of methanol, containing 100 yg (100 ng/yL) of 2,4’-bipyridyl was added prior to GC/SIM/MS analysis. Cigarette Smoke Sample Preparation. A 30-port Borgwaldt smoking machine (using standard smoking conditions of 1 puff/min, 2-s puff duration, puff volume of 35 mL, and smoking to a butt length of 23 mm) was used to collect the cigarette smoke condensate (CSC) from 60 cigarettes. All cigarettes were 85 mm long and were conditioned at 60% relative humidity for 48 h prior to smoking. Smoke was trapped in 200 mL of citrate buffer (pH 4.5) to which 20 mM of ascorbic acid and 100 yg of 2,4’-bipyridyl had been added. The buffer solution was then extracted as above. Capillary Gas Chromatography. The fused silica (FS) Superox-4 (4 X lo6 poly(ethy1ene glycol)) capillary GC column was prepared by our method (33). For GC and GC/MS analyses, separations were performed with a Hewlett-Packard 5840 gas chromatograph, which is an integral part of the Hewlett-Packard 5985B GC/MS system. Capillary GC methodology was first developed using a flame ionization detector (FID). Conditions for cold on-column capillary GC analysis were as follows: a 30 m X 0.3 mm i.d. column; temperature program, 50 “C for 1 min, 50-150 “C at 20 deg/min, 150-250 “C at 4 deg/min; 40 cm/s He flow; 1-pL methanol solution injected, using our laboratory-constructed cold on-column capillary GC inlet (29). Conditions for splitless injection with the Hewlett-Packard 18835Ccapillary inlet system were as follows: 30 m x 0.3 mm i.d. column; temperature program, 55 “C for 1.5 min, 55-150 “C a t 20 deg/min, 150-250 “C at 2 deg/min; 1.5 min purge time; 40 cm/s He flow; 1-yL methanol solution was injected. For samples with low levels of NNN and NNK, the final volume was reduced with a stream of dry N2 and 2 yL was injected. Capillary Gas Chromatography/Selected Ion Monitoring Mass Spectrometry (GC/SIM-MS). The GC/SIM-MS analyses were performed on a Hewlett-Packard 5985B GC/MS system. The FS Superox-4 capillary column was interfaced to the mass spectrometer via our laboratory-constructed open split interface (OSI),operating in the ideal mode (30). The configuration of our OS1 in the ideal mode consisted of a piece of 0.15 mm i.d. vitreous silica (VS) tubing that had been deactivated with Superox-4 (33) and connected the OS1 to the mass spectrometer source. Within the OSI, the entrance end of the VS interface tubing (0.15 mm i.d. X 0.25 mm 0.d.) was placed inside the exit end of the FS capillary GC column (0.3 mm i.d.). The OS1 was continually purged with He. Operation of our OS1 in the ideal mode provided low chromatographic dead volume, low surface activity, and low MS background. The Hewlett-Packard 5985B GC/MS, like most modern instruments, is controlled with a computer data system, which includes a selected ion monitoring program, that enables the user to easily and accurately select ions and dwell times for SIM analyses. Determinations of concentrations of NNN and NNK were performed by use of an internal standard spiking technique, with 2,4’-bipyridyl as the ISTD. Mass spectra (70 eV electron impact) of 2,4’-bipyridyl (molecular weight 156), NNN
Table I. Parameters for GC/SIM-MS Analyses Run Type GC, E1
start time, min run time, min total dwell, ms mass dwell
Run Time 30.0 min
1 (ISTD)
2 (NNN)
3 (NNK)
12.0 3.0 500
21.0 2.5 500
27.0 3.0 500
156.1/500
177.1/500
177.1/500
Table 11. Reproducibility of Response for NNN and NNK
compound
cold on-column cold on-column injection GC/FID injection GC/SIM-MS re1 % re1 % SDc error K , SD error
2,4’-bipyridyl (ISTDjd
1.00
NNN
0.63 0 0.96 0.0173
NNK
’
1.0,o
0 1.8
0.068 0.00231
0.32
0.01155
3.4 3.7
“ K = response factor = (area X/amount X)/(area ISTD/ amount ISTD). * K , mean of three analyses. ‘SD, standard deviation. ISTD, internal standard.
(molecular weight 177), and NNK (molecular weight 207) showed that the ISTD (2,4’-bipyridyl) had a strong molecular ion a t m / z 156; this was chosen for SIM/MS analyses, as was the NNN molecular ion a t m / z 177 and the strong m / z 177 ion of NNK (34). Although the actual mass of an ion can be accurately calculated, in practice a dynamic calibration is done to ensure that the mass to be dwelled upon is indeed at the top of the mass spectral peak. This technique corrects for mass defect and any calibration inaccuracies. The top of each mass spectra peak to be measured was determined by monitoring ion currents around the nominal mass a t 0.1 a m u intervals. The results of this dynamic calibration are a series of peaks on successively adjacent mass chromatograms, one of which has the largest area, and this m / z value, to the nearest 0.1 amu, is the one used for that ion. Table I gives a set of typical operational run parameters. Groups 1, 2, and 3 represent 2,4’-bipyridyl, NNN, and NNK, respectively. Each group, consisting of one ion each in this instance, has a start time, a run time, and a total dwell time for that group. Up to 20 ions may be monitored in each group, but we chose only one ion per group, for maximum sensitivity. The actual masses that gave maximum abundances for SIM/MS were 156.1 for 2,4’-bipyridyl and 177.1 for both NNN and NNK. Studies have shown that the length of the dwell times for each m / z value should be chosen so that a t least 20 cycles per GC peak are obtained (19). We chose our dwell times to exceed this value.
RESULTS A N D D I S C U S S I O N A critical step in both the tobacco and CSC sample preparations was the basic alumina column chromatography (14, 32). Basic alumina, that has been heated prior to column packing, should not be exposed t o ambient air for more than a few minutes, as it will rapidly take u p moisture from the air. This causes changes in its activity and alters the elution of the N-nitrosamines. A gas chromatogram of a standard mixture of NNN, NNK, and 2,4’-bipyridyl separated on a FS Superox-4 column and using a flame ionization detector (FID) is shown in Figure 1. Each peak represents approximately 50 ng of material. Table I1 gives reproducibility data for three consecutive analyses of the standard mixture. Relative percent errors of less than 2% indicated the excellent reproducibility provided by cold on-column injection capillary GC. SIM-MS for determination of component concentrations places several constraints on the analyst. Standards of each component of interest must be available. Studies have shown that maximum sensitivity is achieved when as few ions as possible are monitored and ions specific for the compound
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
567
Table 111. Levels of NNN and NNK in Tobacco Determined by Capillary GC/SIM-MS NNK
!,4'-Dipyridyl
amtof NNN,
amtof NNK,
rglg
rglg -a 0.35 0.30
tobacco type NC 2326 (flue-cured) 2R1 cigarette tobacco V-445 air-cured normal nitrogen sample 1 V-445 air-cured normal nitrogen sample 2
NNN
Less than 0.05 ~
1.00
0.74 0.20 0.16
0.40
d g .
~
Table IV. Comparison of N-Nitrosonornicotine (NNN) Values from Mainstream Cigarette Smoke Condensate UQ I cigarette HPLC-TEA" GC/SIM-MS
commercial nonfilter cigarette commercial filter cigarette Kentucky reference cigarette
0.24 0.31 0.39*
0.30 0.50 0.20'
K. D. Brunnemann et al. (13). *Kentucky reference 1R1 cigarettes. Kentucky reference 2R1 cigarettes.
m A
/ 2,4'-DIPY
R I DY L IISTDI
xz
n
.
I
Figure 2. Cold on-column injection capillary GC/SIM-MS chromatograms: (A) standard mixture of 2,4'-bipyridyl (ISTD; selected ion 156.l),NNN (selected ion 177.1), and NNK (selected ion 177.1); (E) N-nitrosamine fraction from NC 2326 tobacco (note the marking the retention window for NNK); (C) N-nitrosamine fraction from NC 2326 tobacco after spiking with 10 ng/FL of NNK; (D) N-nitrosamine
+
..--..-.. ..-... .
fraptinn frnm V-Ad5 . .- tnharnn
of interest are chosen. Ions chosen for SIM-MS should also be a t high mass with high abuhdance and no interferences. A GC/SIM-MS chromatogram of our standard mixture of 2,4'-bipyridyl, NNN, and NNK is shown in Figure 2A. Reproducibility data were obtained, based upon peak areas for cold on-column capillary GC/SIM-MS of this standard mixture (Table 11). Comparison of the data in Table I1 with similar NNN data obtained for splitless capillary GC/SIM-MS revealed relative percent errors of 3.4% for cold on-column injection and 6.7% for splitless injection. Cold on-column injection was chosen because it gave better reproducibility than splitless injection. Linearity of SIM-MS response in the concentration range of 5-50 ng/hL injected was also determined by varying the amounts of NNN and NNK in our standard mixture, while keeping the amount of ISTD constant. These data showed
that the responses of NNN and NNK vs. the ISTD were linear over this range. The application of this methodology to determine the concentration of NNN and NNK in four tobacco samples is given in Table 111. The samples were also analyzed by the method of Chamberlain et al. (14) and the results obtained were consistent with the data in Table 111. Studies have shown that NNN is present in freshly harvested tobacco at less than 1 ppb and is predominantly formed during tobacco processing ( 4 , 3 5 ) . For instance, it has been shown that NNN was not detected in freshly harvested Burley tobacco but occurred only during and after air curing a t levels of 0.5-1.1 ppm. Thus, postharvest handling of tobacco is a major factor in determining the levels of N-nitrosamines in tobacco products. Comparison of levels of NNN for the air cured V-445 tobacco in Table I11 (0.16-0.20 ppm) with levels reported by Chamberlain et al. (14) for flue-cured V-445 tobacco (100 ppm) illustrates this point. North Carolina (NC) 2326 tobacco was a normal flue-cured variety, 2R1 are standard reference cigarettes manufactured by the University of Kentucky, and V-445 was a flue-cured tobacco variety that was air-cured. The results are graphically presented in Figure 2. Figure 2B shows the capillary GC/SIM-MS chromatogram for the NC 2326 tobacco. Note the prominent NNN peak and the essential absence of NNK (C0.05 pg/g) at its retention window. Figure 2C shows the analysis of the same sample, after spiking with approximately 10 ng/hL of NNK. Figure 2D shows the chromatogram for the determination of NNN and NNK in V-445 sample 1. It should be noted that large extraneous peaks sometimes appear in the SIM mass chromatograms but do not interfer with the determination of NNN or NNK. We subsequently determined values for NNN from mainstream cigarette smoke of several cigarettes and compared them to published values for similar cigarette types obtained by the high-pressure liquid chromatography/TEq method of Brunnemann et al. (13). These data are given in Table IV and show that our methodology yielded values for NNN of the same order of magnitude as those obtained using the HPLC/TEA method.
ACKNOWLEDGMENT The authors are grateful to D. Hoffmann and S. Hecht of the Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY, for their generous gift of the NNK used as a standard in this work.
588
Anal. Chem. 1986, 58,568-572
LITERATURE CITED (1) Druckrey, H.; Preussman, R.; Ivankovic, S.; Schmu, D. 2. Krebsforsch. 1987, 69, 103. Toth, 9.;Shubik. P. Cancer Res. 1987, 27, 43 Griswold, D. P., Jr.; Casery, A. E.; Weisburger, E. K.; Weisburger, J H.; Schnabei, F. M., Jr. Cancer Res. 1988, 619. Hecht, S. S.;Chen, C. 6.; Hoffmann, D. Acc. Chem. Res. 1979, 12, 92. Hoffmann, D.; Rathkamp, G.; Liu, Y. Y. IARC Sci. Pubi. 1974, 9 ,159. Klus, H.; Kuhn, H. fachliche Mltt. Oesterr. Tabakregle 1973, 14, 251. Hecht, S. S.;Ornaf, R. M.; Hoffmann, D. J . Natl. Cancer Inst. 1975, 54, 1237. Bharadwa], V P.; Takayama, S.; Yamada, T.; Tanimura, A. A . Gann 1976, 66, 585. Munson, J. W.; Abdine, H. Anal. Lett. 1977, IO, 777. Klus, H.; Kuhn, H. Fachliche Mltt. Oesterr. Tabakregie 1975, 16, 307. Hecht, S. S.;Ornaf, R. M.; Hoffmann, D. Anal. Chem. 1975, 4 7 , 2046. Hoffmann, D.; Dong, M.; Hecht, S. S. J . Natl. Cancer Inst. 1977, 58, 1841. Brunnemann, K. D.; Adams, J. D.; Ho, D. P. S.; Hoffmann, D. “Proceedings of the 4th Joint Conference on Sensing of Environmental Pollutants”, American Chemical Society: Washington, DC, 1978; p 876. Chamberlain, W. J.; Arrendale, R. F. J . Agrlc. Food Chem. 1983, 31, 909. Hoffmann, D.; Ornaf, R. M.; Hecht, S. S. Science 1974, 186, 265. Henneberg, D. 2. Anal. Chem. 1981, 183,12. Watson, J. T. “Introduction to Mass Spectrometry, Biomedical, Environmental and Forensic Applicatlons”; Raven Press: New York, 1976; p 199. Millard, B. J. “Quantitative Mass Spectrometry”; Heyden, London, 1978.
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RECEIVED for review April 12, 1985. Accepted October 23, 1985. Names of products are included for the benefit of the reader qnd do not imply endorsement or preferential treatment by the USDA.
Preserving Toxicologic Activity during Chromatographic Fractionation of Bioactive Complex Mixtures Arthur L. Lafleur,* Andrew G. Braun, Peter A. Monchamp, and Elaine F. Plummer Department of Applied Biological Sciences, Energy Laboratory a n d Center for Health Effects of Fossil Fuels Utilization, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Four types of chromatographic materials were evaluated for degree of recovery of mutagenlc components during column chromatography. Materlals tested were silica, alumina, Fiorlsll, and cyanopropyl-bonded silica. A combustlon-generated complex Fixture that vas highly characlerlred and known to contaln mutagenlc components was used gs the reference sample. The cyanopropyl materlal was found to be the most efflclent material far mutagen recovery and alumlna proved to be the least efficlent.
Samples obtained from the combustion of fossil fuels are highly complex mixtures containing combustion products, unburned fuel, and insolublg particulates. They are so complex that even very powerful techniques of chemical analysis are severely challenged by them unless the samples are first fractionated into simpler sets of mixtures prior to analysis. Many fractionation schemes for complex combustion-related mixtures have been developed during the past 2 decades (1-5) and a large number of them have been reviewed (1,5). A list of more recently develoRed methods has also been reported (4) and it would appear that the most widely used methods for preliminary fractionation are column adsorption chromatography using silica gel or alumina as sorbents (1). For samples containing components of widely varying polarity such as fossil fuel combustion products and coal-derived synthetic fuels, separations based whole or in part on alumina frac-
tionation have been shown to be simple and efficient (2-4, 6). After implementation of an alumina fractionation method in our laboratory for a number of combustion samples, it became apparent that although the fractionation was efficient in terms of separation of covpound classes, the biological data indicated that much of the applied mutagenic activity of the samples could not be recovered in the fractions. Therefore, the study that is the subject of this report was initiated to determine the effect of sorbent composition on recovery of mutagenic activity using standardized, commercially available adsarption columns.
EXPERIMENTAL SECTION Apparatus. The liquid chromatographic fractionationcolumns were Baker-IO solid phase extraction (SPE) columns obtained from J. T. Baker Chemical Co. The columns were obtained Rrepacked with sorbent except for the alumina column, which was prepared by dry packing an SPE filter column with Woelm neutral alumina (Brockman activity I). The alumina used was obtained from ICN Pharmaceuticals,Inc., and had a particle size distribution.of 50-200 pm (70-290 mesh). The silica gel sorbent had a particle size of 40 pm and a pore size of 60 A, as did the cyanopropyl-bondedsilica material used in the cyano column. The Florisil sorbent had a 100-pm particle size. All columns were packed with 1.0 g of sorbent and had a 6.0-mL solvent reservoir. The columns were constructed of polypropylene and the sorbent material was held in place by a two polyethylene fritted disks of 20-pm porosity. Chemicals and Reagents. The hexane, dichloromethme, and methanol used for fractionation were Ultrapure glass distilled
0003-2700/86/0358-0588$01.50/00 1986 American Chemlcai Society
