Teflon column packed with 1 Silicone Gum Rubber SE-30 on Gas Pack F (60/80 mesh) at a temperature of 121 "C. The injection port temperature was 176 "C and the TC detector was at 223 "C. Helium flow rate was 100 ml/min. The single peak eluted in 225 sec. In the case of mixed Zr and Hf alkoxides, one peak was always obtained no matter what ratio of compounds was used, indicating very similar volatility and sensitivity to the thermal conductivity detector. The two chromatograms show CC14 peaks because it was necessary to rinse the hypodermic syringe with solvent between injections to prevent clogging after repeated use. A small amount of CC14remained in the needle after rinsing, producing a noticeable peak on each injection.
An attempt is being made in our laboratory to synthesize selected Zr- and Hf-alkoxides with sufficient differences in volatility for the purpose of separating Zr and Hf by capillary column gas chromatography. ACKNOWLEDGMENT The authors thank Mrs. Peggy Wifall for her contribution to the gas chromatography of the alkoxides, Daniel Dyer and Lee D. Smithson of the Analytical Branch of the Physics Division, AFML, for help in obtaining the NMR and-mass spectra, respectively,and Mrs. JeanneGwinn for typing the manuscript.
RECEIVED for review December 12, 1968. Accepted June 3, 1969.
Gas Chromatography of Catecholamine Metabolites Using Electron Capture Detection and Mass Spectrometry Erik Anggird and Goran Sedvall Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden
Conditions were studied for the gas chromatography of normetanephrine, metanephrine, J-methoxytyramine, 3-methoxy-4-hydroxyphenyl ethylene glycol, vanillylmandelic acid, and homovanillic acid with electron capture (EC) detection and mass spectrometry. Pentafluoropropionates (PFP) and heptafluorobutyrates were more stable and had higher EC-responses than the corresponding trifluoroacetates. These derivatives were also used to separate the amines on XE-60 and the deaminated metabolites on OV-17. The linear range with the 63NiEC-detector was 0.02-0.8 ng for the PFP-derivatives. Mass spectra were obtained from the perfluoroacylated, trimethylsilylated, and acetylated metabolites.
IN HUMANS the major metabolic pathways for epinephrine, norepinephrine, and dopamine is 0-methylation to metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) and deamination to give vanillylmandelic acid (VMA), homovanillic acid (HVA), and 3-methoxy-4hydroxyphenyl ethylene glycol (MOPEG) ( I , 2). The major separation problems in the gas chromatography of these compounds have been solved by use of various phases and derivatives (3-12). These conditions have been suitable (1) J. Axelrod, Pharmacol. Reu., 39, 751 (1959). (2) I. J. Kopin, Aizesthesiolog.,29, 654 (1968). (3) N. P. Sen and P. L. McGeer, Biochem. Biophys. Res. Comm., 13, 390 (1963). (4) S. Lindstedt, Clin. Chim. Acta, 9, 309 (1964). ( 5 ) E. C. Homing, M. G. Homing, W. J. A. Vanden Heuvel, K. L. Knox, B. Holmstedt, and C. J. W. Brooks, ANAL. CHEM., 36, 1546 (1964). (6) P. Capella and E. C. Homing, ibid., 38, 316 (1966). (7) S. Kawai, T. Nagatser, T. Imanari, and Z . Tamura, Chem. Pharm. Acta, 10, 193 (1964). (8) C. M. Williams and M. Greer, Clin. Chim. Acta, 7,880 (1962). (9) M. G. Homing, K. L. Knox, C. E. Dalgliesh, and E. C. Horning, Anal. Biochem., 17,244 (1966). (10) H. G. Homing, A. M. Moss, and E. C. Homing, Biochim. Biophys. Acta, 148, 597 (1967). (11) C. E. Dalgliesh, E. C. Homing, M. G. Homing, K. L. Knox, and K. Yarger, Biochem. J.,101, 792 (1966). (12) M. Greer, T. J. Sprinkle, and C. M. Williams, Clin. Chim. Acta, 21,247 (1968). 1250
0
ANALYTICAL CHEMISTRY
for work with ionization detectors. Lately the high sensitivity of the electron capture (EC) detector has been used for the analysis of urinary VMA, dopamine, and MOPEG as their trifluoroacetates (13-15). Also pentafluoropropionates and heptafluorobutyrates of related compounds have been prepared and reported to give high EC-responses [16-18). An important recent advance in this field was the development of a new acylating reagent, heptafluoroimidazole (19). The present investigation is focused on the analysis of MN, NMN, 3-MT, VMA, HVA, and MOPEG using gas chromatography with electron capture detection and mass spectrometry. It attempts a systematic study of the formation and stability of perfluoroacyl derivatives of these compounds and a comparison of their EC-responses. Conditions for their gas chromatographic separation were also studied. Further, we investigated the mass spectrometric properties of several derivatives of these compounds to get the basic information for their identification with the combined gas chromatograph-mass spectrometer. EXPERIMENTAL Preparation of Derivatives. The catecholamine metabolites were purchased from the following commercial sources; HVA and 3-MT from Sigma; VMA from K & K Laboratories; NMN and MN from Winthrop Laboratories; MOPEG was from Calbiochem.
(13) S. Wilk, S. E. Gitlow, M. Mendlowitz, M. 3. Franklin, H. E. Corr, and D. D. Clarke, Atzal. Biochem., 13, 544 (1965). (14) D. D. Clarke, S. H. Wilk, S. E. Gitlow, and M. J. Franklin, J. Gas Chromatog., 5,307 (1967). (151 S. Wilk. S. E. Gitlow, D. D. Clarke, and D. H. Paley, Chi. Chim. Acta, 16, 403 (1967). (16) S. Wilk, S. E. Gitlow, M. J. Franklin, and H. E. Corr, ibid., 10, 193 (1964). (17) D. D. Clarke, S. Wilk and S . E. Gitlow, J . Gas Chromatog., 4, 310 (1966). (18) S. Kawai and Z. Tamura, Chem. Pharm. Bull. (Tokyo), 16, 699 (1968). (19) M. G. Homing, A. M. Moss, E. A. Boucher, and E. C. Horning, Anal. Letters, 1 (3, 311 (1968). ~
Table I. Methylene Unit Values for Derivatives of Catecholamine Metabolites on Different Liquid Phases Derivative 3% SE-30 3z UCW-98 3z OV-17 3% XE-60 Compound 15.90 17.28 28.25 TFA 15.91 NMN PFP 16.15 16.09 16.19 26.11 17.11 16.30 25.60 HFB 17 21 16.36 17.50 25.43 TFA 16.38 MN 16.60 16.52 23.41 PFP 16.67 HFB 17.68 17.53 16.70 22.99 16.10 18.46 27.33 TFA 16.08 3-MT 16.26 17.75 25.99 PFP 16.27 17.04 . 17.90 25.81 HFB 17.09 15.05 16.28 20.99 TFA 15.05 VMA-Me 15.38 15.75 20.16 PFP 15.42 16.22 15.94 20.18 HFB 17.20 TFA 14.98 14.94 17.19 20.83 HVA-Me PFP 15.20 15.19 16.96 20.52 15.68 17.16 20.65 HFB 15.69 15.03 21.22 TFA 14.41 ... MOPEG 14.55 20.40 PFP 14.98 ... I
HVA and VMA were dissolved in methanol and converted to the methyl esters with diazomethane. The diazomethane was removed immediately after addition by a stream of nitrogen. Esterification under these conditions is instantaneous. Longer exposure to CH2N2 leads to methylation of phenolic hydroxyl groups (9). MN, NMN, 3-MT, MOPEG, and methyl esters of VMA and HVA were dissolved in redistilled ethyl acetate and an equal volume of the appropriate perfluoro acid anhydride (K & K Chemical Co. and Pierce Chemical Co.) was added. After 15 minutes at room temperature the reagent was removed with a stream of nitrogen and the derivative was dissolved in ethyl acetate. The structures of all derivatives were confirmed by mass spectrometry as described below. Trimethylsilyl ethers (TMSiO) of VMA-Me and HVA-Me were prepared by adding 50 p1 of bis(trimethylsily1)acetamide (BSA, Applied Science Laboratories) to the esters dissolved in tetrahydrofuran at 37 "C for 15 minutes. The reagent mixture was injected directly into the column. Acetylation was performed by dissolving the methyl esters of VMA and HVA in equal volumes (50 pl) of dry pyridine and acetic anhydride. The deuterated acetic anhydride was supplied by Merck, Sharp, and Dohme, Canada. Instrumental Conditions. Two instruments were used for the gas chromatographic analyses. A Varian Model 204 gas chromatograph was connected with one column to a flame ionization detector and the other column to an electron capture detector with 250 pC tritium as the electron source. Columns were of 6-ft X l/s-inch glass packed with 1% SE-30, 1% OV-1, or 1% XE-60 on 100-120 mesh Gas Chrom Q (Applied Sciences). Nitrogen was used as carrier gas. The flow rate was 25-30 ml/minute and the pressure was 3 kg/cm2. Alternatively, an F & M Model 402 gas chromatograph was used. The instrument had two columns, one connected to a hydrogen flame ionization detector and the other to a 63Nielectron capture detector. The carrier gas on the column to the flame ionization detector was nitrogen with a flow rate of about 50 ml/minute and a pressure of 3 kg/cm2. The carrier gas through the column connected to the EC-detector was 10% methane in argon. The "Ni EC-detector was connected to a pulse generator operated with a pulse interval of 50 pseconds. The columns were 6-ft X 4-mm 0.d. glass packed with 3y0 of the liquid phases on 100-120 mesh Gas Chrom Q. For all columns the support was coated with the liquid phase using the fluidization technique (20). Columns were conditioned at maximum temperature for 24 hours. (20) R. F. Kruppa, R. S. Henley, and D. L. Srnead, ANAL.CHEM., 39,851 (1967).
Gas Chromatography-Mass Spectrometry. The derivatives were injected in a LKB Model 9000 combined gas chromatograph-mass spectrometer (21), equipped with a 1% SE-30 column. The carrier gas (helium) had a flow rate of 30 ml/minute. Temperatures were: column 130-170 "C, flash heater 250 "C,molecule separator 260 "C, ion source 290 "C. The ionizing current was 60 pA and the energy of the electrons 22.5 eV. Background spectra were recorded for each occasion and the abundance of background peaks was subtracted from those at the same m/e value in the mass spectra of the analyzed derivative. Determination of Methylene Unit Values. Methylene unit values (9, 11, 22) were calculated by plotting the log retention time of each derivative against the log retention time of two simultaneously injected alkanes having retention times shorter and longer, respectively, than the compound to be characterized. Each value is the mean (Std. dev. i. 0.02) of three injections. Deterniination of Detector Responses. Flame ionization detector (FID) responses and electron capture (EC) responses of the different derivatives were determined relative to an internal standard of lindane (hexachlorocyclohexane). A solution containing both the derivative and lindane in a concentration of 1 mg/ml in ethyl acetate was used to determine the FID response. The same solution diluted 10,000 times (0.1 pg/ml) with redistilled dry ethyl acetate was used to evaluate the EC-response. The EC-response is expressed relative to lindane after correction for the different FID responses of the derivatives. Peak areas were calculated with the use of a Disc integrator or by planimetry. RESULTS AND DISCUSSION Choice of Stationary Phase. Columns were prepared with silicone phases of different selectivity. SE-30 is a nonpolar methylsiloxane. UCW-98, containing methylvinyl groups, was judged to be slightly more selective. OV-17 and XE-60, being phenyl methylsiloxanes and cyanoethylsilicones respectively, are still more polar. Thinly-coated supports (1-3z) were used for high column efficiency (500-700 theoretical plates/foot) and low bleed rate. The separations of trifluoroacetates (TFA), pentafluoropropionates (PFP), and heptafluorobutyrates (HFB) of MN, NMN, 3-MT, VMA-Me, HVA-Me, and MOPEG are sum-
(21) R. Ryhage, ibid., 36,759 (1964). (22) W. J. A. Vanden Heuvel, W. L.,Gardiner,and E. C. Homing, ibid., p 1550. VOL. 41, NO. 10,AUGUST 1969
1251
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Figure 2. Separation of 0.2 ng each of MOPEG, HVA-Me, and VMA-Me
Figure 3. Separation of MN, NMN (0.2 ng), and 3-MT (0.4 ng) as the PFP-derivatives
Conditions: 3% OV-17, 150 "C, 63Nielectron capture detector
Conditions: 3 % OV-17, 140 "C, 6aNielectron capture detector
Conditions: 3% XE-60, 170 "C, 63Ni electron capture detector
marized in Table I. The retention times are expressed as iheir methylene unit (MU) values ( 9 , I I , 22). As expected, the metabolites had increased retention times on the more polar phases relative to the alkanes used as references. With the nonselective phases the elution order was TFA, PFP, and HFB. Using the OV-17 and especially the XE-60 columns the elution pattern was reversed inasmuch as the PFP and HFB derivatives appeared before the TFA. On SE-30 and UCW-98 good separations between the N M N and MN derivatives were obtained. NMN and 3-MT however appeared together. The derivatives of VMA and HVA did not separate appreciably on either SE-30 or UCW98. Table 11. Electron Capture Responses of Perfluoroacylated Derivatives of Catecholamine Metabolites EC-Response (relative to Compound Derivative lindane = 1) NMN TFA 0.17 PFP 0.62 HFB 0.82 MN TFA 0.30 PFP 0.56 HFB 0.74 3-MT TFA 0.15 PFP 0.62 HFB 0.84 VMA-Me TFA 0.32 PFP 0.47 HFB 0.48 HVA-Me TFA 0.07 PFP 0.24 HFB 0.25 MOPEG TFA 0.52 0.87 PFP Phenylethylamine PFP 0.05 HFB 0.09 Phenylethanolamine PFP 0.55 p-Hydroxyphenylethyl amine PFP 0.47 p-Hydroxyphenylethanol amine PFP 0.82 1252
ANALYTICAL CHEMISTRY
The OV-17 column achieved only partial separation of the NMN and M N derivatives. These compounds were, however, well separated from 3-MT (Figure l). VMA-Me, HVA-Me, and MOPEG also separated well on this phase (Figure 2). Somewhat better results were obtained with the PFP and HFB derivatives as compared to the TFA (Table I). XE-60 was the best column for the separation of the perfluoro acyl derivatives of NMN, MN, and 3-MT. Adequate separations were obtained with all the derivatives, the TFA and PFP, however, being somewhat better than the HFB. Figure 3 shows the separation of 0.2 ng each of the PFP derivatives on a XE-60 column using an electron capture detector. For VMA and HVA derivatives, marginal base line separations were obtained on this phase. In conclusion, we would recommend the use of XE-60 columns for the separation of NMN, MN, and 3-MT, preferably as the TFA or PFP derivatives. The best separations of NMN and 3-MT or MN and 3-MT were obtained as PFP and HFB derivatives on OV-17. The methyl esters of VMA and HVA separate best on OV-17 as the PFP or HFB derivatives, MOPEG-PFP separates from the corresponding derivative of all the other compounds on both OV-17 and XE-60. Our results relate well with those of Kawai and Tamura (18) who report good separation of NMN-TFA and MN-TFA on GE-XF 1105, another nitril silicon phase. Evaluation of Electron Capture Detector Responses. The electron capture (EC) responses of the perfluoroacylated derivatives were determined relative to lindane. The results are summarized in Table 11. The EC-responses for all derivatives were very good, confirming the work by Clarke et a1 (I 7) who showed that perfluoroacylated phenolic amines possess good properties for EC-detectors. Our results give, in addition, comparison between TFA, PFP, and HFB derivatives of NMN, MN, 3-MT, MOPEG, VMA-Me, and HVA-Me. For each of these compounds the magnitude of the EC-response increased
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in the order TFA < PFP < HFB. The differences were, however, not as marked as could be expected from the ratios of electronegative atoms (3 : 5 :7). N o great differences in the EC-responses were found between the same derivatives. The order of sensitivity-e.g., for PFP-derivatives were MOPEG > MN, NMN, and 3-MT > VMA > HVA. Substitution on the hydroxyl groups seemed to be the most important factor for the EC-response. This was demonstrated by studying the EC-responses of the PFP derivatives of phenylethylamine, p-hydroxyphenylethylamine (tyramine), phenylethanolamine, and p-hydroxyphenylethanolamine (Table 11). The three latter derivatives gave EC-responses comparable to those of the investigated catecholamine metabolites, whereas the former derivative, possessing only a primary amino group, gave a much smaller response. The sensitivity of the perfluoro acyl derivatives of the catecholamine metabolites to the EC-dectector is such that amounts down to 0.02 ng can be measured, especially using the more sensitive 3H detector. The chromatograms of 0.2-0.4 ng each of the PFP derivative of the metabolites are shown in Figures 1, 2, and 3. The areas of the peaks were
linearly related to the amount of injected derivative over a 30- to 50-fold range. This is exemplified in Figure 4. The linear ranges using PFP derivatives and 63Ni detector were for MOPEG 0.01-0.4 ng, for NMN, MN, and HVA 0.02-0.6 ng, for 3-MT 0.02-0.8 ng, and for HVA 0.02-1 ng. The use of trimethylsilyl ether-heptafluorobutyryl derivatives (TMSi-HFB) of catecholamines and related compounds was recently suggested for their analysis by gas chromatography with EC-detection (19). Our results (Table 11), as well as the earlier data by Clarke et a1 (17), show that the heptafluorobutyramides are relatively insensitive to the EC-detector as compared to the ester derivatives. The TMSi-HFB derivatives might, therefore, only have marginal advantages for use with the EC-detector, although they represent an elegant solution to the problem of preparing derivatives of the catecholamines. Stability of Derivatives. The stability of the derivatives was examined in two solvents, ethyl acetate and hexane. The response relative to an internal standard of an alkane was determined after 1 hour, one day, three days, and one week. The results are shown in Table 111. The TFA derivatives were relatively unstable. Even the simple process of removing the reagents under a stream of nitrogen at room temperature lead to a decrease in the response by about 50%. After three days very little was left of the original derivative. I n contrast, the PFP and HFB derivatives of the catecholamine metabolites could be concentrated under nitrogen without loss and were stable for at least three days in ethyl acetate. The same derivatives in hexane showed a more rapid degradation. The stability of all derivatives was also studied in dilute (1 pg/ml and 0.1 pg/ml) solutions of redistilled dry ethyl acetate using lindane or heptachlor as the internal standard. Essentially similar results to those shown in Table I11 were obtained. The instability of TFA esters is well known (23). It has, however, not been recognized that the PFP and HFB esters are much more stable. For this reason, as well as for the better EC-response, the latter derivatives should be preferred for the analysis of the catecholamine metabolites. (23) E. J. Bourne, C . E. M. Tatlow, and J. C . Tatlow, J . Chem. SOC., 1950, 1367.
Table 111. Stability of Perfluoro Acyl Derivatives in Hexane and Ethyl Acetate Ethyl acetate Hexane Compound derivative One hour One day Three days Seven days One hour One day Three days Seven days 6 NMN TFA 100 84 53 0 100 83 PFP 100 100 101 88 100 10 5 5 HFB 100 103 85 100 30 MN 10 TFA 100 7 100 85 4 PFP 100 104 86 100 99 42 HFB 100 104 100 100 76 3-MT TFA 100 95 96 100 60 30 36 PFP 100 100 100 100 96 HFB 100 96 98 100 98 38 VMA-Me TFA 100 92 56 0 100 100 PFP 100 100 97 66 100 72 HFB 100 100 90 20 100 98 67 20 HVA-Me TFA 100 102 10 100 86 73 PFP 100 99 93 72 100 106 82 3 HFB 100 101 100 104 78 47 '05 90 MOPEG TFA 100 89 78 16 100 99 84 ... PFP 100 103 100 102 97 100 98 90
VOL. 41, NO. 10,AUGUST 1969
1253
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Mass Spectrometry. TFA, PFP, and HFB derivatives of NMN, MN, 3-MT, MOPEG, HVA-Me, and VMA-Me were analyzed. Mass spectra of the acetates (AcO) and trimethylsilyl ethers (TMSiO) of HVA-Me and VMA-Me were also taken. Strong molecular ions were seen in the mass spectra of the perfluoroacyl derivatives of NMN, MN, and 3-MT (Figure 5). Other abundant fragments could be explained through cleavage a and fi to the nitrogen. For NMN and 3-MT the base peak was assigned to the elimination of NH2-acyl from the molecular ion. In derivatives of MN the /CH3 CHZ=N+ residue was present as the base peak. \acyl Abundant fragments in the spectra of both NMN and MN occurred at the same mje values. Thus the base peak in the NMN-PFP spectrum was at 458 mje corresponding to loss of NH2-PFP (Figure 5). With MN-PFP the elimination of NHCH3-PFP gives a strong peak, also at 458 mje. Similarly, abundant peaks are seen at 445 mje in both NMN-PFP and MN-PFP spectra. The base peak in the MN spectra and the abundant molecular ion in the NMN spectra should, however, distinguish the two compounds clearly from each other. The methyl esters of VMA and HVA were analyzed after conversion to the TFA, PFP, HFB, acetyl, and trimethyl silyl (TMSiO) derivatives. The results are shown in Figures 6 and 7. With the perfluoroacyl derivatives (Figure 6) the molecular ion was prominent, being the base peak in HVA and of about 6 0 z relative abundance in VMA. Other strong peaks were assigned to elimination of the carbomethoxy group and in HVA removal of the perfluoroacyl residue.
The mass spectra of the acetyl derivatives were characterized by peaks occurring through elimination of the carbomethoxy group and ketene. The latter fragment is the result of a hydrogen transfer from the acetyl to the remaining part of the molecule when the CH3C0 is split off. These mechanisms were demonstrated by comparison of spectra of acetates prepared with normal and deuterated acetic anhydride. In Figure 7 the spectra of hydrogen and deuterium acetates of VMA-Me are shown. Because the molecule has two hydroxyl groups and three deuteriums are added per acetoxy group, the mje value for the molecular ion is shifted from 296 to 302. The peak due to elimination of CH2=C=0 at 254 mje is shifted to 258 mje in the deuterated derivative. similarly, the fragment at 195 mje (see Figure 7) is increased by four. Together the spectra of H3-acetoxy-VMA-Me and Ds-acetoxy-VMA-Me established the mechanism of the hydrogen transfer in the elimination of the ketene molecule and in addition facilitated interpretation of the fragmentation patterns. The trimethyl silyl ethers (TMSiO) of VMA-Me and HVA-Me also gave well defined spectra. The molecular ion The fragmentation pattern was fairly prominent (9-16 was characterized by loss of the carbomethoxy and of TMSiO groups. In the spectra of perfluoroacylated MOPEG the molecular ion was present at the base peak (Figure 6). Other abundant fragments occurred after elimination of one molecule of perfluoro acid and from the substituted benzyl ion after cleavage of the bond between the carbons of the side chain. The prerequisite for the use of combined gas chromatography and mass spectrometry for the analysis of the catecholamine metabolites would be to find'derivatives which separate on the liquid phases which can be used in conjunction with
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VOL. 41, NO. 10, AUGUST 1969
1255
mass spectrometry. Moreover, the derivatives should give characteristic fragmentation patterns and preferably also an abundant molecular ion. Our results show that perfluoroacylated derivatives of the catecholamines have excellent properties for mass spectrometric analysis. Because fluorine is monoisotopic, abundant isotope peaks are not observed. This is an advantage for comparison with deuterium and 13C-labelled compounds which only give small increments in the nzje value. Furthermore the molecular ion was, in each case, an abundant fragment, usually over l o x , and for HVA-Me and MOPEG it was the base peak (Figure 6). Our results give the basic information for proceeding with the analysis of the catecholamine metabolites in several directions. First they can be analyzed with great sensitivity using an electron capture detector. For biological work they must, however, be fairly extensively purified prior to the GLC analysis. Second, studies on the metabolism of the catecholamines using precursors labelled with stable isotopes are facilitated by the development of methods by which the main metabolites can be examined by combined gas chromatography and mass
spectrometry (24). Identification of small amounts of these compounds in the presence of large amounts of impurities could also be made by the use of the mass spectrometer as a detector, focused on abundant fragments arising from catecholamine metabolites (25). Work is in progress to develop techniques along those lines for the analysis of catecholamine metabolites. ACKNOWLEDGMENT
The technical assistance of Miss Berit Holmberg is gratefully acknowledged. RECEIVED for review December 16, 1968. Accepted March 27, 1969. Study supported by grants from the Delegation for Applied Medical Defence Research No. 278, the Swedish Medical Research Council No. 14X-2211 and 14X-2291, and from the C. B. Nathhorst Foundation. (24) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, ANAL. CHEM., 38, 1549 (1966). (25) C. G. Hammar, B. Holmstedt, and R. Ryhage, Anal. Biochem., 25, 532 (1968).
Quantitative Determination of 9-Methylcarbazoles in Cigarette Smoke Dietrich Hoffmann, Gunter Rathkamp, and Stephen Nesnow Dicision of Encironmental Cancerigenesis, Sloan-Kettering Institute f o r Cancer Research, New York, N . Y . I002l
An analytical method for the determination of 9-methylcarbazoles in cigarette smoke is described. The nonvolatile particulate matter of the smoke collected in solvent is distributed between two pairs of soivents and the resulting concentrate is chromatographed on alumina and subsequently analyzed by gas chromatography. 9-Methyl-14C-carbazole was synthesized and served as internal standard. The mainstream smoke of an 85-mm U.S. nonfilter cigarette contained 103 ng of 9-methylcarbazole, 11.8 ng of 1,9-dimethylcarbazole, 19.9 ng of 2,9- and 3,9-dimethylcarbazole, and 5.7 ng of 4,9-dimethylcarbazole. 9-Ethylcarbazole was identified in a concentration of about 6 nglcigarette. The identification of 9-alkylcarbazoles in cigarette smoke represents their first isolation from a respiratory environment. The lack of biological data at this time does not permit a conclusion as to the possible biological significance of 9-methylcarbazoles in experimental tobacco carcinogenesis.
BIOASSAYS ON MOUSE SKIN with fractions of cigarette smoke condensate have established that the highest carcinogenic and tumor-initiating activity resides in the neutral subfraction BI (1, 2). This subfraction amounts to about 0 . 6 z of dry cigarette “tar” and contains polynuclear aromatic hydrocarbons, chlorinated hydrocarbon insecticides, indoles, and carbazoles (2). More recent studies indicated the presence of 9-methylcarbazoles in one of the biologically active subfractions of BI. Until now, 9-alkylcarbazoles have not been identified in the human respiratory environment (3-5). The 9-alkylcarbazoles differ significantly from other carbazoles in their absorption behaviour. In the method reported here, the alkylcarbazoles are enriched by distribution of the cigarette “tar” between two pairs of solvents and by chromatography on alumina. The resulting concentrate is separated 1256
ANALYTICAL CHEMISTRY
into individual components by gas chromatography; 9-methylIC-carbazole is employed as internal standard for the quantitative determination. EXPERIMENTAL Apparatus. A Perkin-Elmer gas chromatograph Model 800 with dual flame ionization detector was used for the qualitative analysis. The @-radiation was counted with a Nuclear Chicago Scintillation System 720. Ultraviolet absorbance measurements were made with a Cary Model 11 recording spectrophotometer. For the quantitative analysis, we smoked the cigarettes individually with a CSM-10 (Cigarette Components Ltd.) whereas for the isolation of N-alkylcarbazoles we employed a 30-channel automatic smoker with vibrating liquid trap (4, 6). The mass spectra were obtained with a Hitachi-Perkin-Elmer RMU-6D by the Morgan-Schaffer Corporation (Montreal, Canada). The energy of the bombarding electrons was kept at 70 eV. The elemental analyses were carried out by Spang Microanalytical Laboratory, Ann Arbor, Mich. Evaporations were completed at reduced pressure with water bath temperatures below 50 “C. The melting points were determined in sealed capillary tubes. (1) D. Hoffmann and E. L. Wynder, Proc. Am. Assoc. Cancer Res., 7 , 32 (1966). (2) E. L. Wvnder and D. Hoffmann, Science, 162,862 (1968). (3) R. L. Stkdman, Chem. Rea., 68,153 (1968). (4) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco Smoke, Studies in Experimental Carcinogenesis,” Academic Press, New York, N. Y . 1967. ( 5 ) D. Hoffmann and E. L. Wynder in “Air Pollution, Vol. 11” A. C. Stern, Ed., Academic Press, New York 1968, Chapter 20. (6) F. Seehofer, J. E. Miller, and H. Elmenhorst, Beitr. Tabakforsch., 3, 75 (1965).
