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PME RESEARCH LABORATORY, DECEMBER 1979 1 Project Title ANALYTICAL INVESTIGATDONS Period Covered DECEMBER 1979 Written By K. D. BRUNNEMANN ANALYSIS OF TOBACCO- SPECIFIC NITROSAMINES BY GC - TEA Instrumental The tubular oven built recently by J. Bourquin (1) has shown good performance so far. It has been observed that the detector response is most consistent when the cera- mic catalyst is operated at 450°C (2). Gas chromatographic conditions: 7ft xk" o.d., glass co- Lumn, 3'$ SP-2100 on Supelcoport 100-120, oven 175°C, inj. port 230°C, 40 ml He/min. Analytical Procedure a) Mainstream smoke (MS)1: 50 cigarettes are smoked (with an N2 flush every alternating,puff): into two gas wash bottles containing 100 ml ascorbate buffer each,; a Cambridge :____r paw is piace; between the wash bottles and the smoking machine. After every-10 cigarettes, the filter pad is exchanged and combined with the buffer solution. b) Si'destream,smoke (SS): Ten cigarettes are smoke&in- dividualliy in a sidestream cham::er with a"pushing;" air flow at 25 m1/sec. into two gas wash bottles containing 100 ml ascorbate buffer each; a Cambridge filter pad is placediafter the wash bottles. After smoking the Cambridge filter pa&is combined with the buffer solution. c) Tobacco : 25 g Tobacco are extracted overnight with 250 ml ascorbate buffer under shaking. d) Work-up : NNN - 14C is used as internal standard (84000 cpm, 450 ng). The work-up is similar to that of Hoffmann et al (3), with the foll'owing modifications: the CH2CL2 e:ctract was not dried over Na2SO4 but filtered through a C1inEliut column. The solvent used for the column chromatography was 1 % acetone in CH2C12. After eluting 100m1, the NNN - 19'C was detected. It eluted in approx. 200ml ; an additional 200ml were collected to in- clude the NNK. Before making to a final volume (usually 5m1),, dihexylnitrosamine was added to serve as internal standard for the GC. Aliauots were used to account for - 5 -

recovery. For the smoke samples, the first 100m1 eluted from the column contain the volatile nitrosamines. RESULTS AND DISCUSSION Figure 1 depicts gas chromatograms obtained for both to- bacco and~mainstream smoke of the Kentucky IRI reference cigarette. As expected:, both NNN and NNK are detected. Interestingly, nitrosoanatabine (NAtB) is as abundant as is NNN. This has aLso beemobserved in other studies for Fv-nerican (:3,4), French (4,5) and German cigarettes (6) although~no explanation has been found so far. Con- sidering that the major precursor for NNN is nicotine (7), it i~s not clear from where the NAtB derives since the typical tobacco alkaloid distribution reported (8) is nicotine (194,5 %), anatabine (2,7 %):, nornicotine (0,96 $)~ and anabasine (0y35 %). In order to evaluate our new method, we compared the Kentucky IRI! data with those of Hoffmann's group (3)) and Galliaher (9). Table L summarizes the data. Since we had only a qualitative trace of NAtB available to deter- mine the retention time, our data for NAtB are estimated, based on the same TEA response as for NNN. Table 1 Tobacco-specific Nitrosamines in IRI. A comparison. FTR' D. Hoffmann D. Briggs NNN 1.02 01.63 1.26 Tobacco NNK 0.54 01.13 0.60 (ppm) NAtB 1.2* 01.62 1.37 NN 0.17,0 01.390~ 0.298 MS NNK 0.120 01.160, 0.202 (,ug/cig.) NAtB 0.18* 01.5301 0.550 NNN 0.135 01.210, 0.165 . SS NNK - 0.240, 0.534. (,ug/cig.) NAtB - 0.190 0.148 * estimated: When analyzing other tobacco samples, we observe&NAtB only in Kentucky 2RI tobacco but not in ribs (Table 2). F - 6 -

, 1 I 1 1 Table 2 Tobacco-specific Nitrosamines in tobacco (ppm). Tobacco NNY NNK' NAtB 2RI 1.4 01.77 1.4* 3483'Ribs 7.3 4.8 not present 3508 Ribs 2.6 0.92 not present 8206 Ribs 2.0 - not present * estimated We also noted that if the Cambridge filter was not combi- ned withithe buffer, significantly lower values were ob- served (MS, IRI) or no nitrosamines were detected at all (SS,IRI). This does not exclude the possibility of pos- sible artefact formation on the filter pad. We pLan to persue this finding. In addition we attempted to use the new Thermosorb cartridge (10) for trapping NNN in SS. .. When extracted with 5ml CH2C12-Acetone 1:1, we detected no tobacco-specific nitrosamines at all. According to B. Spiegelhalder, (the European representative of Thermo Electron), the Thermosorb cartridge was not intended for non-volatile nitrosamines but rather to cover the vola- tili~ty range from DMN to ni!trosomorpholine (11). The recovery off NNN-14C for smoke samples ranged from 78'to 96 $; for tobacco extracts we recovere&from 47 to 75 %. Fin,: lly we explcred .inzther the volatile nitrosamines can be determined simoultaneously in smoke samples using the same work-up. We thus used the first 100m1 of the column fraction that eluted before NNN-14C. The data are summa- rized in Table 3. Table 3 Volatile Nitrosamines (ng/cig.) Kentucky lRl Sample DMN DPY MS, with Cambridge filter 9'.1 9.2 MS, without Cambridge filter 7.1 2.2 SS, with Cambridge filter 325 85 SS, without Cambridge filter 296 79' SS, with Thermosorb cartridge 221 4,6 Hoffmann (12)i reported 9.01 ng DMN and 6. 6 ng NPY for MS Ken- tucky IRI. We pian to investigate the efficiency of the ascorbate traps with Thermosorb:cartridges since we al- so obtain lower values for volatile nitrosami~nes when the Cambridge filter is not combined with the buffer solution. - 7 -

References PME Research Laboratory, Monthly Report, Oct. 1979, p.9 PME!Research Laboratory, Monthly Report, Nov. 1979, p.5 D. Hoffmann et al, Cancer Research, 39,2505 (1979) D. Hoffmann et al, 6th IARC Meeting, Budapest, 16-19 Oct. 1979 J.J. Piade et al;"33rd TCRC, Lexington, Ky, 29-31 Oct. 1979 C. RuhI et al, paper subm. to Beitr. Tabakforsch. S.S. Hecht et al, J. Natl. Cancer Inst., 60, 819, (11978) H. E•lmenhorst, Bu1L. Inf. Coresta, Abstr. S-13, p. 120 (1978). D. Briggs, Gallaher, personal communication Dec. 21, 1979' D. Fine et al, 6th IARC Meeting, Budapest, 16-19 Oct. 1979 B. Spiegelhalder, personal communication, Dec• 21, 19'79 D. Hoffmann et al, Cancer Research, 37, 3218 (1977) - 8 - ~;b

"Il i) !'J Ol I ~fr T 1 FIGURE 1. GC-TEA chromatograms of tobacco-specific nitrosamines. Left: Tobacco extract,lRl, right: Mainstream smoke, 1R1 - 9 -