ANALYST FEBRUARY 1986 VOL. 111 139 Prevention of Artifactual Formation of Nitrosamines During the Analysis of Baby Bottle Rubber Nipples Nrisinha P. Sen and Stephen W. Seaman Food Research Division Food Directorate Health Protection Branch Health and Welfare Canada Ottawa, Canada KIA OL2 and Santosh C. Kushwaha Scientific and Laboratory Services Division Product Safety Branch Department of Consumer and Corporate Affairs Ottawa Canada KIA OC9 It has been found that considerable amounts of nitrosamines may be formed as artifacts during the analysis of rubber nipples by a method that involves Soxhlet extraction of the samples with dichloromethane. The extent of such formation was monitored by incorporating morpholine as a marker amine and studying the formation of nitrosomorpholine which varied between 9 and 80 ng per analysis depending on the type of sample analysed and the brand of dichloromethane used.The problem could be minimised by pre-testing dichloromethane for its N-nitrosation potential and by incorporating propyl gallate an N-nitrosation inhibitor, in the method. Keywords Volatile nitrosamine determination; artifactual formation; gas - liquid chromatography - thermal energy analysis; rubber nipple analysis; propyl gallate During the past few years considerable effort has been spent by various researchers in developing sensitive and specific methods for the determination of trace levels of N-nitros-amines (mainly volatile nitrosamines) in baby bottle rubber nipples and pacifiers.l-4 The reason for this intense interest in this field stems from the fact that most of the nitrosamines detected in these products are potent carcinogens in labora-tory animals5 and that trace amounts of these chemicals can migrate to liquid infant foods or infant saliva thus posing a potential health hazard to infants using these products.For a detailed background on the subject the reader is advised to consult earlier publications on this topic. 1,276-8 Like all other trace analyses the analysis of rubber nipples for volatile nitrosamines is a complex and difficult task. The analyst has to pay attention to the usual problems such as developing efficient extraction and clean-up techniques, avoiding contamination from reagents and glassware and developing sensitive and specific detection methods.The situation is further complicated by the fact that some of the nitrosamines for which the analysis is carried out may be formed as artifacts during work-up of the samples or during final analysis (e.g. in the hot injector of the gas chromato-graph) of the extract by gas - liquid chromatography (GLC). This can happen because the rubber products themselves may contain the necessary precursors (amines and nitrosating agents) for nitrosamine formation or nitrogen oxide gases (NO,) present in the air can enter into the system and nitrosate the amines in the nipple extracts. For this reason, additional precautions have to be taken and safeguards have to be incorporated in the methods to minimise or eliminate such formation. Krull et al.9 discussed various ways of minimising artifactual formation during analysis and emphas-ised the importance of correctly determining the concentra-tion of carcinogenic nitrosamines in consumer products.The first evidence that artifactual formation of nitrosamines might pose a problem during analysis of rubber nipples was observed by Sen et a1.3 while comparing two different methods of analysis. It was noted that one of the methods that incorporated a nitrosation inhibitor and employed a simpler extraction technique (avoiding Soxhlet extraction) gave much lower values for nitrosamines in some samples than those obtained by another method2 that did not include any nitrosation inhibitor and employed Soxhlet extraction with dichloromethane. Although the latter method had been tested2 for artifactual formation by adding (after Soxhlet extraction) nitrite and morpholine to nipple extracts and analysing for possible formation of N-nitrosomorpholine it was felt that the test was inadequate because the nitrosamine precursors were not added at the start of the analysis.Therefore the possibility of artifactual formation in the latter method could not be completely ruled out; the need for further work was suggested. In this paper we present further evidence to indicate that artifactual formation of nitrosamines can indeed take place during the extraction of rubber nipples with dichloromethane. An improved method is suggested that greatly alleviates the problem. Experimental Apparatus A Model 502 thermal energy analyser (TEA) (Thermo Electron Corp.Waltham MA USA) coupled to a Varian VISTA 6000 gas chromatograph was used for the determina-tion of volatile nitrosamines. The gas chromatographic col-umns and operating conditions were similar to those reported previously.7 The TEA was operated in the GLC mode with a stainless-steel cold trap immersed in liquid nitrogen and a furnace temperature of 475 "C. The vacuum chamber pressure was ca. 1.8 Torr. Reagents and Standards Glass-distilled dichloromethane (DCM) was purchased from three suppliers. Of these two were sold as nitrosamine-free reagents and for convenience these will be referred to as brands A and C. The third (brand B) was of the regular glass-distilled variety and contained cyclohexene as a preser-vative. It is understood that the DCM of brands A and C did not contain any known preservative but they may have been processed by special techniques.As commercial DCM had previously been shown10 to be contaminated with N-nitroso-morpholine each bottle of DCM was redistilled from an all-glass apparatus and tested for nitrosamine contamination before use. All other reagents used were of analytical-reagent grade. To ensure the absence of nitrosamine contamination a reagent blank was used with each batch of new reagents and analysed by GLC - TEA. Highly activated basic alumina (IC 140 ANALYST FEBRUARY 1986 VOL. 111 Nutritional Biochemicals Cleveland OH USA) was pre-pared by heating overnight at 500 "C cooling in a desiccator and then storing in a stoppered flask. It was used without any addition of water for the purification of certain batches of DCM as will be discussed later.Ascorbyl palmitate and propyl gallate were obtained from ICN K & K Laboratories, Plainview NY USA and Eastman Chemical Products, Kingsport TN USA respectively. Morpholine was obtained from BDH Chemicals Poole UK. Dilute standards (ca. 10 pg ml-1 of each in ethanol) of a mixture of seven volatile nitrosamines and a separate standard of N-nitrosodipropylamine (100 pg ml-1) were purchased from Thermo Electron Corp. The mixture consisted of the following N-nitrosodimethylamine (NDMA) N-nitroso-diethylamine (NDEA) N-nitrosodipropylamine (NDPA) , N-nitrosodibutylamine (NDBA) N-nitrosopiperidine (NPIP) N-nitrosopyrrolidine (NPYR) and N-nitrosomor-pholine (NMOR) .The solutions were appropriately diluted with DCM before use. NoteReference to a brand or company name does not constitute endorsement by the Consumer and Corporate Affairs of Canada or by Health and Welfare Canada over others of a similar nature not mentioned. Samples All of the samples except one were purchased locally during October - December 1984 or procured by the Inspector of the Department of the Consumer Corporate Affairs. These are identified by capital letters (D H F etc.). Only a few samples of brand H from a recent (February 1985) production lot were obtained directly from the manufacturer. Those identified by lower-case letters (a e etc.) were left-over samples from an AOAC collaborative study in which we participated during June - July 1984.Caution-As all of the nitrosamine standards mentioned above are potent carcinogens extreme precaution should be taken when working with or handling the chemicals. Contact with skin or inhalation of vapour must be avoided. All solutions containing nitrosamines should be destroyed by a well established procedure11 before disposal. Analysis of Rubber Nipples Method 1 The method was essentially the same as that described by Havery and Fazio2 but later modified to include the addition of 2 g of barium hydroxide to prevent foaming during distillation. Basically it consisted of (a) overnight soaking of the cut nipple pieces with DCM followed by Soxhlet extrac-tion (b) distillation of the DCM extract from an aqueous alkaline solution (c) re-extraction of the aqueous distillate (containing the volatile nitrosamines) with DCM (d) concen-tration of the DCM extract to a small volume (ca.1 ml) using a Kuderna - Danish concentrator and a micro-Snyder column (for the final concentration step to 1 ml) and (e) GLC - TEA analysis of the final extract. In this study 1.0 ml of NDPA (10 ng ml-1) was added as an internal standard to each sample at the beginning of the analysis. This was carried out only to check the performance of the method; the final results were not corrected for percentage recoveries of NDPA. Also in some instances, 1 mg of morpholine (in 1 ml of DCM brand B) was added at the start to the sample as a marker amine to monitor the extent of artifactual formation of nitrosamines. Method 2 The method has been described in detail elsewhere.3 In summary it consisted in overnight extraction of the sample with DCM in the presence of 100 mg of ascorbyl palmitate as an N-nitrosation inhibitor rinsing of the nipple pieces using a special extraction technique that did not involve Soxhlet extraction concentration of the extract to 1 ml as in method 1 and final analysis by GLC - TEA.Testing the Effect of Different Brands of DCM on Nitrosamine Levels in Rubber Nipples as Determined by Method 1 As nitrosamine levels may vary from nipple to nipple such tests were always carried out in pairs using half of a nipple or using aliquots from a composite mixture of cut nipple pieces. Also everything else such as size of the distillation and Soxhlet flasks size of the Soxhlet extractor and other reagents was kept constant.The size of the apparatus was kept as small as possible so as to avoid an excessive reduction in volume of DCM from the Soxhlet extraction flask (at the bottom) that, otherwise could have caused overheating. The paired experi-ments were carried out simultaneously in the same fume hood. This ensured identical atmospheric conditions such as NO, levels that may have an influence on the formation of nitrosamines during Soxhlet extraction. A 1-ml volume each of NDPA internal standard and morpholine marker amine were also routinely added at the start to check the perfor-mance of the method and to monitor the artifactual formation of NMOR respectively. Prior to this the samples were analysed without any addition of morpholine to ensure the absence of pre-formed NMOR in them.Morpholine blanks (1 mg) were also run in the same manner with different lots and brands of DCM the only difference being that no nipples were present in these tests. The Soxhlet extracts in these instances were concentrated directly (omit-ting the aqueous distillation) and analysed by GLC - TEA. Testing the Effect of Propyl Gallate (PG) While carrying out this test all the factors including the DCM solvent were kept constant. Half of a nipple was analysed by the above method2 and the other half by the same method but with added PG (100 mg). The stoppered flask containing the nipple pieces PG and DCM was gently shaken (to aid in dissolving PG) overnight in the dark while the other (without PG) was allowed to sit in the dark without shaking as specified in the original method.2 The other steps were unchanged.Results and Discussion The data (Table 1) indicate that the levels of nitrosamines detected in a sample of rubber nipple by method 1 can vary widely depending on the type of DCM used for the analysis. The variation in results due to the use of different DCM could not all have been due to random errors because duplicate results (using brand B DCM) run under such controlled conditions usually were within 10%. Therefore the difference (considered to be significant if 3 _+30%) in the two sets of results in Table 1 was probably caused by artifactual forma-tion the extent of which varied from brand to brand of DCM and to a smaller extent from bottle to bottle of DCM within the same brand.This theory was also supported by the fact that there was a concomitant rise in the artifactual formation of NMOR from added morpholine for a particular DCM used for the analysis. As neither the morpholine nor the nipples (when analysed alone) contained any detectable amount of NMOR the NMOR detected in these experiments must have formed as an artifact. Similar conclusions could also be drawn from the results for the morpholine blanks (Table 2). Some typical chromatograms obtained from these experiments are shown in Fig. 1. Although the exact nature of the nitrosating agent(s) responsible for the artifactual formation of nitrosamines in the above method is not known it is well established that DCM is an excellent medium for N-nitrosation.12313 Both inorgani ANALYST FEBRUARY 1986 VOL. 111 141 Table 1. Effect of different brands of DCM on the levels of nitrosamines in various nipples and that of artifactually formed NMOR in the presence of added morpholine Brand of nipple e . . . . e . . . . b . . . . b . . . . e . . . . e . . . . H . . . . H . . . . H . . . . H . . . . H . . . . H . . . . H . . . . Blanks . . * . . . * . . . . . . . . . . . . . . . . . . . . . . . Brand of DCMused for analysis B C B A B A B A A passed through basic A purified by sulphamic alumina acid wash and alkali wash, then dried over anhydrous sodium sulphate B C C passed through basic A B or C (without added alumina morpholine) Nitrosamines detected in nipples/ I % kg-l NDEA,5.5 NDEA 11.7 Negative NDMA,55.3;NDEA,4.3; NDBA,4.8;NPYR,6.7; NMOR,3.5 NDEA,3.9;NMOR,2.3 NDEA 10.0; NMOR 2.2 NDMA 9 NDMA 83 NDMA 38.9 NDMA,8.2 NDMA,8.3 NDMA 19 NDMA,7.9 All negative * When analysis was carried out with 1 mg of added morpholine as a marker amine.t Not included. 10.9 74.3 41.8 11.5 11.8 23.5 11.2 NMOR Recovery of formed as NDPA internal an artifact*/ng standard % 9.2 -$ 67.8 -- 97.5 100 75 80 95.2 96.3 103 80.2 83.3 93.8 75.7 Table 2. Artifactual formation of NMOR from added morpholine in blank runs by method 1 NMOR formed from 1 mg of added morpholine/ng DCM A 30.6 47 > 60 11.5 (after special processing) * * See text.DCM B 16.9 22.9 41.8 14.5 14.5 12.4 DCM C 13.7 15.7 19.6 80.3 38.6 31.3 24.1 14.2 (after special processing) * 59.6 nitrite and NO gases can efficiently nitrosate secondary amines in DCM.12J3 The nitrosating agents could have originated in several ways. Firstly they could have been present in the nipples1.14 or formed as a result of thermal degradation during Soxhlet extraction. Transnitrosation by organic nitro or nitroso compounds present in the nipples might have been partly responsible for the artifactual formation of nitrosamines observed in this study. This was demonstrated by analysing (by method 1) a 5-g aliquot of cut nipple pieces (brand H) with 1 mg of added N-nitrosodiphenylamine a well known rubber curing ingre-dient,l4 and showing increased formation of NDMA during the analysis (10000 pg kg-1 compared with 13 pg kg-1 in the absence of added N-nitrosodiphenylamine) .This suggested that there was enough amine precursors present in the sample that reacted with N-nitrosodiphenylamine which is an excel-lent transnitrosating agent to produce the excess of NDMA. From the excessively high result obtained in the above experiment one would expect the formation of substantial amounts of NDMA even in the presence of much smaller t fn C 0 a 2? L c 0 al U c z I NDMA - 4 8 ’ NDMA 83 p.p.b. 96.3% ecovery II I I 4 8 1 Ti me/m in 98.4% recovery 0 I 0 4 8 1 2 Fig. 1. Typical chromatograms showing the effect of special process-ing of DCM on the results obtained by method 1.1 Nitrosamine standards; 2 a nipple (brand H) analysed using DCM of brand A (before special processing or clean-up); a total of 74.3 ng of NMOR was formed from the added morpholine marker amine; 3 direct injection of morpholine solution showing the absence of NMOR; 4, DCM of brand A (before special processing) reagent blank taken through all the steps of method 1 (without morpholine marker amine); and 5 above nipple analysed using specially processed (sulphamic acid and alkaline washes etc.) DCM from same bottle as above; only 11.5 ng of NMOR were formed. Percentage recoveries refer to those with added NDPA internal standard. For details see text and Table 142 ANALYST FEBRUARY 1986 VOL. 111 amounts (e.g.W100 pg) of N-nitrosodiphenylamine. Alter-natively NO and O2 in the air could have entered the system during Soxhlet extraction. According to Mirvish,13 nitrosation of certain amides by NO in DCM is about 30000 times faster than that by nitrous acid in aqueous solution. Therefore even trace amounts of NO gases in the air could form significant amounts of nitrosamines because the reaction is almost quantitative. The fact that NMOR could form in the absence of rubber nipples (e.g. morpholine blanks) led us to speculate that NO, gases in the air might be partly responsible for such artifactual formation. As the extent of such formation varied with different brands of DCM (run side by side) the existence of some other factors such as catalysts (e.g.trace amounts of HCl) or of other nitrosating agents in the DCM was a possibility. Other possible mechanisms include (a) participa-tion of certain transition metals (e.g. zinc dithiocarbamates are used as additives in rubber manufacture) as catalysts of N-nitrosation14.15 and (b) formation of amine - NO Drago complexes followed by oxidation by O2 (in the air) to the nitrosamines. 16 As reagent blank determinations (Fig. 1) carried out according to the protocol of method 1 cannot distinguish a good from a bad DCM (both give negative blanks) we have developed a procedure that allows one to pre-test the DCM. This is done by carrying out a morpholine blank determination as described under Experimental (Table 2). The greater the extent of NMOR formation in such a test the greater will be the chance of artifactual formation of nitrosamines during analysis of rubber nipples using the particular DCM.The best commercially available glass-distilled DCM tested gave a value of ca. 10 ng of NMOR in such tests. Therefore it is recommended that any DCM that gives a significantly higher value for NMOR formation (say >15 ng) should not be used for such analyses otherwise the results in extreme instances (e.g. sample H Table 1) could be inflated by as much as 950%. To our knowledge the possibility of this happening has not been investigated or reported previously. It should be emphasised that neither of the DCMs of brand A or C was of sub-standard quality. Both were of glass-distilled varieties and both were redistilled and tested to be nitrosamine free before use.Also both gave negative reagent blanks (Fig. 1D) when taken through all the steps of method 1. Therefore without a knowledge of the data presented here, an analyst would have no justification for rejecting them for use in the analysis of rubber nipples for nitrosamines. As all the DCMs were redistilled before use the responsible nitrosating agent or catalyst in the DCM must be volatile and should not be removed by distillation. Therefore alternative methods were developed to purify DCM. A 1-1 volume of brand C DCM (initially forming 25-30 ng of NMOR in the morpholine test) was purified by passage through a column containing 50 g of highly activated (activity I) basic alumina. The purified DCM on re-testing formed only 14 ng of NMOR.Similar treatment through alumina however was only partially effective for a sample of brand A DCM that initially formed >60 ng of NMOR in the morpholine test (Table 2). This DCM was further purified by shaking vigorously for 5 min in a separating funnel with 1% sulphamic acid (prepared in 0.5 M H2S04) back-washing with 1 M KOH solution and drying over anhydrous sodium sulphate . The treatment greatly improved the DCM which on re-testing gave a very low value of 11.5 ng of NMOR in the morpholine test (Table 2). These two specially purified DCMs were further tested for the analysis of rubber nipples and the results were compared with those obtained with unpurified DCM from the respective bottles (Table 1). The results (Table 1 nipple H DCM of brands A and C) clearly indicate a noticeable reduction in the artifactual formation of NMOR and also lower results for the nipples compared with those obtained with the corresponding unpurified DCM.The respective new (with purified DCM) results were also comparable to those obtained with the brand B DCM the best commercially available. The fact that it was possible to purify two poor lots of DCM and obtain results comparable to those obtained with a third brand (B) of good DCM lends further support to the conclusion that the results obtained by method 1 are subject to extreme variations depending on the quality of DCM used for the analysis. Therefore the importance of pre-testing DCM before starting the analysis cannot be overemphasised.Next the possibility of similar artifactual formation of nitrosamines that could be occurring even with the best DCM, i.e. brand B was investigated. In previous studies with cured meats,I7 fried bacon,17 beer18 and rubber nipples,3 research-ers have recommended incorporation of N-nitrosation inhibi-tors in the analytical protocols in order to minimise such formation. After some preliminary trials with various inhibi-tors (ascorbic acid ascorbyl palmitate a-tocopherol pyrrole and propyl gallate) propyl gallate (PG) was selected for this purpose because it gave the most consistent results. The results of several analyses in which duplicate halves of rubber nipples were analysed with or without PG are presented in Table 3. As can be seen from the data the inclusion of 100 mg of PG in method 1 gave much lower values for nitrosamines in most instances suggesting artifactual formation of nitros-amines in its absence.In a few instances this was also confirmed by adding morpholine as a marker amine (Fig. 2). PG also reduced the formation of NMOR. It should be emphasised that this phenomenon was not observed with all nipples tested (e.g. samples D F H6 and i). This was particularly noticeable with the most recent nipple of brand H (H6). Probably the nitrosamines in these instances were already present in the nipples or the necessary precursors were absent (as a result of the introduction of improved rubber curing practices). The extent of inhibition of nitrosamine formation in the presence of PG varied depending on the brand of DCM used and also with the type of nipple.Even with the best commercially available DCM (brand B) one could obtain a result that was 375% higher (Table 3 sample H4) if PG was omitted. In an extreme instance (e.g. sample H5 with brand A DCM) the difference was 700%. The addition of PG did not in any way affect the performance of the method nor did it affect the recoveries of added nitrosamines. In the presence of PG, the recoveries of all seven volatile nitrosamines (see Reagents and Standards) added to rubber nipples at ca. 20 pg kg-1 levels and also that of NDPA internal standard added to all samples were excellent (8&100./,). This ruled out the possibility of any loss of nitrosamines due to breakdown or interaction with PG. A few analyses were carried out (using brand H nipple) in which PG or N-nitrosodiphenylamine was added at various stages of method 1.The results suggest that in the absence of PG artifactual formation of nitrosamines can take place during both DCM extraction and alkaline distillation. As information regarding the detailed composition of various nipples was not easily available it was difficult to investigate the problem more thoroughly. Further work might be desirable to understand fully the mechanism of artifactual formation observed in this study. In a few limited instances the results obtained by the improved method 1 (with PG and pre-testing DCM) were compared with those obtained by method 2 (Table 4). They were in excellent agreement. In a previous study3 with rubber nipples it had been observed that method 1 (without PG) could give results higher than or comparable to (never lower than) those obtained by method 2 which included an N-nitrosation inhibitor.The findings presented in this paper have been helpful in explaining the reasons behind these discrepancies. The occasional higher results previously ob-served with method 1 were probably due to artifactual formation. This was also substantiated by the data in Fig. 3 ANALYST FEBRUARY 1986 VOL. 111 143 Table 3. Inhibition of artifactual formation of nitrosamines by PG during analysis of rubber nipples for volatile nitrosamines Brand of nipple * HI . . . . Hz . . . . H3 . . . . H4 . . . . H5 . . . . H6$ . . . . a . . . . . . a . . . . . . a . . . .. . F . . . . . . g . . . . * . i . . . . . . e . . . . . . d . . . . . . D . . . . . . Volatile nitrosamines detected DCM brand used for analysis Nitrosamine CLgkg-lt PG/pg kg- 1 t Amount by method 1/ Amount by method 1 with . . C NDMA 60 (81.3%) 13.6 (93.6%) . . c NDMA 78.1 (90.3%) 15.4 (96.9%) . . B NDMA 9.8 (86.5%) 4.0 (98.0%) . . B NDMA 13.5 (76.7%) 3.6 (76.7%) 107 (85%) 14.0 (93.8%) . . A NDMA . . B NDMA 2.0 (goo/,) 2.0 (85%) NDEA 1.5 1.5 NMOR 4.0 4.0 . . c NDMA 79.1 (85.4%) 10.3 (94.4%) NDMA 14.5 (92.2%) 5.9 (87.5%) . . B . . B NDMA 8.5 (100%) 3.6 (100%) . . B NDBA 181 (102.7%) 167 (89.3%) NPIP 39.4 36.3 . . B - Negative (86.7%) Negative (92%) . . B NDBA 81.9 99.4 . . B NDEA . . B NDEA 15.0 (81%) 5.6 (77%) 3.2 (75.6%) 2.6 (85%) .. B NDBA 87.7 (94%) 79.3 (94.5%) NPYR 13.7 12.3 NMOR 10.9 12.3 * Different subscripts indicate samples from different lots or different nipples from the same lot. t Figures in parentheses represent recoveries of NDPA internal standard. $ Obtained directly from the manufacturer in February 1985. Table 4. Comparison of results obtained by improved* method 1 with those obtained by method 2 NDMA detected/pg kg-17 Brand of Improved nipple Method 1 method 1 Method 2 a 8.5 (100%) 3.6 (100%) 3.3 (97%) a 14.5 (92.2%) 5.9 (87.5%) H 18.9 (89.2%) 5.1 (82.5%) 5.9 (97.5%) H$ 71.4 (82.1%) - 9.7 (98.5%) * Using brand B DCM and including 100 mg of PG. t Figures in parentheses represent percentage recoveries of NDPA $ Using brand C DCM. internal standard.( a ) NDMA 14. e 1.p.b. 92.2% ecovery NDMA 5.9 p.p.b. 4 8 12 0 4 8 Timelmin Fig. 2. GLC - TEA chromatograms (a) a nipple of brand a analysed by method 1 using DCM of brand B; (b) the same nipple analysed in the presence of PG. About 24.3 and 12.6 ng of NMOR were formed, respectively from the morpholine added in each instance (a) Method 2 I I 9.7 p.p.b. recovery u-0 4 8 12 1 6 0 4 8 12 Ti me/m in Fig. 3. GLC - TEA chromatograms of a ni ple (brand H see Table 4) analysed by (a) method 2 and (b) metgod 1 (with mor holine marker amine added in each instance) using DCM of brand 8. Both the NDMA levels detected in the nip le and the amounts of NMOR formed were higher with method 1. d e GLC column and conditions were slightly different from those in Figs.1 and 2 hence the slight difference in retention times which show increased formation of NMOR in method 1 compared with method 2. It has been previously shown3J8 that the use of a micro-Snyder column (instead of blowing down in a stream of nitrogen) for the final concentration from 4 to 1 ml and the use of a Graham condenser which is more efficient than a straight-jacket condenser during the distillation step can give consistently good recoveries of the volatile nitrosamines. It is therefore recommended that both of these modifications also be incorporated in the improved version of method 1. Finally it should be emphasised that this work should not be viewed as an undue criticism of method 1. The main objective was to determine the cause of variations in results produced by method 1 and to improve it.Basically it is a good method that needed refinement. With the modifications suggested above and those mentioned earlier (i. e. pre-testing DCM and including PG) the method would improve greatly 144 ANALYST FEBRUARY 1986 VOL. 111 and should give more precise and accurate results. Also the effect of PG on nitrosamine levels detected by method 1 should be tested for any new product not analysed before. This will offer a safeguard against any possible catalytic influence it might have on nitrosamine formation in the presence of new rubber curing agents. 1. 2. 3. 4. 5. 6. 7. 8. References Spiegelhalder B. and Preussmann R. ZARCSci. Publ. 1982, No. 41 231. Havery D.C. and Fazio T. Food Chem. Toxicol. 1982,20, 939. Sen N. P. Seaman S. W. Clarkson S. G. Garrod F. and Lalonde P. IARC Sci. Publ. 1985 No. 57 51. Billedeau S. M. Thompson H. C. Jr. Miller B. J. and Wind M. L. 98th Annual AOAC Meeting October 29-November 2 1984 Washington DC Abstract No. 252. Preussmann R. and Stewart B. W. in Searle C. E. Editor, “Chemical Carcinogens,” Second Edition ACS Monograph No. 182 American Chemical Society Washington DC 1984, p. 643. Babish J. G. Hotchkiss J. H. Wachs T. Vecchio A. J., Guttenmann W. H. and Lisk D. J. J . Toxicol. Environ. Health 1983 11 167. Sen N. P. Kushwaha S. C. Seaman S. W. and Clarkson, S. G. J. Agric. Food Chem. 1985,33,428. Osterdahl B.-G. Food Chem. Toxicol. 1983 21 755. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Krull I. S. Fan T. Y. and Fine D. H. Anal. Chem. 1978, 50 698. Eisenbrand G. Spiegelhalder B. Janzowski C. Kann J., and Preussmann R. ZARCSci. Publ. 1978 No. 19 311. Castegnaro M. Eisenbrand. G Ellen G Keefer L. Klein, D. Sansone E. B. Spincer D. Telling G. and Webb K. S . , in “Laboratory Decontamination and Destruction of Nitros-amines in Laboratory Wastes,” ZARC Sci. Publ. 1982 No. 43. Angeles R. M. Keefer L. K. Roller P. P. and Uhm S. J., ZARCSci. Publ. 1978 No. 19 109. Mirvish S . in Magee P N. Editor “Nitrosamines and Human Cancer,” Banbury Report No. 12 Cold Spring Harbor Laboratory New York 1982 p. 227. International Agency for Research on Cancer “IARC Mono-graphs on the Evaluation of the Carcinogenic Risk of Chem-icals to Humans The Rubber Industry,” IARC Lyon 1982, Chapter IV p. 89. Croisy A. F. Fanning J. C. Keefer L. K. Slavin B. W. and Uhm S.-J. IARC Sci. Publ. 1980 No. 31 83. Hansen T. J. Croisy A. F. and Keefer L. K. ZARC Sci. Publ. 1982 No. 41 21. Hotchkiss J. H. Libbey L. M. Barbour J. F. and Scanlan, R. A. IARCSci. Publ. 1980 No. 31 361. Sen N. P. Seaman S . and Bickis M. J. Assoc. Off. Anal. Chem. 1982 65 720. Paper A51236 Received July lst 1985 Accepted September 16th 198