NITROSAMINE DETERMINATION 221 Table I Comparison of Thermo Electron Corp.'s with Max Factor's Results. Thermo Electron Corp. Sample Results NDE1A Found Max Factor's Results Total Nitrosamine as NDE1A a 3200 ppb 2600 ppb b 1400 1900 c 180 270 d 60 40 bottle, 10 ml of ethylene dichloride are added. This is a "keeper" solvent. This is necessary to prevent a volume change of the color solution. Then we place an 8-cc vial containing 2 ml of color solution into the 4 oz. bottle. To the 4 oz. bottle 7 ml of 3% hydrobromic acid with 0.2% bromine in glacial acetic acid is added taking care none of this gets into the 8-cc vial. The bottle is quickly capped using a cap with a teflon or polyethylene liner. The volatile nitrosyl bromide or nitric oxide formed (3,5) diffuses over into the color vial. Thus we have concentrated our entire amount of isolated nonvolatile "nitrosamine" from the 2.5 gm sample into 2 ml of color solution. Walters has shown this reaction to be quite specific for nitrosamines (4). A recent paper by Fine shows that we may have either N-nitroso or C-nitroso compounds (5). However, he pointed out that C-nitroso compounds are so unstable in most cases that they have yet to be reported as present in the environment. We allow the unit to stand at room temperature in the dark for two days (optimum time found). The color vial is removed and its absorbance at 520-5 nm is measured. Blank and standards must be run daily. We have found the method successful on cosmetic products, raw materials, grilled hot dogs, cutting fluids, and beer. We have assayed samples sent to Thermo Electron Corporation and have found good correlation with their findings. Our technique suffers the same problem as Thermo Electron Corp's procedure in that loss of the nitrosamine does occur during the isolation procedure and we are working to improve this. The same procedure is used for "nitrite" determination: One gram of most raw materials is added directly to the 4 oz. bottle and you proceed from the point of adding the ethlyene dichloride as the "keeper" solvent. Eighteen to twenty samples plus standards can be run by one person in eight hours. Table II Example of Calibration Curve Using NDE1A Concentration of NDE1A (ug) Absorbance 0 .024 1 .185 2 .248 3 .362 4 .445 5 .613

222 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS DISCUSSION We have modified Walter's denitrosating solution, (i.e. 3% Hydrobromic acid in glacial acetic acid), when learned that: 1) aged solutions were yielding higher amounts of color 2) that bromine denitrosates nitrosamine (6). With this modification of adding 0.2% bromine to the hydrobromic acid in glacial acetic acid we increased our color or sensitivity 3-to 4-fold. We have not tried to identify what nitrosamine is actually present, but further work can be done with the sample after the color vial is removed since the sample has not been contaminated. The resulting amine(s) that are present probably can be identified by following Eisenbrand's procedure of forming either the heptafluorobutyryl or dansyl chloride derivative of the remaining amine(s) (7). REFERENCES (1) L. C. Thomas and G.J. Chamberlin, Nitrite Method I in Colorimetric Chemical Analytical Methods 8th ed., The Tintometer Ltd., Salisbury, England. p281 (1974). (2) I.E. Rosenberg, J. Gross, T. Spears, and U. Caterbone, Methodology development for the determination of nitrite and nitrosamines in cosmetic raw materials and finished products. J. Soc. Cosmet. Chem. 30, 127-135 (May/June 1979). (3) M. J. Downes, M. W. Edwards, T. S. Elsey, and C. L. Walters, Determination of a non-volatile nitrosamine by using denitrosation and a chemiluminescence analyser, Analyst 101, 742-748 (Sept. 1976). (4) E. M. Johnson and C. L. Walters, The specificity of the release of nitrite from N-nitrosamines by hydrobromic acid, Analytical Letter, 4 (6), 383-386 (1971). (5) I. S. Krull, E. U. Goff, G. G. Hoffman, and D. H. Fine, Confirmatory methods for the thermal energy determination of N-nitroso compounds at trace levels, Anal. Chem. 51, 1706-1709 (Sept. 1979). (6) R. F. Eizember, K. R. Vogler, R. W. Souter, W. N. Cannon, and P.M. Wege II, Destruction of Nitrosamines. Treatment of nitrosamines with various acids and halogens, j. Org. Chem. 44 (5), 784-786 (1979). (7) G. Eisenbrand, Determination of volatile nitrosamines at low levels in food by acid-catalysed denitrosation and formation of derivatives from the resulting amines. IARC Scientific Publication No. 3, P. Bogovski, R. Preussmann, E. A. Walker and W. Davis, Eds., International Agency for Research on Cancer, Lyon (1972) pp 64-70.