j. Soc. Cosmet. Chem., 31,237-252 (September/October 1980) Analysis of nitrosamines in cosmetic raw materials and finished product by high pressure liquid chromatography IRA E. ROSENBERG, JOHN GROSS, and TONY SPEARS, Clairol Incorporated, 2 Blachley Road, Stamford, CT 06902, and PETER RAHN, IVaters Associates, Milford, MA 01757. Received March 31, 1980. Synopsis The analysis of N-nitrosodiethanolamine (NDE1A) in di- and triethanolamine and selected alkanolamides of diethanolamine using both normal and reverse phase high pressure liquid chromatography is described. The possibility of extending the method to cosmetic finished products is demonstrated. Accuracy and precision of the methods, along with limits of detection, approach those achieved by a thermal energy analyzer. The use of a radial compression separation system for achieving increased baseline resolution is discussed. INTRODUCTION From the beginning of the early 1950's, nitrosamines have gained considerable scientific attention and, to date, a majority of those tested have been shown to cause cancer in laboratory test animals (1-4). In most areas where nitrosamines have been found, both the amine and nitrosating source were known. For example, nitrite, a preservative for the prevention of bbtulism when used in cured meat products, gave rise to low levels of nitrosamines. In the metal industry, sodium nitrite and di- and tri-ethanolamine are present in commercially available cutting fluids. The findings of Fan and co-workers that these formulations contained N-nitrosodiethanolamine (NDEIA) was a surprise only because the nitrosamines formed in a product with a basic pH (5). The scope of nitrosamine formation has been further enhanced by the finding that formaldehyde can catalyze the formation of NDEIA above pH 7 (6). These findings clearly indicated that nitrosation could occur in acidic, neutral, or basic media. In 1977, Fan and Fine presented data showing the presence of trace amounts of NDEIA in some cosmetics, creams, and lotions (7). These findings were significant for several reasons. First, although amines and their derivatives are important formula ingredients for the cosmetic chemist, nitrite or nitrosating agents are rarely used in cosmetics. Second, when preservatives which can liberate nitrite such as 2-bromo- 2-nitro-l,3-propanediol (Bronopol) were present, the cosmetic products contained high 237

238 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS levels of NDE1A. Recent studies indicate that Bronopol can act as a nitrosating agent for diethanolamine (8). Third, NDE1A was separated by LC and detected by a thermal energy analyzer which had previously only been coupled to a gas chromatograph to determine volatile nitrosamines. Numerous methods for the trace analysis of nitrosamines have been developed and published (9). However, the majority of nitrosamines studied were volatile and presented little separation problems from the matrix. The cosmetic chemists problem is more difficult since the non-volatile trace contaminant (NDEIA) in the cosmetic product is often in a multi-complex emulsion system. The complexity of a finished cosmetic formulation and possible trace levels of NDE1A, originating from the raw materials, indicate that the NDE1A analysis must start with the raw materials used to formulate these products. Since the cosmetic raw materials di- and triethanolamine can readily form NDEIA by nitrosation, the latter more slowly than the former, these raw materials were the first to be studied for nitrosamine contamination. NDEIA and other polar nitrosamines are very amenable to separation by reverse phase chromatography using either water or water/alcohol mobile phases (10). The water/al- cohol mobile phase provides good solubility for the raw material matrices eliminating the need to perform multiple isolation steps prior to analysis. The thermal energy analyzer's inability to handle mobile phases or samples containing water are alleviated by quantitating the nitrosamines by a UV detector. Rahn (10) has reported that NDEIA in the ethanolamines can be analyzed by ttPLC while Rosenberg and co-workers have reported on the analysis of NDE1A in Lauramide DEA by HPLC (11). The major difference in the two methods was the design of the UV detector employed for quantitation. The difference between a fixed wavelength and a variable wavelength detector will be detailed later in the discussion section. The data presented in this report is the result of a joint study between laboratories on the applicability of HPLC for the routine analysis of NDE1A. Both normal and reverse phase high pressure liquid chromatography coupled with UV detection for the isolation, identification, and quantitation of NDEIA in other cosinetic raw •naterials and a prototype product have been explored. The precision and accuracy of the method and the extension of the method into the part per billion (ppb) range is reported. It is our intention to also give the reader some insight as to how UV detection compares with that of a thermal energy analyzer for the quantitative analysis of polar nitrosamines such as NDE1A. EXPERIMENTAL A. APPARATUS AND REAGENTS The liquid chromatographic separations were performed on a Waters Associates Model 204, equipped with two M6000A Solvent Delivery Systems, a Model U6K Universal Injector, a Model 440 Fixed Wavelength Detector operated at 254 nm, a Model 660 Solvent Programmer, and a M730 Data Module or a Brinkman Model 2541 recorder. The Variable Wavelength Detector used was a Perkin-Elmer LC-75. Where indicated, a Waters Associates Radial Compression Separation System (RCSS) was