N- zyxwv Nitrosodiethanolamine Revisited? zy Jan R. Andersen, Walther Batsberg, Lars Carlsen, Helge Egsgaard and Elfinn Larsen Chemistry Department, Rise, National Laboratory, DK-4000 Roskilde, Denmark Alexander Senning Department of Chemistry, University of Aarhus, DK-8000 Arhus C, Denmark N-nitrosodiethanolamine is believed to be a weakly carcinogenic chemical, and as it occurs widely-in consumer products for example-it may constitute a significant hazard to humans. However, the chemical evidence concerning the identity, purity and properties of N-nitrosodiethanolamine is incomplete, and this casts some doubt on the basis of the current interest in this substance. In the present paper a purification procedure of synthetic N-nitrosodiethanolamine based zyxwvut on high-performance liquid chromatography is given. Other frac- tionation procedures such as gas liquid chromatography, ambient pressure column chromatography and distillation are shown to be inadequate. The purity and identity of purified N-nitrosodiethanolamine is established by means of electron impact and field ionization mass spectrometry, including metastable defocusing and collision induced decomposition techniques. Furthermore, 'H and ''C nuclear magnetic resonance and, to a lesser extent, infrared and ultraviolet spectroscopy are used. Deuterium labelled analogues of N- nitrosodiethanolamine and the parent diethanolamine are employed in rationalizing the results obtained. INTRODUCTION During the last decade there has been increasing concern about the exposure of man to carcinogenic substances. Amongst these, the N-nitroso compounds are perhaps the best known and the most frequently referenced examples of such a class of related chemicals because of their apparent omnipresence.' Recently, however, a single compound belonging to this class, N-nitrosodiethanolamine (l), attracted much attention, as it was found as a secondary constituent in cuttin fluids,' as well as in cosmetics and skin-care products. zyxwvu 4 zyxwvut 0- CH2 zyxwvutsrqpo - CHz- OH 0 CHz-CH2-0H \ +/ \ / N=N ++ N-N \ CH2-CH2-0H CHz - CH2-OH \ zyxw 1 of the prepared materiaL7 Later works utilizing the same synthetic procedure have so far been unable to supply any conclusive evidence as to the properties of authentic 1. It is especially remarkable that 1, in contrast to structurally related nitrosamines (e.g. N- nitrosodiethylamine' and N-nitros~morpholine~), exhibits an extremely complicated 60 MHz 'H NMR ~pectrum.~ Nevertheless, this has been taken into account in identifying 1, without commenting on the relationship between the observed and the expected spectrum. Even the high resolution electron impact (EI) mass spectrometricdata obtained from 1 did not provide unequivocal proof of the structure, because the molecu- lar ion had only a relative intensity of 0.5'/0, and the fragmentation pattern was not immediately inter- pretati~e.~ With these thoughts in mind we conducted a study on the purification, and on the physicochemical properties of 1. Compound 1 is believed to be weakly car~inogenic,~ but in our opinion the identity and purity of 1 was never established in any detail. In connection with this a recent report suggested that it may not be 1, but rather its mono nitrite ester, that is responsible for the analytical results obtained earlier on cutting fluid^.^ Together with other unresolved questions-such as the risk of obtaining false positive indications with the thermal energy analyser often used to detect nitrosamines,6 and the possibility that the weak carcinogenicity being observed was actu- ally caused by a preparation contaminated with an extraneous carcinogen-this poses serious doubts as to the very basis of the current interest in 1. The original synthetic procedure, for example, yield- ing 1 as a yellow, undistillable oil, is lacking in detail, and the sparse characterization of the product-two ultraviolet absorptions and a crude elemental analysis- may leave questions concerning the identity and purity EXPERIMENTAL Sample preparation and purification N-Nitrosodiethanolamine (1) was prepared as described by Pre~ssman.~ The crude product was purified by means of high performance liquid chromatography (HPLC), see Fig. 1, as no other method available to us proved to be satisfactory. Vacuum distillation resulted in extensive degradation, and even the small fraction of distillate obtained was impure as judged by its 13C NMR spectrum. Normal column chromatography on silicagel was also inappropriate (13C NMR), and gas liquid chromatography on a non-polar column resulted in total degradation as indicated by on-line mass spectrometry. (A Perkin-Elmer F 11 gas chromatograph interfaced with an all glass two-stage jet separator to the mass spectrometer. A 1 m x 3 mm i.d. glass column packed 4- Abbreviation: TSP = trimethylsilylpropionate @ Heyden & Son Ltd, 1980 CCC-0306-042X/80/0007-0205$03.00 BIOMEDICAL MASS SPECTROMETRY, VOL. 7, NO. 5, 1980 205