Effects of buffer composition on water stability of alkylating agents. The example of N-ethyl-N-nitrosourea

The half-life of N-ethyl-N-nitrosourea (ENU) in water solution was measured at pH 6.1, 6.8 and 7.3 using buffer systems characterized by differences in quality and quantity of the component species. The decay of the concentration of ENU as a function of time was detected both indirectly, by measuring its residual capability of inducing alkali-labile sites in CHO DNA, and directly, by measuring its residual concentration by means of 1H-nmr spectroscopy. Under controlled pH, temperature, and buffering conditions, the rate of hydrolytic loss of ENU measured by the biological and chemical experiments compared quite well, whereas at constant pH and temperature, it was substantially dependent upon the concentration of the buffer. This was attributed to the occurrence of general base catalysis promoted by the nucleophilic anions present in the buffer systems. This conclusion, largely supported by analysis of previously reported data concerning the kinetics of ENU in water, suggests that buffering conditions may play a role in determining the actual levels of reactive substrates in in vitro assays and, therefore, that different buffering conditions may lead to poorly reproducible results.