Measurement of carbamoylating activity of nitrosoureas and isocyanates by a novel high-pressure liquid chromatography assay

Abstract

A new assay for carbamoylating activity using reversed phase liquid chromatography (RPLC) is described which offers several advantages over existing assays. A comparison was made of the extent of carbamoylation of the amino group of 5'-amino-5'-deoxythymidine (5'-AdThd) by chloroethyl isocyanate and a series of six nitrosoureas upon incubation (37°) at pH 7.4 in phosphate-buffered saline. Determinations can be made in less than 30 min, whereas more than 24 hr are required in the paper chromatography method of Wheeler et al. [ Cancer Res. 34, 194 (1974)]. There is a very high coefficient of correlation ( r = 0.986, N = 7, P < 0.001) between these two methods of analysis which allows compounds assayed by the new procedure to be compared to the data compiled through the earlier assay. The application of the new assay to the analysis of the rate of carbamoylating activity for these agents is presented, and a mechanistic basis for the marked differences among these structurally similar compounds is discussed.