HPLC determination of N-nitrosomethylurea in biological fluids by reaction with 4-nitrothiophenol.

A simple and sensitive method for the determination of N-nitrosomethylurea (NMU) in biological fluids was developed. The principle is based on the formation of 4-nitrothiophenol methyl thioether (NTP-Me) from 4-nitrothiophenol (NTP) and diazomethane formed from MNU by adding alkali. After sodium hydroxide solution was added to the mixture of NMU in aqueous solution and diethyl ether, the aqueous layer was removed and NTP was added to the ether layer in which diazomethane had been trapped. NTP-Me thus formed was determined by HPLC. This method was successfully applied to the analysis of NMU in biological samples with some clean-up. Stomach and intestine contents and blood samples from guinea-pigs were cleaned up by use of a kieselguhr column followed by heptane-water partition. The recoveries of NMU from the stomach and intestine contents were about 95% and that of NMU from blood samples was about 80%. The detection limit of NMU was 0.5 nmol/g for the stomach contents and 1 nmol/g for the intestine contents and blood samples.