Two sensitive detection systems are described for the quantitative determination of a nonvolatile nitrosamine, nitrosoproline. One procedure involves denitrosation followed by derivatization of the amino product, proline, with 7-chloro4nitrobenzo-2-oxa-l,3-diazole (NBD-Cl). The highly fluorescent NBD-proline compound formed is then identified and quantitated by either thin-layer chromatography or high-pressure liquid chromatography(HPLC).
In the second system,the volatile methyl ester of the intact nitrosoproline is prepared, then detected by gas-liquid chromatography (GLC), and confirmed by combined gas-liquid chromatography and mass spectrometry (GLC-MS).
Both methods permit the quantitative detection of less than 10 ng of nitrosoproline. However, the HPLC fluorescence technique is approximately ten times as sensitive as the.GLC method.
LNTRODUCTION
Trace quantities of N-nitrosopyrrolidine (NPyr) have been consistently detected and confirmed in fried bacon'. Since this compound is carcinogenic to several species of test animals2, several studies have been carried out to identify the amino precursors involved in its formation3-5.
Kinetic data on the nitrosation of pyrrolidine in model systems6 and the fact that pyrrolidine is not normally found free in animal tissue, suggest that other precursors might be involved in NPyr production. Huxel et aZ.' reported that NPyr was formed at elevated temperatures from nitrite and proline. Pensabene et aL5 showed that the decarboxylation of nitrosoproline (NPro) yielded NPyr at temperatures cIose to those normally recommended for frying bacon. Proline could be a precursor for NPyr since it is a natural component of animal tissue. Bacon contains co. 2 % collagen (dry weight basis) and collagen is composed of more than 10% bound proline'. Gray and Dugan4 also suggest that collagen might be the precursor for NPyr in fried bacon. While NPro has been isolated and identified in uncooked bacons, at present there is no satisfactory quantitative method for its determination.