Thermospray liquid chromatographic-mass spectrometric analysis of mutagenic substances present in tryptophan pyrolysates

During protein pyrolysis, as can occur when broiling meat or fish, mutagenic substances are formed, as shown by in vitro mutagenicity assays. Some of the most active compounds have been shown to originate from tryptophan (Trp). Hundreds of grams of Trp had to be used previously to study the formation of these compounds by classical separation and detection methods. Studies have been made of the formation of two active heterocyclic amines, 3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by heating Trp at different temperatures and for different periods at time. Advantage was taken of the high selectivity and sensitivity of thermospray liquid chromatography-mass spectrometry coupling, which permitted the use of much smaller amounts (10 g) of starting material. These conditions permit a more accurate control of the pyrolysis temperature and the method of extraction can be shortened and simplified. The results show that Trp-P-1 and Trp-P-2 were already formed at 250°C. These substances were detectable in the low ppb range, i.e., less than the threshold levels necessary to elicit a positive response in the Ames test under standard conditions.