Amphetamine adducts of melanin intermediates demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of hair as a matrix for the determination of a history of drug abus e is becoming increasingly widespread. Melanin has been shown to play a key role in the incorporation of drugs in hair. The mechanism of this incorpor ation and the nature of the interaction remains poorly understood. Cationic drugs, such as amphetamine, are thought to be ionically bound to melanin; however, their inextricability has led to the suggestion that they may be c ovalently bound to a great degree. Identification of covalent adducts remai ns elusive due to the insoluble polymeric nature of melanin. We succeeded i n identifying several such adducts by matrix-assisted laser desorption/ioni zation-time-of-flight mass spectrometry (MALDI-TOF) analysis of the product s of in vitro synthesis of melanin in the presence of amphetamine. Amphetam ine was incubated with L-DOPA and mushroom tyrosinase under a stream of oxy gen. After 1 h, a signal at m/z 281.1324 (n = 1, R = H) was observed. After 2 h, the major adduct mass visible in the spectrum was at m/z 470.1074. Th is appeared to be derived from the monodecarboxylation of a minor adduct at m/z 514.1245 (n = 2, R = CO2H). A totally decarboxylated adduct was also o bserved at m/z 426.1448 (n = 2, R = H). These were identified as amphetamin e adducts of indole quinones. Corroboration of their identity was obtained by observing the mass shifts with deuterated L-DOPA and amphetamine analogu es. Accurate mass measurements using the reflectron mode of the MS showed t hat the smaller adduct was within 14 ppm, and the larger adducts were withi n 70 ppm of their theoretical monoisotopic masses. Postsource decay experim ents agreed with our structural assignments.