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 abuse is becoming increasingly widespread. Melanin has been shown to play a key role in the incorporation of drugs in hair. The mechanism of this incorporation 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 covalently bound to a great degree. Identification of covalent adducts remains elusive due to the insoluble polymeric nature of melanin. We succeeded in identifying several such adducts by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) analysis of the products of in vitro synthesis of melanin in the presence of amphetamine. Amphetamine was incubated with l-DOPA and mushroom tyrosinase under a stream of oxygen. 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. This appeared to be derived from the mono-decarboxylation of a minor adduct at m/z 514.1245 (n = 2, R = CO2H). A totally decarboxylated adduct was also observed at m/z 426.1448 (n = 2, R = H). These were identified as amphetamine adducts of indole quinones. Corroboration of their identity was obtained by observing the mass shifts with deuterated l-DOPA and amphetamine analogues. Accurate mass measurements using the reflectron mode of the MS showed that the smaller adduct was within 14 ppm, and the larger adducts were within 70 ppm of their theoretical monoisotopic masses. Postsource decay experiments agreed with our structural assignments.