Nicotine and cotinine adducts of a melanin intermediate demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Pigmentation is a major factor in the incorporation of many drugs into hair . In an attempt to elucidate potential mechanisms of drug-melanin interacti on, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deutera ted analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS d eterminations were carried out on each sample to provide a mean and standar d error for the masses of interest. Internal calibration allowed accurate m ass measurement of the products. A careful comparison of the spectra of sam ples prepared both with and without drug indicated the presence of masses c orresponding to the protonated drug, melanin oligomers, and nicotine or con tinine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). A dditional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and o bserving that the masses shifted accordingly. Structures of the adducts wer e further confirmed by select ion gating and postsource decay analysis.