Dehn D L, Claffey D J, Duncan M W, Ruth J A
Program in Molecular and Environmental Toxicology and Biochemical Mass Spectrometry Facility, School of Pharmacy, The University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Chem Res Toxicol. 2001 Mar;14(3):275-9. doi: 10.1021/tx000205l.
Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, 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 deuterated 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 determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis.
色素沉着是许多药物掺入毛发的一个主要因素。为了阐明药物与黑色素相互作用的潜在机制，在烟碱（我们已证明其与黑色素有大量相互作用）和可替宁（烟碱的主要代谢产物）存在的情况下体外合成黑色素。真黑素的前体左旋多巴被酪氨酸酶氧化并寡聚化。以10:1的左旋多巴与药物比例将烟碱、可替宁和/或它们的氘代类似物加入到寡聚化反应混合物中。60分钟内形成黑色沉淀。在60、120和360分钟时从孵育混合物中取出等分试样。对每个样品进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）测定，以提供感兴趣质量的平均值和标准误差。内部校准允许对产物进行精确的质量测量。对有药物和无药物制备的样品光谱进行仔细比较，结果表明存在与质子化药物、黑色素寡聚物以及单体黑色素中间体多巴醌（DOPAQ）的烟碱或可替宁加合物相对应的质量。通过在反应中使用烟碱和左旋多巴的氘代类似物并观察到质量相应移动，为药物-黑色素加合物的存在提供了额外支持。通过选择离子门控和源后衰变分析进一步证实了加合物的结构。
