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Matrix-assisted laser desorption/ionization (MALDI) and fast-atom bombardment (FAB) mass spectrometry experiments were applied to the study of the early stages of the oligomerization reaction of dopamine with mushroom tyrosinase. Ultrafiltration was employed to remove the enzyme at various reaction times, to prevent possible attachment of the protein to the highly reactive intermediates. Two sets of five samples each, obtained at different reaction times, in one case immediately lyophilized and in the other left to react under an oxygen stream for 24 h before lyophilization, were compared. FAB showed the presence of various species and of these, that at m/z 305 increased in abundance with reaction time in immediately lyophilized set of samples only. Accurate mass measurements and tandem mass spectrometric experiments indicated the structure of a dopamine protonated dimer for this ion. MALDI measurements showed that all samples were composed of clusters of oligomers differing in degree of oligomerization. Oligomerization increases with reaction time, resulting in the formation of species at 2643-2911 Da. These clusters in turn were formed of species with a different degree of oxidation, detected in both sets of samples.

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Both TT and FT levels have cyclic variations throughout the menstrual cycle, being lowest at mid-cycle (14-16 cycle days), concomitant with the highest LH and FSH concentrations, and higher before and after mid-cycle phase, coinciding with the lowest circulating LH/FSH levels. Since TT and FT levels in the plasma have cyclic changes, our study: (i) suggests that a consumption of serum serotonin precursors takes place concomitant with gonadotrophin release during menstrual cycle; (ii) may represent an in vivo model to investigate this relationship in women in different physiopathological conditions.

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Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry together with collisional activation (CA) experiments were applied to the study of the oxidation pathway of dopamine by tyrosinase. In order to prevent attachment of the protein to the highly reactive intermediates, ultrafiltration was employed to remove the enzyme at different reaction times. FAB, privileging molecular species formation, was successfully used for identification of transient intermediates and their relative concentrations with respect to time, directly in the reaction mixture. The presence of isobaric molecular species made chromatographic separation necessary. Further EI mass spectrometry and collision spectroscopy led to structural identification of pure components. Of these, dopamine-o-quinone, leucoaminochrome, and aminochrome semiquinone were characterized for the first time as real intermediates in dopamine melanogenesis, in agreement with previous hypotheses. This approach elucidated the pathway of dopamine melanogenesis.