Horizontal linear paper chromatography was utilized to achieve the semiquantitative separation of adrenochrome-adrenolutin mixtures. Data obtained by the spectrophotofluorometric assay of bands eluted from these chromatograms show evidence of the formation of a 1:l complex of the two components.
s A FACET of a continuing study of the photo-A degradative mechanisms of epinephrine, chromatographic separations of its degradation products were undertaken with a view toward quantitative estimation of the various components present. Heacock and his group uscd an adsorption method to separate the oxidative degradation products of epinephrine (1). Heacock and Mahon reported the separation of adrenochrome, adrenolutin, and 5,Gdihydroxy-N-methylindole on acetylated paper (2). The accepted mechanism of oxidation and subsequent degradation of epinephrine was first elucidated about 19.50 (3-G). An expanded scheme has been published by Harrison (7).
Adrenolutin, one of the degradation products, is characterized by an intense apple-green fluorescence. This fluoresence has been utilized as the basis for the assay of epinephrine, adrenochrome, or adrenolutin (8-1 I).
EXPERIMENTAL
Adrenochrome Preparation-Adrenochrome was prepared by the silver oxide oxidation of epinephrine, using a modification of the method of Somkaite (12), which was derived from that of Sohotka and Austin (13). Oxygen was displaced from the adrenochrome solution during crystallization by placing a small piece of dry ice in the solution. In order to minimize the tendency of moisture from the air to condense in the -40' solution, a cover filled with dry ice was placed over the container holding the crystallizing solution, m.p. 125" dec.' Adrenolutin Preparation-All operations were performed under a carbon dioxide atmosphere using solutions prepared with doubly distilled carbon dioxide saturated water unless otherwise stated. These solutions ranged in concentration from 0.05 to 1.0 mg./ml.
An amount of adrenochrome equivalent to the concentration of adrenolutin desired was placed in a carbon dioxide filled 10-ml. flask. Nitrogen saturated 0.05 N tertiary sodium phosphate solution