Identity of the “Pink Spot”

Abstract

A NUMBER of authors have now reported the occurrence of “pink spot” in the urine of schizophrenics¹. Its identification as β-3,4-dimethoxyphenylethylamine (DMPE) rests on paper chromatographic evidence and a melting point provided by Friedhoff and Van Winkle² together with the gas chromatographic evidence of Sen and McGeer³. The pink spot produced from DMPE by successive treatments with ninhydrin and Ehrlich's reagent is evanescent and some 5–10 µg are required on the paper after chromatography in order to be certain of its occurrence. Drug metabolites can. seriously interfere with its determination by this technique. We find that DMPE is readily detected by its reaction with formaldehyde on paper⁴, and that it can be separated by high voltage electrophoresis from urinary metabolites of ‘Largactil’ (2,000 mg/day), ‘Stelazine’ (540 mg/day), ‘Nardil’, ‘Disipal’ (300 mg/day), barbiturates and haloperidol (2.25 mg/day). We have examined urines from patients during treatment with these drugs (at the doses quoted), and find that their metabolites give no fluorescence with formaldehyde and do not interfere with the formaldehyde fluorescence of added DMPE. (‘Tofranil’ metabolites, however, had a native fluorescence which may interfere at high doses.) Even when overloading of the paper has caused tailing of the DMPE and overlap with metabolite spots, the fluorescence still develops normally. The fluorophore can be eluted with 0.4 N sodium hydroxide in methanol, and after acidification with hydrochloric acid it can be measured quantitatively in a fluorimeter at 360 mµ activation/460 mµ fluorescence. In case of tailing on the electrophoresis paper, the eluted fluorophore can be re-run on thin layer plates of silica gel using n-butanol : water : acetic acid (60 : 20 : 20), or isopropanol: methyl acetate : water : concentrated ammonia (35: 45: 15: 5), in order to separate it from traces of drug metabolites or other urinary constituents.