LC–MS for Identification and Elucidation of the Structure of In-Vivo and In-Vitro Metabolites of Atropine

Abstract

In-vivo and in-vitro metabolism of atropine has been investigated by use of a highly specific and sensitive LC–MSn method. Feces, urine, and plasma samples were collected separately after ingestion of 25 mg kg−1 atropine by healthy rats. Rat feces and urine samples were cleaned by liquid–liquid extraction and by solid-phase extraction (on C18 cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Atropine was incubated, in vitro, with homogenized liver and with intestinal flora from rats. The metabolites in the incubation solution were extracted with ethyl acetate. These pretreated samples were then analyzed by reversed-phase high-performance liquid chromatography on a C18 column with methanol–ammonium acetate (2 mm, adjusted to pH 3.5 with formic acid), 70:30 (v/v), as mobile phase. Detection was by on-line MSn. Identification and elucidation of the structure of the metabolites were achieved by comparing molecular mass (ΔM), retention-times, and full-scan MSn spectra with those of the parent drug. Ten new metabolites (aponoratropine, apoatropine, hydroxymethoxyatropine, trihydroxyatropine, dimethoxyatropine, dihydroxymethoxyatropine, hydroxydimethoxyatropine, trihydroxymethoxyatropine, dihydroxydimethoxyatropine, and tropic acid) were identified in rat urine after ingestion of atropine. Nine metabolites (nortropine, tropine, aponoratropine, apoatropine, noratropine, hydroxyatropine, hydroxyatropine N-oxide, hydroxymethoxyatropine, and tropic acid) and the parent drug were detected in rat feces. Five metabolites (nortropine, tropine, tropic acid, apoatropine, and hydroxyatropine) and the parent drug were detected in rat plasma. Only two metabolites (apoatropine and noratropine) were detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (tropine and tropic acid) and dehydrated metabolite apoatropine were found in the rat intestinal flora incubation mixture.