Characterization of in vivo and in vitro metabolic pathway of anisodamine by liquid chromatography-tandem mass Spectrometry

In vivo and in vitro metabolisms of anisodamine were investigated using a highly specific and sensitive LC-MSn method. Feces, urine, and plasma samples were collected individually after ingestion of 25 mg/kg anisodamine to healthy rats. Rat feces and urine samples were cleaned up by a liquid-liquid extraction and a solid phase extraction procedure (C-18 cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Anisodamine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubation solution were extracted with ethyl acetate. Then, these pretreated in vivo and in vitro samples, were injected into a reversed-phase C-18 column with mobile phase of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on-line MS' system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (AM), retention times and full scan MS' spectra with those of the parent drug. Fifteen new metabolites (aponoranisodamine, apoanisodamine, methoxyanisodamine, hydroxy-methoxyanisodamine, trihydroxyanisodamine, dimethoxyanisodarnine, dihydroxy-methoxyanisodamine, tetrahydroxyanisodamine, hydroxy-dimethoxyanisodamine, trihydroxy-methoxyanisodamine, dihydroxy-dimethoxyanisodamine, tetrahydroxy-methoxyanisodamine, trihydroxy-dimethoxyanisodamine, dihydroxy-trimethoxyanisodamine, and hydroxy-tetramethoxyanisodamine) were identified in rat urine after ingesting anisodamine. Seven metabolites (nor-6 beta-hydroxytropine, 6 beta-hydroxytropine, tropic acid, aponoranisodamine, apoanisodamine, noranisodamine, and anisodamine N-oxide) and the parent drug were detected in rat feces. Six metabolites (nor-6 beta-hydroxytropine, 6 beta-hydroxytropine, tropic acid, apoanisodamine, hydroxyanisodamine, and anisodamine N-oxide) and the parent drug are detected in rat plasma. Only apoanisodamine was detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (6 beta-hydroxytropine and tropic acid) and the dehydrated metabolite of anisodamine were found in the rat intestinal flora incubation mixture.