Fengmei Han

Analysis of scopolamine and its eighteen metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method is described for the determination of scopolamine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC-MS/MS). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of scopolamine. After extraction procedure, the pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol/ ammonium acetate (2mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MS/MS system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. The results revealed that at least 18 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, hydroxyscopolamine N-oxide, p-hydroxy-m-methoxyscopolamine, trihydroxyscopolamine, dihydroxy-methoxyscopolamine, hydroxyl-dimethoxyscopolamine, glucuronide conjugates and sulfate conjugates of norscopolamine, hydroxyscopolamine and the parent drug) and the parent drug existed in urine after ingesting 55mg/kg scopolamine to healthy rats. Hydroxyscopolamine, p-hydroxy-m-methoxyscopolamine and the parent drug were detected in rat urine for up 106h after ingestion of scopolamine.

Characterization of in vivo and in vitro Metabolic Pathway of Anisodamine by Liquid Chromatography‐Tandem Mass Spectrometry

Abstract 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 (C18 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 C18 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 MSn system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention times and full scan MSn spectra with those of the parent drug. Fifteen new metabolites (aponoranisodamine, apoanisodamine, methoxyanisodamine, hydroxy‐methoxyanisodamine, trihydroxyanisodamine, dimethoxyanisodamine, 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‐6β‐hydroxytropine, 6β‐hydroxytropine, tropic acid, aponoranisodamine, apoanisodamine, noranisodamine, and anisodamine N‐oxide) and the parent drug were detected in rat feces. Six metabolites (nor‐6β‐hydroxytropine, 6β‐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 (6β‐hydroxytropine and tropic acid) and the dehydrated metabolite of anisodamine were found in the rat intestinal flora incubation mixture.

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSn spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.

Identification of sophoridine and its metabolites in rat urine by liquid chromatography-tandem mass spectrometry.

Sophoridine is an important quinolizidine alkaloid which has wide pharmacological effect including antiarrhythmia, antitumor, immunological enhancement, and immunosuppression. In this work, a rapid, sensitive and dependable liquid chromatographic–electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of sophoridine and its metabolites in rat urine after administration of a single dose of sophoridine (12 mg/kg) by vena caudalis injection. The rat urine samples were purified by use of a C18 solid-phase extraction cartridge, then chromatographically separated by a reversed-phase C18 column (Aichrom ReliAsil C18, 5 μm, 2 mm × 150 mm i.d.) with 70:30(v/v) methanol-0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) as mobile phase and detected by an on-line ESI ion trap MS-MS detector. Identification and structural elucidation of the metabolites were performed by comparing the molecular weights, retention-times, and MS-MS spectra of them with those of the parent drug. As a result, six phase I metabolites of sophoridine (N-oxide metabolite, mono-hydroxylation metabolite, hexa-dehydrogenation metabolite and three di-dehydrogenation metabolites) were identified in rat urine for the first time.