Phase I and II metabolites of benztropine in rat urine and bile

Abstract

1. Following oral administration of benztropine (10 mg/kg, body weight), the phase I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4′-hydroxybenztropine, N-desmethyl-4′-hydroxybenztropine, 4′-hydroxybenztropine N-oxide and methoxy-4′-hydroxybenztropine, together with unmetabolized benztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry (EI GC/MS). microcolumn LC-electrospray mass spectrometry (ES LC/MS) and hplc followed by MS analysis.

2. The mass spectra and chromatographic properties of isolated N-desmethylbenztropine, benztropine N-oxide and tropine were confirmed by comparison with authentic reference standards.

3. Sufficient quantities of 4′-hydroxybenztropine and N-desmethyl-4′-hydroxybenztropine were isolated from the urine by tlc and examined by ¹H-nmr, ES/MS and EI/MS. The structure of the methoxy-4′-hydroxybenztropine metabolite was determined by EI/MS. 4′-Hdroxybenztropine N-oxide was identified by reacting it with a reducing agent, titanous chloride, to form 4′-hydroxybenztropine, which was then confirmed by comparing its EI/MS and ES/MS behaviour with a previously isolated and ¹H-nmr-authenticated sample.

4. In addition, four intact glucuronide conjugates of benztropine were also characterized in bile and urine as phase II metabolites, including 4′-O-glucuronylbenzotropine. N-desmethyl-4′-O-glucuronylbenztropine, methoxy-4′-O-glucuronylbenztropine and 4′-O-glucuronylbenztropine N-oxide by hplc followed by ES/MS analysis. These results provide the first direct evidence of the presence of these metabolites of benztropine in rat.