Sensitive and specific liquid chromatographic–tandem mass spectrometric assay for atropine and its eleven metabolites in rat urine

A sensitive and specific method is described for the simultaneous determination of atropine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC–MSn). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of atropine. After extraction procedure the pretreated samples were separated on a reversed-phase C18 column using a mobile phase of methanol/ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70: 30, v/v) and detected by an on-line LC–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. The results revealed that at least eleven metabolites (N-demethyltropine, tropine, N-demethylatropine, p-hydroxyatropine, p-hydroxyatropine N-oxide, glucuronide conjugates and sulfate conjugates of N-demethylatropine, p-hydroxyatropine and the parent drug) and the parent drug existed in rat urine after ingesting 25 mg/kg atropine. p-Hydroxyatropine and the parent drug were detected in rat urine for up 106 h after ingestion of atropine.