Abstract: AIM To study the phase II metabolites of phenoprolamine hydrochloride (DDPH) in rat bile. METHODS DDPH was administered by ip to bile duct-cannulated rats. Bile samples were collected before drug administration and up to 12 h after drug administration. After being purified and enriched with C-18 SPE columns the rat bile samples were analyzed by LC/DAD/MSD to identify the peaks of phase II metabolites. The fractions of phase II metabolites were prepared by HPLC and treated with β-glucuronidase, and then were purified and enriched with C-18 SPE columns and analyzed by LC/DAD/MSD. The corresponding reference standards of DDPH phase I metabolites were analyzed by LC/DAD/MSD under identical conditions. RESULTS The peaks M₇, M₈ and M₉ in the chromatograms of rat bile samples were the phase II metabolites of DDPH and the enzymatic hydrolysates of M₇, M₈ and M₉ were 1-(2,6-dimethyl-4-hydroxyphenoxy)-2-(3,4-methoxyphenylethylamino)-propane (M₃), 1-(2,6-dimethyl-3-hydroxyphenoxy)-2-(3,4-methoxyphenyl ethylamino)-propane (M₂) and 1-(2,6-dimethylphenoxy) -2-(3-methoxy-4-hydroxyphenylethyl-amino)-propane (M₁) respectively. CONCLUSION β-1-O-3,5-dimethyl-4--2-methyl-2-(3,4-dimethoxy-phenylethylamino)-ethoxy-phenyl-glucuronic acid (M₇, glucuronide of M 3), β-1-O-2,4-dimethyl-3-2-methyl-2-(3,4-dimethoxy-phenylethylamino)-ethoxy-phenyl-glucuronic acid (M₈, glucuronide of M₂) and β-1-O-2-methoxy-4-1-methyl-2-(2,6-dimethylphenoxy)-ethylamino-ethyl-phenyl-glucuronic acid (M₉, glucuronide of M₁) were the phase II metabolites of DDPH in rat bile.