Identification of ractopamine hydrochloride metabolites excreted in rat bile

1. Rats dosed orally with 2.85 +/- 0.30 mg [14C]ractopamine HC1 [(1R*, 3R*), (1R*, 3S*)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)- 1-methylpropyl]-amino]-methyl]([U-14C]benzenemethanol)hydrochloride] containing 1.44 +/- 0.15 microCi radioactivity excreted 58 +/- 7% of the administered radioactivity in the bile within 24 h. Absorption and excretion of radioactivity was rapid as 55% of the administered radiocarbon was excreted into the bile during the first 8-h collection period. 2. Radioactivity excreted in rat bile was partitioned by XAD-2 column chromatography and reverse-phase hplc into at least seven different crude metabolite fractions; metabolites representing approximately 76% of the biliary radioactivity were isolated and identified from four of the crude metabolite fractions. 3. Approximately 46% of the biliary radioactivity was identified as a sulphate-ester, glucuronic acid diconjugate of ractopamine. Identification was based on 1H-nmr and negative-ion FAB-ms spectroscopy. Enzymatic and chemical hydrolysis of the sulphate-ester followed by co-chromatography of the hydrolysis products with synthetic ractopamine mono-glucuronides, established the site of sulphation at the C-10' phenol (phenol attached to carbinol) and glucuronidation at the C-10 phenol (phenol attached to methylpropyl amine) of ractopamine. 4. A metabolite representing approximately 6% of the biliary radioactivity was identified as a ractopamine mono-sulphate conjugate by using mass spectral and 1H-nmr techniques. Sulphate was conjugated at the C-10' phenol of ractopamine and was not stereospecific. 5. Approximately 25% of the biliary radioactivity was identified as ractopamine mono-glucuronides. The major site of glucuronidation was at the C-10 phenol, but ractopamine glucuronidated at the C'-10 phenol was also present.