Identification of ractopamine hydrochloride metabolites excreted in rat bile

Abstract

1. Rats dosed orally with 2 ± 85 ± 0 ± 30mg [¹⁴C]ractopamine HC1 [(1R*,3R*), (1R* ± 3S*)-4-hydroxy-α-[[[3-(4-hydroxyphenyl)-1-methylpropyl]-amino]-methyl]([U-¹⁴C]ben-zenemethanol)hydrochloride]containing 1·44 ± 0·15 μCi-radioactivity excreted 58 ± 7% of the administered radioactivity in the bile within 24h. 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.