title: Identification of novel metabolites of colchicine in rat bile facilitated by enhanced online radiometric detection.
abstract: Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatography-accurate radioisotope counting. The known 2- and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.
authors.affiliation: Drug Metabolism and Pharmacokinetic Department, Merck Research Laboratory, Boston, MA 02115, USA. lin_xu2@merck.com
authors.forename: Lin
authors.initials: L
authors.lastname: Xu
authors.forename: Bruce
authors.initials: B
authors.lastname: Adams
authors.forename: Valentina V
authors.initials: VV
authors.lastname: Jeliazkova-Mecheva
authors.forename: Laird
authors.initials: L
authors.lastname: Trimble
authors.forename: Gloria
authors.initials: G
authors.lastname: Kwei
authors.forename: Andreas
authors.initials: A
authors.lastname: Harsch
mesh_terms: D000818:Animals; D001646:Bile / Q000737:chemistry* / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003078:Colchicine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D011874:Radiometry / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D003078:Colchicine