title: Characterization of benzimidazole and other oxidative and conjugative metabolites of brimonidine in vitro and in rats in vivo using on-line HID exchange LC-MS/MS and stable-isotope tracer techniques.
abstract: The characterization of brimonidine metabolites presents some challenges since brimonidine and its metabolites generate few structurally informative fragment ions in the LC-MS/MS spectra. The objective of the current study is to use on-line hydrogen/deuterium (H/D) exchange LC-MS/MS and stable-isotope tracer techniques to further characterize unknown brimonidine metabolites in vitro and in vivo. Brimonidine and D4-brimonidine were co-incubated in rat and human microsomes and rabbit aldehyde oxidase in vitro. In addition, the urine was collected from rats co-administered orally with brimonidine and D4-brimonidine. The hepatic microsomal and urinary metabolites were then characterized by H/D LC-MS/MS system. In addition to previously characterized 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine, the results show that oxidation occurs at quinoxaline ring producing oxo-hydroxybrimonidine and hydroxyquinoxaline metabolites. The hydroxyquinoxaline metabolite was only observed in microsomal incubations with hydroxylation at the 7- or 8- position. The dehydro-hydroxybrimonidine metabolites were characterized as 2-oxo or 3-oxo -4', 5'-dehydrobrimonidine. A novel metabolite ((4-bromo-lH-benzoimidazol-5-yl)-imidazolidin-2-ylidene-amine) of benzimidazole derivative of brimonidine in rats in vivo was identified and confirmed with reference standard. In conclusion, on-line H/D exchange LC-MS/MS and stable-isotope tracer techniques are useful for the characterization of brimonidine metabolites.
authors.affiliation: Department of Drug Safety Evaluation, Allergan, 2525 Dupont Dr., Irvine, CA 92612, USA. ni_jinsong@allergan.com
authors.forename: J
authors.initials: J
authors.lastname: Ni
authors.forename: J
authors.initials: J
authors.lastname: Rowe
authors.forename: T
authors.initials: T
authors.lastname: Heidelbaugh
authors.forename: S
authors.initials: S
authors.lastname: Sinha
authors.forename: A
authors.initials: A
authors.lastname: Acheampong
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000737:chemistry / Q000378:metabolism; D042931:Aldehyde Oxidase / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism; D000068438:Brimonidine Tartrate; D003903:Deuterium; D006801:Humans; D006859:Hydrogen; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D011810:Quinoxalines / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
chemical_list: D000316:Adrenergic alpha-Agonists; D001562:Benzimidazoles; D011810:Quinoxalines; D000068438:Brimonidine Tartrate; D006859:Hydrogen; D003903:Deuterium; D042931:Aldehyde Oxidase