title: Analytical power of LLE-HPLC-PDA-MS/MS in drug metabolism studies: identification of new nabumetone metabolites.
abstract: Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date. In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE-HPLC-PDA and achiral LLE-UHPLC-MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC-MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite. To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE-HPLC-PDA-MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA).
authors.affiliation: Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Heyrovského 1207, CZ-500 02 Hradec Králové, Czech Republic. nobilis@uebf.cas.cz
authors.forename: Milan
authors.initials: M
authors.lastname: Nobilis
authors.forename: Jiří
authors.initials: J
authors.lastname: Mikušek
authors.forename: Barbora
authors.initials: B
authors.lastname: Szotáková
authors.forename: Robert
authors.initials: R
authors.lastname: Jirásko
authors.forename: Michal
authors.initials: M
authors.lastname: Holčapek
authors.forename: Chamseddin
authors.initials: C
authors.lastname: Chamseddin
authors.forename: Thomas
authors.initials: T
authors.lastname: Jira
authors.forename: Radim
authors.initials: R
authors.lastname: Kučera
authors.forename: Jiří
authors.initials: J
authors.lastname: Kuneš
authors.forename: Milan
authors.initials: M
authors.lastname: Pour
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D002074:Butanones / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D000077430:Nabumetone; D009280:Naphthaleneacetic Acids / Q000032:analysis / Q000378:metabolism*; D011355:Prodrugs; D051381:Rats; D017208:Rats, Wistar; D013057:Spectrum Analysis / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C584289:3-hydroxynabumetone; D000894:Anti-Inflammatory Agents, Non-Steroidal; D002074:Butanones; D009280:Naphthaleneacetic Acids; D011355:Prodrugs; C044612:6-methoxy-2-naphthylacetic acid; D000077430:Nabumetone