title: Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method.
abstract: Identification, characterization and structure elucidation of human metabolites of drug candidates is crucial for the pharmaceutical industry to assess their activity against the therapeutic target of interest and potential toxicological effects. It often requires in vitro synthesis of microgram quantities of metabolites of interest with enzymatic preparations, pre-concentration of the reaction mixture by solid phase extraction (SPE), metabolite isolation using HPLC systems coupled to fraction collectors prior to nuclear magnetic resonance characterization. The method reported herein is a rapid and simple technique using solely off-line mixed phase anionic exchange lipophilic SPE cartridges to selectively isolate glucuronide and sulfate metabolites from their parent compound. This approach capitalizes on the pKa differences between the parent compound, devoided of acidic moieties, and the negatively charged glucuronide and/or sulfate metabolites. Once loaded on the SPE cartridge, the incubation mixture is washed successively with a basic aqueous solution, methanol to elute the non-anionic parent compounds, and then with an acidic methanolic solution to protonate and recover the phase II conjugates. Over 100 microg (>95% purity) of 17 alpha-ethynylestradiol-3-glucuronide and 6-gingerol-4'-glucuronide were successfully isolated using this technique, as well as glucuronide and a sulfate conjugates of 1-{4'-[(1R)-2,2-difluoro-1-hydroxyethyl]biphenyl-4-yl}cyclopropanecarboxamide (DHBC) synthesized in-house. Their structures were confirmed by Ultra Performance Liquid Chromatography coupled to Quadrupole-Time of flight (UPLC-QTof) and nuclear magnetic resonance analysis.
authors.affiliation: Merck Frosst Centre for Therapeutic Research, 16711 Transcanada Hwy, Kirkland, Québec, Canada H9H 3L1. sebastien_gagne@merck.com
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gagné
authors.forename: Julie
authors.initials: J
authors.lastname: Laterreur
authors.forename: Louiza
authors.initials: L
authors.lastname: Mahrouche
authors.forename: Dan
authors.initials: D
authors.lastname: Sørensen
authors.forename: Jacques Yves
authors.initials: JY
authors.lastname: Gauthier
authors.forename: Vouy Linh
authors.initials: VL
authors.lastname: Truong
authors.forename: Nathalie
authors.initials: N
authors.lastname: Chauret
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Lévesque
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods; D020719:Glucuronides / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D050217:Metabolic Detoxication, Phase II; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods*; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000838:Anions; D020719:Glucuronides; D012997:Solvents