Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method.
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.
Gagné
Sébastien
S
Merck Frosst Centre for Therapeutic Research, 16711 Transcanada Hwy, Kirkland, Québec, Canada H9H 3L1. sebastien_gagne@merck.com
Laterreur
Julie
J
Mahrouche
Louiza
L
Sørensen
Dan
D
Gauthier
Jacques Yves
JY
Truong
Vouy Linh
VL
Chauret
Nathalie
N
Lévesque
Jean-François
JF
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
D000838:Anions; D020719:Glucuronides; D012997:Solvents