Liquid chromatography/mass spectrometry and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz.
Efavirenz (Sustiva) is a potent and specific inhibitor of the HIV-1 reverse transcriptase and is approved for the treatment of HIV infection. The metabolism of efavirenz in different species has been described previously. Efavirenz is primarily metabolized in rats to the glucuronide conjugate of 8-OH efavirenz. Electrospray ionization-liquid chromatography/mass spectrometry analyses of bile samples from rats dosed with either efavirenz or with 8-OH efavirenz revealed three polar metabolites, M9, M12, and M13, with pseudomolecular ions [M-H](-) at m/z 733, 602, and 749, respectively. The characteristic mass spectral fragmentation patterns obtained for metabolites M9 and M13 suggested that these were glutathione-sulfate diconjugates, and the presence of a glutathione moiety in metabolite M9 was confirmed by liquid chromatograpy/nuclear magnetic resonance (NMR) analysis of bile extracts. Metabolite M12 was characterized by liquid chromatography/mass spectrometry as a glucuronide-sulfate diconjugate. Unambiguous structures of M9, M12, and M13 were obtained from one-dimensional proton and carbon NMR as well as proton-proton (correlated spectroscopy, two-dimensional shift correlation), proton-carbon heteronuclear multiple quantum correlation, and long-range proton-carbon (heteronuclear multiple bond correlation) correlated two-dimensional NMR analyses of metabolites isolated from rat bile. The mass spectral and NMR analyses of M10, which was isolated from rat urine, suggested a cysteinylglycine-sulfate diconjugate. The isolation of these polar metabolites for further characterization by NMR was aided by mass spectral analyses of HPLC fractions and solid phase extraction extracts during the isolation steps. The complete characterization of these novel diconjugates demonstrates that further phase II metabolism of polar conjugates such as sulfates could take place in vivo.
Mutlib
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Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Company, Stine-Haskell Research Center, Newark, Delaware 19714, USA. abdul.mutlib@dupontpharma.com
Chen
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Nemeth
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Gan
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Christ
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D000480:Alkynes; D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism*; D048588:Benzoxazines; D001646:Bile / Q000737:chemistry / Q000378:metabolism; D001711:Biotransformation; D002853:Chromatography, Liquid; D003521:Cyclopropanes; D004151:Dipeptides / Q000378:metabolism / Q000652:urine; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000378:metabolism*
D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D004151:Dipeptides; D010078:Oxazines; D018894:Reverse Transcriptase Inhibitors; C028505:cysteinylglycine; D054303:HIV Reverse Transcriptase; C098320:efavirenz