Use of a trapping agent for simultaneous capturing and high-throughput screening of both "soft" and "hard" reactive metabolites.
Glutathione (GSH) has been widely used for in vitro trapping and subsequently detecting reactive metabolites using liquid chromatography-mass spectrometry. A major drawback of GSH is its low trapping efficiency for "hard" reactive metabolites such as reactive aldehydes. In the present study, a bifunctional trapping agent (gamma GSK, gamma-glutamylcysteinlysine) is investigated as an alternative of GSH for simultaneous trapping both "hard" and "soft" reactive metabolites. In microsomal incubations, soft and hard reactive metabolites are captured by conjugation to the free thiol and the amine group of gamma GSK, respectively, resulting in formation of stable peptide adducts. Similar to GSH conjugates, all gamma GSK adducts derived from both soft and hard reactive metabolites contain a gamma-glutamyl moiety and, thus, undergo a neutral loss of 129 Da under collision-induced dissociation. As a result, an NL MS/MS scan can be utilized as a generic method for rapid detecting of both hard or soft reactive metabolites. As demonstrated by a number of model compounds, this approach, in combination with the isotope trapping technique, is reliable, sensitive, and efficient and can be potentially utilized as a high-throughput method for screening and rapid identification of both soft and hard reactive metabolites. In comparison with other methods, this approach is highly efficient and suitable in drug discovery for screening a wide variety of compounds for different reactive metabolites.
Yan
Zhengyin
Z
Division of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, Spring House, Pennsylvania 19477, USA. zyan@prdus.jnj.com
Maher
Noureddine
N
Torres
Rhoda
R
Huebert
Norman
N
D003408:Cresols / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005663:Furans / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008861:Microsomes / Q000201:enzymology; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D003408:Cresols; D005663:Furans; D009842:Oligopeptides; C522713:glutamyl-cysteinyl-lysine; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; C077977:cresol; C039281:furan