Screening for xenobiotic electrophilic metabolites using pulsed ultrafiltration-mass spectrometry

Citation
D. Nikolic et al., Screening for xenobiotic electrophilic metabolites using pulsed ultrafiltration-mass spectrometry, COMB CHEM H, 2(3), 1999, pp. 165-175
Citations number
28
Categorie Soggetti
Chemistry & Analysis
Journal title
COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING
ISSN journal
13862073 → ACNP
Volume
2
Issue
3
Year of publication
1999
Pages
165 - 175
Database
ISI
SICI code
1386-2073(199906)2:3<165:SFXEMU>2.0.ZU;2-3
Abstract
A pulsed ultrafiltration-mass spectrometric screening assay has been develo ped to generate and identify electrophilic metabolites of xenobiotic compou nds formed by hepatic cytochrome P450 enzymes. This assay would be suitable for the early identification of potentially toxic compounds during the ini tial phase of drug development. Rat liver microsomes were trapped by an ult rafiltration membrane in a stirred flow-through chamber, and substrates for microsomal cytochrome P450 including hydroxychavicol, 3-methylindole, cypr oheptadine and 2-tert-butyl-4,6-dimethylphenol were flow injected individua lly through the chamber along with the cofactors, NADPH and glutathione. Me tabolites and glutathione conjugates were detected on-line using electrospr ay mass spectrometry. Alternatively, the ultrafiltrate was concentrated on a reversed phase HPLC column and analyzed using electrospray LC-MS or LC-MS -MS to separate and characterize isomeric metabolites and metabolites prese nt at low concentration. Enzymatic activation of each xenobiotic substrate produced highly electrophilic metabolites such as quinones, quinone methide s and imine methides that reacted with glutathione on-line to produce gluta thione conjugates which were detected by using electrospray mass spectromet ry. Although epoxides such as cyproheptadine epoxide were generated, it is likely that these compounds were insufficiently reactive to form glutathion e conjugates in the absence of cytosolic glutathione S-transferases. Pulsed ultrafiltration-electrospray mass spectrometry offers an efficient method for in vitro formation and mass spectrometric characterization of activated microsomal drug metabolites and is suitable for use during the drug discov ery process for the early identification and screening out of potentially t oxic lead compounds.