Screening for xenobiotic electrophilic metabolites using pulsed ultrafiltration-mass spectrometry

A pulsed ultrafiltration-mass spectrometric screening assay has been developed to generate and identify electrophilic metabolites of xenobiotic compounds formed by hepatic cytochrome P450 enzymes. This assay would be suitable for the early identification of potentially toxic compounds during the initial phase of drug development. Rat liver microsomes were trapped by an ultrafiltration membrane in a stirred flow-through chamber, and substrates for microsomal cytochrome P450 including hydroxychavicol, 3-methylindole, cyproheptadine and 2-tert-butyl-4,6-dimethylphenol were flow-injected individually through the chamber along with the cofactors, NADPH and glutathione. Metabolites and glutathione conjugates were detected on-line using electrospray 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 present at low concentration. Enzymatic activation of each xenobiotic substrate produced highly electrophilic metabolites such as quinones, quinone methides and imine methides that reacted with glutathione on-line to produce glutathione conjugates which were detected by using electrospray mass spectrometry. Although epoxides such as cyproheptadine epoxide were generated, it is likely that these compounds were insufficiently reactive to form glutathione 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 discovery process for the early identification and screening out of potentially toxic lead compounds.