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
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.