Glucuronides of hydroxylated metabolites of amitriptyline and nortriptyline isolated from rat bile

Polar conjugates were isolated from the bile of rats given amitriptyline (AT, unlabeled or labeled with 14C), nortriptyline (NT), or 10-hydroxy (10-OH) derivatives of the drugs. The procedure involved extraction on a column of polystyrene resin, elution with methanol, and separation by preparative TLC followed by reversed phase HPLC. Individual metabolites were characterized by NMR spectroscopy and fast atom bombardment mass spectrometry and by enzymatic or acid deconjugation with subsequent identification of aglycones and glucuronic acid. Conversely, they were compared with conjugates obtained from hydroxy compounds by incubation with rat liver microsomes and UDP-glucuronic acid. Glucuronides isolated from the bile of rats given AT were derived from 2-OH-AT, (E)- and (Z)-10-OH-AT, 2-hydroxy-3-methoxy- (or 3-hydroxy-2-methoxy) AT, 10, 11-(OH)2-AT, and some of the N-demethylated analogues of these compounds. In most cases, 10-OH compounds form two diastereoisomeric glucuronides produced from the enantiomeric alcohols; 10, 11-(OH)2 metabolites occur as cis- and trans-isomers that are conjugated with glucuronic acid. Administration of synthetic (E)- and (Z)-10-OH-AT and -NT leads to the excretion of their glucuronides along with conjugates formed after demethylation and/or introduction of a second OH group. NT gives rise to 2-OH-NT glucuronide besides those conjugates derived from (E)-10-OH-NT. No glutathione conjugates could be detected.