Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

Abstract

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and β-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of β-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of β-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.