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

Abstract

AbstractA 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%.