This Application Note demonstrates: The use of the Agilent 1200 Rapid Resolution LC (RRLC) system for high resolution separation of metabolites from an in-vitro metabolism experiment. The use of the Agilent 6520 QTOF mass spectrometer for the acquisition of data for computer assisted metabolite identification. The use of the Agilent MassHunter Metabolite identification software for highly productive identification of expected metabolites. The results of the Metabolite ID data analysis for expected metabolites of the pharmaceutical drug nefazodone
This document describes a workflow for the computer-assisted identification of metabolites from pharmaceutical drugs. It specifically uses the Agilent 1200 Rapid Resolution LC (RRLC) system and the Agilent 6500 Series Accurate Mass QTOF mass spectrometer to analyze the metabolism of the drug nefazodone.
The note details the experimental setup, including sample preparation and instrument parameters for high-resolution separation and mass spectrometry. It explains the data analysis method within the MassHunter Metabolite ID software, which utilizes multiple cooperative algorithms to compare metabolite samples against control samples to identify new compounds and assign metabolic reactions.
| LC System | Agilent 1200 Series Rapid Resolution LC system |
|---|---|
| Mass Spectrometer | Agilent 6500 Series Accurate Mass QTOF mass spectrometer |
| Column | ZORBAX SB-C18, 2.1 x 150 mm, 1.8 um particle size |
| Flow rate | 0.5 mL/min |
| Mass range | 100-1000 |
| Fragmentor voltage | 200 V |
The Agilent MassHunter Metabolite identification software is used for highly productive identification of expected metabolites.
The pharmaceutical drug Nefazodone is used as a benchmark because it undergoes extensive, well-documented metabolism.
Compounds that are new or increased by at least 2-fold in the metabolized sample compared to the control are considered potential metabolites.