This application note describes how to use the Agilent 1200 Series Rapid Resolution LC system and Agilent ZORBAX Rapid Resolution High Throughput columns to separate compounds in complex samples, how to calculate the empirical formula of unknown compounds from high mass accuracy data acquired with the Agilent 6210 time-of-flight and teh Agilent 6510 Q-TOF mass spectrometer, how to use the Agilent MassHunter Profiling software to quickly and computer-assistedly analyze complex mass spectrometry data, and how to use the Agilent MassHunter Profiling software to analyze the differences in related samples in different applications.
This document describes a method for the differential analysis of complex samples, such as natural product extracts or drug metabolism reactions, using Agilent 6210 time-of-flight and Agilent 6510 quadrupole time-of-flight mass spectrometers. The analysis utilizes the Agilent MassHunter Profiling software to identify unknown compounds and examine differences between sample groups based on accurate mass data.
The study presents two case examples: the identification of pseudoginsenoside F11 in American ginseng versus Asian ginseng, and the detection of hydroxylated buspirone metabolites in a drug metabolism reaction. The text details the experimental setup, including the Agilent 1200 Series LC system and specific MS operating conditions, as well as the data processing workflow using Molecular Feature Extractor and Profiling software.
| Mass accuracy range | single-digit ppm mass error |
|---|---|
| Column type | Agilent ZORBAX SB C18, 2.1 x 150 mm, 1.8 pm particle size |
| Flow rate | 0.5 mL/min |
| Column temperature | 50 °C |
| DAD wavelength | 220 nm +4 nm, reference 360 nm + 16nm |
| Dry gas flow | 10 L/min |
| Dry Temp | 300 °C |
| Nebulizer pressure | 50 psi |
| Fragmentor voltage | 200 V |
| Skimmer voltage | 60 V |
| Capillary voltage | 3500 V |
| Scan range | 200-1300 |
The Agilent MassHunter Profiling software is used for the differential analysis of TOF and QTOF data.
The study uses the Agilent 6210 time-of-flight MS and the Agilent 6510 QTOF mass spectrometer.
The average relative mass accuracy for pseudoginsenoside F11 in the American ginseng sample group is 1.53 ppm.
671 molecular features were identified and displayed in the mass against retention time plot.