Agilent MassHunter Unknowns Analysis

Overview

• Purpose: Set up a method in Agilent MassHunter software for quick screening of unknown samples.
• Key features:
  • Deconvolution to detect individual components.
  • Comparison of components with mass spectral libraries.
  • Outputs include a list of sample components, deconvoluted spectra, and match factors.

Software Installation

• MassHunter Unknowns Analysis is part of the MassHunter software suite.
• Installed automatically with MassHunter quantitative analysis software.
• Accessing the program:
  • If not on desktop, navigate to Agilent MassHunter quantitative analysis tools.
  • Use the "Setup Desktop Icons" application to enable MassHunter Unknowns Analysis.

Co-eluting Compounds

• Chromatographic peaks can consist of multiple co-eluting compounds.
• Example: One peak in the Total Ion Chromatogram (TIC) may include three compounds.
• Goal of Deconvolution:
  • Separates co-eluting components.
  • Creates a pure spectrum for each component.

Setting Up a Method

1. Open MassHunter Unknowns Analysis Program:
   • Double-click on the icon to launch the software.
2. Create New Analysis:
   • Click on the new analysis icon in the upper left corner.
   • Navigate to the raw data file location.
   • Enter a name for the analysis and click "Create".
3. Add Samples:
   • Select samples to add to the sample table.
   • Use "Browse to Copy" for files from different locations.
   • Change data file types as needed (e.g., from sample to matrix blank).

Method Parameters Setup

• Peak Detection Method:

  • Focus on deconvolution for identifying components.
  • Options to exclude M/z values from background noise.
  • Filters for peak area or height to exclude small components.

• Deconvolution Parameters:

  • Retention time window size factor controls grouping of extracted ion signals.
  • Larger values yield fewer components; smaller values yield more.
  • Default for GC-MS is typically set around 75.

• Library Search Settings:

  • Specify libraries for the method.
  • Include customized and commercial libraries (e.g., NIST).
  • Adjust search function settings (forward/reverse search).
  • Utilize retention time criteria to reduce false positives:
    • Requires a retention calibration file (CSV format).

• Compound Identification Settings:

  • Set minimum match factor threshold for identification.
  • Define lower limit for M/z values for spectral matches.
  • Specify the search process for multiple libraries.

• Blank Subtraction Settings:

  • Change data file type to blank or matrix blank to subtract compounds found in blanks from the sample analysis.

Analyzing Results

• Results Review:

  • Sample table on the left shows identified components and library matches.
  • Components table displays retention time, compound name, match factor, and more.
  • Chromatogram on right displays TIC signal and identified components.
  • Deconvoluted spectra and library spectra are shown.

• Example of Using Retention Time Calibration:

  • Identify if a compound (e.g. delta isomer of hexachlorocyclohexane) has a poor retention index match.
  • Use "Show Alternative Hits" to find better matches based on retention time.

Data Export and Reporting

• Options to export data or generate reports:
  • Export to CSV, library, or perform quantitative analysis.
  • Generate reports using available templates.

Conclusion

• Provided an introduction to setting up and analyzing unknown samples using Agilent MassHunter Unknowns Analysis.