Processing EI GC-MS Data in Chromatogram Window NIST26

The NIST 26 Mass Spectral Search Program introduces an integrated workflow for EI GC-MS data processing that combines chromatographic review, AMDIS deconvolution, and library searching within a single environment. This approach allows analysts to move efficiently from raw chromatographic data to compound identification while maintaining full control over filtering, review, and reporting.

Although the integrated workflow incorporates AMDIS technology, the standalone AMDIS application remains available and offers additional capabilities such as comparison of multiple files and review of multiple library hits for the same component.

Download Handout in PDF HERE

Before You Begin

Before processing data, users should be familiar with the basic configuration and navigation of the NIST 26 Chromatogram Window. The software contains extensive built-in documentation that can significantly shorten the learning curve.

To access the most useful help resources:

1. Open Help → Help Topics
2. Select Index
3. Search for Chromatogram
4. Review:
   • Chromatogram Tab documentation
   • GC/MS Chromatogram Tips
   • GC/MS Chromatogram Help pages

These resources provide detailed explanations of the interface and processing options.

Step 1 – Select the Processing Mode

If both EI and tandem MS versions of NIST software are installed, first select the appropriate spectrum type. This simplifies the user interface and ensures that only relevant options are displayed.

Typical options include:

• EI
• Tandem MS
• Any

For EI GC-MS data, select EI before opening files.

Step 2 – Open the GC-MS Data File

To begin analysis:

Analyze → EI Input File

The software supports multiple vendor formats, including Agilent data files. After selecting the data file, choose the libraries that will be searched.

Common library selections include:

• Mainlib (NIST Main Library)
• Replib (NIST Replicate Library)
• Wiley Libraries
• User-created libraries

Searching multiple libraries simultaneously can improve identification coverage and confidence.

Step 3 – Run AMDIS Deconvolution and Library Search

Once the file and libraries are selected, NIST performs:

• Automated deconvolution
• Spectral extraction
• Library searching
• Preliminary identification

The resulting screen contains:

Chromatogram Window (Top)

Displays chromatographic peaks identified by circles corresponding to deconvoluted components.

Results Table (Bottom)

Displays:

• Retention time
• Abundance
• Library match factors
• Identified compounds
• Additional search statistics

Selecting a peak automatically highlights the corresponding result entry. Conversely, selecting a result highlights the associated chromatographic peak.

Step 4 – Examine Library Matches

For each detected component:

• The upper spectrum displays the unknown spectrum.
• The lower spectrum displays the best library match.

A butterfly comparison plot allows rapid visual assessment of spectral agreement. Analysts should always visually inspect this comparison rather than relying solely on match factors.

Unlike standalone AMDIS, the integrated chromatogram window displays only the best library hit for each component. Additional searching can still be performed through NIST library search tools if required.

Step 5 – Improve Visualization

Complex samples often contain many low-abundance components hidden near the baseline.

A useful technique is switching abundance display from:

Linear → Logarithmic Scale

Benefits include:

• Enhanced visibility of trace components
• Better comparison of concentration differences
• Easier review of low-level analytes

This adjustment is performed through the chromatogram display properties.

Step 6 – Apply Filters to Reduce Complexity

NIST 26 provides several filtering tools that help focus on the most relevant identifications.

Match Score Filter

Allows highlighting or displaying only identifications above a chosen score threshold.

Example:

• Score > 900
• High-confidence identifications highlighted
• Lower-confidence matches remain available if needed

The software clearly reports how many hits remain after filtering.

Max2Med Filter

Max2Med represents:

Maximum abundance ÷ Median abundance of the query spectrum

This metric provides an indication of spectral quality. According to the handout:

Library search results tend to become less reliable when Max2Med approaches 10.

Using a minimum Max2Med threshold can remove lower-quality identifications and simplify review.

Merge Duplicate IDs

Deconvolution sometimes generates multiple component markers for the same chromatographic peak.

The Merge Duplicate IDs option suppresses duplicate identifications occurring within a specified retention-time window (typically about 2 seconds), making chromatograms easier to interpret.

Step 7 – Sort and Select Components of Interest

The results table is highly interactive. Analysts can:

• Sort by abundance
• Sort by retention time
• Sort by match score
• Sort by analyte name

The workflow demonstrated in the tutorial uses:

1. Sort by abundance
2. Select compounds above a chosen abundance threshold
3. Create a filtered list
4. Re-sort by retention time for systematic review

Keyboard shortcuts improve efficiency:

• Shift + Click → select ranges
• Ctrl + Click → add/remove individual entries

This flexible workflow allows analysts to focus only on relevant components.

Step 8 – Review Selected Components

After defining a list of compounds of interest:

1. Sort by retention time.
2. Select the first component.
3. Use keyboard arrow keys to move sequentially through the list.
4. Review each butterfly spectrum comparison.
5. Decide whether each identification should be retained in the final report.

This systematic review process helps ensure that reported identifications are chemically and spectrally reasonable.

Step 9 – Export Results to Excel

Once the final list has been created:

1. Select desired components.
2. Right-click.
3. Choose Copy Selected Hits to Clipboard.
4. Paste directly into Excel.

This allows:

• Custom report generation
• Data editing
• Further calculations
• Distribution to clients or collaborators

A common workflow is exporting all components above a defined abundance threshold and then performing final report formatting in Excel.

Step 10 – Save Your Configuration

One of the most valuable productivity features is configuration management.

After customizing:

• Filters
• Libraries
• Display settings
• Sorting preferences
• Processing parameters

Save the configuration as an INI file.

To do this:

1. Switch to another tab (such as Lib Search).
2. Open the File menu.
3. Select Save Configuration.

Saved configurations can later be restored, eliminating the need to recreate complex workflows for different projects. NIST also provides several default configuration files with the software.

Key Takeaways

• NIST 26 integrates AMDIS deconvolution and library searching into a streamlined chromatographic review environment.
• Multiple libraries can be searched simultaneously, including NIST, Wiley, and user-created libraries.
• Log-scale abundance displays improve visualization of trace compounds.
• Filters such as Match Score, Max2Med, and Merge Duplicate IDs simplify complex datasets.
• Retention-time-based review combined with butterfly spectrum inspection provides an efficient identification workflow.
• Results can be exported directly to Excel for reporting.
• Saving configurations dramatically improves productivity and consistency across projects