MassFinder Identification

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MassFinder 4: Tutorial

This is the second lesson of the MassFinder 4 tutorial. You will learn how to identify and label peaks and how to mark unknown peaks. You will also be given a first demonstration of the value of retention indices.

Contents

Scientist's responsibilities

MassFinder 4 has a powerful and reliable search engine and employs the two-dimensional algorithm considering both spectrum similarity and retention index. However, there are many cases, where the first library hit MassFinder presents is not the correct identification, e.g. in case of mixtures of compounds in co-eluting peaks, overloaded spectra, bad low-intensity spectra or wrong retention indices due to mismatched temperature programs in gas chromatography can make an identification difficult or misleading. If the compound is not in the library, MassFinder suggest similar spectra as possible matches.

In summary, please note the two important points:

  • Do not trust MassFinder blindly. The scientist as user of MassFinder is solely responsible for identifying and assigning spectra. MassFinder is only a tool to make this process as reproducible, reliable and convenient as possible and it supports and facilitates the documentation and presentation of your assignments.
  • Always consider not only the first library hit, but also the next few hits. Check how closely the retention indices match. Compare the adjacent scans and their respective search results.

Step 1: Viewing more library matches

On the previous tutorial page you learned how to navigate through the GC/MS and you probably enjoyed the instantaneous library hits displayed on the right side.

Please again play around navigating through the GC/MS and this time more closely watch the right panel displaying the library search results. In the status bar at the bottom of the window MassFinder displays how many library matches were found.

Analogous to navigating the GC by using the mouse wheel turning up or down, you may turn the mouse wheel over the right library panel to move to the second-best hit and so on. If the top or bottom spectrum remains empty, there are no more better or worse hits. The library hits are always sorted by match quality.

If MassFinder has found no hits at all for a given GC scan, the right library panel remains empty. MassFinder only displays hits that are at least fairly related to the spectrum or have a very close retention index. You will learn later how to edit these thresholds yourself (see MassFinder Options).

Each library entry displays on the right side the chemical name, the associated retention index, the stored molecular weight calculated from the molecular formula and in the upper left corner the match quality.

Step 2: Assign names to identified peaks

If you decide to accept a library hit as identification for a GC scan, you can easily assign the name of the library entry to the GC scan just by clicking anywhere on top of the highlighted library spectrum. This copies the substance name of the library entry to the highlighted scan and marks the assigned peak with a yellow arrow and its retention time in the chromatogram profile.

If you want to undo the assignment, just click again on the same library spectrum (or twice on any other library spectrum).

Please try to mark several of the peaks with library assigments. Most peaks of oil2122.mfg can be identified without ambiguity.

Step 3: Zoom into mass spectra

Sometimes you might want to zoom into mass spectra, e.g. to check for isotopic patterns or very small signals. You can use the same mouse operations for viewing mass spectra as for navigating the GC/MS, i.e.

  • zooming by selecting a box with the left mouse button
  • shifting by dragging the mass axis with the left mouse button
  • stretching by dragging the mass axis with the right mouse button
  • reset to default mass range by double-clicking on the mass axis

Please try all these possibilities and become familiar with them. Please note that the abundance axis displays the zoom factor.

Please also note that little '+' appear at the left or right side of the mass axis if mass signals exist that are not currently displayed. Please try to shift and stretch the axis and observe the indicators.

Step 4: Butterfly comparisons

Please note the little icon in the upper left corner of the highlighted library entry; it is called the butterfly icon. If you click on this icon, the current target GC/MS mass spectrum and the current library entry are displayed head-to-tail (top = library, bottom = scan).

Many people feel butterfly presentations facilitate comparing spectra with each other. You need to try for yourself whether this is true for you. If you click the butterfly icon a second time the normal view is restored. You may scroll through different library entries while being in butterfly mode.

Step 5: Marking unknown peaks

Naturally, there are cases where no perfect library match exists, e.g. discovering novel constituents or handling compounds not covered by the library. To mark such interesting scans as unknown, just click anywhere on top of the highlighted scan (center spectrum in left panel). This gives a name to the unknown spectrum containing helpful information for discussing the peak (base peak, possible molecular ion, retention index) and marks the unknown peak with a red arrow and its retention time in the chromatogram. If you want to undo the assignment, just click again on the same scan. You can turn assigned scans to unknown scans just by clicking on the highlighted scan and vice versa.

We recommend to assign and mark all interpreted peaks to avoid repeating the identification process when opening the GC/MS run the next time. All marks and assignments can be saved with the chromatogram as well as exported in graphical or text form to document the assignments (you will learn how to do this later).

Step 6: Editing the names of unknown peaks

MassFinder performs assignments conveniently with only one mouse click and gives reasonable names to identified peaks or informative text to unknowns. However, you may also edit the name of any GC scan manually if desired.

Please press the letter 'n' as mnemonic for name and a input dialog box will pop up. Enter any text you like and press return or click OK. If the scan already had a name assigned, the input box will display the current text and let you edit it. The list box offers all previously used labels for your convenience, so if you frequently find the same unidentified peaks it is easy to repeatedly assign the same labels. You may double-click on any list box entry to accept that label. The little grey cross in the lower left corner allows you to delete the currently selected entry from the list box.

Please note, that all edited names are always marked with a red arrow in the chromatogram. If you edit a yellow marked peak it will turn red after editing.

Hint: This is the first of only a few keyboard commands you will have to learn. All keyboard commands can be viewed by clicking on the online help button in the upper right corner of the main window.

Step 7: Jumping from peak to peak

You can select the next or previous marked scan (yellow or red arrow) by pressing the '<' and '>' keys. This enables you to swiftly move through your processed chromatogram.

Hint for German users: Since the German keyboard layout is different with regards to the '<' and '>' keys, there is an option to reverse the direction of both keys for a more logical and intuitive feeling. We recommend that German users activate this option (see MassFinder Options).

Step 8: A first glance on the value of retention indices

Please move to the peak of g-Muurolene, scan 763, retention time 11.54 min, RI 1480. MassFinder will display g-Muurolene as first library hit and the status bar will tell you it is the first hit out of a list of 6 ("hit 1/6").

The library RI value of 1474 matches quite well with the GC/MS value of 1480 and g-Muurolene is indeed the correct identification. For this identification and all other library searches you have done so far in this tutorial, MassFinder took into account both the mass spectral similarity as well as the retention index.

Now turn off MassFinder's retention index feature, so that only mass spectral similarity is taken into account.

  • Click with the left mouse button on the 'RI1' field in the status bar at the bottom of the main window.
  • Alternatively, you can press the key 'r' (or 'R') to toggle the retention feature on and off.

If the retention index feature is activated, the text 'RI1' appears in black, if deactivated it is greyed. Each time you toggle the feature on or off, MassFinder repeats the library search and displays the results. In this example, MassFinder finds 6 results with RI turned on and 39 results with RI turned off.

Please adjust the library panel so that MassFinder displays the first three library hits while RI turned off (this is done by turning the mouse wheel while hovering over any library spectrum). The right panel should look like the screen shot on the right. Observe how very similar these three spectra are: There is no chance to securely distinguish these three mass spectra from each other. Conclusion: In this mode (RI turned off) no identification is possible.

However, if you manually take the retention indices into account (g-cadinene RI 1507, g-muurolene RI 1474 and g-amorphene RI 1492) it is easy to deduce that neither g-cadinene nor g-amorphene can be the correct identification.

Please try turning the RI feature on and off for all yellow marked peaks. Each time observe how many and how similar hits are displayed while turned off and how few hits are displayed if turned on.

Summary: The retention index feature enables you to distinguish compounds with very similar mass spectra and to correctly identify peaks otherwise impossible to assign.

You can read more details and explanations on how retention indices work in our Retention index guide.

Step 9: Save the processed run

Please click on the save GC/MS button to open a standard Windows save dialog. You may enter a new name and click OK. MassFinder now saves the processed GC/MS run including the yellow and red labels.


Continue with the next part of the tutorial: MassFinder Autoreport

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