Mass Frontier can automatically predict possible fragments from a chemical structure using general rules and fragmentation libraries, along with primary determination of the structural plausibility of generated ions. The prediction of mass spectra is hindered by the difficulty of predicting thermochemical data, thermodynamic stability of product ions and reaction rates. However, generated fragments and their m/z values can be used for creating so-called “Bar Code” spectra. A bar code spectrum contains peaks at predicted m/z values with identical (maximal) intensity.

To create a bar code spectrum:

• Click on Bar Code Spectrum button in any Fragments & Mechanisms window.

The created bar code spectrum will be placed in a Database Manager window. Bar code spectra are automatically linked with their original Fragments & Mechanisms windows. If you click any bar code peak in Database Manager the program displays corresponding fragment(s), along with its (their) formation mechanism(s). This link will remain in place as long as the corresponding Fragments & Mechanisms window exists.

Bar code spectra can be used in several strategies for investigating spectra-structure relationships. The primary purpose of generating bar code spectra is that they allow the possibility to identify spectral differences in structurally similar compound classes, for which mass spectra are not available. To study fragmentation dissimilarities between structurally-related compounds, it is far easier and quicker to compare two or more bar code in Database Manager than manually compare fragments and their m/z ratios between Fragments & Mechanisms windows.

For example if you are interpreting an unknown spectrum and you have established two structural proposals, in order to find out which structure belongs to the unknown spectrum you should first draw both structures separately in Structure Editor and generate fragments for both structures. Then create bar code spectra and place them in the same Database Manager window. After that you can compare the bar code spectra in the Compare Spectra tab in Database Manager. In the Difference Spectrum pane you should see the specific peaks that this pair of spectra do not have in common. You can then compare these specific peaks with the unknown spectrum and take a closer look at the fragmentation mechanisms of these peaks. If a specific peak is present in the unknown spectrum and the mechanism of formation seems to be simple and plausible you can select the most likely structure. This approach, using bar codes, is far superior to simply comparing explained peaks, as it can be applied to a large number of structural proposals simultaneously.

Experimental and Bar Code spectra of Pyrrolidine[2,1-c]-2H,5H-1,4-benzodiazepin-2,5-dione, 1,3-dihydro- (Electron impact mode). The first bar code spectrum was generated using 1ppm mass precision (monoisotopic mass settings). In this case the abundance is proportional to the number of generated m/z values that fit into single integer m/z value. The second bar code spectrum was generated using unit mass precision.

The generated fragments can be used in many ways, for example, with bar code spectra, for fragment comparison of different structures, or for export to Microsoft Excel. The automated generator can predict a large number of theoretically possible fragments, so you might need to extract only selected fragments for use in the mentioned methods. To do so you can mark fragments (m/z values) that should be either considered or ignored during further processing.

Three types of mark can be used in the Fragments & Mechanisms module:

1. Set (Only fragments with a given m/z value will be considered)

2. Exclude (All fragments with a given m/z value will be ignored)

3. Default (No m/z value marked)

The Set mark has the highest priority. If you mark one or more m/z values - “Set” only these will be considered. If you mark one or more fragments - “Exclude” then these fragments will not be considered. If no fragments are marked (by default) then all the fragments will be considered.

The fragment marks apply to Bar Code spectra, Fragments Comparator window and copying to Excel.

To mark an m/z value:

• Select the m/z value and click the Set or Exclude buttons, or

• Click the appropriate check box for an m/z value in the m/z list (on the right side of the window). The check box can have one of three states: Set , Exclude , or Default

To delete a mark:

• Select the marked m/z value and then click one of the mark buttons

• Or, click the check box of the m/z value once or twice until the check box is in the default state

• Or, if you want to delete all marks, click the Default button in the bottom right corner of the window.