The Fragmentation Library™ module includes a full-featured graphical editor for entering and editing fragmentation reactions. To open Reaction Editor, simply select the Reaction Editor tab in the top left corner of the Fragmentation Library™ module.

The Reaction Editor buttons on the module buttons bar can be detached and placed anywhere in the program. Some drawing actions can be called up from pop-up menus by right clicking the mouse.

To add a new structure:

• Double click on the drawing canvas

• Or, click on the Add/Edit Structure button on the button bar

• Or, click the right mouse button on a blank canvas and choose the New Structure pop-up menu item

Structure Editor will then appear, where you can draw your fragment. This Structure Editor is a dialog window, which means you cannot open another window unless it is first closed. To place your fragment on the canvas, click the OK button. The fragment can be moved anywhere on the canvas by dragging the structure with the mouse.

To edit an existing structure:

• Double click on a fragment

• Or, select the structure and press the Add/Edit Structure button

• Or, point the mouse cursor at a structure, right click the mouse button and choose the Edit Structure pop-up menu item

Structure Editor will then appear. To confirm your changes, click the OK button. To reject the changes, click Cancel.

To add a straight reaction arrow:

• Click on the Draw Straight Arrow button and then point and click the mouse cursor on the canvas at the position you want to place the arrow

• Or, point the mouse cursor at a structure, right click the mouse button and choose the New Arrow pop-up menu item

In order for the expert system to correctly extract mechanisms, the structures in a reaction scheme must be properly connected by arrows. The system considers standalone structures and disconnected arrows as errors and ignores them. If a structure is connected with at least one arrow, the selection squares are displayed in green . If a structure is not connected, the selection squares are displayed in yellow . The same color-coding applies to arrows. If an arrow is connected with a structure, this arrow end will be shown in green. If the arrow end is not connected, it will appear in yellow.

To help you properly connect the drawing objects, small red selection squares will appear around the structure or arrow when you move them if the object is close enough to connect.

The arrow caption in Fragmentation Library window can be edited by double clicking on the arrow and entering the new formatted text into the annotation dialog box.

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In addition to a fragmentation scheme, text data can also be maintained in a particular database record. To enter or edit text data, select the Data Editor tab in the top left corner of the Fragmentation Library module. There are four available fields:

• Record activity

• Source of fragmentation scheme (journal, author, etc.)

• Experimental technique and instrument info

• Comment

Data Editor fields can be edited at any time, even for an existing record. To keep the changes, the record must be saved. Changes to Active Record check box are immediately updated in the database and do not need to be saved.

HighChem Fragmentation Library™ and Fragmentation Library™ are trademarks of HighChem, Ltd., Slovak Republic

Mass Frontier applies the mechanisms of fragmentation reactions to a user provided structure and automatically predicts fragmentation reactions for the given compound. However, some mechanisms may be erroneous or too general and the expert system may apply them even though they are not desired. To exclude a particular record from the generation of fragments, Active Record can be unchecked.

This option is especially useful for stopping the use of library reactions consisting of small fragments whose mechanisms can coincidently fit to virtually any compound. This can cause the generation of an extremely large number of fragments, which dramatically slows the generation process and makes reviewing the predicted fragments difficult.

Record activity can be changed in the Data Editor’s Active Record check box or in the “A” column of the record grid. These two check boxes are linked, so they always work together.

To activate or deactivate all the selected records, right click on them and choose the Activate All Selected Records or Deactivate All Selected Records pop up menu item.

To sort active and inactive records in a library grid, click on the column header. To restore the ID number ordering, click on the ID header tab in the library grid. It is possible to sort any column by clicking on its grid column header. To display the reverse order, click again on the header.

By default, the Fragmentation Library window shows all the library records in a corresponding library grid. If you need to list a subset of library records, you can hide selected records. Simply right click with the mouse button on the selected records and select the Hide Selected Records item from the pop-up menu. To restore the complete list of library records, click on the Reset button in the Search tab.

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To make a change permanent in Fragmentation Library, it must be saved.

To save changes in Reaction Editor or Data Editor:

• Click on any prior or following record to the edited record in the record grid

• Or, click the line up or line down key

• Or, click on the Library button and choose the Save Record To Library pop-up menu item

When saving a record, the software performs three sequential actions:

1. The fragmentation scheme in Reaction Editor is checked for formal correctness. If the software detects an error, you will be prompted to either save the scheme as it is, or return to the record to correct the problem.

2. A fragmentation mechanism for every single unimolecular reaction in the fragmentation scheme is extracted using advanced algorithms. This process can take several seconds for complex reactions.

3. The reaction scheme, additional text, numerical data, and the extracted mechanisms are stored in the database.

If the check for formal correctness encounters an error, you will be prompted to either save the record as it is, or go back and make appropriate changes. If you save a scheme with just a single error, the reaction mechanisms will not be extracted and the entire record will be ignored in the prediction of fragments and mechanism feature. You can return to an erroneous record at any time to fix such a problem and so enable the record for the fragmentation prediction.

Reaction symbols along with reaction abbreviations will only appear if a scheme is correctly saved.

HighChem Fragmentation Library™ and Fragmentation Library™ are trademarks of HighChem, Ltd., Slovak Republic

At the core of the automated prediction of fragmentation and rearrangement pathways is a computer-based system for the extraction of unimolecular decomposition mechanisms from user provided reactions. This complex software system decodes the underlying principle of fragmentation mechanisms from reaction drawings and builds a knowledge base of fragmentation events. This unique system works fully automatically and replaces the need for the tedious manual input of atom-atom correspondence in precursor and product ion pairs. A computer learning procedure has been developed to allow the processing of specific fragmentation details. Similarly, in experimental mechanistic studies, labeled or generic structures can be used to direct the desired dissociation route. By means of deuteria or substituents (R) participation, the decoding algorithm unambiguously extracts the underlying mechanism.

Since many reactions stored in a fragmentation library follow general fragmentation rules, which can be predicted by the use of preprogrammed unimolecular reactions, the library distinguishes between class specific mechanisms and general fragmentation reactions. After a reaction has been saved into the database, the system attempts to identify a general fragmentation rule and assigns the relevant reaction symbol above the arrow. The reaction symbol of every reaction in Fragmentation Library window can be edited by double clicking on the arrow and entering the new formatted text into the annotation dialog box.

As regards fragmentation prediction, the determination of the preferred ionization site is as important as a detailed knowledge of the fragmentation mechanism. The system supports the virtual generation of charged molecules based on library ionization reactions using the exact location of the positive or negative charge, or the unspecified charge location symbol that can be assigned to a structure drawing.

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Mass Frontier’s expert system automatically extracts fragmentation mechanisms from a reaction drawing after a scheme has been saved. If a reaction follows one of the pre-programmed general fragmentation rules, the arrow will be captioned with the particular rule abbreviation.

Even if a reaction is formally correct, it may not be possible to derive a reaction mechanism from a user provided drawing. This can occur if the user enters an unfeasible fragmentation mechanism or because the unimolecular reaction is incomprehensible to the expert system. If a mechanism cannot be extracted, the reaction arrow will have a cross through it. In this case, the mechanism is reduced to the exact precursor and product structures and only the identical neutral or ionic precursor is matched with the user provided structure in the fragmentation prediction process.

The software will sometimes be able to decode a mechanism from a drawn reaction but the atom-matching procedure will be unable to find the corresponding atom counterparts on both sides of the reaction leading to partially recognized mechanisms. When this occurs, the reaction arrow will appear with a small line through it at right angles. This kind of reaction can only be used for fragment prediction for some input structures according to the fragmentation algorithms decision. So do not be surprised if a partially recognized reaction mechanism is not selected for fragments prediction, even when your input fragment looks similar to the precursor in the library reaction.

To overcome such a problem with unrecognized or partially recognized mechanisms, we recommend trying to decompose complex one-step mechanisms into a number of simple reaction steps and then save these in a fragmentation library.

HighChem Fragmentation Library™ and Fragmentation Library™ are trademarks of HighChem, Ltd., Slovak Republic

The primary purpose of library reactions is to create a database of fragmentation and rearrangement mechanisms that can be applied to user provided structures to predict the decomposition pathways occurring in a mass spectrometer. The library mechanisms are a significant extension to the general fragmentation rules which may not cover all the complex processes for a broad spectrum of ionization and ion activation techniques. The usefulness of library mechanisms is the flexibility it gives for altering predicted fragmentation pathways and entering highly specific mechanisms that apply for a limited class of compounds. Since the current version contains around 19,000 reactions, the fragmentation predictability is much higher than if only general rules apply. This is especially true for low energetic experiments such as ESI or APCI that often yield complicated skeletal rearrangements and unusual ring closures.

Any recognized and active mechanism that has been saved into a library serves as a Knowledge Base for the prediction of fragmentation pathways. So, after you have drawn and saved a reaction into any fragmentation library, you can use the mechanism template for prediction fragmentation pathways for any structure that the derived mechanism can be applied to. The range of applicability depends on many factors, but structurally similar compounds with a common ring scaffold usually exhibit identical fragmentation mechanisms.

If a fragmentation reaction was predicted using a library reaction, you can double click on the Lib arrow caption to see the corresponding mechanism. Both template and generated fragmentation mechanisms will be displayed in red in the Fragmentation Library window and the Fragments & Mechanisms window.

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Fragmentation libraries are fully searchable by various criteria. Search criteria can be combined to narrow your search. All searchable items can be found in the Fragmentation Library window in the Search tab. To conduct a search, simply type the desired query in relevant search box or select one of the predefined options and click on the Search button. The search results will be listed in the grid box in the lower part of the window.

(Sub)Structure search allows you to select all the reactions in a library that match all the reaction precursor or product structures with your query (sub)structure. The search results display the query substructure in red in the library structure, in the same manner as for the Substructure Search procedure in Database Manager.

Since fragmentation libraries largely contain ionic structures that can undergo resonance reactions, Mass Frontier offers a unique resonance substructure search. This feature ensures the correct retrieval of all resonance structures, even if the query structure is in a different resonance form than the library structure. This feature works fully automatically, so the user need not worry about the particular resonance state of the ionic structures. However, be aware of this functionality when reviewing search results, as the query and library structures in positive search results may appear to be different if the resonance reaction are possible. In this case, do not consider this difference as an error, but rather be aware that there may be complex resonance variations.

It is possible to ignore charges and radicals in (sub)structure searches if their location is ambiguous. When searching structures with an unspecified charge location or substituents, be sure to review the search rules that apply.

HighChem Fragmentation Library™ and Fragmentation Library™ are trademarks of HighChem, Ltd., Slovak Republic

At HighChem, we have been collating fragmentation mechanisms published in all the available printed media dedicated to mass spectrometry for a number of years and these have been entered into our computer system. Each reaction, along with the chemical structures, has been manually drawn in Reaction Editor and saved in the HighChem Fragmentation Library™, which currently contains around 100.000 individual reactions. Fragmentation pathways are accompanied with complementary information such as the title, authors and source of the information. This library collection serves as a knowledge base for the prediction of fragmentation pathways from user provided structures.

To ensure the data is of the highest quality, fragmentation mechanisms have been rigorously evaluated in two stages: manual and automatic. The manual evaluation includes accuracy and plausibility assessments of reaction mechanisms and consistency checking between fragment masses and peak m/z values, if the spectrum was available. The automatic evaluation includes simple element, charge and radical consistency checks on both sides of the reaction, in addition to newly developed algorithms for complex electron mapping that has revealed formally erroneous mechanisms. Numerous problems and errors regarding mechanisms were uncovered by both stages and either appropriate corrections were made, or these mechanisms were excluded from the library.

Please note that HighChem owns all the intellectual property rights regarding reactions data and reaction drawings in the HighChem Fragmentation Library™ and they may not be copied, translated, distributed, published or used in connection with other systems without  the written approval of HighChem, Ltd., Slovak Republic.

HighChem Fragmentation Library™ and Fragmentation Library™ are trademarks of HighChem, Ltd., Slovak Republic