Finnigan Xcalibur

®

HighChem©: Mass Frontier™ Software

Revision A XCALI-97073

Written by Robert Mistrik. Finnigan™ is a trademark of and Xcalibur® is a registered trademark of Thermo Electron Corporation. Mass Frontier™ is a trademark of HighChem©. Microsoft®, Windows®, and Windows NT® are registered trademarks of Microsoft Corporation.

Technical information contained in this publication is for reference purposes only and is subject to change without notice. Every effort has been made to supply complete and accurate information; however, Thermo Electron Corporation assumes no responsibility and will not be liable for any errors, omissions, damage, or loss that might result from any use of this manual or the information contained therein (even if this information is properly followed and problems still arise). This publication is not part of the Agreement of Sale between Thermo Electron Corporation and the purchaser of a GC/MS or LC/MS system. In the event of any conflict between the provisions of this document and those contained in Thermo Electron Corporation’s Terms and Conditions, the provisions of the Terms and Conditions shall govern. System Configurations and Specifications supersede all previous information and are subject to change without notice.
Printing History: Revision A printed in June 2003. Software Version: Xcalibur 1.2, 1.3, and 1.4

The products of Thermo Electron San Jose are produced under ISO 9001 accredited quality management systems.

Published by Technical Publications, Thermo Electron Corporation, San Jose, California. Copyright© 2003 Thermo Electron Corporation. All rights reserved. Printed in the United States of America.

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Contents Xcalibur ________________________________________________________________________

Contents

Read This First............................................................................................................................ vii
Changes to the Manual and Online Help .............................................................................................. viii Abbreviations .......................................................................................................................................... ix Typographical Conventions .................................................................................................................. xiii Data Input ............................................................................................................................ xiii Boxed Information............................................................................................................... xiv Topic Headings..................................................................................................................... xv Reply Cards........................................................................................................................................... xvi

Introducing Mass Frontier............................................................................................................1
1.1 General Information...................................................................................................................... 2 System Requirements ............................................................................................................. 3 Installation .............................................................................................................................. 4 Program Limitations............................................................................................................... 6 Modules Overview........................................................................................................................ 8

1.2

Structure Editor ...........................................................................................................................11
2.1 Structure Editor Window ............................................................................................................ 12 Reduced Structure Editor Window....................................................................................... 14 Restoring Defaults ................................................................................................................ 14 Opening and Saving Structures ............................................................................................ 15 Structure Data Formats......................................................................................................... 15 Structure Layout.......................................................................................................................... 16 Text.............................................................................................................................................. 18 Templates: Selecting Atoms and Bonds..................................................................................... 19 Atom Properties .......................................................................................................................... 21 Bond Properties........................................................................................................................... 22 Copying Structures...................................................................................................................... 23 Pasting Structures........................................................................................................................ 24 Moving, Resizing, Rotating, and Mirroring Structures .............................................................. 25

2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

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2.10 2.11 2.12 Cleaning Structures......................................................................................................................26 Checking Structures.....................................................................................................................27 MS Calculations ..........................................................................................................................28

Spectra Manager..........................................................................................................................29
3.1 Open Spectra Manager Window..................................................................................................31 Records in Spectra Manager .................................................................................................31 Additional Information Associated with a Record ...............................................................33 Mass Differences .........................................................................................................................36 Comparing Spectra ......................................................................................................................37 Cutting, Copying, and Pasting Records.......................................................................................38 Data Exchange between Microsoft Excel and Spectra Manager.................................................39 Exporting Data to Microsoft Excel .......................................................................................40 Importing Spectra from Microsoft Excel ..............................................................................41 Using Microsoft Excel as a Spectrum Editor........................................................................42 Structures in Spectra Manager.....................................................................................................43 Working with Spreadsheets .........................................................................................................46 Search Utilities ............................................................................................................................47 Spectrum Search ...................................................................................................................49 (Sub)Structure Search ...........................................................................................................51 Name Search .........................................................................................................................54 Molecular Formula Search....................................................................................................54 Molecular Mass Search.........................................................................................................54 ID Number Search ................................................................................................................54 CAS Number Search.............................................................................................................55 Search Constraints.................................................................................................................55 Mass Spectral Data Exchange Between Modules .......................................................................57

3.2 3.3 3.4 3.5

3.6 3.7 3.8

3.9

Library Utilities............................................................................................................................59
4.1 4.2 4.3 4.4 NIST/EPA/NIH Mass Spectral Database.....................................................................................60 Library Installation ......................................................................................................................61 Creating User Libraries ...............................................................................................................63 Uninstall Library..........................................................................................................................65

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4.5 4.6 4.7 4.8

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Adding Records to a User Library .............................................................................................. 66 Deleting Library Entries.............................................................................................................. 68 Changing Structures in Libraries ................................................................................................ 70 Opening and Saving Spectra, Structures, and Spectra References ............................................. 71

Fragments & Mechanisms ..........................................................................................................73
5.1 Features ....................................................................................................................................... 74 General Fragmentation and Rearrangement Rules............................................................... 74 Charge Localization Concept ............................................................................................... 74 Unimolecular Linear Reaction Mechanisms ........................................................................ 74 Even-Electron Rule .............................................................................................................. 75 Bond Cleavages Only ........................................................................................................... 75 Ionization Methods............................................................................................................... 75 Formally Possible Solutions ................................................................................................. 75 Fragmentation, Rearrangement and Resonance Reactions ......................................................... 76 Starting Generation ..................................................................................................................... 79 Fragments & Mechanisms Window............................................................................................ 82 Reaction Restrictions .................................................................................................................. 84 Ionization & Cleavage Page ................................................................................................. 84 H-Rearrangement Page......................................................................................................... 86 The Resonance Page............................................................................................................. 87 The Additional Page ............................................................................................................. 89 The Sizes Page...................................................................................................................... 90 Generated Fragments Linked with Spectrum.............................................................................. 92 Eliminating Generated Fragments Not Present in a Spectrum ................................................... 93 Simulation of MSn Experiments ................................................................................................. 94 Unexplained Peaks...................................................................................................................... 95 Too Many Proposed Fragments for a Peak ................................................................................. 97 Bar Code Spectra ...................................................................................................................... 101 Marking Fragments ................................................................................................................... 104

5.2 5.3 5.4 5.5

5.6 5.7 5.8 5.9 5.10 5.11 5.12

Fragments Comparator.............................................................................................................105

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Contents

___________________________________________________________________ Finnigan Xcalibur Mass Spectra Classification ......................................................................................................109
7.1 7.2 7.3 7.4 Spectra Classification ................................................................................................................ 110 Principal Component Analysis (PCA)....................................................................................... 112 Neural Networks (Self-Organizing Maps) ................................................................................ 114 Spectra Transformation ............................................................................................................. 115

Spectra Classifier .......................................................................................................................117
8.1 8.2 8.3 Spectra Classifier Window ........................................................................................................ 118 Classifying Mass Spectra...........................................................................................................122 Maintaining Groups of Spectra..................................................................................................124

Spectra Projector .......................................................................................................................125
9.1 9.2 9.3 9.4 9.5 9.6 Generating Spectra Projector Module .......................................................................................126 Spectra Projector Window .........................................................................................................127 3D Projection Mode...................................................................................................................129 Opening and Saving of Classification Results ..........................................................................130 Accessing Spectra from Spectra Projector ................................................................................131 Adding External Spectrum ........................................................................................................132

Neural Networks ........................................................................................................................133
10.1 10.2 10.3 Generating Neural Networks Module........................................................................................135 Neural Networks Window .........................................................................................................136 Working with Neural Networks.................................................................................................138

GC/LC/MS Processor ................................................................................................................139
11.1 11.2 11.3 11.4 GC/LC/MS Processor Window .................................................................................................141 Data File Formats ......................................................................................................................142 Opening Chromatograms...........................................................................................................143 TIC Page ....................................................................................................................................144

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11.5 11.6 11.7 11.8 11.9

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Info Page ................................................................................................................................... 145 Spectra Averaging ..................................................................................................................... 146 Background Subtraction............................................................................................................ 147 Processing Extracted Spectra .................................................................................................... 148 Selected Ion Chromatogram...................................................................................................... 149

11.10 Automated Component Detection and Spectra Deconvolution ................................................ 151 11.11 Processing Xcalibur MSn Data ................................................................................................. 156

Monoisotopic Mass Settings ......................................................................................................159

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Read This First
Welcome to HighChem©: Mass Frontier™, which is a part of Xcalibur®, the Thermo Electron Finnigan™ mass spectrometry data system! This Xcalibur manual provides you with information about how to use Mass Frontier Software for mass spectral interpretation. The Mass Frontier Software manual includes the following topics: Introducing Mass Frontier provides an overview of the product and describes installation and system requirements. Structure Editor gives basic information about chemical structures and how to edit your software displays. Spectra Manager provides instructions about comparing spectra and library search parameters. Library Utilities describes how to manage your custom or commercial libraries databases. Fragments & Mechanisms gives information about the chemistry of mass fragmentation. Fragments Comparator provides instructions about processing and comparing fragments generated in the Fragments & Mechanisms windows. Mass Spectra Classification describes the mass classification methods available in Mass Frontier Software. Spectra Classifier gives instructions about retrieving and organizing spectra. Spectra Projector provides instructions about displaying the results of a Principal Component Analysis by projecting the mass spectra as points on a two-dimensional plane or a three-dimensional twistable space. Neural Networks explains how to generate the Neural Networks module and describes the components of the Neural Networks window. GC/LC/MS Processor lists tools for displaying your chromatographic and spectral data. Monoisotopic Mass Settings describes how to change the monoisotopic mass settings used for all calculated masses displayed in Mass Frontier Software.

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Read This First Changes to the Manual and Online Help

_____________________________________ Finnigan Xcalibur

Changes to the Manual and Online Help
To suggest changes to this manual or the online Help, please send your comments to: Editor, Technical Publications Thermo Electron San Jose 355 River Oaks Parkway San Jose, CA 95134-1991 U.S.A. You are encouraged to report errors or omissions in the text or index. Thank you.

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Read This First Xcalibur _____________________________________________________________ Abbreviations

Abbreviations
The following abbreviations are used in this and other manuals and in the online Help. A ac ADC AP APCI API ASCII b B baud rate °C CD CD-ROM cfm CI CIP cm cm3 CPU CRC CRM <Ctrl> d Da DAC dc DDS DEP DS DSP ampere alternating current analog-to-digital converter acquisition processor atmospheric pressure chemical ionization atmospheric pressure ionization American Standard Code for Information Interchange bit byte (8 b) data transmission speed in events per second degrees Celsius compact disc compact disc read-only memory cubic feet per minute chemical ionization carriage and insurance paid to centimeter cubic centimeter central processing unit (of a computer) cyclic redundancy check consecutive reaction monitoring control key on the terminal keyboard depth dalton digital-to-analog converter direct current direct digital synthesizer direct exposure probe data system digital signal processor

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Read This First Abbreviations _________________________________________________________ Finnigan

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EI EMBL <Enter> ESD ESI eV f °F .fasta file FOB ft FTP g G GC GC/MS GND GPIB GUI h h HPLC HV Hz ICIS ICL ID IEC IEEE in. I/O k K KEGG kg

electron ionization European Molecular Biology Laboratory enter key on the terminal keyboard electrostatic discharge electrospray ionization electron volt femto (10-15) degrees Fahrenheit extension of a SEQUEST search database file free on board foot file transfer protocol gram giga (109) gas chromatograph; gas chromatography gas chromatograph / mass spectrometer electrical ground general-purpose interface bus graphical user interface hour height high-performance liquid chromatograph high voltage hertz (cycles per second) Interactive Chemical Information System Instrument Control Language inside diameter International Electrotechnical Commission Institute of Electrical and Electronics Engineers inch input/output kilo (103, 1000) kilo (210, 1024) Kyoto Encyclopedia of Genes and Genomes kilogram

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Read This First Xcalibur _____________________________________________________________ Abbreviations

l L LAN lb LC LC/MS LED µ m m M M+ MB MH+ min mL mm MS MS MS/MS MSn m/z n NCBI NIST OD Ω p Pa PCB PID P/N P/P

length liter local area network pound liquid chromatograph; liquid chromatography liquid chromatograph / mass spectrometer light-emitting diode micro (10-6) meter milli (10-3) mega (106) molecular ion Megabyte (1048576 bytes) protonated molecular ion minute milliliter millimeter mass spectrometer; mass spectrometry MSn power: where n = 1 MSn power: where n = 2 MSn power: where n = 1 through 10 mass-to-charge ratio nano (10-9) National Center for Biotechnology Information (USA) National Institute of Standards and Technology (USA) outside diameter ohm pico (10-12) pascal printed circuit board proportional / integral / differential part number peak-to-peak voltage

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Read This First Abbreviations _________________________________________________________ Finnigan

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ppm psig RAM RF RMS ROM RS-232 s SIM solids probe SRM SSQ TCP/IP TIC Torr TSQ u URL V V ac V dc vol w W WWW

parts per million pounds per square inch, gauge random access memory radio frequency root mean square read-only memory industry standard for serial communications second selected ion monitoring direct insertion probe selected reaction monitoring single stage quadrupole transmission control protocol / Internet protocol total ion current torr triple stage quadrupole atomic mass unit uniform resource locator volt volts alternating current volts direct current volume width watt World Wide Web

Note. Exponents are written as superscripts. In the corresponding online Help, exponents are sometimes written with a caret (^) or with e notation because of design constraints in the online Help. For example: MSn (in this manual) MS^n (in the online Help) 105 (in this manual) 10^5 (in the online Help)

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Read This First Xcalibur ___________________________________________________Typographical Conventions

Typographical Conventions
Typographical conventions have been established for Thermo Electron San Jose manuals for the following: • • • Data input Boxed information Topic headings

Data Input
Throughout this manual, the following conventions indicate data input and output via the computer: • • • • • Messages displayed on the screen are represented by capitalizing the initial letter of each word and by italicizing each word. Input that you enter by keyboard is represented in bold face letters. (Titles of topics, chapters, and manuals also appear in bold face letters.) For brevity, expressions such as “choose File | Directories” are used rather than “pull down the File menu and choose Directories.” Any command enclosed in angle brackets < > represents a single keystroke. For example, “press <F1>” means press the key labeled F1. Any command that requires pressing two or more keys simultaneously is shown with a minus sign connecting the keys. For example, “press <Shift> - <F1>” means press and hold the <Shift> key and then press the <F1> key. Any button that you click on the screen is represented in bold face letters and a different font. For example, “click on Close”.

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Boxed Information
Information that is important, but not part of the main flow of text, is displayed in a box such as the one below.
Note. Boxes such as this are used to display information.

Boxed information can be of the following types: • Note – information that can affect the quality of your data. In addition, notes often contain information that you might need if you are having trouble. Tip – helpful information that can make a task easier. Important – critical information that can affect the quality of your data. Caution – information necessary to protect your instrument from damage. CAUTION – hazards to human beings. Each CAUTION is accompanied by a CAUTION symbol. Each hardware manual has a blue CAUTION sheet that lists the CAUTION symbols and their meanings. DANGER – laser-related hazards to human beings. It includes information specific to the class of laser involved. Each DANGER is accompanied by the international laser radiation symbol.

• • • •

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Read This First Xcalibur ___________________________________________________Typographical Conventions

Topic Headings
The following headings are used to show the organization of topics within a chapter:

Chapter 1 Chapter Name
1.2 Second Level Topics
Third Level Topics
Fourth Level Topics
Fifth Level Topics

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Read This First Reply Cards __________________________________________________________ Finnigan

Xcalibur

Reply Cards
Thermo Electron San Jose manuals contain one or two reply cards. All manuals contain a Customer Registration / Reader Survey card and some contain a Change of Location card. These cards are located at the front of each manual. The Customer Registration / Reader Survey card has two functions. First, when you return the card, you are placed on the Thermo Electron San Jose mailing list. As a member of this list, you receive application reports and technical reports in your area of interest, and you are notified of events of interest, such as user meetings. Second, it allows you to tell us what you like and do not like about the manual. The Change of Location card allows us to track the whereabouts of the instrument. Fill out and return the card if you move the instrument to another site within your company or if you sell the instrument. Occasionally, we need to notify owners of our products about safety or other issues.

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Chapter 1 Introducing Mass Frontier
HighChem©: Mass Frontier is a part of Xcalibur®, the Finnigan mass spectrometry data system. Mass Frontier 3.0 is a software package for the management, evaluation and interpretation of mass spectra and chromatograms. This software provides a number of useful tools for processing and organizing mass spectral data. Because of the large volume of information a mass spectrometer can produce, management capabilities are essential in mastering your analytical workloads. In contrast to other software systems, Mass Frontier offers many sophisticated features for the efficient interpretation of mass spectra even where an unknown is not contained in your user-contributed or commercially available library. Many of its features are innovative and unique, so you will need to take a few minutes to familiarize yourself with them. In addition, we will try to show you some new ways to review your data and how to use the power of Mass Frontier to interpret mass spectra. This introduction contains the following topics:
• •

General Information Modules Overview

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1.1 General Information
Mass Frontier is based on ten modules, which are accessible as windows. All the modules are seamlessly integrated within an intuitive multidocument interface (MDI). This means that all the windows are located in your program desktop. Three of the ten modules (Fragments & Mechanisms, Neural Networks, and Spectra Projector) cannot be directly opened from the program desktop by clicking a button or menu item; they must be generated from user-supplied input data. Although each of the modules is independent, the program can automatically establish a link between several modules. For example, the program can make a link between the Spectra Manager window and the Fragments & Mechanisms module. In this case the peaks in a mass spectrum, displayed in the Spectra Manager module, are linked with massto-charge ratios in the Fragments & Mechanisms module. Records in Spectra Manager can also be linked with objects in the Spectra Classifier and Spectra Projector modules. A simple double-click on these objects gives the spectra (Spectra Classifier) or spectrum (Spectra Projector) that are linked with a particular record in Spectra Manager. In addition, spectra, structures, and library entries can be exchanged between modules via the Copy and Paste commands. General information includes the following topics:
• • •

System Requirements Installation Program Limitations

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Introducing Mass Frontier

System Requirements
• • • • • • • • • •

Microsoft® Windows® NT 4.0 (Service Pack 3) or higher with Xcalibur 1.2 Microsoft Windows 2000 Professional (Service Pack 2) with Xcalibur 1.3 Microsoft Windows XP Professional with Xcalibur 1.4 PC with Pentium 133 MHz or higher processor 64 MB of RAM (128 MB recommended) SVGA Monitor 50 MB free hard disk space Microsoft Office 97, 2000 (SR-1), or XP Microsoft Internet Explorer 4.01 (SP 1) or higher Xcalibur 1.2, 1.3, or 1.4 installed with local user access

Note. Mass Frontier 3.0 supports both the 2002 and 1998 versions of the NIST Library. If you install the 2002 version, you will need to download the Mass Frontier SR1 from http://www.highchem.com/mfd.htm.

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Installation
Before you install Mass Frontier software, make sure the Xcalibur data system is present on your PC, Mass Frontier cannot be activated without it. If you have installed Mass Frontier without Xcalibur being present, you should uninstall Mass Frontier, then install Xcalibur and reinstall Mass Frontier. To install Xcalibur and Mass Frontier software, do the following: 1. Insert the CD labeled Xcalibur Core Data System Software into your CD ROM drive. 2. Choose Start | Run from your Windows NT Desktop. 3. Type D:\setup.exe, and click OK. (Substitute the appropriate letter of your CD ROM drive for D.) 4. Follow the instructions on the screen until you reach Finish. You can now install the Mass Frontier software. 5. The Mass Frontier CD-ROM is AutoPlay-enabled. Insert the CD-ROM and the setup program will start automatically. 6. Follow the Mass Frontier software loading instructions located on the back of the plastic CD-ROM box. If the AutoPlay feature is not enabled, open the Control Panel window, and click Add | Remove Programs. Then, follow the installation instructions that appear on the screen. 7. After the installation procedures are completed and you first start Mass Frontier, the license page will appear displaying your system software Serial Number. See Figure 1. Record this number (or highlight the number with the cursor and use the Copy command). Fax this number (or use the Paste command to paste the number into an e-mail message) to Thermo Electron San Jose to obtain your software access code Activation Key for Mass Frontier. Every system that Mass Frontier is loaded on requires its own activation key, as they are not transferable. If you have purchased multiple copies of Mass Frontier, start the loading procedure on each system to obtain the software serial numbers for each one.

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Introducing Mass Frontier

Figure 1. License page, showing the serial number and the types of Activation Key available

8. Send your request for Mass Frontier Activation Access Codes along with all software serial numbers and the following information: Your Name Company Name Address Your Fax or E-mail Address To: Fax: Thermo Electron San Jose (408) 965-6120

Email: license.finnigan-lcms@thermo.com 9. When you receive your activation key, record it in a visible place (for example, write it with a marker on your software CD and in the manual). Type your activation key in the box provided on the loading screen and continue with the loading procedure. Remember that if you wish to load the software on another PC system, you will require another activation key. 10. If you are using the 2002 Version of the NIST Library, you need to download and install the Service Release of Mass Frontier (SR1) from the following location: http://www.highchem.com/mfd.htm.

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When the installation is complete, you can find the new HighChem Mass Frontier program directory by choosing Start | Programs | HighChem. To add HighChem Mass Frontier to the Xcalibur Home Page window Tool menu and/or the Qual Browser window Tool menu, choose Tools | Add Tools from the appropriate Xcalibur window. The Add Programs to Tool Menu dialog box will appear. The HighChem Mass Frontier program is installed at the following location: C:\Program Files\HighChem\Mass Frontier 3.0\MassFrontier.exe

Program Limitations
Mass Frontier offers a number of advanced features. However, you should be aware of the following limitations:

Mass Frontier deals primarily with small organic structures rather than peptides and other biologically related molecules. The Structure Editor and other modules dealing with structures have a limit of 127 nonhydrogen atoms per structure. If you try to exceed this number a message box will appear reminding you of this limitation. Mass Frontier is designed for pure substances only. Mixtures are not accepted. The program considers a mixture to be two or more structures, depicted in the same window, which are not connected by a bond (represented as a line). If you try to generate fragments and mechanisms from a mixture, the message box alerts you that this action is not permitted. The Check Structures option also detects mixtures as an error. However, library utilities support "mixtures", to ensure backward compatibility with commercial libraries. Mixtures may also be added to a user library. Fragments & Mechanisms can be generated in three modes: Electron Ionization (EI) mode, Protonation mode [M+H]+, and Chemical Ionization (CI) mode. Protonation mode includes the following “soft” ionization techniques: Electrospray Ionization (ESI), Atmospheric Pressure Ionization (API) and Field Desorption Ionization (FD). Mass Frontier offers the ability to select reagent gases for Chemical Ionization. However, the relative ionization potentials of reagent gases cannot be modified. Negative ionization is not supported. Since “soft” ionization techniques are mainly low energy experiments, which often yield complex skeletal and “random” rearrangements, the predictability of these fragmentation and rearrangements processes is not as high as attained by electron ionization.

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Introducing Mass Frontier

Mass Frontier is designed exclusively for neutral and single charged molecules. As a consequence you can only attach the charge symbol (+ or -) to one atom. The Generation of Fragments and Mechanisms command currently only supports neutral molecules and cations. However, all the other modules support anions. Biradicals are not supported by any module. Mass Frontier supports single-unit mass spectra with a mass-to-charge ratio range of 1 to 3000 mass units. Mass spectra containing peaks with a mass-to-charge ratio greater than 3000 are not displayed. Only 800 peaks per spectrum can be displayed at the same time. If a spectrum contains more than 800 peaks, the program will select the 800 most prominent peaks.

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1.2 Modules Overview
Mass Frontier consists of the following modules: Structure Editor, Spectra Manager, GC/LC/MS Processor, Spectra Classifier, Fragments Comparator, Isotope Pattern, Periodic Table, Spectra Projector, Neural Networks, and Fragments & Mechanisms. See Figure 2. The Spectra Projector, Neural Networks, and Fragments & Mechanisms modules cannot be opened directly from program desktop. These modules must be generated from user-supplied data.

Periodic Table Isotope Pattern Fragments Comparator Spectra Classifier GC/LC/MS Processor Spectra Manager Structure Editor
Figure 2. Mass Frontier modules menu

Structure Editor is an easy-to-use, full-featured structure drawing tool. Structure Editor automatically calculates the mass of a selected fragment and the corresponding loss. Many different kinds of chemical structure created in this module can be used throughout the program. Spectra Manager provides a number of convenient ways for organizing and processing mass spectra, chemical structures, and libraries. A user-friendly spreadsheet format is provided for data handling. Advanced database query and search features give the user instant access to the information needed for rapid compound identification. User libraries containing chemical structures of ions, radicals, isotopes, and optically active compounds can be created at the click of a mouse. This module supports data exchange with Microsoft Excel.

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GC/LC/MS Processor has been designed for the convenient processing of mass spectral scans by users of hyphenated chromatographic techniques such as GC-MS or LC-MS. Powerful component detection and spectra deconvolution algorithms allow the automated extraction of individual spectra from complex chromatographic data files. This module also provides visual tools for selecting particular spectra from MSn experiments according to selected criteria. Spectra Classifier allows the user to retrieve and organize spectra intended for classification. Because spectra can be classified according to various criteria (structural, physical, and other properties), it is useful to organize the spectra into different groups. Such groups of spectra can be visually represented in different ways (using colors, symbols, and numbers), which highlight similarities or dissimilarities among the user-chosen spectral groups. Fragments Comparator shows a series of fragments in a table format. Columns are made up of individual compounds and rows show either massto-charge ratios or the structures of fragments. This module allows an effective comparison of the product ions of analogous molecules. Isotope Pattern displays the relevant isotopic profile, whenever a structure or fragment is selected in the program. Periodic Table allows you to display the terrestrial isotopic abundance of elements and their multi-atomic isotopic profiles. Spectra Projector displays the results of Principal Component Analysis (PCA). Mass spectral data can be classified using 2D or 3D projections in which each point represents a spectrum. If the class membership of an unknown spectrum needs to be determined, it can be opened or pasted into an existing projection. Neural Networks is the second classification strategy available in Mass Frontier. Mass spectra are classified using a powerful method called selforganizing maps (SOM), which are a special class of neural network. If two or more spectra activate the same neuron, we can assume that the corresponding compounds will exhibit similar physical or chemical properties, or biological activities. The Fragments & Mechanisms module is an expert system for the automated generation of fragments and detailed fragmentation and rearrangement mechanisms from a user-supplied chemical structure. This module consists of a system that includes a comprehensive set of known reaction mechanisms, allowing automated prediction at an expert level. This module can be generated either from Structure Editor or Spectra Manager module.

Introducing Mass Frontier

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Chapter 2 Structure Editor
Mass spectra reflect the structural features of molecules. Structural information is essential for the interpretation and the investigation of structure-spectrum relationships. Mass Frontier incorporates the structure drawing tool - Structure Editor, which permits the interactive handling of all kinds of structural information. Structure Editor is a full-featured structure drawing tool for editing, importing, exporting, and checking chemical structures. Structure Editor is the gateway to three other modules in this program: Spectra Manager, Fragments & Mechanisms, and Isotope Pattern. The chapters dealing with these modules assume that you are already familiar with how to draw high quality structures. This chapter contains the following topics:
• • • • • • • • • • • •

Structure Editor Window Structure Layout Text Templates: Selecting Atoms and Bonds Atom Properties Bond Properties Copying Structures Pasting Structures Moving, Resizing, Rotating, and Mirroring Structures Cleaning Structures Checking Structures MS Calculations

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Structure Editor Structure Editor Window ____________________________________________________

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2.1 Structure Editor Window
The Structure Editor Window is shown in Figure 3.

Open Structure Save Structure Print Structure Undo Redo

Generate Fragments & Mechanisms (Sub)Structure Search Structure Layout Check Structure Clean

Single Bond Double Bond Triple Bond Hydrocarbon Chain Benzene Ring Six Membered Ring Five Membered Ring n-Membered Ring Templates Atom Properties Bond Properties Positive Charge Radical Text Remark Cut

Mirror Rotate Resize Select All Delete Paste Copy

Figure 3. Structure Editor Window, showing names of icon commands

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To open the Structure Editor window, do either of the following:
• •

Structure Editor

Click on the Structure Editor button Frontier window (see Figure 2).

in the toolbar of the main Mass

Choose Tools | Structure Editor in the menu bar of the main window (see Figure 2).

If you open a structure by choosing File | Open | Structure, Structure Editor will start automatically. Select the file type *.mol.

Note. Only one Structure Editor window can be opened at any one time in the program. If you click the Structure Editor button, or choose Tools | Structure Editor, and Structure Editor is already open, this window becomes active.

To begin drawing a chemical structure in Structure Editor, click one of the buttons on the vertical toolbar. Every time you click one of these buttons the shape of the cursor changes, giving you a visual indication of the engaged drawing mode. The vertical buttons, in contrast to the horizontal buttons, are not represented in the menu. Because using buttons is easier and faster than using menu items, the best methods for drawing chemical structures in Structure Editor are explained mainly via the buttons. If the function of a button is not apparent from its appearance, simply point the cursor at the button to display a tool tip. We recommend trying out all the various features on a test structure.

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Structure Editor Structure Editor Window ____________________________________________________

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Reduced Structure Editor Window
When you reduce the size of the Structure Editor window, the button array on your desktop changes. See Figure 4. The reduced Structure Editor window is more compact, but retains all the buttons on the toolbars. A smaller window might prove advantageous if your program desktop is filled with other modules.

Figure 4. Structure Editor window, reduced size

Restoring Defaults
In the Structure Editor’s default state, all buttons are switched off, and no atom or bond is selected. The plain arrow cursor indicates that the Structure Editor is in default state. To restore the default state of the editor, do either of the following:
• •

Click on the Default button Right-click the mouse

in the upper left corner, or

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Structure Editor

Opening and Saving Structures
To open or save a structure, do either of the following:
• •

Click on the Open Structure button in the Structure Editor window.

or the Save Structure button

Choose File | Open | Structure or File | Save | Structure in the Mass Frontier window.

If you are opening a file that contains more than one structure, only the first structure in the file is loaded into Structure Editor. Mass Frontier is a 32-bit application, so you can use long names to save structures. You can even save structures by their actual names (for example, 1-Amino-2-hydroxyindane.mol).

Structure Data Formats
Structure Editor supports two kinds of structure formats: MDL MOL-files, with the .mol extension, and HighChem MCS format (Maximal Compressed Structure), with the .mcs extension. These formats are also supported in the Spectra Manager module. Templates are stored in MCS-format, using the extension .tml. One of Mass Frontier’s particularly useful features is the ability to restrict a search by a set of structural constraints, the so-called Good-Bad list. For example, you can instruct the program to conduct a library search comparing an unknown spectrum only with the spectra of ketones. This feature provides an endless range of possibilities with which to target your search results. The Good-Bad structures are stored in the \Constraints directory, and the structures are saved in MCS-format. The program automatically retrieves all MCS-structures from the \Constraints directory and puts them in a Good-Bad list box in the Constraints dialog box. To learn more about the structure search constraints refer to Chapter 3, Spectra Manager.

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Structure Editor Structure Layout __________________________________________________________

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2.2 Structure Layout
Mass Frontier is a layout powerhouse. For structures, as well as for other objects, you can change almost anything that it is practically possible to change. Every layout setting change also affects printing and copying to the Clipboard, (the one exception is background color, which only affects the screen display in the Mass Frontier program.) The various layout items allow you to tailor the graphics to your individual report or publication needs. By default, the symbols for hydrogen atoms attached to carbon atoms are not displayed. See Figure 5 (a). If you want to display them, do the following [See Figure 5 (b).]: 1. Click on the Structure Layout button .

2. Select the Show Carbon Symbols check box in the Atom tab page. Hydrogen atoms are only displayed for carbons if the Show Carbon Symbols check box is selected. Otherwise, corresponding hydrogens are displayed for heteroatoms only.

Note. If you draw nonisotopic explicit hydrogen atoms these are removed in the Fragments & Mechanisms window. See Figure 5 (c). They can make the mechanism network unclear, especially for complex hydrogen rearrangement steps.

(a) OH

(b)
HC HC

OH C CH CH CH

(c)
H

OH H

H H

H

Figure 5. Structures, showing different displays of hydrogens

The structure layout settings apply to all structures in Mass Frontier simultaneously. This means that if you change a structure layout item, all structures in the Structure Editor, Spectra Manager, Fragments & Mechanisms, and Fragments Comparator modules are affected.

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Structure Editor

Note. If you do not have a color printer and are printing in black and white, and you have set bright colors for bonds or atoms, the lines and fonts may appear indistinct. To avoid this problem, specify darker colors for all structural items, including spectra, chromatograms, and mechanisms.

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Structure Editor Text ____________________________________________________________________

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2.3 Text
Structure Editor offers the possibility of labeling a structure or displaying a text note on the screen or on the printout. See Figure 6. To enter a text note, do the following: 1. Click on the Text button .

2. Click anywhere in the drawing area to place the text. 3. Type the desired text, and confirm the text by clicking outside the text area or press any button in Structure Editor. It is possible to create up to 127 separate text notes. If you want to change the font, color, size, or background of the text notes, you can do this in the Structure Layout window on the Text tab.

Note. Text notes are not associated with structures. As a result, the Open, Save, Copy, and Paste actions only apply to structure(s). When these actions are applied, the text notes are ignored even if a structure is selected together with a text. Additionally, structure-handling routines such as resizing, rotating, or mirroring can only be performed on structures.

Figure 6. Structure labeled with its chemical name

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Structure Editor Selecting Atoms and Bonds

2.4 Templates: Selecting Atoms and Bonds
If you click the Templates button , the Template dialog box will appear. Mass Frontier comes with more than 200 predefined templates. To insert a template structure into Structure Editor: 1. Select a group of templates in the directory spin box. You can do this using the arrow keys on the keyboard or clicking the appropriate name of the group. 2. Click on any atom or bond, depending on whether you want to attach the template to an atom or a bond of a structure in Structure Editor. 3. The Template dialog box will disappear, and you can place or attach the chosen template. 4. Switch off the template button, or restore the default state of the Structure Editor by clicking the default button .

Mass Frontier makes it easy for you to create your own group of templates or add a structure to an existing group. The templates are organized by directory. The template root directory is \Templates. Every group of templates is stored in a separate subdirectory of the template root directory. Subdirectories are named after compound groups (for example: Steroids). The files within each subdirectory are named after actual structures using the extension .tml (for example, Cholesterol.tml). When you save a structure for template purposes select the Template (.tml) format in the Save As Type list box in the Save Structure dialog box. To build your own templates, follow these steps: 1. Draw a template structure. 2. Create or select a subdirectory in the directory \Templates. 3. Save the template structure in the subdirectory created or chosen in step 2 by selecting the Template (.tml) format in the Save Structure dialog box. Any modification that you make to a structure applies only to the selected atoms or bonds. In addition, when a (Sub)Structure search is initiated, the program automatically uses the selected (sub)structure in Structure Editor (see the Spectra Manager chapter for more details). Before you select one or more atoms you should restore the default state of the editor. To select a group of atoms that are next to each other, do the following: Hold down the mouse button, and drag a rectangle around the atoms you want to select.

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Structure Editor Templates: Selecting Atoms and Bonds________________________________________

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The usual Windows convention for selecting multiple items applies. To select atoms at different locations, use the keyboard Shift key. You can select a group of atoms that are not adjacent in either of the following ways:
• •

While holding down the Shift key, click on the atoms you want to select. While holding down the Shift key, hold down the LEFT mouse button and drag a rectangle around the atoms you want to select.

If you want to select all of the atoms and bonds in the structure, do either of the following:
• •

Click on the Select All button

, or choose Edit | Select All.

Double click anywhere in the draw area within Structure Editor, except on atoms and bonds.

Structure Editor offers two selection modes: Rectangle Selection and Lasso Selection. See Figure 7. To choose the selection mode that fits your needs, do either of the following:
• •

Click on the Default Mode button and choose the appropriate selection mode from the popup menu. Click anywhere in the draw area within Structure Editor, and click the Rectangle or Lasso Selection menu item on the popup menu that will appear in the draw area.

Lasso Selection

Rectangle Selection

Figure 7. Lasso and rectangle selection in Structure Editor

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9tyytrfyÁWhfwtg†ƒ __________________________________________________________ Atom Properties

Structure Editor

2.5 Atom Properties
To change the charge state or isotope of an atom, or to change the element entirely, use the Atom Properties dialog box. To open the Atom Properties dialog box, use either of the following methods:
• •

Select the Atom Properties button want to change.

, and then click on the atom you

Restore defaults, and then double-click on the atom you want to change.

In the Atom Properties dialog box you can make the changes by clicking the appropriate element button, charge and radical check box, or nucleon number edit box.

Note. The changes carried out in the Atom Properties dialog box only affect a single atom.

If you want to change an element that has a single character symbol (for example, C, H, D [deuterium], N, O, B, F, K, P, S, I, V, W, Y, or U), a faster method is available, as follows: Select all the atoms that you want to change, and press the appropriate letter key on the keyboard. All the selected atoms are transformed into the element you have chosen. Chlorine (Cl) or bromine (Br) atoms can be set in a similar way: While holding down the Shift key, select all the atoms that you want to change, and press either the C (for chlorine) key or B (for bromine) key on the keyboard.

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Structure Editor Bond Properties ___________________________________________________________

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2.6 Bond Properties
The Bond Properties dialog box includes bond multiplicity, bond style, and bond color. To change the multiplicity of a bond, click on the bond you want to change. , , or , and then click

To change the color or optical orientation of a bond, use the Bond Properties dialog box. To open the Bond Properties dialog box, do either of the following:
• •

Click on the Bond Properties button want to change.

, and click on the bond you

Restore defaults, and double-click on the bond you want to change.

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Structure Editor

2.7 Copying Structures
Mass Frontier supports extensive use of the Windows Clipboard for the exchange of structural information between modules. In addition, you can use the Copy and Paste commands inside Structure Editor. To draw larger structures efficiently, we recommend taking advantage of Copy and Paste commands. See Figure 8. To copy a structure or part of a structure to the Clipboard, do the following: 1. Select the structure or part of structure you want to copy. 2. Click on the Copy button , or choose Edit | Copy.

Note. Only the atoms that have been selected and their associated bonds will be copied!

In addition to structure exchange between modules, Mass Frontier allows structure export to other programs that deal with structural information. When you copy a structure, Mass Frontier automatically copies two different formats to the clipboard: structural information in MOL format and graphics in windows metafile format. If you paste a structure into the structure editing software, the MOL format will be used. If you paste the structure into any text editor, spreadsheet or any other program that works with graphics, the graphical information will be used. All these actions occur automatically, so you need not worry which format is used.

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Structure Editor Pasting Structures _________________________________________________________

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2.8 Pasting Structures
If you have copied a structure or fragment anywhere in the program, you can paste it to Structure Editor. If necessary, the structure can be changed or corrected and then returned to where it originated. This is especially useful for structure elucidation. For example, you can copy a structure from the Spectra Manager window, paste it to Structure Editor, make the required changes, and then move it back to Spectra Manager. If the spectrum and the structural proposal are not consistent, the process can be repeated. To paste a structure to Structure Editor, do either of the following:
• •

Click on the Paste button Choose Edit | Paste.

.

If you have copied a structure or fragment in a program other than Mass Frontier, you can only paste this structure if the external structure drawing software supports MOL format, and this format is activated. The majority of structure drawing tools support MOL format and usually have this format activated by default. When pasting a structure from an external source, it may sometimes appear larger in Mass Frontier than in the original software. If this should happen, simply reduce the size of the structure using the Resize tool.

O

Spectra Manager
O

Structure Editor

Fragments & Mechanisms

Figure 8. Structure exchange via Copy/Paste commands

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9tyytrfyÁWhfwtg†ƒ _____________________________ Moving, Resizing, Rotating, and Mirroring Structures

Structure Editor

2.9 Moving, Resizing, Rotating, and Mirroring Structures
Structure Editor provides four handling routines for moving, resizing, rotating, and mirroring structures. As before, you must first select the atoms or bonds you want to reconfigure. To move a structure, do the following: 1. Point the mouse cursor at any selected atom or bond. 2. Hold down the mouse button, and drag the selected structure to the new location. 3. Release the mouse button to drop the selected structure at the new location. To resize a structure, do the following: 1. Click on the Resize button Structure | Resize. in Structure Editor, or choose

2. Drag one of the small rectangles on the structure edge until the new size is achieved. 3. Release the mouse button.

Note. If you drag one of the diagonal rectangles, the aspect ratio will be kept constant during structure resizing.

The Rotate Structure option allows you to rotate a structure in any direction. The center of rotation, indicated by a small circle with a cross in the middle , can be moved to any location. To rotate a structure, do the following: 1. Click on the Rotate button , or choose Structure | Rotate. 2. Move the center of rotation to the desired position by dragging the circle with a cross. 3. Drag any of the small rectangles on the structure edge to achieve the new angular position. To make a mirror image of your structure, do the following: 1. Click on the Mirror button , or choose Structure | Mirror. 2. Click on one of the small rectangles on the structure edge.

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Structure Editor Cleaning Structures ________________________________________________________

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2.10 Cleaning Structures
The Clean function allows you to achieve a professional look to your structures. See Figure 9. With Mass Frontier you can clean up the entire structure or only part of a structure. For example, you can restrict cleaning to certain functional groups, while the main skeleton remains intact. However, the algorithm of cleaning 2D structures is a particularly difficult mathematical problem with a multitude of possible solutions. As a result, this function may, in some complicated cases, lead to structures you will not be completely satisfied with. If this should occur, simply use the Undo function. To clean a structure, do the following: 1. Select the structure or part of the structure. 2. Click on the Clean button , or choose Structure | Clean.

O

1.

O

2.

O N

N

N

Figure 9. Structures, showing the results of using the Clean command

Note. If you want to clean only part of a structure, the selected atoms must be connected. Otherwise, a message box will appear reminding you that it is only possible to clean connected atoms.

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Structure Editor

2.11 Checking Structures
Structure Editor comes complete with a function for checking chemical structures. See Figure 10. The Check Structure command searches for formal errors and unusual structural features. If a structure is formally incorrect, or Structure Checker considers there is some doubt about its validity, a Structure Check Results dialog box will appear with a list of errors and warnings. When this window is closed, the program automatically selects the atoms and bonds that are considered to be incorrect. As mentioned in the topic, Program Limitations, structures that are not connected are considered to be mixtures, and are reported as errors.
OH O

Figure 10. Structure, showing the result of the Check Structure command

To check a structure, do either of the following:
• •

Click on the Check Structure button Choose Structure | Check.

.

Note. The Check Structure option cannot perform quantum mechanical or thermodynamical calculations concerning possible structure stability.

After finishing a structure drawing, you should always check it for errors before proceeding. Once the Fragments & Mechanisms generation is initiated, a structure is automatically checked for errors. If any error is discovered, the program prevents you from continuing with the generation.

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Structure Editor MS Calculations___________________________________________________________

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2.12 MS Calculations
When you select a part of a structure, Structure Editor automatically displays the molecular formula and molecular mass of the selected atoms in the status bar of Structure Editor, together with the corresponding loss. You may find this diagram and information useful for ensuring consistency between a mass spectrum and a chemical structure. See Figure 11.

Figure 11. Structure Editor, showing the result of selecting part of a structure

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Chapter 3 Spectra Manager
Spectra Manager is a module for managing spectral and structural information. This module provides powerful library maintenance utilities that enable you to create and organize spectral libraries with chemical structures. In addition, because the program supports ion and radical structures, you can also create MS/MS libraries. Advanced library query and search features provide access to the information needed for compound identification, and can help you interpret unknown spectra. A flexible set of search restrictions is available to target your search results, which is especially useful when you are dealing with large libraries. Spectral and structural data are clearly organized in spreadsheet-like windows, together with a variety of supplementary information. Only your system resources limit the number of Spectra Manager windows that can be open simultaneously. This means unconstrained flexibility in handling spectral and structural data. It is easy to move spectrum-structure pairs from one Spectra Manager window to another, which allows you to organize your libraries, search results, and other data in the way you need with a minimum of difficulty. Spectra Manager provides a simple, customizable tool for creating reports, which can be printed directly or can be copied and pasted to a word processor for more advanced reports. For each record in Spectra Manager you can view the mass spectrum; a list of peaks; the compound identification information, and the neutral losses spectrum if the molecular mass is available; or you can compare two spectra. This information can be viewed by selecting the appropriate tab. This chapter contains the following topics:
• • • • • •

Open Spectra Manager Window Mass Differences Comparing Spectra Cutting, Copying, and Pasting Records Data Exchange between Microsoft Excel and Spectra Manager Structures in Spectra Manager

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Spectra Manager ________________________________________________________________________

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• • •

Working with Spreadsheets Search Utilities Mass Spectral Data Exchange Between Modules

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Spectra Manager Open Spectra Manager Window

3.1 Open Spectra Manager Window
To open a Spectra Manager window, choose either of the following options:
• •

Click on the Spectra Manager button window.

in the toolbar of the main

Choose Tools | Spectra Manager in the menu bar of the main window.

To open a spectrum or spectra from a file, choose File | Open | Mass Spectrum. The Open Mass Spectra dialog box will appear. Choose a spectrum from the Spectra directory. The spectrum is then loaded into Spectra Manager. Start any search from the Search menu, and if a spectrum or structure is found, a Spectra Manager window will open containing the search results. See Figure 12. If an empty Spectra Manager window has already been opened, you cannot open another one. Every Search dialog box has a Merge Results Into Active Spectra Manager check box. As the check box name indicates, if you select this check box, the search results will be merged at the end of the spreadsheet in the active Spectra Manager window. If this check box is not selected, a new Spectra Manager window will be opened, and the search result records will be added to it. This topic contains the following topics:
• •

Records in Spectra Manager Additional Information Associated with a Record

Records in Spectra Manager
In this chapter we will often use the term record. A single record in Spectra Manager contains one spectrum, with a structure (if available), and complementary information associated with the spectrum-structure pair. Each record is visually represented as a single line in the spreadsheet. The hand icon always points to the active record. The active record is also highlighted on the spreadsheet. The structure, spectrum, and any other information are displayed for the active record only. The spectrum and structure associated with the active record are displayed in the upper half of the window.

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Spectra Manager Open Spectra Manager Window ______________________________________________

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Spectrum associated with active record

Structure associated with active record

Figure 12. Spectra Manager window, showing the Mass Spectrum page

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Spectra Manager Open Spectra Manager Window

Additional Information Associated with a Record
In addition to the mass spectrum and chemical structure, each record can include various compound identification information including the chemical name, nominal molecular mass, molecular formula, ID number, CAS registry number, origin, and synonyms. This additional information is contained on the Info page associated with the record. See Figure 13. The Info page contains the following eight elements:
• • • • • • • •

Library/File provides information about the source of the spectrum. Name contains the chemical name of the compound. Mol. Mass contains the molecular mass (molecular weight). ID Number provides a library access number. Formula contains the molecular formula. CAS Number provides a Chemical Abstracts Service registry number. Origin gives information about the origin or the “author” of the spectrum. The Synonyms text box lists synonyms of chemical names (for commercial libraries only).

The user can enter the chemical name, CAS number, and origin. The molecular mass and molecular formula are calculated automatically from the structure, where available. When the structure is not available, and your records originate from a JCAMP or MSP file that contains the molecular mass and the formula, this information will be displayed. The ID number is issued by the program and may not be changed. Synonyms are only available for entries in the NIST library.

Figure 13. Spectra Manager window, showing the Info page

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Spectra Manager Open Spectra Manager Window ______________________________________________

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The items whose titles are in bold can be changed directly by typing the desired information into the edit box.

Tip: When you want to save a spectrum, or a reference to a spectrum, you can copy the name of the compound from the Info page by highlighting the name and typing <Ctrl>-C. The name can be pasted to a Save Mass Spectra dialog box in the File Name text box by clicking the text box and typing <Ctrl>-V. See Figure 14.

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Spectra Manager Open Spectra Manager Window

Figure 14. Spectra Manager window, showing the spectrum name copied, then pasted, to the File Name text box of the Save Mass Spectra dialog box

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Spectra Manager Mass Differences __________________________________________________________

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3.2 Mass Differences
The Mass Differences page in Spectra Manager allows you to select any peak and see the mass differences to the right and left (possible parentdaughter ions). The m/z scale can be moved using the track bar at the top of the page. If molecular mass info is available, the zero value of the m/z scale automatically starts at the molecular mass, i.e. a standard neutral loss spectrum is displayed. In this case a blue selection bar in the track bar marks the shift of the m/z scale with respect to molecular mass. The graphic can be enlarged to allow you to see the number for less prominent peaks when there are a large number of peaks close to each other.

Figure 15. Spectra Manager window, showing the Mass Differences page

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9tyytrfyÁWhfwtg†ƒ ________________________________________________________Comparing Spectra

Spectra Manager

3.3 Comparing Spectra
The Compare Spectra page in Spectra Manager allows you to compare two spectra. See Figure 16. The bottom spectrum is from the active record in the spreadsheet. The middle spectrum displays the difference between the top and bottom spectra. The top spectrum can be added from an active record by clicking the Add button, or it can be pasted from the Clipboard by clicking the Paste button. The peaks in the top and bottom spectra have different colors, therefore, the color of a Difference Spectrum peak is taken from the spectrum that has the greater intensity at a particular mass-tocharge ratio.

Figure 16. Spectra Manager window, showing the Compare Spectra page

Note. After a spectrum search has been carried out, the search spectrum is automatically pasted to the top spectrum in the Compare Spectra page to allow viewing of the peak differences of spectra in the hit list and query spectrum.

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Spectra Manager Cutting, Copying, and Pasting Records_________________________________________

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3.4 Cutting, Copying, and Pasting Records
Records can be copied or moved from one Spectra Manager window to another by using the Cut, Copy, and Paste commands. Records can also be copied or moved to different locations within a spreadsheet in the same Spectra Manager window. This permits clear organization and maintenance of your experimental data or search results. The Cut, Copy, and Paste commands apply to selected records only. Because the spreadsheet allows the selection of multiple records, you can efficiently copy or move a large number of records at once. To cut, copy, or paste a record, do the following: 1. Select one or more records in the spreadsheet. 2. If Spectra Manager is an active window, click the Cut button, the Copy button, or the Paste button in Spectra Manager. Alternatively, choose Edit | Cut, Edit | Copy, or Edit | Paste on the menu bar of the main window. To select more than one record, choose either of the following:
• •

Click on the first record you want to select, then type and hold down <Shift> while clicking the last record. Point the mouse arrow at the first record you want to select, click the left mouse button, drag the cursor to the last record, and then release the mouse button.

If you cut or copy a record, the mass spectrum (in graphic format) is copied to the Windows Clipboard and to the record. If you select more than one record, only the graphic of the spectrum in the first record is copied to the Clipboard. This graphic spectrum representation can then be pasted to any Windows application. Graphics are copied to the Windows Clipboard in the newest 32-bit format (enhanced windows metafile), which is sometimes not supported by older 16-bit applications. A single spectrum copied in the GC/LC/MS Processor can be pasted to Spectra Manager. In this way you can extract spectral scans, and move them to Spectra Manager for further processing.

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9tyytrfyÁWhfwtg†ƒ _____________________ Data Exchange between Microsoft Excel and Spectra Manager

Spectra Manager

3.5 Data Exchange between Microsoft Excel and Spectra Manager
There are three basic types of data you can exchange between Microsoft Excel and Mass Frontier: text tables, graphics, and mass spectra in table format. The data can be exchanged using copy and paste commands. Mass Frontier does not support the import or export of native Excel files (*.xls) directly into Spectra Manager. However, Excel documents can be embedded into Mass Frontier (Microsoft Office | Open Microsoft Excel Document menu item) and data can be exchanged via the Clipboard. In this case a new window will open and the Excel menu and toolbars will appear.

Note. Embedded Microsoft Excel or Word windows have their own main menus (displayed above the Mass Frontier menus) and buttons (displayed below Mass Frontier buttons). Microsoft Office controls (menus and buttons) are only visible if an Excel or Word window is active. These controls do not contain Open and Save commands. If you want to open or save an Office document, you must use the Microsoft Office menu item on the Mass Frontier main menu.

Mass Frontier controls

Microsoft Office controls

Figure 17. Menus and toolbars belonging to Mass Frontier and Microsoft Office

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Spectra Manager Data Exchange between Microsoft Excel and Spectra Manager _____________________

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Exporting Data to Microsoft Excel
You can export three types of data from Spectra Manager to Microsoft Excel: text tables, graphics, and mass spectra in numerical table format. Because all these types can be accessed at the same time, you must specify the type of data you want to move to Excel. To export Info tab data: 1. Choose the Info tab (Info tab data must be visible). 2. Click the small Copy Selected Tab button in the top right corner of the Info tab control. 3. Paste the data by clicking the Paste button in Excel.

To export mass spectrum or mass differences graphics: 1. Choose the Mass Spectrum or Mass Differences tab (a Mass Spectrum or Mass Differences spectrum must be visible). 2. Click the small Copy Selected Tab button in the top right corner of the Mass Spectrum tab control. 3. Paste the graphic by clicking the Paste button in Excel.

To export a mass spectrum in numerical table format: 1. Choose the Data tab (an m/z and abundance table must be visible). 2. Click the small Copy Selected Tab button in the top right corner of the Data tab control. 3. Paste the table by clicking the Paste button in Excel.

To export records data in the spreadsheet: 1. Select one or more records in the spreadsheet. 2. Click Copy Selected Rows button right to Spreadsheet tab control. 3. Paste the data into Excel by clicking the Paste button.

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Main Copy button Copy Selected Tab

Spectra Manager

Copy Selected Rows in Speadsheet
Figure 18. Spectra Manager window, showing the three different copy buttons

Importing Spectra from Microsoft Excel
Mass spectra in table format stored in Excel can be imported to Spectra Manager via the Clipboard. Spectral tables can be organized horizontally or vertically. In order to correctly interpret m/z values and abundance you should follow one of these conventions: 1. If the spectral table is vertical, the first column must be the m/z value and the second must be abundance. 2. If the spectra are oriented vertically and the first column is abundance and the second column is the m/z value, you should caption the first row of the first column “Abundance” and the first row of the second column “m/z”. 3. If the spectral table is oriented horizontally, the first row must be the m/z value and the second row must be abundance. 4. If your spectra are oriented horizontally and your first row is abundance and the second row is the m/z value, you should caption the first column of the first row “Abundance” and the first column of the second row “m/z”.

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5. More than one spectrum can be imported at a time. In this case your first column or row, depending on the orientation, must be the m/z values and all the other columns (or rows) must be abundance.

To import spectra from Excel to Mass Frontier: 1. Make sure your table actually contains mass spectra. 2. Select the table you want to export to Mass Frontier. 3. Click the Copy button in Excel or if your document is embedded, click the Copy button on the Excel toolbar. 4. Click the Paste button in the Spectra Manager window. Mass Frontier supports standard tables with separated numbers, so spectra can also be imported from other programs.

Using Microsoft Excel as a Spectrum Editor
Microsoft Excel can be used as a spectrum editor for Mass Frontier by using the export and import features. This tool is useful if, for example, you have an experimental spectrum with a number of noise peaks at high m/z values that you want to delete, or you want to extract part of a spectrum that is important to your report or presentation. For obvious reasons you should not add, delete or alter prominent peaks.

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Spectra Manager

3.6 Structures in Spectra Manager
Chemical structures are used at every stage in the Spectra Manager module, and can easily be added to corresponding mass spectra. A particularly useful feature of Mass Frontier is its ability to create user libraries with structures including isotopes, ions, radicals and optically active compounds. Structures in Spectra Manager can be used in connection with the Fragments & Mechanisms module to check the consistency of a mass spectrum and chemical structure. Spectra Manager allows you to perform structure elucidation through modification of input structure and regeneration of product fragments. A structure can be added to Spectra Manager in either of the following ways:
• •

Paste the structure Load the structure from an external file

A structure which you intend to paste into Spectra Manager can be copied from anywhere in the program. To paste a structure into Spectra Manager, use the Paste Structure button in the top right corner of the Mass Spectrum page. The Copy and Paste buttons on the toolbar are intended for records, not for single structures. To paste a structure to a record, do the following: 1. Activate the record into which you want to paste the structure. 2. Paste the structure by clicking the Paste Structure button. If a structure has been added to a record, or an existing structure has been replaced, the word Updated will appear at the bottom of the structure pane. If anything has been changed in the record, including the structure, a small circle is displayed in the ID Num. column in the spreadsheet. After you add or change a structure, the new molecular formula and molecular mass are automatically calculated and updated.

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Spectra Manager Structures in Spectra Manager _______________________________________________

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Copy & Paste Record(s)

Copy & Paste Structure

Symbols of added or changed structure
Figure 19. Spectra Manager window, showing the Mass Spectrum page

As mentioned previously, the second method of importing structures to Spectra Manager is to load them from an external MOL-file. In contrast to the pasting of a structure, when you load from an external file you can add structures to more than one record at a time. This allows you, for example, to assign a large number of structures to library spectra when importing an external library to Mass Frontier. To import structures from a file, do the following: 1. Select the records to which you want to add structures. 2. Load the structures by choosing File | Open | Structure. When adding more than one structure to your records, the structures are added in the same order as they are present in the file, from the first to the last selected record. If the number of structures in an external file is greater than the number of selected records, structures are added only to the selected records. If the number of selected records is greater than the number of structures in the file, all the structures are added, and some records remain without structures.

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Spectra Manager

Note. A record must contain a mass spectrum. The presence of a structure is optional. Therefore, a structure can only be pasted or loaded to an existing record.

After assigning a structure to a record, the structure is only temporarily associated with the spectrum. To make the link between a spectrum and structure permanent, do either of the following:
• •

Add the updated record to a user library by choosing Library | Add To Library. If the structure you want to change is from a library record, choose Library | Change Structure In Library.

When you save a reference to a record using File | Save | Spectra References, the updated structures are not saved. Sometimes you might need to simultaneously preview all structures, rather than record information in the spreadsheet. To display a grid of structures, select the Structures tab next to the Spreadsheet tab. In this window the structures are organized in small cells. If you need to enlarge a structure, you can freely resize any structure cell by dragging the edges of the column or row headers. See Figure 23 and Figure 26.

Note. In the Structure pane, you can select only one record at a time.

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Spectra Manager Working with Spreadsheets __________________________________________________

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3.7 Working with Spreadsheets
The lower pane of the Mass Spectrum page displays a spreadsheet. See Figure 20. The spreadsheet is organized on a row basis. Each record is represented by a single row. The columns contain supplementary record information. In the spreadsheet you can move columns by dragging the appropriate column header to a new position. If you want to move a row, you must use the Cut and Paste commands, as Click and Drag commands from the mouse are not supported.

Figure 20. Spectra Manager window, showing the Spreadsheet and Structures pane

When you open a Spectra Manager window, the spreadsheet will be empty. You can add records to it by conducting a search, by opening spectra or references, or by pasting records or standalone spectra. For an active record, the associated spectrum and structure (optional) appear in the upper half of the window. The hand cursor in the first column indicates which record is active. You can select more than one record, but the row with the hand icon is always the active one. Remember that a record must contain a mass spectrum, but the presence of a structure is optional. Therefore, a structure can only be pasted or loaded to an existing record. A Spectra Manager window can contain 999 records, and you can open as many Spectra Manager windows as your system allows.

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Spectra Manager

3.8 Search Utilities
Several query and search features are available for retrieving spectra and structures from libraries. Mass Frontier allows you to search every library simultaneously. The following search options are available:
• •

Spectrum Search searches for the library spectrum most closely matching an unknown spectrum. (Sub)Structure Search searches for an exact match for the query structure (structure search) or searches for an exact match for the structure subset (substructure search). Name Search performs an incremental name search. Molecular Mass Search searches for compounds with a specified molecular mass (unit resolution only). Formula Search searches for compounds with a specified molecular formula. ID Number Search searches for library entries with a specified identification number. CAS Number Search searches for library entries with a specified Chemical Abstract Service registry number.

• • • • •

To start a search, do the following: 1. Click on Search in the menu bar of the main window. 2. Choose the command for the specific search you want to conduct. A dialog box will appears. See Figure 21 for a typical search dialog box. 3. When the dialog box appears, you can select the active library or libraries to be searched. If the search is successful, the results (in a hit list) are stored in the spreadsheet in the Spectra Manager window.

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Spectra Manager Search Utilities____________________________________________________________

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Figure 21. Library Search dialog box, showing the Spectrum page

Every Search dialog box has a Merge Results Into Active Spectra Manager window check box. If the check box is selected, the search results will be stored in the spreadsheet of an active Spectra Manager window. If you deselect this box a new Spectra Manager window will open, containing the 7 search results. When the checkbox is disabled, there is no active Spectra Manager window, and a new window will be opened.

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Spectra Manager

Spectrum Search
Note. This program uses the NIST library search algorithm developed by Stephen E. Stein. This algorithm is based on the optimized dot-product function together with an additional term, based on ratios of peak intensities. Extensive testing, carried out using a wealth of examples, has shown that this algorithm is one of the most efficient commercially implemented algorithms. A detailed description of the algorithm can be found in Stein, S. E., Scott, D. R.; J. Am. Soc. Mass Spectrom. 1994, 5, 859-866 and Stein S. E.; J. Am. Soc. Mass Spectrom. 1994, 5, 316323.

A query spectrum can originate from Spectra Manager or from GC/LC/MS Processor. Before you start a search you must select a spectral scan or deconvoluted component spectrum in a chromatogram. In addition, a query spectrum can be pasted from the Clipboard to the search dialog box. In this case the spectrum can only be copied to Mass Frontier. The Search Spectrum option allows you to choose between Identity and Similarity searches. Identity searching locates a library spectrum that closely matches an unknown. Similarity searching retrieves spectra of library entries of similar compounds when the unknown is either not in the library or its spectrum is distorted so that a reliable match is not possible. After a spectrum search has been carried out, the search results (hit list) are stored in the spreadsheet. The result of the spectrum search is a hit list of 100 records. After a search has been completed, a Match column is automatically added to the spreadsheet. See Figure 22. The Match factor is a number from 1 to 999 that specifies the degree of similarity between the query spectrum and the library reference spectrum. A Match factor of 999 means a perfect match. To draw your attention to a match greater than 930, a Lightning icon is displayed in the Match column in the spreadsheet. If the Identity search does not provide an acceptable match (i.e. the unknown has not been positively identified), a Similarity search can be used. In this case the algorithm does not use the high mass peak index, but employs wider abundance ranges. The hit list resulting from a similarity search can be of value in deducing structure, especially in establishing a structural proposal for an unknown spectrum.

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Spectra Manager Search Utilities____________________________________________________________

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Figure 22. Mass Spectrum page of the Spectra Manager window, showing Match factors

To establish a structural proposal for an unknown, you can switch to the Structures page. See Figure 23. From the hit list of similar compounds you may recognize some common structural features, which are displayed in the structures grid. You can copy the structures to Structure Editor and put the pieces of the structural “puzzle” together and so create an initial structure. This structure can be pasted back into a Spectra Manager window to the record holding the unknown spectrum. A comparison of the peaks in the spectrum with generated fragments can provide valuable information about the consistency of the proposed structure and unknown spectrum. If the mass-to-charge ratios of the fragments do not match the spectrum, the structure can be modified, and this procedure can be repeated. However, if the structures in the hit list are highly diverse and dissimilar, this approach needs to be combined with other methods.

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Figure 23. Structures page of the Spectra Manager window

(Sub)Structure Search
A very important feature for retrieving library entries are structure and substructure searches. See Figure 24. The structure search is the most straightforward method for finding compounds in a library. Since the rules of systematic nomenclature need not necessarily lead to a unique name for each compound, searching by name is, in many cases, ineffective. It is much easier to draw or import a structure query than to type a complicated name. It is generally easier for a chemist to deal with structures rather than names or CAS numbers. •
NH2

NH2

HO

O

OH HO

O

OH

OH OH

HO

OH

Negative structure search result

Positive structure search result

Figure 24. Structures, showing equivalent and non-equivalent results of structure searches

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Spectra Manager Search Utilities____________________________________________________________

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While a structure search provides an exact match of query and library structure, a substructure search retrieves compounds that contain a common structural subset, called substructure. See Figure 25. The exact substructure must be embedded in each molecule retrieved. An exact match in structure and substructure searches has a notable exception: stereo bonds are ignored. Optical activity does not play a significant role in mass spectrometry. All other structural features such as bond multiplicity, charge and radical position, atom state, and skeletal arrangement must match exactly. Therefore, atoms with isotopes, charges, or radicals must have exact counterparts for both the structure and substructure search. A structure or substructure query can be taken from Structure Editor, Spectra Manager or a fragment copied in the Fragments & Mechanisms window.

Figure 25. Library Search dialog box, showing the results of a substructure search

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A Substructure search can be used for a variety of purposes. It allows you to study mass spectra of structurally related molecules. If you want to positively identify an unknown, or you need help in interpreting a mass spectrum, compounds retrieved by substructure search can provide analogies to the fragmentation processes in the spectrum under consideration. See Figure 26.

Spectra Manager

Figure 26. Structures page of the Spectra Manager

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Spectra Manager Search Utilities____________________________________________________________

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Name Search
The name search option provides incremental search capabilities. As you type a compound name, the program suggests possible names that match the text you have already typed. The chemical structure is displayed for the highlighted name.

Molecular Formula Search
The molecular formula search option allows you to search for all compounds with a specific molecular formula. Lower case can be used to enter the formula, unless this would lead to an ambiguous query (for example, si could be interpreted as Si or SI), so the correct case must be used to avoid misinterpretation. Parentheses are supported. For example, if you enter (CH3)3CCOOH, the program automatically transforms the query to C5H10O2.

Molecular Mass Search
In this program the term molecular mass is used to mean “nominal molecular mass” (often incorrectly called nominal molecular weight). The nominal molecular mass is the sum of the integral masses of the most abundant isotopes. Only integer values of molecular mass can be used.

ID Number Search
Each library entry is individually numbered. In this mode you can search for a single ID number or for a range of ID numbers. The ID search dialog box contains a From text box and a To text box to input the ID range. If you want to retrieve a single ID number, the To text box can be left blank. The maximal ID number that can be found is displayed in the Max. ID box. This is the only search mode that allows you to search in only one library at a time.

Note. The ID number never changes. For example, if you delete a record with ID = 1, the remaining records are not shifted, and record number one remains empty.

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CAS Number Search
The Chemical Abstract Service (CAS) registry number search option is only available for the NIST ’98 library. You should not use dashes when entering CAS numbers.

Search Constraints
All searches, except Name Search, can be restricted by a set of constraints. The dialog boxes of these searches contain a panel of option buttons for activating constraints and a button for editing search constraints. If you click the Edit button, the Search Constraints dialog box will appear, which allows you to search selected libraries for matches with a set of specific criteria. Four constraint types are available, arranged in group boxes, as follows: Molecular Mass range, Number of Atoms range, Allowed Elements list, and Good-Bad list. Searches conducted with activated constraints can be timeconsuming because each library entry is examined to find those that match the criteria you have set. Search constraints are especially useful when you are dealing with large libraries, and you want to retrieve hits that are of interest for your specific problem. The Good list (required substructures) allows you to focus your search results on the particular compound classes you are most interested in. The Bad list (forbidden substructures) eliminates all structures from a hit list containing unwanted functional groups. For example the Good-Bad List can be used in a search of acids with a specific molecular formula, or you can search for spectra similar to an unknown, with the requirement that ketones do not appear in the hit list. The following figures exemplify a substructure search using the Search Constraints option. Figure 27 shows a Search Constraints dialog box with C, N, and O set as allowed elements. The Good-Bad list is set to require esters (good) and to disallow tertiary amines (bad). The substructure query is the triphenyl group. The search results, with the restrictions described above, are shown in Figure 28.

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Figure 27. Search Constraints dialog box, showing allowed elements and functional group

Figure 28. Structures page of the Spectra Manager window, showing the results of a library search using the constraints specified in Figure 27

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Spectra Manager

3.9 Mass Spectral Data Exchange Between Modules
Spectra Manager serves as a gateway for several modules. See Figure 29. Mass spectra can be imported directly from a file, GC/LC/MS Processor, or Xcalibur. Spectra can also be exchanged between Spectra Manager modules. In addition, Mass Frontier is able to automatically establish a link between Spectra Manager records and other modules that need to access spectral or structural data. This ability to link equivalent spectral information allows you to access the original data that was supplied as input information for one of the many interpretation methods available in Mass Frontier. The direct feedback between source (records and mass spectra) and results (mechanisms, bar code spectra, groups of spectra, and projections) is particularly useful as it helps you keep track of all the modules that originate from a single source. This feature makes it easier to employ more modules simultaneously allowing for more sophisticated problem solving approaches. Establishing a link between Spectra Manager and a particular module, and how to exploit this, is fully detailed in the chapters dealing with these modules.

Xcalibur

Fragments & Mechanisms

GC/LC/MS Viewer

Spectra Projector Spectra Manager Neural Networks

Microsoft Excel

Spectra Classifier

Figure 29. Exchange of mass spectral information between Mass Frontier modules, Microsoft Excel, and Xcalibur

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Chapter 4 Library Utilities
Mass Frontier provides convenient ways for creating and maintaining mass spectral libraries with chemical structures. To help you visually distinguish between libraries, each library has its own icon. The user can choose an icon for user libraries. Main and replicate icons from the NIST library (1998 and later versions) are assigned automatically by the program and cannot be changed. Up to sixteen libraries can be installed at a time. Mass spectra stored in libraries are in unit resolution only. The user libraries are stored in NIST format. Therefore, libraries compatible with the NIST format that were created by different programs can also be installed in Mass Frontier. Since the original NIST Library format did not support structures for user libraries, the library structures are stored in an internal format. However, the library structures can be exported very easily in MOL-format, even if they were created commercially. This chapter contains the following topics:
• • • • • • • •

NIST/EPA/NIH Mass Spectral Database (NIST ’98 Library and later versions) Library Installation Creating User Libraries Uninstall Library Adding Records to a User Library Deleting Library Entries Changing Structures in Libraries Opening and Saving Spectra, Structures, and Spectra References

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Library Utilities NIST/EPA/NIH Mass Spectral Database________________________________________

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4.1 NIST/EPA/NIH Mass Spectral Database
Mass Frontier can work either as a standalone application, or can be combined with the NIST ’02 Library or the NIST ’98 Library. If a NIST Library was not simultaneously installed with Mass Frontier, the library can simply be incorporated in the program. See the topic: Library Installation. The NIST ‘02 Library meets the highest quality criteria for a collection of mass spectral and structural information. The library contains 174,948 spectra from 147,198 compounds. The most important feature of this NIST Library is that it contains 147,194 high quality structures (99.997%). The NIST ’98 Library contains 129,136 spectra from 107,886 compounds. The most important feature of this NIST Library is that it contains 107,829 high quality structures (99.9%). Since Mass Frontier is completely oriented towards chemical structures, the combination of Mass Frontier and the NIST Library is the logical choice for a modern mass spectrometry lab.

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Library Utilities

4.2 Library Installation
If you have purchased the NIST library, or the library was provided with your MS system, the library can be installed in Mass Frontier from CDROM, or from hard disk. The procedure described below for the installation of an NIST Library also applies to any user library in NIST format. Please note if you install the library from CD-ROM by copying files to your hard drive, you must deactivate the Read-only file attributes for all files before installation in Mass Frontier. To install a user or NIST Library into Mass Frontier, choose Library | Install Library on the main menu. The Install Library command displays the Install Library dialog box. See Figure 30. When the Install Library dialog box appears, follow these installation steps:

Figure 30. Install Library dialog box

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Library Utilities Library Installation _________________________________________________________

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1. Select the library on drive X:. 2. Click on the button Find Libraries on X:. The program will scan the drive for libraries. If a library is found, go to the next step. 3. Select the library you want to install. If you select NIST Main library, the NIST Replicate library will be installed automatically. An NIST Replicate library cannot be installed as a standalone library without a Main library. 4. Choose an icon for the library. For an NIST Library, the program will assign an icon that cannot be changed. 5. Click on the Install Library button. If the library has been successfully installed, the icon, the name, and the path of the library are displayed in the List of Installed Libraries grid in the Install Library dialog box.

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Library Utilities

4.3 Creating User Libraries
Mass Frontier allows you to create user libraries with structures by using the Create User Library dialog box. See Figure 31. To create a user library, choose Library | Create User Library on the main menu. When the Create User Library dialog box appears, follow these steps: 1. Enter or select the directory for the user library. We recommend accepting the default directory. 2. Enter the library name. Only the characters a-z, A-Z, and 0-9 can be used. 3. Select a library icon. 4. Click on the Create & Install User Library button.

Figure 31. Create User Library dialog box

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Library Utilities Creating User Libraries _____________________________________________________

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If the library has been successfully created, the icon, the name, and the path of the library are displayed in the List of Installed Libraries grid. The program creates an individual subdirectory for each new library with the name of the library. The full library path is displayed in the Directory column. An unlimited number of user libraries can be created, but no more than sixteen libraries can be installed in Mass Frontier at the same time. If you require more libraries to be installed, you can uninstall a library you do not need temporarily, and create or install the library which you want to use.

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Library Utilities

4.4 Uninstall Library
Experimental data, as well as reference spectra, can be stored in your user libraries. The creation of a user library is a very easy task, so feel free to organize your mass spectral and structural data in user libraries. Since there is a limitation of sixteen libraries, sometimes you may need to uninstall a library. If you uninstall a library, the library is not deleted. The library reference is merely removed from the program without any loss of spectral or structural information. An uninstalled library can be reinstalled in seconds. Mass Frontier allows you install or uninstall a library as many times as you wish. To uninstall a library, do the following: 1. Choose Library | Uninstall Library to display the Uninstall Library window. See Figure 32. 2. Select the library you want to uninstall. 3. Click on the Uninstall button.

Figure 32. Uninstall Library dialog box

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Library Utilities Adding Records to a User Library _____________________________________________

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4.5 Adding Records to a User Library
Mass Frontier provides a simple way of adding records (spectra, structures, and compound identification information) to a user library. Using Mass Frontier, you can create libraries with chemical structures that include ions, radicals, isotopes and optically active compounds. A spectrum you wish to add to a library cannot contain more than 800 peaks and the mass-to-charge ratio may not be greater than 3000. A single structure accompanying a spectrum in a user library cannot contain more than 127 non-hydrogen atoms. To add one or more records from Spectra Manager to a user library, do the following: 1. On the spreadsheet, select the record you want to add to the user library. 2. Choose Library | Add To Library. 3. When the Add To Library dialog box appears, select the library you want to add to the record by choosing the appropriate option button. See Figure 33. 4. Click on Add.

Note. Only selected records in a Spectra Manager spreadsheet can be added to a user library.

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Library Utilities Adding Records to a User Library

Figure 33. Add To Library dialog box

You might want to check whether the structure associated with the spectrum that you want to add to a user library is already present in the library. Mass Frontier allows you to perform redundancy checks by choosing the Check option button in the Check Structure Redundancy pane. When this option is selected, the program compares each structure to be added to the library with structures in the selected library. This option, of course, only makes sense if the structure is present. Otherwise, the option is ignored.

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Library Utilities Deleting Library Entries _____________________________________________________

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4.6 Deleting Library Entries
Deleting a library record causes the information to be irreversibly lost. There is no undo function for deleted library records. You can only delete library entries from a user library. If you want to delete one or more records, do the following: 1. Retrieve the record you want to delete using any search. 2. Select the record on the Spectra Manager spreadsheet. 3. Choose Library | Delete From Library. 4. When the Delete From Library dialog box appears, carefully review the spectra and structures. See Figure 34. 5. If you want to delete all the records displayed in Preview, choose the Delete Without Confirmation option button. If you want to delete records one at a time, select the Confirm Each Spectra/Structure Pair Before Deletion option button. 6. Click on Delete.

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Library Utilities Deleting Library Entries

Note. If you delete a record from a library, the remaining records keep their original ID Numbers. This means the ID number of a deleted record remains unoccupied, and you cannot add a record to it.

Figure 34. Delete From Library dialog box

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Library Utilities Changing Structures in Libraries ______________________________________________

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4.7 Changing Structures in Libraries
If you have strong evidence of an erroneous chemical structure in a user or a commercial library, you can replace the structure. However, you should be very careful, especially when changing a structure in a commercial library. We recommend making a note of the ID number of the library record, because you might want to reconsider your decision later. To change a structure in a library record, do the following: 1. Retrieve a single record from the library to a Spectra Manager spreadsheet. 2. Make this record active (the hand cursor will point to the active record). 3. Paste the new structure into this record. 4. Choose Library | Change Structure in Library.

Note. Molecular Mass and Formula searches disregard changed structures. Due to the internal library format, these two search options consider only original information.

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Library Utilities

4.8 Opening and Saving Spectra, Structures, and Spectra References
Mass Frontier supports three file formats for spectra: JCAMP, MSP, and Xcalibur RAW format. The first two formats are ACSII files, and the third is a raw file format that is opened and saved in MOL-format. Spectra Manager can save and open references to spectra contained in a library or in external files. When saving references to a file, the spectra are not physically stored. Only information about the spectra location is saved. For example, you can save library search results as references to records in the library. You may also wish to save groups of spectra as references, which can be submitted for classification using the Spectra Classifier and Spectra Projector modules. The reference files are ASCII files with a .ref extension.

Note. You cannot save a reference to a spectral scan that originates from an average of scans or from a background subtraction that was produced in GC/LC/MS Processor, since such spectrums are not physically stored in a file. In this case you must save the spectrum and not the reference to it.

To open a spectrum or the reference to a spectrum, do either of the following:
• •

Click on the Open button in Spectra Manager, and choose Open Mass Spectra or Open Spectra Reference. Choose File | Open | Mass Spectra or File | Open | Spectra Reference.

To open a structure, select the record to which you want to add a structure, then do either of the following:

To load the structures, click the Open button, then choose Open Spectra Reference. The Open Spectra Reference dialog box will appear. Select a file, then click Open. Choose File | Open | Spectra Reference. The Open Spectra Reference dialog box will appear. Select a file, then click Open.

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Chapter 5 Fragments & Mechanisms
One of Mass Frontier’s most outstanding features is the automated generation of possible fragments at an expert level. The feature includes complete fragmentation and rearrangement mechanisms, starting from a user-supplied chemical structure. The Fragments & Mechanisms module provides information about basic fragmentation and rearrangement processes that can occur in a mass spectrometer.
• • • • • •

Generated fragments and corresponding mechanisms can be particularly useful for the following activities: Checking consistency between a chemical structure and its mass spectrum Confirming library search identifications Recognizing the structural differences between spectra of closely related compounds Interpreting the spectra of isotopically labeled compounds Illustrating the structure-spectra relationship for educational purposes

This chapter contains the following topics:
• • • • • • • • • • • •

Features Fragmentation, Rearrangement, and Resonance Reactions Starting Generation Fragments & Mechanisms Window Reaction Restrictions Generated Fragments Linked with Spectrum Eliminating Generated Fragments Not Present in a Spectrum Simulation of MSn Experiments Unexplained Peaks Too Many Proposed Fragments for a Peak Bar Code Spectra Marking Fragments

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5.1 Features
The Fragments & Mechanisms module is based on an expert system, which uses a mathematical approach for the simulation of unimolecular iondecomposition reactions. Therefore, it is important to understand the features and limitations of the system, and what the user can and cannot expect from this module. This expert system, which generates possible fragmentation and rearrangement pathways, is based on the following assumptions:

General Fragmentation and Rearrangement Rules
The system only predicts reaction pathways that are based on general fragmentation and rearrangement rules. Compound specific mechanisms (“random” rearrangements) that cannot be applied generally are not included. Although this might seem to be a disadvantage, this feature can be used in combination with a substructure search for the identification of specific compound classes. Fragmentation and rearrangement rules that apply in Mass Frontier are described in the next section.

Charge Localization Concept
Every ion-decomposition reaction that is generated is based on the charge localization concept. The program exactly determines the location of the charge site in all precursor and product ions. Mass Frontier internally generates resonance reactions, which are not displayed by default. Note that charge sites can be moved to distant locations by these reactions, and in some complicated structures it may appear that the charge localization concept is violated. If a reaction step is not clear, you can instruct the program to display mechanisms along with resonance reactions.

Unimolecular Linear Reaction Mechanisms
Mass Frontier only generates unimolecular reactions. The reaction pathways are displayed as linear reaction mechanisms, which incorporate one intermediate left-hand site and one intermediate right-hand site for each reaction step. Therefore, ionic products are only included in reaction pathways, and neutral fragments are not displayed.

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Even-Electron Rule
Reaction mechanisms are generated in accordance with the Even-Electron rule. The Even-Electron rule states that the homolytic bond cleavages of an even-electron ion are energetically unfavorable. Therefore, Mass Frontier never generates radical cations from an even-electron ion.

Bond Cleavages Only
Fragments can be generated only from bond cleavages. Bond creation is not supported, with the exception of an H-X bond in hydrogen rearrangements, where X is any element. Therefore, this expert system does not include ring contractions, cyclization, or skeletal and non-hydrogen rearrangements.

Ionization Methods
Mass Frontier supports Electron Ionization M+ ,, protonation [M+H]+, and chemical ionization (CI) methods. Negative ionization is not supported.

Formally Possible Solutions
The mechanisms generated by Mass Frontier contain formally possible reaction steps. The determination of the stability of product ions from thermodynamic data or rates of reaction is not performed. When evaluating generated mechanisms a simple rule should be kept in mind: Short and uncomplicated reaction pathways are more favorable than complex mechanisms involving complicated multi-step hydrogen rearrangements.

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5.2 Fragmentation, Rearrangement and Resonance Reactions
Mass Frontier uses fragmentation, rearrangement, and resonance reactions that can be applied generally. Table 1 shows some basic unimolecular reactions that are used by the program to generate possible reaction pathways. Table 2 shows the reaction formalism used in Mass Frontier.

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9tyytrfyÁWhfwtg†ƒ dddddddddddddddddddddddddddddddddddddddddddddddddd Fragmentation, Rearrangement and Resonance ReactionsÁ

Fragments & Mechanisms

Table 1. Fragmentation, rearrangement, and resonance reactions

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Table 2. Reaction formalism used in Mass Frontier

α π σ

Alpha cleavage Pi electron ionization Sigma electron ionization Charge stabilization Non-bonding electron ionization Electron sharing Inductive cleavage Protonation Hydrogen radical loss Hydride abstraction Radical site rearrangement Charge site rearrangement (α, β) Charge site rearrangement (γ) Charge-remote rearrangement Hydrogen shift to adjacent position Radical resonance

cr -ees i +H+ -H• -H: rHA rHB rHC rHR rH1,2 rr

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5.3 Starting Generation
Fragmentation and rearrangement pathways can be generated from any usersupplied structure, including ions and isotopically labeled compounds. The structure originates from the Structure Editor, the Spectra Manager, or from a Fragments & Mechanisms window. Before the generation is performed, the program checks the input structure for errors. If any errors are found, a message box will appear and informs you about the errors. The generation is then aborted. When you start a generation from Structure Editor, in contrast to the copy function or substructure search, the structure does not have to be selected. The program assumes that the complete structure is intended as input for the generation. If a generation is started from the Spectra Manager window, the program automatically links the generated fragments in the Fragments & Mechanisms window with the corresponding spectrum in the Spectra Manager window. Peaks that have the same mass-to-charge ratio as the generated fragments are highlighted in the color that is set in the Spectra Layout dialog box (red by default). Selecting a highlighted peak reveals all the pathways leading to it. To start a generation of possible fragmentation and rearrangement pathways, do either of the following:

Click on the Generate Fragments and Mechanisms button in Structure Editor, Spectra Manager, or Fragments & Mechanisms window. Make the Structure Editor or Spectra Manager active, and then choose Tools | Fragments & Mechanisms.

During the generation of possible fragmentation and rearrangement pathways, the Generation of Fragments & Mechanisms dialog box is displayed. See Figure 35.

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Figure 35. Generation of Fragments & Mechanisms dialog box

A combo box in the Already Generated m/z group box stores mass-tocharge ratios of ions that have already been generated. A list of ions can be displayed by clicking the down-arrow. The total number of ions generated is displayed next to the combo box. While a generation is in progress, the Reactions Limit bar gives you an approximate indication of how many temporary internal reactions have been generated from a particular structure. Large and structurally complicated molecules can produce an enormous number of reactions. The generated reactions consume a large amount of memory, so there is a limit to the number of temporarily generated reactions. If the reactions limit is reached, the generation stops, and a message box will appear to remind you of this fact. Even if the generation is stopped, the fragments and mechanisms generated up to that point are displayed. The system that generates the fragments and mechanisms is designed in such a way that the fragments generated first are the most important. Therefore, if a generation is stopped the most important fragments are the most likely to have been generated. However, if you are missing an important fragment, and believe this is due to the generation being interrupted, you can increase the number of internal reactions in the Reaction Restriction window on the Size page. A very important message is displayed in the Info group box, as follows: Mass Frontier is a multi-threading application, so you can continue using other modules while the reactions are being generated. The multi-threading strategy allows concurrent performance of more than one task. For example, while reactions are being generated, you can search libraries or analyze your spectra. You can also run two or more generations of fragments and mechanisms at the same time.

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The Pause button allows you to temporarily interrupt generation to redirect processing power to other tasks that are running simultaneously in Mass Frontier, or in Windows. Clicking on the Finish button stops a generation. Fragments and their production mechanisms generated up to that point are displayed. The Cancel button can be used to abort a generation. Sometimes it takes several seconds for a window to disappear after a generation has been aborted.

Fragments & Mechanisms

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5.4 Fragments & Mechanisms Window
When all the reaction-generating processes are complete, a Fragments & Mechanisms window is displayed. See Figure 36. Open Fragments & Mechanisms Save Fragments & Mechanisms Print Fragment or Mechanism Copy Mechanism Copy Selected Fragment Copy List of Fragments Ionization Method Display Fragments Only Display Mechanisms

Default m/z values Set m/z value Excluded m/z value Generate “Bar Code” spectrum Compare Fragments Show m/z values for explained peaks only Generate Fragments & Mechanisms from Selected Fragment (MS/MS) Mechanism Layout Possible Fragments for selected m/z value

Figure 36. Fragments & Mechanisms window

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The Fragments & Mechanisms window displays complete mechanisms of ion-decomposition reactions or fragments. To display the desired mechanism or fragment for a particular mass-to-charge ratio, choose the appropriate mass-to-charge ratio tab, or use the combo box in the Mechanism Layout dialog box, described later. A Fragments & Mechanisms window contains three Copy buttons (Figure 36). The first one is used to copy mechanisms in graphic format (Enhanced Windows Metafile). The second one is used to copy a selected fragment. The copied fragment can then be pasted to Structure Editor, to Spectra Manager in Mass Frontier, or to any Windows application in graphic format (Enhanced Windows Metafile). The third Copy button is for copying a list of fragments, which can then be pasted into a Fragments Comparator window or Microsoft Excel.

Fragments & Mechanisms

Note. Only a selected fragment can be copied to the Clipboard.

Several possible isobaric fragments can be generated for a particular massto-charge ratio. If more than one fragment is generated for a particular mass-to-charge ratio, a hand pointer will appear with the caption, Select possible fragments with m/z xy. The hand pointer moves from right to left to capture your attention. The isobaric fragments and their corresponding mechanisms can be displayed by clicking the numbered buttons next to the hand pointer. The fragments are sorted according to the simplicity of their production mechanism. So, fragment No.1 is produced by the simplest (shortest) mechanism. The isobaric fragments are usually isomers of the same fragments with a different charge, radical, or π-bond location.

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5.5 Reaction Restrictions
Reaction Restrictions can be used to change the default settings of the ionization method and of the basic fragmentation and rearrangement mechanisms. To display the Reaction Restrictions dialog box, choose Options | Reaction Restrictions. See Figure 37. The Reaction Restrictions dialog box contains the following five pages: Ionization & Cleavage, H-Rearrangement, Resonance, Additional, and Sizes.

Ionization & Cleavage Page
On the Ionization & Cleavage page, you can choose between the ionization mode Electron Ionization (EI) that produces M+ • ions and the protonation mode that produces [M+H]+ ions. The protonation mode represents “soft ionization” techniques such as Chemical Ionization (CI), Electrospray Ionization (ESI), Fast Atom Bombardment (FAB), and other techniques that produce a protonated molecular ion. The chemical ionization option offers three basic ionization reactions: protonation, hydride abstraction and adduct formation. In addition, you can select one of the six most common reaction gases: methane (CH4), hydrogen (H2), isobutane (i-C4H10), ammonia (NH3), water (H2O), and nitrogen monoxide (NO).

Figure 37. Reaction Restrictions dialog box, showing the Ionization & Cleavage page

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When you compare generated fragments and mechanisms with a mass spectrum, you should always choose the correct ionization method. The program will show a warning message if the reaction restrictions are set for protonation techniques, and you are attempting to compare generated fragments with a spectrum from the NIST library that only contains EI spectra. However, if the spectrum is from a file, the mass spectrum type and ionization techniques are not checked for consistency. So be sure to use the correct settings. There are two possible reasons for changing the default setup of cleavages, rearrangements, and other reactions. First, if you are missing an important peak, you can generate it by activating rearrangements and reactions that are switched off (by default). Second, you might want to simplify a mechanism by deactivating rearrangements or cleavages that cause redundant reaction steps. Bond cleavage on aromatic systems can serve as an example of the first case. See Figure 38. By default, cleavage on an aromatic ring is not activated. However, when you deal with small aromatic compounds, you should activate bond cleavage on aromatic rings by selecting the Cleavage checkbox in the Allowed on Aromatic Systems group box on the Additional page. See Figure 41. For example, when you generate fragments and mechanisms of phenol, the important fragment corresponding to the peak m/z 66 can be generated only if cleavage on aromatic systems has been activated. Cleavage on aromatic systems is not active by default. (This default setting is due to the fact that huge numbers of fragments can be generated from large aromatic compounds and the aromatic bond is a very strong bond).

Fragments & Mechanisms

OH


OH

O

O

rHA

α

- CO

m/z 66 m/z 94 m/z 94 m/z 94

Figure 38. Structures of phenol, showing bond cleavage on an aromatic system

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H-Rearrangement Page
The H-Rearrangement page contains controls for setting three basic hydrogen rearrangements. See Figure 39. The hydrogen rearrangements that involve radical (odd electron ions) rHA are set by default for hydrogen transfer from a steric optimal atom, usually from a γ-atom (McLafferty rearrangement). However, if you suspect an unusual rearrangement, you can specify that hydrogen transfer occur from an α, β, γ or δ atom.

Figure 39. Reaction Restrictions dialog box, showing the HRearrangement page

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Resonance Page
Mass Frontier generates fragmentation and rearrangement mechanisms along with electron shift reactions (resonance reactions). Since these reactions may, even for small structures, cause a huge number of byproducts, the resonance reactions are not depicted by default. To keep the reaction network clear and simple, the program performs a reduction of the reaction complexity by not displaying resonance reactions. Therefore, elementary reaction steps that include resonance reactions are merged into a single step. An inexperienced mass spectrometrist might have problems in understanding such reduced mechanisms because several reaction steps are merged. If such a problem with unclear elementary reaction steps should arise, you can force the system to display all the resonance reactions by clicking the Yes option button in the Display Resonance Reactions group box. See Figure 40.

Figure 40. Reactions Restrictions dialog box showing the Resonance page

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If you have established restriction settings that you wish to apply frequently to a specific compound class, you can save the current reaction restrictions to a file (*.rrs file extension) by clicking the Save button in the Reaction Restrictions dialog box. When you open a reaction restriction file by clicking the Open button, the dialog box adopts the restrictions saved in the file. You must then press the OK button to make these restrictions active in Mass Frontier.

Note. All changes in the Reaction Restrictions dialog box take effect after the regeneration of fragments and mechanisms. The existing Fragments & Mechanisms windows are not affected by changes to Reaction Restriction. In addition, the changes in the Reaction Restrictions dialog box affect all subsequent generations, unless you restore default settings. Therefore, when changing the settings, keep in mind that the default reaction restrictions should be restored when the changes are no longer needed.

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Additional Page
The Reaction Restrictions dialog box contains the Additional page. See Figure 41. For more information see text under the Ionization and Cleavage page.

Figure 41. Reaction Restrictions dialog box, showing the Additional page

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Sizes Page
The Sizes page allows the user to limit the size and complexity of a reaction pathway generation. The Reaction Steps Max Number box gives the number of cascaded fragment reactions. Increasing this number could exponentially increase the number of fragments produced for a given reaction path. Generally, this number should be kept small, and if additional fragments need to be generated, individual fragments can be selected by the user and used as starting points for additional reactions.

Figure 42. Reaction Restrictions dialog box, showing the Sizes page

Large and structurally complicated molecules can produce an enormous number of reactions. Because generated reactions take up a large amount of memory, the number of temporarily generated reactions is limited. The system that generates the fragments and mechanisms is designed in such a way that the fragments that are generated first are the most important. If the reactions limit is reached, the generation will be stopped, and a message box will appear as a reminder of this fact. Even if the generation is stopped, the fragments and mechanisms generated up to that point will be displayed. Therefore, when a generation is stopped, the most important fragments will probably already have been generated. However, if you are missing an important fragment, and you believe this is due to the generation being interrupted, you can increase the number of internal reactions in the Reactions Limit box. See Figure 43.

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Fragments & Mechanisms

Figure 43.

Generation of Fragments & Mechanisms dialog box, showing the Reactions Limit box

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5.6 Generated Fragments Linked with Spectrum
Mass Frontier offers the ability to link generated fragments with a mass spectrum. If you start a generation of fragments and mechanisms from Spectra Manager, the generated fragments are automatically linked with peaks in a spectrum according to their mass-to-charge ratios. So Mass Frontier helps you to explain peaks in a spectrum. After a generation, highlighted (“explained”) peaks are displayed in a different color (red by default) in the original mass spectrum. Selecting a highlighted peak reveals all the mechanisms leading to it. If an “unexplained” peak is likely to be an isotopic peak of an “explained” peak, this is depicted in a third color (green by default). Selecting such a peak reveals all the mechanisms leading to the inferred fragment which can produce this isotopic profile. One very important issue needs to be discussed further. The inability to predict energies and barriers in ionized molecules prevents the prediction of all the peaks in a mass spectrum. In addition, Mass Frontier only includes general fragmentation and rearrangement mechanisms. Thus, fragment predictability usually ranges between 50-90%. However, a prominent unexplained peak can be of special value for the interpretation or identification of an unknown. An unexplained peak can indicate a compound-specific mechanism that occurs in molecules with similar structural features, or with a common substructure. There are a number of mechanisms that have only been observed in a specific group of compounds and cannot be applied generally when proposing fragmentation and rearrangement pathways. If you suspect a compound-specific mechanism of fragment formation, you should verify your assumption by conducting a substructure search and then comparing the explained and unexplained peaks in the spectra retrieved by the substructure search.

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Fragments & Mechanisms Eliminating Generated Fragments Not Present in a Spectrum

5.7 Eliminating Generated Fragments Not Present in a Spectrum
The Fragments & Mechanisms module shows all mass-to-charge ratio values that have been generated with given Reaction Restrictions settings. If the generated fragments are linked with a spectrum (i.e. the fragments were generated from a still existing Spectra Manager window which contains the given spectrum), you can eliminate the mass-to-charge ratio values that do not have corresponding peaks in the spectrum. Mass Frontier will, in some cases, generate a large number of theoretically possible fragments with a variety of mass-to-charge ratio values. It is, therefore, useful to have displayed only those fragments that can be linked with a spectrum (“explained” peaks). To eliminate mass-to-charge ratio values that cannot be linked with peaks in a given spectrum: Click the Show m/z Values For Explained Peaks Only button .

If you eliminate mass-to-charge ratio values that do not correspond to a spectrum, these values will not be permanently deleted. When the button is in the down position, Mass Frontier removes these values from the tab control or from the combo box depending on the selected settings of the mass-to-charge ratio selector (Mechanism Layout dialog box). To restore the original state, click the button to its up position. All generated mass-tocharge ratio values will again be listed.

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5.8 Simulation of MSn Experiments
Mass Frontier allows the simulation of MSn experiments. See Figure 44. The fragments and mechanisms can be generated not only from neutral compounds, but also from ions. You can even select a product fragment (parent ion) in a Fragments & Mechanisms window, and start the generation from there. An unlimited number of consecutive secondary-ion decomposition reactions can be simulated.

Figure 44. Fragments & Mechanisms window, showing the simulation of an MSn experiment

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Fragments & Mechanisms

5.9 Unexplained Peaks
The fact that a peak cannot be explained by Mass Frontier, because the corresponding fragment was probably formed by a compound specific mechanism, can in some cases be surprisingly helpful in the identification of the characteristic structural groupings that gave rise to the peak. For example, phthalates produce a characteristic ion with m/z 149, which is formed by a highly specific mechanism. See Figure 45. The peak at m/z 149 is easily recognized as a contaminant from elasticized polymers. Mass Frontier is not able to explain this peak since its corresponding fragment is formed by an unusual hydrogen rearrangement and cyclization mechanism. To distinguish between a “random” unexplained peak and a compoundspecific peak, you need to find some examples in the library. You can retrieve compounds that contain a phthalate group as a common substructure by using a substructure search. Even after the generation of fragments and mechanisms of the retrieved examples, the prominent peak corresponding to the phthalate group remains unexplained in the majority of cases. For example, a phthalate with a functional group at position 3, 4, 5, or 6 will have its prominent peak shifted to higher masses by the mass of this functional group. Such an unexplained prominent peak, present in the spectra of structurally similar compounds, can be a strong indicator of a compound-specific fragmentation process. This information can serve as evidence toward the identification of the substructure under investigation.

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100

149

O
75

OH
50

O O O
263

O

O
25 41 65 0 40 60 80 100 120 140 160 180 200 220 240 57 77 104 133 207

O
260 280

100

149

O
75

OH
50 43 25 85 97 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 113 141 57 71 279 307

O O O O

O
167

100 43 75

323

O O

O

O OH
O O O
425 611 509 400 450 500 550 600 650 694 700

50

O
57 29 69 50 100 95

O

25

O

O

O O O O

149 150 200

252 250 300

341 350

0

Figure 45. Spectra and structures of phthalate fragments

This approach can be used for the identification of compound-specific mechanisms and their resulting fragments, in cases where a prominent peak in the spectra of structurally related compounds cannot be explained by the program.

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Fragments & Mechanisms

5.10 Too Many Proposed Fragments for a Peak
For a molecule that contains a larger number of locations for possible reaction initiation (heteroatoms and π-bonds), Mass Frontier can generate several theoretically possible ions with identical mass-to-charge ratios. Usually, these ions are isomers of the same fragment, but sometimes they are structurally different. If the program generates two or more different fragments with identical mass value, you need to choose the correct one. As before, a substructure search can provide the necessary information. The following example shows how to select the most likely fragment from several possible candidates. The spectrum of 2,3,3a,4,5,6-Hexahydro-10methyl-1H-pyrazino[3,2,1-j,k]carbazole exhibits a base peak at m/z 198 (M-28). See Figure 46. For this peak, Mass Frontier selected twenty-two possible ions that are mostly isomers of two fragments.
100 198

M-28
75 226 50 99 39 0 51 50 60 65 70 79 91 105 128 80 90 100 110 120 130 140 154 150 160 170

N NH
182 180 190 200 211 210 220

25

30

40

230

Figure 46. Spectrum of 2,3,3a,4,5,6-Hexahydro-10- methyl-1Hpyrazino[3,2,1-j,k]carbazole

The first fragment is formed by a Retro-Diels-Alder reaction involving an α-bond on a carbazole group. The second fragment is the result of ethylene loss from a piperazine group. See Figure 47. The way to determine the most likely fragmentation process for the formation of the fragment m/z 198 (M-28) is discussed next.

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1. •
π
N NH N NH

α
N NH

α
N NH

m/z 226

m/z 226

m/z 198

2.
π
N NH


N NH

α
N

α
N NH NH


m/z 226

m/z 226

m/z 198

Figure 47. Two possible mechanisms of fragment formation

First we begin a substructure search of the investigated structure, excluding the methyl group, to determine whether the spectra of the retrieved compounds also contain a base peak or a prominent peak at M-28. See Figure 48.

100

200

100

91

183

HO
75

M-28
N NH

75

N N

50

50 65 39

274

25 40 51 0 40 50

63 77 60 70 80 89 100 115 127 145 154

25 227 44 0 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 40

51 77 60 80 115 127 102 100 120 140 154

167 211 180 200 220 240 260

M-28
302

160

280

300

Figure 48. Spectra from a substructure search

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When the peak M-28 is confirmed as the characteristic peak of this compound class, we need to retrieve two different groups of spectra. See Figure 48. The first group of retrieved spectra shows compounds with a common substructure that are involved in the first mechanism. See the spectra on the left side of Figure 49. Similarly, the second group of spectra shows compounds with a common substructure that are involved in the second mechanism. 1 .
NH
100 143

Fragments & Mechanisms

2 .

N NH

100

121

75

M-28
NH
115 28 39 40 51 50 63 60 70 77 84 89 80 90 102 100 110 120 128 130 140 140 154 150 160

Cl
75 231

50

50

N NH

25 167

25 36 51 0
30 170

67 75 60 70 80

92 98 90

111

167

197 202

0

40

50

100 110 120 130 140 150 160 170 180 190 200 210 220 230

100

177

100

193

264

75

Cl NH
41 51 57 63 115 75 71 70 84 89 80 90 142 154

M-28
205

75 72

N
50

50

N
25 85 0 70 80 90 132 115 152

220 165 179 249

25

0 40 50 60 100 110 120 130 140 150 160 170 180 190 200 210

100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

100 183 75

211

100

43

O O N F N N
83 109 137 153 183 254

M-28
N
168

75

50

50 70

H2N

25 41 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 77 84 90 51 63 106 196 115 128 154

25

0 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260

Figure 49. Two groups of spectra, showing compounds with common substructures that are involved in two different mechanisms

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Two separate substructure searches of 1,2,3,4-Tetrahydrocarbazole and Piperazine result in the retrieval of two groups. We can then see which group yields a prominent peak at M-28. These two groups of spectra prove that the peak m/z 198 in the spectra of 2,3,3a,4,5,6-Hexahydro-10-methyl-1H-pyrazino[3,2,1-j,k]carbazole is formed by a Retro-Diels-Alder reaction (Mechanism 1).

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5.11 Bar Code Spectra
Mass Frontier can automatically predict possible fragments from a chemical structure using general fragmentation and rearrangement rules, 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, the thermodynamic stability of product ions, and reaction rates. However, generated fragments and their mass-to-charge ratio values can be used for creating so-called “Bar Code” spectra. A bar code spectrum contains peaks at predicted mass-to-charge ratio values with identical (maximal) intensity. See Figure 50.
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Figure 50. Experimental and Bar Code spectrum of Pyrrolidine[2,1-c]-2H,5H-1,4benzodiazepin-2,5-dione,1,3-dihydro- (Electron Ionization mode)

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Mass Frontier allows you to create bar code spectra from predicted fragments. To create a bar code spectrum: Click on the Bar Code Spectrum button Mechanisms window. in any Fragments &

The created bar code spectrum will be placed in a Spectra Manager window. Bar code spectra are automatically linked with their original Fragments & Mechanisms windows. If you click any bar code peak in Spectra 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 spectrastructure 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 Spectra Manager than manually compare fragments and their massto-charge ratios between Fragments & Mechanisms windows. For example, if you are interpreting an unknown spectrum and you have established two structural proposals, you need to do the following: 1. Draw both structures separately in Structure Editor. 2. Then generate fragments for both structures, in order to find out which structure belongs to the unknown spectrum. 3. Next create bar code spectra, and place them in the same Spectra Manager window. 4. Then compare the bar code spectra in the Compare Spectra page in Spectra Manager. In the Difference Spectrum box, 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.

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The following example demonstrates how to identify the correct structure for an unknown spectrum. Let us assume two structures are suggested: 1Butanone, 1-phenyl- and 1-Propanone, 2-methyl,1-phenyl-. A comparison of their bar code spectra shows that the peak at m/z 120 is present in the unknown spectrum and only one of the bar code spectra. The formation mechanism for the peak m/z 120 reveals a classical sequence of McLafferty rearrangement and α-cleavage that is very common for 1-butanones or larger ketones. See Figure 51.

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Figure 51. Postulating structures from isobaric possibilities using Bar Code spectra

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5.12 Marking Fragments
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 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 marks can be used in the Fragments & Mechanisms module:
• • •

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

Note. Any changes you make to marks are instantly reflected in the Bar code spectrum. Once a list of fragments with specific marks has been sent or copied to the Fragments Comparator, subsequent changes to marks are ignored. The same applies for a fragment list copied to Excel.

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.

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Chapter 6 Fragments Comparator
The Fragments Comparator module allows you to process and compare fragments generated in Fragments & Mechanisms windows. The fragments can originate from different structures. This module support fragments generated using any ionization method (see Figure 37). Using fragment marks in the Fragments & Mechanism window, it is possible to export only a selected set of fragments into the Fragments Comparator module. The fragments are organized in columns. Each column represents a set of fragments provided by an associated Fragments & Mechanisms window. The Fragments Comparator module can hold an almost unlimited number of fragment sets, limited only by system resources. The Fragments Comparator was designed as an integral part of the Fragments & Mechanisms module. If you double-click any fragment in the Fragments Comparator module, the associated mechanisms will appear in the Fragments & Mechanisms window.

Note. The Fragments Comparator is only able to recall mechanisms that are present in an open Fragments & Mechanisms window. If you close the associated Fragments & Mechanisms window, the link will be lost.

The comparison feature is especially useful when analyzing the fragmentation products of structurally related compounds. Common fragments point toward a common substructure in terms of fragmentation. The most interesting are however the fragment differences. They can indicate fragments along with corresponding peaks in a spectrum that are characteristic for the distinction of structural details. Predicted m/z values of fragments that are different for structurally related compounds should attract your attention when examining spectral differences in Spectra Manager in the Compare Tab.

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The Fragments Comparator window consists of Table and Structures views. Table view lists the numerical m/z values of fragments and Structures view displays structural drawings of possible fragments. Because the Fragments & Mechanisms module can generate several isobaric isomers for a single m/z value, the Structures view imports only the first fragment for each generated m/z value. Fragments are imported into these two views simultaneously but the information is managed independently. If a column in one view is moved or deleted, this action does not affect the other view.

Note. All fragments are compared by m/z values using monoisotopic mass settings. The fragments are usually predicted in several isomeric forms, therefore a structural comparison would not be practicable. Because the fragments are compared by m/z values the calculated precision, defined in the monoisotopic mass settings, has a significant influence on the comparison results.

Figure 52. The Fragments Comparator window, showing Table and Structures tab

Both Table and Structures views are divided into two parts. The left part contains columns of imported fragments for each Fragments & Mechanisms window. The right part contains three columns that show three types of comparison results. The first result column shows all available fragments (logical OR), the second column shows all common fragments (AND), and third column shows different fragments (NAND). Columns can be moved, in both views. The columns for imported fragments can be deleted.

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In table view it is possible to select a column, or a part of a column, and copy the data. The m/z values can then be pasted into Excel or any spreadsheet program. In Structure view the cell can be resized with the Cell Size track bar.

Fragments Comparator

Note. The Fragments Comparator can only display structures of fragments if the associated Fragments & Mechanisms window is still open. If you close the associated Fragments & Mechanisms window, the corresponding column of fragments in the Structures tab will be removed.

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Chapter 7 Mass Spectra Classification
Computer methods of analyzing mass spectral data center on three fundamental methodologies: library search techniques, expert system procedures and classification methods. The first two methodologies were described in previous chapters. In Mass Frontier, classification methods are introduced that close the triangle of computer oriented methods for interpreting mass spectral data. Classification is a powerful enhancement of library search and fragmentation prediction methods. The computeroriented methods available in Mass Frontier complement each other, but are based on different principles. This provides possibilities for creating alternative strategies and enables highly effective data interpretation. In contrast to standard statistical software packages, Mass Frontier allows the direct application of the classification methods to mass spectral data. All pre-processing and data transformation procedures, which are essential for the utilization of classification methods, are carried out automatically. This ensures the classification methods can be used correctly, even by those with no knowledge of the multivariate statistic. This chapter contains the following topics:
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Spectra Classification Principal Component Analysis (PCA) Neural Networks (Self-Organizing Maps) Spectra Transformation

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7.1 Spectra Classification
The primary goal of spectra classification is to find correlation between the properties of compounds and their mass spectra. Because physical and chemical properties and the biological activities of chemical compounds are, to a large extent, a function of molecular structure, the results of classification analysis reflect structural features that are determined by the fragmentation ions which appear in a mass spectrum. From the user viewpoint, the important advantage of classification methods is the fact that the user does not require detailed knowledge of the complex spectrastructure relationship to get satisfactory results. Classification strategy in Mass Frontier is based on a user-friendly graphic representation of the results, which can be easily viewed on the screen. See Figure 53. Mass Frontier contains two classification methods: Principal Component Analysis (PCA) and Self-Organizing Maps (SOM), which is a special class of Neural Networks. These methods are based on different principles and allow the user to explore complex data from various perspectives. PCA uses multivariate statistics and Neural Networks are based on competitive learning.

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Figure 53. Classification of mass spectra in Mass Frontier

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In the multivariate statistic each spectrum can be considered as a single point in an n-dimensional space, with the intensities being the coordinates of this point. A dimension (axis) of that space represents the mass-to-charge ratio of the considered peak. Therefore, the dimensionality is determined by the mass-to-charge ratio value of the last peak in the spectrum. For example, the EI spectrum of hydrogen exhibits two peaks at m/z =1 (intensity 2%) and m/z = 2 (100%). This spectrum can be viewed as a point in a two-dimensional space with the coordinates [2, 100]. In reality, we deal with spectra that have a far higher dimensionality than two. If the dimensionality is too high, or several coordinates are equal to zero (usually a mass spectrum does not have peaks at every mass-to-charge ratio value), the classification methods may not provide the results we require. Therefore, a reduction of dimensionality is carried out either before a spectrum is placed in n-dimensional space, or during the classification process. The basic hypothesis of multivariate statistical methods is the assumption that the distance between points (spectra) in an n-dimensional space is related to a relevant property of the compounds that represent these points. If the points are close enough to form a cluster or a separated region, we assume that the compounds that correspond to these points exhibit common or similar properties. To ensure that the results of the classification methods have statistical significance, a large number of spectra (usually one or more groups, each with 10-1,000 spectra) should be placed in the same ndimensional space. See Figure 54. Then, we apply classification methods, with various parameters, in order to evaluate these points (spectra). The objective of a classification process is to separate these points (spectra) into two or more classes according to the desired structural or other properties.

Mass Spectra Classification

m/z 3 m/z 2 Mass Spectrum 1 Mass Spectrum 2

m/z 1

Figure 54. Representation of two spectra as points in a 3-dimensional space

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7.2 Principal Component Analysis (PCA)
Mass Frontier now offers the classification method called Principal Component Analysis (PCA). The central idea of principal component analysis is to reduce the dimensionality of a data set in which there are a large number of interrelated (i.e. correlated) variables, while retaining as much as possible of the variation present in the data set. In the case of mass spectrometry, the data set consists of the mass spectra of different compounds. The mass spectra are expressed as the intensities of individual mass-to-charge ratios (i.e. variables). The aim of PCA is to find a new coordinate system that can be expressed as the linear combination of the original variables (mass-to-charge ratios) so that the major trends in the data are described. Mathematically, PCA relies upon eigenvalue/eigenvector decomposition of the covariance or the correlation matrix of the original variables. PCA decomposes the data matrix X as the multiplication of two matrices P (the matrix of new coordinates of data points) and TT (transposition of the coefficients matrix of the linear combination of the original variables): X = P u TT Generally, it is found that the data can be adequately described using far fewer coordinates, also called principal components, than original variables. PCA also serves as a data reduction method and a very good visualization tool. When the data points are plotted in the new coordinate system, the relationships and clusters are often more apparent than when the data points are plotted with the original coordinates. See Figure 55.

Figure 55. Geometrical interpretation of PCA

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The axes of the new coordinate system – principal components p1 and p2 – are created as linear combinations of the original axes. New coordinates (PC - principal components) are orthogonal (perpendicular) to each other. It is clear, that there is greater variation in the direction of p1 than in either of the original variables, but very little variation in the direction of p2. The first PC describes the direction of the greatest variation in the data set, the second PC describes the direction of the second greatest variation, and so on, for data sets with more than two variables.

Mass Spectra Classification

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7.3 Neural Networks (Self-Organizing Maps)
Self-Organizing Maps (SOM), sometimes called Kohonen networks, are a special class of neural networks. A self-organizing map consists of neurons placed at the nodes of a two-dimensional lattice. The neurons become selectively activated to various input mass spectra or classes of spectra in the course of a competitive learning process. The neurons compete among themselves to be activated or excluded. SOM can be considered as a nonlinear generalization of PCA. The principal goal of self-organizing maps is to transform a set of ndimensional input spectra into a discrete two-dimensional map, and to display this transformation in a user-readable fashion. Each input spectrum presented to the network activates a neuron according to a complex set of interrelationships between spectra. To ensure that the self-organizing process has a chance to develop properly, the networks should be exposed to a number of different spectra. Therefore, in Mass Frontier a minimum of 10 spectra must be used in a self-organizing process. A detailed description of self-organizing maps can be found in the comprehensive text book: Haykin S., Neural Networks. Second Edition. 1999, 443-483, Upper Saddle River, NJ: Prentice Hall.

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Mass Spectra Classification

7.4 Spectra Transformation
It has been shown that various mathematical transformations of mass spectra can increase classification efficiency. Better separation of classes can be achieved in some cases if transformed spectra, instead of original spectra, are submitted for classification. In addition, some transformation procedures reduce the number of variables and lower the dimensionality of the spectral space, which shortens the computing time. Because the most common neutral loss is 14 (loss of CH2), the logical spectra transformation is into modulo-14 spectra, which thereafter can be used as input data for PCA. Modulo-14 spectra are calculated as the sum of peak heights at mass-to-charge ratio values shifted by 14. Each modulo-14 spectrum has 14 dimensions (transformed mass-to-charge ratio values) that are significantly lower than regular spectra. Mass Frontier offers modulo-14 transformation with (A1) or without (A2) normalization of such spectra. The classification of mass spectra assists the interpretation of structurally related compounds. Because the characteristic peaks in spectra of structurally related compounds can be shifted due to the presence of various substituents, it can be difficult for classification methods to recognize structural similarity. This difficulty can be overcome by transforming spectral data into auto-correlation spectra. The auto-correlation function: A(∆x) =

œ f ( x ). f ( x + ∆ x ) dx

is suitable for detecting periodicity in a series of spectra. In Mass Frontier, you can choose auto-correlation transformation with (B1) or without (B2) normalization of mass spectra. Since auto-correlation does not reduce the space dimensionality and requires computing time to be calculated, a classification that uses this transformation is the most time-consuming procedure among the transformation methods. Mass Frontier allows the user to submit original (not transformed) spectra (C1) to classification as well.

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Mass Frontier offers the following spectra transformations:
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Modulo-14 spectra (normalized) Modulo-14 spectra (not normalized) Auto-correlation spectra (normalized) Auto-correlation spectra (not normalized) Original spectra (normalized)

A1 A2 B1 B2 C1

No general rule exists concerning the selection of an appropriate mass spectrum transformation. Classification methods can be employed for a broad range of problems, and each of them may need a different spectrum transformation. Your objective should be to find the transformation that provides the best separation of classes. The only reliable way to find the best transformation for particular groups of spectra, is to experiment with all of them. Subsequently, the transformation which provides you with the most information will be your first choice when dealing with comparable data.

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Chapter 8 Spectra Classifier
Mass Frontier provides tools for the classification of mass spectra. Three modules, Spectra Classifier, Spectra Projector and Neural Networks are included in Mass Frontier to retrieve, organize, and classify mass spectra and display the results. Similarly to other modules described in previous chapters, Spectra Classifier, Spectra Projector and Neural Networks are seamlessly integrated within the program interface, allowing information exchange between the modules. Mass Frontier can be used for the classification of spectra according to physical or chemical properties (point of origin, toxicity, aromaticity, etc.). For spectra that cannot be found in a library, classification can involve identification of substructure types or compound classes (structure elucidation) in order to establish and confirm structural proposals. Classification can also be useful in cases when only structurally related compounds need to be retrieved from a complex chromatography scan (metabolite research). This chapter contains the following topics:
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Spectra Classifier Window Classifying Mass Spectra Maintaining Groups of Spectra

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8.1 Spectra Classifier Window
The Spectra Classifier module allows the user to retrieve and organize spectra, which can then be submitted to principal component analysis (PCA) and self-organizing maps (SOM). Spectra are organized into groups that can have their own names and graphic representations. When spectra are organized into groups, it is easier to distinguish between the classes of spectra that may arise from the submitted groups of spectra after the classification process.

Note. In classification analysis it is important to distinguish between two terms: groups and classes. It does not make sense to speak about classes prior to the classification process. Classes can only be assigned after the classification method (PCA or SOM) clearly shows the occurrence of clusters or regions that consist of spectra with the desired properties. Prior to classification, we can only allocate spectra to groups according to userselected criteria, i.e. a priori information. Often the objective of the classification process is to obtain classes that closely resemble groups of spectra submitted to classification.

The Spectra Classifier window is the gateway to PCA and SOM. Any spectra that you wish to classify must first be sent or pasted to the Spectra Classifier window. A spectrum or spectra are automatically assigned to a group. Mass Frontier automatically gives a name to a new group, which can be easily renamed. The program also assigns a graphic representation to each group that is prepared for classification. Up to 255 groups of spectra can be added to Spectra Classifier and each group can consist of an unlimited number of spectra. Conversely, it is also possible for a group to only contain a single spectrum. The points are given symbol types and colors according to settings in Classification Layout. These settings only show group membership and have no influence on the results of the analysis. To open the Spectra Classifier window:
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Choose the Tools | Spectra Classifier menu item to open an empty Spectra Classifier window

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Only one Spectra Classifier window can be opened at any one time in the program. If you click the Spectra Classifier button, or choose the Tools | Spectra Classifier menu item, and Spectra Classifier is already open, this window becomes active. To choose a spectrum for classification: 1. Select one or more records in the Spectra Manager window, or select one or more scans or components in the GC/LC/MS Processor window. 2. Click on the Add Selected Records To Spectra Classifier button (Spectra Manager), or click the Add Selected Scans Or Components To Spectra Classifier button (GC/LC/MS Processor). In addition to the above method, you can also use copy and paste commands to add spectra to the Spectra Classifier module. There are two panes in the upper half of the Spectra Classifier window. The left one contains groups of spectra that have been sent from the Spectra Manager window. You can fill this pane successively with the spectra you have chosen for classification. Each group of spectra is visually represented as a single line in the left or right pane. The right pane contains the groups of spectra that will actually be classified if you click the Classify Now button. You can move a group from one pane to another by clicking the Add and Remove button. The two text box setup allows you to have spectra available in the left text box, ready for inclusion in future classification runs. See Figure 57.

Spectra Classifier

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Move Selected Group Up Move Selected Group Down Paste Spectra Show Spectra Source in Spectra Manager Show Spectra Source in GC/LC/MS Processor Classification Layout Groups of spectra sent from Spectra Manager or GC/LC/MS Processor Groups of spectra about to be classified

Editing name of selected group of spectra Preview of selected group of spectra
Figure 57. Spectra Classifier window

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If you select a line (group) in either of the two text boxes, all corresponding records will be previewed in the grid that displays spectra, structures and additional information. If you double-click a line (group) in either of the two text boxes, the window from which the corresponding spectra originate, will automatically open, and these records will be selected.

Spectra Classifier

Note. Spectra Classifier can only hold spectra that are still present in the original Spectra Manager or GC/LC/MS Processor windows. In addition, once you have created a group of spectra in Spectra Classifier, you must not move the spectra from the original Spectra Manager window or clear the component spectra from a chromatogram in GC/LC/MS Processor. If you violate either of these conditions, the group of spectra concerned will be removed from Spectra Classifier.

Spectra Classifier contains five option buttons for the selection of the transformation that should be applied to the spectra (see the Mass Spectra Classification Chapter). Due to the long names of transformation techniques, only short code names are displayed. However, if you point the cursor at a code name, the full name will be displayed in a small tool tip box. You can choose from two classification methods: Principal Component Analysis and Neural Networks. If you choose PCA, you can set the number of principal components that should be calculated. If the Neural Networks is your method of choice, the lattice size can be set in two ways; either the user can assign the x and y size, or the program can set the optimal size automatically (recommended method). Once the data have been prepared you can start the classification. This can be done by clicking the Classify Now button. All groups of spectra listed in the right pane will be classified according to the chosen options (transformation and classification method). After the classification is completed, the results are displayed either in a Spectra Projector window (PCA) or in a Neural Networks window (SOM). It must be stressed again that while a classification is being processed, you can continue working with the program, as Mass Frontier is a multi-threading application.

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8.2 Classifying Mass Spectra
A simple example should help clarify how the classification of mass spectra can be carried out. Suppose we wanted to classify the derivatives of nicotine and caffeine. We have an EI spectrum of an unknown compound that we suspect is either a metabolite of nicotine or caffeine, and we need to find out which of these two compound classes it belongs to. To complete this task we need to retrieve a sufficient amount of spectra for each group (nicotines and caffeines), which will then be submitted to a principal component analysis to form clusters of each group. We can do this by performing a substructure search, where the search query will be the structure of nicotine and caffeine. If sufficient amounts of spectra for each group are found (more than 10 spectra), do the following: 1. Select these records and add them, separately for each group, to the Spectra Classifier. 2. Assign an appropriate name to each group using the Selected Group of Spectra edit box. 3. Select a line in the left pane of Spectra Classifier and click the Add button. 4. Repeat this for the next group. Mass Frontier automatically assigns a graphic representation to each group. This is shown in the right pane for each line. See Figure 58. You can easily change the graphic representations of groups using the Menu | Projection Layout menu item. Please note that the colors and type of graphic representation are only used to distinguish the groups in the plots and have absolutely no influence on the results of the analysis. When all the groups of spectra you want to classify are in the right window, click the Classify Now button to launch the classification process. After the generation is finished, a new Spectra Projector window will be displayed.

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Spectra Classifier

Add selected records to Spectra Classifier

Substructure search

Select all records

Figure 58. Retrieving data for a classification task

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8.3 Maintaining Groups of Spectra
The Spectra Manager chapter details how Mass Frontier allows the saving and opening of references to records present in the Spectra Manager spreadsheet with the extension *.ref. This feature is especially useful for maintaining groups of spectra that are sent from Spectra Manager to the Spectra Classifier window. If you have retrieved spectra for classification, it is useful to save these records as references, rather than save the spectra as ASCII files (JCAMP or MSP). In contrast to JCAMP or MSP files, a reference file stores the locations, where all the relevant information about each spectrum is saved (scan, structure, experimental conditions, etc.). In addition, reference files are easy to manipulate, and you can add or remove one or more records. Deconvoluted spectra from GC/LC/MS Processor cannot be saved as references.

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Chapter 9 Spectra Projector
The Spectra Projector module displays the results of a Principal Component Analysis. In Spectra Projector, mass spectra are projected as points on a two-dimensional plane or a three-dimensional twistable space, according to user-selected principal components. The objective of classification analysis is to find classes of spectra on the projection that exhibit common or similar properties. Spectra Projector also allows the user to place an external spectrum onto a projection in order to examine its class membership. Classification can also be applied to spectra that cannot be distributed a priori into groups. In this case, we should classify only one group of spectra, and attempt to find points on the projection that represent compounds with the desired properties. Spectra Projector allows the user to select such points, in order to examine their spectra and structures. This chapter contains the following topics:
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Generating Spectra Projector Module Spectra Projector Window 3-D Projection Mode Opening and Saving of Classification Results Accessing Spectra from Spectra Projector Adding External Spectrum

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9.1 Generating Spectra Projector Module
A Spectra Projector window cannot be directly opened from the program desktop. It must be generated from the Spectra Classifier module, by selecting Principal Component Analysis option and clicking the Classify Now button. See Figure 59. You cannot alter classification results once they have been generated. If you want to remove a spectrum (point) from a projection plane, you must remove this spectrum from the input data, and then launch a new classification process. An unlimited number of Spectra Projector windows can be opened at any given time in the program.

Figure 59. Generating a Spectra Projector window from Spectra Classifier

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Spectra Projector 9tyytrfyÁWhfwtg†ƒ ____________________________________________________ Spectra Projector Window

9.2 Spectra Projector Window
Spectra Projector displays spectra as points on a 2D plane or in a 3D space. A combination of two principal components, which make up the 2D projection plane, can be selected in the tab control. See Figure 60. If you are viewing PCA results using the 3D projection mode, you can select a combination of the three associated principal components. If you click, for example, the 2D projection tab captioned “2-5”, the spectra will be projected onto the plane of the 2nd and 5th principal components. The number of tabs is determined by the number of principal components that have been selected in the Spectra Classifier module. Spectra Projector displays a tab for every possible combination of the selected principal components. For example, if you have generated Spectra Projector with 3 principal components, 3 tabs will be available (1-2, 2-3, and 1-3). For the 5 (default) principal components, 10 tabs will be available (1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5). The same principle applies to the 3D projection mode. Please note that in the 3D mode the number of combinations of principal components for more than five components is significantly higher in comparison to 2D. Therefore, if you want to analyze your spectra in the 3D mode, you should not use more than five principal components. The number of principal components can be set in the Spectra Classifier window (see 8.1) prior to the PCA calculation.

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Open Projections Save All Projections Print Projection Copy Projection 3D Projection mode Show Axes Select Spectra and show their origin

Legend of Group Members

Paste External Spectrum Open External Spectrum Projection Layout Projection of the 1st and 2nd Principal Components

External Spectrum

Figure 60. Spectra Projector window

The Status bar in a Spectra Projector window is divided into three parts. The left part informs you which principal components have been selected. The middle part displays the type of spectra transformation that has been utilized for classification. The right part shows how many spectra have been classified (the total number of spectra from all groups). Spectra Projector allows you to enlarge any region of the projection plane independently, for each combination of principal components, by using the left mouse button. To get the original scale, simply click the Zoom Out button, which will appear in the top left corner of the projection plane after any change of scale. In the 3D mode it is not possible to enlarge the projection.

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Spectra Projector 9tyytrfyÁWhfwtg†ƒ _________________________________________________________ 3D Projection Mode

9.3 3D Projection Mode
The principle of PCA classification is the reduction of the dimensionality of a spectral space to a level comprehensible to the human eye. Since a paper or screen plot is inherently 2D, the method of choice is usually 2D PCA. However, if a 3D projection on a 2D computer screen is accompanied with motion, the user can perceive this simulation as 3D space. To increase the space feeling the 3D plots are interactive, i.e. the plot can be rotated with the mouse. Spectra Projector allows interactive 3D viewing of your data. The 3D projection mode used in Mass Frontier stems from the idea that spectra classification of complex data set in a 3D space can be more effective and reliable than a simple 2D plot. This idea is based on the wellrecognized fact that the human brain is incredibly efficient in visual pattern recognition in 3D space and can still outperform any computer system. When analyzing PCA results you should not rely entirely on 2D plots. Two or more clusters which overlap in a 2D plot may be separated in 3D space or, on the other hand, two seemingly separate clusters discernable in a 2D plot may appear in 3D mode to be too close together to be considered as two different objects. To view PCA results in 3D mode, do the following: 1. Click the 3D Projection button .

2. Click anywhere in the PCA plot with the left mouse button and move the mouse to rotate the plot interactively. Note that the arrow cursor will be replaced by a circle during the dragging step. To better appreciate the three-dimensionality of the space you can display the x-, y-, and z- axes by clicking the Show Axes button, which is next to the 3D Projection button. See Figure 60.

Note. In the 3D projection mode it is not possible to enlarge a part of the plot. However, as in the 2D mode, the selection of particular spectra and the display of their origins is supported.

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9.4 Opening and Saving of Classification Results
Mass Frontier allows you to open and save projection planes that have been generated with particular initial settings (transformation method and number of principal components). To open or save spectra projection planes, do either of the following:
• •

Click on the Open Spectra Projections or Save Spectra Projections button. Choose the File | Open | Spectra Projections or the File | Save | Spectra Projections menu item

Projection planes are saved as graphics, without the possibility of recalling the original spectra, which are displayed as points. If you need to access the spectra with structures from projection planes, you must save the references to records in Spectra Manager, as described in the Spectra Classifier chapter. Refer to topic 8.3 on page 124. In this case the classification must be regenerated from the original data set that was saved as references. However, it is possible to add an external spectrum. Refer to topic 9.6 on page 132.

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Spectra Projector 9tyytrfyÁWhfwtg†ƒ ________________________________________Accessing Spectra from Spectra Projector

9.5 Accessing Spectra from Spectra Projector
It is an important part of classification analysis to know which spectrum is represented by a point on a projection plane. As Mass Frontier links corresponding modules, spectra with structures or chromatographic components can be recalled from any projection plane. To access a single spectrum, scan, or deconvoluted component:

Double-click a point on the projection. The linked module will be displayed on top of all other windows, the corresponding record or component will be selected, and the spectrum will be shown

To access several spectra in a region:
• •

Select a region with the mouse cursor while holding down the RIGHT mouse button Or, click the Select Spectra And Show Their Origin button and then select a region with the mouse cursor while holding down the LEFT mouse button

If the selected spectra originate from Spectra Manager, you will be prompted as to whether the records with spectra and structures should be copied to the last active Spectra Manager window, or to a new one. If your spectra originate from a chromatogram the corresponding scans or components will appear in the same color as they appear in the PCA plot.

Note. Spectra Projector is only able to recall records that are present in a Spectra Manager window. If you close the Spectra Manager window that is the source of the input data for classification, the link between a point and its spectrum will be interrupted. The program automatically warns you if you try to close a Spectra Manager window that is linked with one or more Spectra Projector window(s). In addition, to keep the links between points and spectra intact, you must not move records in, or between, Spectra Manager windows using cut & paste commands. If the data originated from GC/LC/MS Processor, the window must still be open.

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9.6 Adding External Spectrum
The objective of principal component projections of mass spectra is to find classes of compounds with common or similar properties. Sufficiently separated classes can then be used to investigate the class membership of an unknown spectrum. Spectra Projector allows you to add an external spectrum, which was not used for classification, to the projection plane. If the added spectrum is clearly projected into a particular class region, you can assume that this compound has similar, usually structural, properties. To demonstrate this feature, we will continue with the example from the Spectra Classifier chapter. We have retrieved two groups of spectra: nicotine and caffeine derivatives. We want to examine an unknown metabolite that we assume originated either from nicotine or caffeine. After we have prepared the two groups of spectra in Spectra Classifier, we can generate a Spectra Projector window. See Figure 61. The figure below shows that the two groups are separated into clusters. We can then add the unknown metabolite to the projection plane, by pasting or opening its spectrum into Spectra Projector. The projection of this external spectrum clearly shows that it belongs to the nicotine class (squares). This result can then be finally confirmed or rejected using the fragmentation pattern of nicotine.

Unknown Metabolite

Figure 61. Projection of unknown metabolite into the nicotine class

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Chapter 10 Neural Networks
The Neural Networks module displays classification results from SelfOrganizing Maps (SOM), which are a special class of neural network. A self-organizing map is a network of neurons, arranged in the form of a twodimensional lattice. The size of a lattice can either be calculated automatically or defined by the user. During a classification neurons become selectively activated to various input spectra as a result of a competitive learning process. Refer to topic 7.3 on page 114. The objective of classification analysis using SOM is to find classes of spectra on the map that exhibit common or similar properties. If one or more spectra activate the same neuron, we can assume the spectra belong to a common class. In this case the spectra should exhibit certain similarities. In addition, spectra that activate neighboring neurons, and those neurons that have low Euclidian distance between each other (shown by border line thickness), can also be considered as related. In neural networks each mass spectrum must always activate a neuron and this spectrum is shown on the particular neuron. Neurons are displayed as rectangles on the screen. Spectra are represented as symbols or numbers and are placed onto neurons. Since the spectra are located in discrete objects the interpretation of SOM is relatively easy, as, in contrast to PCA, you do not have to deal with diffuse clusters. However, it may happen that larger numbers of neurons are activated by a single spectra and this advantage is lost. Spectra that activate the same neuron belong, in terms of classification, to the same pattern. Therefore, all spectra drawn inside a neuron box are equal for classification purposes and their graphical positions within a neuron are irrelevant.

Note. The SOM classification method exhibits one atypical feature you should be aware of. Different results are produced for an identical data set if the input data is processed in a different order. This order sensibility is an inherent feature of neural networks and NOT a result of faulty algorithms.

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This chapter contains the following topics:
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Generating Neural Networks Module Neural Networks Window Working with Neural Networks

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Neural Networks 9tyytrfyÁWhfwtg†ƒ ____________________________________________ Generating Neural Networks Module

10.1 Generating Neural Networks Module
Neural Networks are generated from the Spectra Classifier module. Similarly as in Spectra Projector, Neural Network cannot be directly opened from the program desktop. Once the network has been generated, classification results cannot be altered. If you want to remove a spectrum (point) from a network, you must remove this spectrum from the input data, and then launch a new classification process. An unlimited number of Neural Networks windows can be opened at any given time in the program.

Figure 62. Generating a Neural Networks window from Spectra Classifier

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Neural Networks Neural Networks Window ____________________________________________________ 9tyytrfyÁWhfwtg†ƒ

10.2 Neural Networks Window
A Neural Networks window displays a topological map of neurons that are drawn as rectangle. Every neuron has a minimum of two and maximum of six neighboring neurons. Spectra are displayed as symbols or numbers within neurons. Open Neural Networks Save Neural Networks Print Neural Networks Copy Neural Networks Classification Layout Open External Spectrum Paste External Spectrum Show Distances between Neurons Select Spectra and show their origin

Show Legend

Spectrum Distance between neurons Neuron
Figure 63. Neural Networks window

Neuron activated by external spectrum

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Neural Networks 9tyytrfyÁWhfwtg†ƒ _____________________________________________________ Neural Networks Window

Although neurons are displayed in a regular 2D lattice, the actual Euclidian distances between neurons vary. To show the approximate distances between neighboring neurons, the borders of the neuron are shown using different thicknesses. The line thickness is proportional to the distance between immediate neighbors. The thinner the line, the closer together are the neurons. See Figure 63. To show neuron distances: Click the Show Distances between Neurons button .

Information about neuron distances can be useful when classifying an unknown spectrum. The distances show how a neuron has been activated by an unknown correlates with neighboring neurons. The distances can also suggest that spectra from a common may have been activated by multiple close neurons. The Status bar in a Spectra Projector window is divided into three parts. The left part informs you about the lattice dimension. The middle part displays the type of spectra transformation that has been utilized for classification. The right part shows how many spectra have been classified (the total number of spectra from all groups). Neural Networks module allows you to enlarge any region of the lattice, by using the left mouse button. To return to the original scale, simply click the Zoom Out button, which will appear in the top left corner of the lattice after any change of scale.

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10.3 Working with Neural Networks
Neural Networks windows have some identical functionality with the Spectra Projector window that is described in the previous chapter. For further information, refer to the following topics: 9.4 Opening and Saving of Classification Results 9.5 Accessing Spectra from Spectra Projector 9.6 Adding External Spectrum

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Chapter 11 GC/LC/MS Processor

Note. Mass Frontier 3.0 now incorporates all the chromatogram and spectral viewing features of Qual Browser in Xcalibur 1.4. The user will find that using Qual Browser allows the use of mass filters for viewing MSn spectra and the correct depiction of chromatograms acquired with data dependent scanning. To activate Mass Frontier from inside Xcalibur Qual Browser, place the mouse cursor on the spectrum of interest and click the right mouse button. Select Export/Mass Frontier and the spectrum will immediately be transferred to the Spectra Manager module in Mass Frontier.

This module enables the convenient extraction and processing of mass spectral scans from GC/MS or LC/MS files and from MSn data. Component detection and spectra deconvolution techniques are available. This module also offers spectral averaging and background subtraction features. Two types of chromatograms can be displayed: Total Ion Chromatogram (TIC) and Selected Ion Chromatogram (SIC). The SIC feature permits the user to display individual mass chromatograms of ions that are characteristic for a specific compound of interest. Mass spectral scans, deconvoluted components, and various types of MSn data can be classified using PCA and SOM. Spectra can be searched in a library for positive compound identification, or the spectra can be copied to the Spectra Manager module for further processing and archiving. Several chromatograms can be opened simultaneously. Fully customizable chromatogram and mass spectrum layouts are available. GC/LC/MS Processor allows you to copy chromatograms with extracted spectra for importing into reports, spreadsheets, or other Windows programs. Xcalibur’s MSn data is displayed in tree structures allowing the user to clearly view the dependencies. Using the built-in filter, it is possible to extract and process the MSn spectra of interest. This module does not include target analysis, and automatic quantitation of ions is not available. This chapter contains the following topics:
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GC/LC/MS Processor Window Data File Formats

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• • • • • • • • •

Opening Chromatograms TIC Page Info Page Spectra Averaging Background Subtraction Processing Extracted Spectra Selected Ion Chromatogram (SIC) Automated Component Detection and Spectra Deconvolution Processing Xcalibur MSn Data

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ _________________________________________________ GC/LC/MS Processor Window

11.1 GC/LC/MS Processor Window
Mass Frontier can keep up to ten GC/LC/MS Processor windows open.

Open New GC/MS, LC/MS or MSn File Save Spectrum Print Window Copy Window and Sel. Spectra Chromatogram Layout Spectrum Layout Search Spectrum Spectra Classifier

Components Detection and Spectra Deconv. Selected Ion Chromatogram (SIC) Set Background Subtraction Scans Cancel Background Subtraction Spectra Average Select Scans or Comp. Clear Chromatogr. Show Scan Points

Figure 64. GC/LC/MS Processor window

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11.2 Data File Formats
Mass Frontier supports various data file formats for importing GC/MS and LC/MS files, including the following:
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Xcalibur (*.raw) Finnigan LCQ, GCQ, ITS40 and Magnum (*.ms) netCDF (*.cdf) JCAMP -DOS, Windows and UNIX (*.jcd)

Files of these types can be imported to GC/LC/MS Processor, but they cannot be exported. Single scans can be saved in JCAMP or MSP format. Mass Frontier supports centroid type data for mass spectra. Centroid mass spectra are displayed as a bar graph. Profile type data is not supported. Due to various netCDF standard implementation some *.cdf files may not be readable in Mass Frontier.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ _____________________________________________________ Opening Chromatograms

11.3 Opening Chromatograms
To load a GC/MS or LC/MS file to a GC/LC/MS Processor window, do the following: 1. Click on the GC/LC/MS Processor button on the speed bar, or choose File | Open | Chromatogram. The Open GC/LC/MS File dialog box will appear. 2. Select the Chromatograms directory. Then select a File name, a file type, and an extension in the Files of type combo box. 3. Click Open. Mass Frontier allows you to display chromatograms that contain up to 30,000 scans. If you open a file that contains more than 30,000 scans, the chromatogram is displayed up to the retention time corresponding to Scan Number 30,000. The remaining scans are ignored, and are not displayed. The program does not allow you to open GC/LC/MS files from a CD-ROM. Files on a CD-ROM should be copied to the hard drive to make them available for import. In addition the file attribute “Read Only” must be removed. To change the file attribute, do the following: 1. Select all the files whose attributes you wish to change In Windows Explorer 2. Click the selected files with the RIGHT mouse button and a pop-up menu will appear 3. Select Properties in the pop-up menu 4. Check out the Read Only attribute check box that is located at the bottom of the Properties dialog window Mass Frontier comes with a GC/MS and an Xcalibur MSn demonstration file, which are located in the /Chromatograms directory.

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11.4 TIC Page
Total Ion Current (TIC) chromatograms are displayed on the TIC page (Figure 64). Both the retention time and the intensity axis can be zoomed by holding down the left mouse button and dragging a rectangle around the region you want to re-scale. To un-zoom the TIC, click the Zoom Out button in the top right corner of the chromatogram display area. To select a scan from the TIC, click anywhere in the chromatogram pane. The scan corresponding to the selected retention time will be displayed on the Scan page. A vertical line on the TIC page indicates the active scan (purple by default). To move the scan point to the next or previous scan, click one of the two arrow buttons. The status bar at the bottom of the GC/LC/MS Processor window displays retention times (Scan tR) and the scan numbers of active scans.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ _________________________________________________________________ Info Page

11.5 Info Page
The Info page of the GC/LC/MS Processor window shows additional information saved in a data file. See Figure 65. Each GC/MS or LC/MS file format contains a different list of items describing the sample, the instrument and experiment conditions, and other information. Mass Frontier extracts all the additional information from the file, and displays it on the Info page.

LC / MS DATAFILE 10 Dec 98 4:07 am LCQ

Figure 65. Info page of the GC/LC/MS Processor window

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11.6 Spectra Averaging
Mass Frontier allows the extraction of the average mass spectrum from several scans. To display a mass spectrum averaged from several scans, click the Spectra Average button. Then, drag your cursor in the shape of a rectangle around the scan region you want to average. See Figure 66. The marked region will be displayed in the same color as the active scan line (purple by default). The average spectrum will be displayed on the Scan page (Figure 64). To cancel the spectra average mode and restore the single scan mode, click anywhere in the chromatogram pane.

Figure 66. TIC page, showing a selected region of several scans to be averaged

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ ______________________________________________________ Background Subtraction

11.7 Background Subtraction
To eliminate the background signal from an active scan or from the average of scans, a manual background subtraction should be carried out. Mass Frontier allows you to specify the location of two representative background scans by clicking the Manual Background Subtraction button and then clicking the scan point in the chromatogram pane. A vertical line, displayed in green, indicates which scans were selected as background. The resulting spectrum is displayed on the Scan page (Figure 64). To cancel a background subtraction, click the Cancel Background Subtraction button. The Selected Ion Chromatogram (SIC) can help you to choose the two most representative background scans. If you would want to see the effects of background subtraction on a scan, copy both the extracted and the original spectra to Spectra Manager and then compare them using the Compare Spectra page. Background subtraction can be used for component detection and deconvolution purposes. Refer to topic 11.10 on page 151.

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11.8 Processing Extracted Spectra
A spectrum obtained from the average of scans, or from scans with a subtracted background, can be further processed in Spectra Manager or it can be directly searched for in a library from GC/LC/MS Processor. To transfer the extracted spectrum to Spectra Manager, use the Copy command. After clicking the Copy button in the GC/LC/MS Processor window, the mass spectrum can be pasted to a Spectra Manager window. In addition, if you click the Copy button in the GC/LC/MS Processor, the chromatogram graphic together with the spectrum from the Scan page can be copied to Windows Clipboard. They are then available for use when creating reports in any Windows application. The GC/LC/MS Processor window contains a Search button allowing you to directly search a library from the spectrum on the Scan page. To search for an extracted spectrum in a library, click the Search button in the GC/LC/MS Processor window. The Library Search dialog box will appear. See Figure 21.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ ___________________________________________________Selected Ion Chromatogram

11.9 Selected Ion Chromatogram
Mass Frontier enables you to display a chromatogram for a selected ion in a different color. The selected ion chromatogram (SIC) is sometimes called an individual or single ion chromatogram, ion profile, or selected ion monitoring (SIM) in Xcalibur. The program can display up to three selected ion chromatograms per window. See Figure 67. To display an SIC for a particular mass-to-charge ratio, do the following: 1. Click on the spectral peak of interest on the Scan page. 2. Select the Data tab and click the m/z value in the mass-to-charge ratio table. 3. Click the Selected Ion Chromatogram button .

In either case a dialog window will appear in which you can change or add the mass-to-charge ratio and confirm your choice. This dialog window allows you to select a color for a particular m/z value. Mass Frontier extracts the SIC from the original file, and this process can be time-consuming for chromatograms with a large number of scans. However, because Mass Frontier is a multithreading application, you can still use other windows during the SIC extraction. A selected ion chromatogram is very useful feature for verifying automated component detection and spectra deconvolution results. An SIC helps you to quickly recognize mixture components in a peak region. An SIC of model peaks should be examined for any component you may wish to use in further analysis. Remember that some structural (alkanes) or optical isomers produce almost identical mass spectra and even if you can clearly see two or more maxima in a peak region, an SIC may not reveal a multicomponent profile. An SIC can help you to determine whether or not the composition of the background changes over the course of a run. To view the background profile, extract the SIC of a base peak, or prominent peak, from a scan which is clearly in a non-peak region. If the SIC of a background peak has a variable profile around the peak you are focusing on, choose two scans with different SIC profiles for background subtraction.

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GC/LC/MS Processor Selected Ion Chromatogram __________________________________________________ 9tyytrfyÁWhfwtg†ƒ

Note. SIC peaks can appear larger on the screen than the corresponding TIC peaks. In reality SIC signals are disproportionally lower than TIC signals, especially if the data was not acquired using the selective ion monitoring mode. It would be impractical to examine minute SIC peaks displayed together with TIC on the screen. Therefore, SIC data are normalized to the TIC signals and visually enlarged for display purposes.

Figure 67. GC/LC/MS Processor, showing selected ion chromatograms of ions with m/z 240, 228, and 227

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ ________________________ Automated Component Detection and Spectra Deconvolution

11.10 Automated Component Detection and Spectra Deconvolution
Mass Frontier incorporates a novel automated system for detecting chromatographic components in complex GC/MS or LC/MS runs and extracting mass spectral signals from closely coeluting components (deconvolution). Mass Spectra obtained after deconvolution can be searched in libraries or classified using principal component analysis and neural networks. This system involves the combined use of the following procedures: 1. Noise examination and signal filtering. 2. Baseline definition and demarcation of chromatographic peaks. 3. Background scan determination and background subtraction. 4. Component candidate detection and model ion selection (m/z). 5. Correlation of model ion profiles and component confirmation or rejection. 6. Calculation of exact component retention time. 7. Spectra deconvolution using linear algebra. The Component Detection and Spectra Deconvolution system works fully automatically. The system is designed for broad types of chromatographic runs, for both GC/MS and LC/MC analyses, for clean and nosy signals, and for simple and for more complex chromatograms. However, some parameter changes may be needed to optimize the system for specific applications. See Figure 68. This automated procedure is designed for small and medium size organic compounds and should not be used for the processing of proteins, oligonucleotides, peptides, or other biomolecules. Currently, Mass Frontier does not support component detection and spectra deconvolution of data files obtained by MSn. To start automated component detection and the spectra deconvolution procedure: 1. Click on the Components Detection and Spectra Deconvolution button . 2. When the parameters setup dialog window appears, change the settings if required, and then click the OK button.

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Figure 68. Parameters setup window for components detection and spectra deconvolution procedure

The Components Detection and Spectra Deconvolution setup dialog window contains various parameters that can be optimized for specific types of analysis. See Figure 68. Please note that these parameters are interdependent, so a change of one parameter can also affect algorithms linked with other parameters. Threshold of total signal: The program uses a different threshold level from the one given in the data file. In especially noisy chromatograms, setting the threshold higher can reduce the number of false positive results. However, if you feel the algorithm is too restrictive and is missing some chromatographic peaks, you can lower the default value. Minimum Model Ion Abundance: The algorithms search for spectral peaks that have the most rapid rise and fall of signal in a peak region. This peak is called “model ion” in the program. To eliminate random fluctuations there is a minimum abundance value that a model ion should exhibit.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ ________________________ Automated Component Detection and Spectra Deconvolution

SIC Shape Similarity of Model Ions: To selectively distinguish between model ions of common and different component proposals the program correlates the SICs for all the model ions in a peak region. The shape similarity parameter allows you to influence this procedure. The lower the value, the more components will be detected and vice versa. Component Spectra Difference Factor: To eliminate different model ions that may be employed for a single component, the spectra of positively identified components must show some degree of dissimilarity. This degree is the Spectra Difference Factor. This can be changed if your run contains components with similar spectra (alkanes and derivates) or if you see deconvoluted spectra that probably originate from an identical component. TIC Smoothing: This option should be only used when very noisy data needs to be analyzed. The program employs a exponential filter similar to the analogue RC filter. Background Subtraction: If you choose the Automatic option, the program attempts to find a region of relatively constant signal intensity before and after every peak and sets two background scans there. If you want to use the Manual option, set the background scans using the Set Background Subtraction Scan button before starting the detection and deconvolution process. Two manual background scans can be set anywhere in the chromatogram. If you choose the None option, background subtraction will not be performed. Retention Time Range: Detection and deconvolution calculations are timeconsuming processes. The computing time needed depends on a number of factors, but the most significant are the number of scans and the number of mixture peaks. To speed up your work you can select only a part of a chromatogram to be analyzed. Other regions will be ignored. Clicking the OK button will begin the calculation. Detected components are marked with a triangle in the original chromatogram. Because the program calculates the precise retention time (tR) of each component the component triangle can appear between scan points.

Note. To display a deconvoluted spectrum of a component rather than of a scan, click the triangle that marks the detected component in the chromatogram pane. To display an original scan at the position of a detected component, you should click above the triangle.

If you select a component by clicking its triangle, a deconvoluted spectrum will appear in the spectrum pane. This spectrum can be processed in the same way as any spectra in the program, for example, searched in a library or copied to Spectra Manager.

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If you move the mouse cursor over a component triangle, a tool tip rectangle will inform you about the component number, precise retention time and model ion m/z value that were used in the automated detection and deconvolution processes. See Figure 69.

Figure 69. GC/LC/MS Processor window, showing detected components and a deconvoluted spectrum for the selected component

To process detected components in Spectra Manager or in Spectra Classifier modules, you must first select them. To select one or more components, do the following: 1. Click on the Select Scans or Components button .

2. Select the desired components with the mouse cursor while holding down the LEFT mouse button. To select all detected components in a run, do the following: 1. Click anywhere in the chromatogram pane with the RIGHT mouse button and a pop-up menu will appear. 2. Click on the Select All menu item.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ ________________________ Automated Component Detection and Spectra Deconvolution

The program makes a strict distinction between original scans and detected components and does not mix them. When selecting a chromatographic region, components are preferred over scans. If you select a chromatographic region that contains components, only these components will be selected. If you select a region that does not contain any components, all scans in that region will be selected.

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GC/LC/MS Processor Processing Xcalibur MSn Data ________________________________________________ 9tyytrfyÁWhfwtg†ƒ

11.11 Processing Xcalibur MSn Data
GC/LC/MS Processor allows you to view and process Xcalibur MSn files. If this type of data is opened in GC/LC/MS Processor, a tab control on the right side of the window will appear. There are two tabs: MS/MS Tree and Spectra Selector. See Figure 70. The MS/MS Tree tab lists in a tree view control all available scans along with average, composite and total composite spectra. The information display in the tree view should be identical with the one displayed in the MSn Browser (Qual Browser) in Xcalibur. The Spectra Selector tab incorporates a useful feature that allows you to extract spectra from a run with the desired criteria. You can for example extract all MS3 composite spectra within a visible peak region. In both tabs if you click a scan item, a corresponding scan will be selected in the chromatogram pane and its mass spectrum will be displayed in the spectrum pane. If you click the Average, Composite or Total composite item, only the spectrum will be displayed. Because these spectra are calculated from several scans and therefore do not have a single corresponding scan the chromatographic scan line will not be displayed. Using the <Shift> and <Ctrl> keys it is possible to select more than one item. The logic of using these keys is similar to the one used in Excel or Windows Explorer. Multi-selected items can be copied and pasted into Spectra Manager or Spectra Classifier. Using GC/LC/MS Processor and Spectra Classifier you can classify any MSn spectra. This is a unique feature among commercial software products.

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GC/LC/MS Processor 9tyytrfyÁWhfwtg†ƒ _________________________________________________Processing Xcalibur MSn Data

Figure 70. GC/LC/MS Processor window, showing MS/MS Tree and Spectra Selector tabs

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Chapter 12 Monoisotopic Mass Settings
All calculated masses displayed in Mass Frontier are monoisotopic masses. The monoisotopic mass of an ion is the mass of the isotopic peak which is composed of the most abundant isotopes of its elements. Because the program’s calculations only support single-charged ions (z = 1), the calculated exact masses (not measured) in structure based modules are equal to their mass value. To open a Monoisotopic Mass Settings dialog window, do the following: Choose Option | Monoisotopic Mass Settings from the main menu You can set the precision (tolerance) using four options: ppm, mili-massunit, decimal digits and unit mass. These settings apply to the Fragments & Mechanisms, Fragment Comparator and Structure Editor modules. All other modules use m/z values in unit mass precision.

Note. Changing the Monoisotopic Mass Settings does not affect fragments that have already been calculated (the Fragments & Mechanisms and Fragments Comparator modules). If you require fragments with a new setting, you must regenerate these fragments.

Figure 71. Monoisotopic Mass Settings dialog window

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Index

Finnigan Xcalibur ________________________________________________________________ Index

A
Already Generated Mass To Charge Ratio group box, 80 AutoPlay feature, 4

G
GC/LC/MS Processor, 139 background subtraction, 147 data file formats, 142 Info page, 145 opening chromatograms, 143 processing extracted spectra, 148 selected ion chromatogram, 149 spectra averaging, 146 Total Ion Current page, 144 GC/LC/MS Processor Window, 141

B
bar code spectra, 101

C
Check Structures option, 6 Compare Spectra page, 37

I
importing data from Microsoft Excel, 41

D
data exchange between Microsoft Excel and Spectra Manager, 39

L
Library Utilities, 59 add records to user library, 66 changing structures, 70 creating user libraries, 63 deleting entries, 68 installation, 61 opening and saving spectra, structures, and spectra references, 71 uninstall, 65 limitations, 6

E
exporting data to Microsoft Excel, 40

F
fragment comparison, 105 Fragments & Mechanisms, 6, 73 bond cleavages, 75 charge localization concept, 74 eliminating fragments, 93 even-electron rule, 75 features, 74 formally possible solutions, 75 fragmentation reactions, 76 fragments linked with spectra, 92 general fragmentation and rearrangement rules, 74 H-Rearrangement page, 86 ionization methods, 75 reaction restrictions, 84 Resonance page, 87 simulation of mass spec experiements, 94 Sizes page, 91 starting generation, 79 too many peaks, 97 unexplained peaks, 95 unimolecular linear reaction, 74 Fragments & Mechanisms window, 82 Fragments Comparator window, 106

M
marking fragments, 104 Mass Differences page, 36 Mass Frontier Full Activation Access Codes, 5 installation, 4 software, 1 system requirements, 3 mass spectra classification, 109, 110 mathematical transformation, 115 neural networks (self-organizing maps), 114 principal component analysis (PCA), 112 Spectra Classifier module, 117 Spectra Projector module, 117 mass spectral data, 1 MDI, 2 Merge Results Into Active Spectra Manager check box, 31 Microsoft Excel, 39 exporting to, 40 importing data from, 41 using as a spectrum editor, 42 Microsoft Windows Installer, 4

_________________ Finnigan Xcalibur HighChem: Mass Frontier Software______________

I

Index

________________________________________________________________Finnigan Xcalibur
modules Fragments & Mechanisms, 9 Fragments Comparator, 9 GC/LC/MS Processor, 9 Isotope Pattern, 9 Neural Networks, 9 Periodic Table, 9 Spectra Classifier, 9 Spectra Manager, 8 Spectra Projector, 9 Structure Editor, 8 Monoisotopic dialog window, 159 Monoisotopic Mass Settings, 159 Info page, 33 mass spectral data exchange, 57 molecular formula search, 54 molecular mass search, 54 name search, 54 records, 31, 33 search constraints, 55 search utilities, 47 spectrum search, 49 structures, 43 working with spreadsheets, 46 Spectra Manager structures, importing, 44 Spectra Manager window, 31 Spectra Projector, 125 3D projection mode, 129 accessing spectra and structures, 131 adding external spectrum, 132 generating, 126 opening and saving results, 130 Spectra Projector window, 127 Status bar, 128 spectrum editor, using Microsoft Excel, 42 Structure Editor, 6 atom properties, 21 bond properties, 22 checking structures, 27 cleaning structures, 26 copying structures, 23 drawing tools, 24 layout, 16 mirroring structures, 25 moving structures, 25 MS calculations, 28 pasting structures, 24 resizing structures, 25 rotating structures, 25 templates, atoms and bonds, 19 text, 18 Structure Editor module, 11 Structure Editor window, 12 data formats, 15 opening and saving structures, 15 reduction, 14 restoring defaults, 14

N
Neural Networks, 133 generating, 135 working with, 138 Neural Networks window, 136 Status bar, 137 NIST library icons, 59 NIST/EPA/NIH Mass Spectral database, 60

R
Reactions Limit bar, 80 rearrangement reactions, 76 resonance reactions, 76

S
Search dialog box, 31 software access code, 5 Spectra Classifier classifying mass spectra, 122 maintaining groups of spectra, 124 Spectra Classifier window, 118 Spectra Manager, 29 (sub)structure search, 51 assigning a structure to a record, 45 CAS number search, 55 cutting, copying and pasting records, 38 ID number search, 54

II ______________ Finnigan Xcalibur HighChem:

Mass Frontier Software _________________

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