Xcalibur High Chem Mass Frontier Software

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.

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fold
Contents
Finnigan 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
1.2 Modules Overview........................................................................................................................ 8
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
2.2 Structure Layout.......................................................................................................................... 16
2.3 Text.............................................................................................................................................. 18
2.4 Templates: Selecting Atoms and Bonds..................................................................................... 19
2.5 Atom Properties .......................................................................................................................... 21
2.6 Bond Properties........................................................................................................................... 22
2.7 Copying Structures...................................................................................................................... 23
2.8 Pasting Structures........................................................................................................................ 24
2.9 Moving, Resizing, Rotating, and Mirroring Structures .............................................................. 25
Thermo ______________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

ELECTRON CORPORATION i
Contents
___________________________________________________________________ Finnigan Xcalibur
2.10 Cleaning Structures......................................................................................................................26
2.11 Checking Structures.....................................................................................................................27
2.12 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
3.2 Mass Differences .........................................................................................................................36
3.3 Comparing Spectra ......................................................................................................................37
3.4 Cutting, Copying, and Pasting Records.......................................................................................38
3.5 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
3.6 Structures in Spectra Manager.....................................................................................................43
3.7 Working with Spreadsheets .........................................................................................................46
3.8 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
3.9 Mass Spectral Data Exchange Between Modules .......................................................................57
Library Utilities............................................................................................................................59
4.1 NIST/EPA/NIH Mass Spectral Database.....................................................................................60
4.2 Library Installation ......................................................................................................................61
4.3 Creating User Libraries ...............................................................................................................63
4.4 Uninstall Library..........................................................................................................................65
ii _________________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
ELECTRON CORPORATION
Contents
4.5 Adding Records to a User Library .............................................................................................. 66
4.6 Deleting Library Entries.............................................................................................................. 68
4.7 Changing Structures in Libraries ................................................................................................ 70
4.8 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
5.2 Fragmentation, Rearrangement and Resonance Reactions ......................................................... 76
5.3 Starting Generation ..................................................................................................................... 79
5.4 Fragments & Mechanisms Window............................................................................................ 82
5.5 Reaction Restrictions .................................................................................................................. 84

Ionization & Cleavage Page ................................................................................................. 84
H-Rearrangement Page......................................................................................................... 86
The Resonance Page............................................................................................................. 87
The Additional Page ............................................................................................................. 89
The Sizes Page...................................................................................................................... 90
5.6 Generated Fragments Linked with Spectrum.............................................................................. 92
5.7 Eliminating Generated Fragments Not Present in a Spectrum ................................................... 93
5.8 Simulation of MSn Experiments ................................................................................................. 94
5.9 Unexplained Peaks...................................................................................................................... 95
5.10 Too Many Proposed Fragments for a Peak ................................................................................. 97
5.11 Bar Code Spectra ...................................................................................................................... 101
5.12 Marking Fragments ................................................................................................................... 104
Fragments Comparator.............................................................................................................105
Thermo ______________ Finnigan Xcalibur HighChem: Mass Frontier Software ________________ iii
Contents
___________________________________________________________________ Finnigan Xcalibur
Mass Spectra Classification ......................................................................................................109

7.1 Spectra Classification ................................................................................................................ 110
7.2 Principal Component Analysis (PCA)....................................................................................... 112
7.3 Neural Networks (Self-Organizing Maps) ................................................................................ 114
7.4 Spectra Transformation ............................................................................................................. 115
Spectra Classifier .......................................................................................................................117

8.1 Spectra Classifier Window ........................................................................................................ 118
8.2 Classifying Mass Spectra...........................................................................................................122
8.3 Maintaining Groups of Spectra..................................................................................................124
Spectra Projector .......................................................................................................................125

9.1 Generating Spectra Projector Module .......................................................................................126
9.2 Spectra Projector Window .........................................................................................................127
9.3 3D Projection Mode...................................................................................................................129
9.4 Opening and Saving of Classification Results ..........................................................................130
9.5 Accessing Spectra from Spectra Projector ................................................................................131
9.6 Adding External Spectrum ........................................................................................................132
Neural Networks ........................................................................................................................133

10.1 Generating Neural Networks Module........................................................................................135
10.2 Neural Networks Window .........................................................................................................136
10.3 Working with Neural Networks.................................................................................................138
GC/LC/MS Processor ................................................................................................................139

11.1 GC/LC/MS Processor Window .................................................................................................141
11.2 Data File Formats ......................................................................................................................142
11.3 Opening Chromatograms...........................................................................................................143
11.4 TIC Page ....................................................................................................................................144
iv ________________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
Contents
11.5 Info Page ................................................................................................................................... 145
11.6 Spectra Averaging ..................................................................................................................... 146
11.7 Background Subtraction............................................................................................................ 147
11.8 Processing Extracted Spectra .................................................................................................... 148
11.9 Selected Ion Chromatogram...................................................................................................... 149
11.10 Automated Component Detection and Spectra Deconvolution ................................................ 151
11.11 Processing Xcalibur MSn Data ................................................................................................. 156
Monoisotopic Mass Settings ......................................................................................................159
Thermo ______________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________ v

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.
Thermo ________________ Finnigan Xcalibur HighChem: Mass Frontier Software _____________ vii
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.
viii _______________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

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

ELECTRON CORPORATION ix
Read This First
Abbreviations _________________________________________________________ Finnigan Xcalibur
EI electron ionization
EMBL European Molecular Biology Laboratory
<Enter> enter key on the terminal keyboard
ESD electrostatic discharge
ESI electrospray ionization
eV electron volt
f femto (10-15)
°F degrees Fahrenheit
.fasta file extension of a SEQUEST search database file
FOB free on board
ft foot
FTP file transfer protocol
g gram
G giga (109)
GC gas chromatograph; gas chromatography
GC/MS gas chromatograph / mass spectrometer
GND electrical ground
GPIB general-purpose interface bus
GUI graphical user interface
h hour
h height
HPLC high-performance liquid chromatograph
HV high voltage
Hz hertz (cycles per second)
ICIS Interactive Chemical Information System
ICL Instrument Control Language
ID inside diameter
IEC International Electrotechnical Commission
IEEE Institute of Electrical and Electronics Engineers
in. inch
I/O input/output
k kilo (103, 1000)
K kilo (210, 1024)
KEGG Kyoto Encyclopedia of Genes and Genomes
kg kilogram
x _________________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
Read This First
Finnigan Xcalibur _____________________________________________________________ Abbreviations
l length
L liter
LAN local area network
lb pound
LC liquid chromatograph; liquid chromatography
LC/MS liquid chromatograph / mass spectrometer
LED light-emitting diode
µ micro (10-6)
m meter
m milli (10-3)
M mega (106)
M+ molecular ion
MB Megabyte (1048576 bytes)
MH+ protonated molecular ion
min minute
mL milliliter
mm millimeter
MS mass spectrometer; mass spectrometry
MS MSn power: where n = 1
MS/MS MSn power: where n = 2
MSn MSn power: where n = 1 through 10
m/z mass-to-charge ratio
n nano (10-9)
NCBI National Center for Biotechnology Information
(USA)
NIST National Institute of Standards and Technology
(USA)
OD outside diameter
Ω ohm
p pico (10-12)
Pa pascal
PCB printed circuit board
PID proportional / integral / differential
P/N part number
P/P peak-to-peak voltage
Thermo ________________ Finnigan Xcalibur HighChem: Mass Frontier Software _____________

ELECTRON CORPORATION xi
Read This First
Abbreviations _________________________________________________________ Finnigan Xcalibur
ppm parts per million

psig pounds per square inch, gauge
RAM random access memory
RF radio frequency
RMS root mean square
ROM read-only memory
RS-232 industry standard for serial communications
s second
SIM selected ion monitoring
solids probe direct insertion probe
SRM selected reaction monitoring
SSQ single stage quadrupole
TCP/IP transmission control protocol / Internet protocol
TIC total ion current
Torr torr
TSQ triple stage quadrupole
u atomic mass unit
URL uniform resource locator
V volt
V ac volts alternating current
V dc volts direct current
vol volume
w width
W watt
WWW 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)
xii ________________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
Read This First
Finnigan 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”.
Thermo ________________ Finnigan Xcalibur HighChem: Mass Frontier Software ____________

ELECTRON CORPORATION xiii
Read This First
Typographical Conventions _______________________________________________ Finnigan Xcalibur
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.
xiv _______________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
Read This First
Finnigan 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
Thermo ________________ Finnigan Xcalibur HighChem: Mass Frontier Software _____________ xv

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.
xvi _______________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

Thermo
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
_________________Finnigan Xcalibur HighChem: Mass Frontier Software _____________ 1

General Information ________________________________________________________ 'JOOJHBO9DBMJCVS
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 multi-
document 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 mass-
to-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
2 _____________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

'JOOJHBO9DBMJCVS _______________________________________________________ General Information
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.

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.

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: Thermo Electron San Jose
Fax: (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.

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 non-
hydrogen 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.

• 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.

Modules Overview _________________________________________________________ 'JOOJHBO9DBMJCVS
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.

'JOOJHBO9DBMJCVS ________________________________________________________ Modules Overview
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 mass-
to-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 self-
organizing 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.

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
_________________Finnigan Xcalibur HighChem: Mass Frontier Software ____________11

Structure Editor
Structure Editor Window ____________________________________________________ 'JOOJHBO9DBMJCVS
2.1 Structure Editor Window

The Structure Editor Window is shown in Figure 3.
Open Structure Generate Fragments & Mechanisms
Save Structure (Sub)Structure Search
Print Structure Structure Layout
Undo Check Structure
Redo Clean
Single Bond Mirror

Double Bond Rotate
Triple Bond Resize
Hydrocarbon Chain Select All
Delete
Benzene Ring
Paste
Six Membered Ring
Copy
Five Membered Ring
Cut
n-Membered Ring
Templates
Atom Properties
Bond Properties
Positive Charge
Radical
Text Remark
Figure 3. Structure Editor Window, showing names of icon commands
12 ____________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

Structure Editor
'JOOJHBO9DBMJCVS ____________________________________________________ Structure Editor Window
To open the Structure Editor window, do either of the following:
• Click on the Structure Editor button in the toolbar of the main Mass
Frontier window (see Figure 2).
• 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.

Structure Editor
Structure Editor Window ____________________________________________________ 'JOOJHBO9DBMJCVS
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 in the upper left corner, or
• Right-click the mouse

Structure Editor
'JOOJHBO9DBMJCVS ____________________________________________________ Structure Editor Window
Opening and Saving Structures

To open or save a structure, do either of the following:
• Click on the Open Structure button or the Save Structure button

in the Structure Editor window.
• 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.

Structure Editor
Structure Layout __________________________________________________________ 'JOOJHBO9DBMJCVS
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) OH (c) OH
C H H
HC CH
HC CH
CH 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.

Structure Editor
'JOOJHBO9DBMJCVS __________________________________________________________ Structure Layout
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.

Structure Editor
Text ____________________________________________________________________ 'JOOJHBO9DBMJCVS
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

Structure Editor
'JOOJHBO9DBMJCVS _______________________________________ Templates: 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.

Structure Editor
Templates: Selecting Atoms and Bonds________________________________________ 'JOOJHBO9DBMJCVS
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

Structure Editor
'JOOJHBO9DBMJCVS __________________________________________________________ Atom Properties
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 , and then click on the atom you
want to change.
• 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.

Structure Editor
Bond Properties ___________________________________________________________ 'JOOJHBO9DBMJCVS
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 , , or , and then click

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

Structure Editor
'JOOJHBO9DBMJCVS ________________________________________________________ Copying Structures
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.

Structure Editor
Pasting Structures _________________________________________________________ 'JOOJHBO9DBMJCVS
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.
Spectra Manager
Structure Editor Fragments & Mechanisms
Figure 8. Structure exchange via Copy/Paste commands

Structure Editor
'JOOJHBO9DBMJCVS _____________________________ Moving, Resizing, Rotating, and Mirroring Structures
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 in Structure Editor, or choose

Structure | Resize.
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.

Structure Editor
Cleaning Structures ________________________________________________________ 'JOOJHBO9DBMJCVS
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.

Structure Editor
'JOOJHBO9DBMJCVS _______________________________________________________ Checking Structures
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
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.

Structure Editor
MS Calculations___________________________________________________________ 'JOOJHBO9DBMJCVS
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

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.
• 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

Spectra Manager
________________________________________________________________________ 'JOOJHBO9DBMJCVS
• Working with Spreadsheets

• Search Utilities
• Mass Spectral Data Exchange Between Modules

Spectra Manager
'JOOJHBO9DBMJCVS _____________________________________________ 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 in the toolbar of the main

window.
• 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.

Spectra Manager
Open Spectra Manager Window ______________________________________________ 'JOOJHBO9DBMJCVS
Spectrum associated Structure associated

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

Spectra Manager
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

Spectra Manager
Open Spectra Manager Window ______________________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Manager
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

Spectra Manager
Mass Differences __________________________________________________________ 'JOOJHBO9DBMJCVS
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 parent-
daughter 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

Spectra Manager
'JOOJHBO9DBMJCVS ________________________________________________________Comparing Spectra
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-to-
charge 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.

Spectra Manager
Cutting, Copying, and Pasting Records_________________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Manager
'JOOJHBO9DBMJCVS _____________________ Data Exchange between Microsoft Excel and 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

Spectra Manager
Data Exchange between Microsoft Excel and Spectra Manager _____________________ 'JOOJHBO9DBMJCVS
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).
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).
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.

Spectra Manager
'JOOJHBO9DBMJCVS _____________________ Data Exchange between Microsoft Excel and Spectra Manager
Main Copy button Copy Selected Tab
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”.

Spectra Manager
Data Exchange between Microsoft Excel and Spectra Manager _____________________ 'JOOJHBO9DBMJCVS
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.

Spectra Manager
'JOOJHBO9DBMJCVS _______________________________________________Structures in 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.

Spectra Manager
Structures in Spectra Manager _______________________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Manager
'JOOJHBO9DBMJCVS _______________________________________________Structures in 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.

Spectra Manager
Working with Spreadsheets __________________________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Manager
'JOOJHBO9DBMJCVS ___________________________________________________________ Search Utilities
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.

Spectra Manager
Search Utilities____________________________________________________________ 'JOOJHBO9DBMJCVS
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.

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, 316-
323.
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.

Spectra Manager
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.

Spectra Manager
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 OH O
O OH
HO
OH OH HO OH
Negative structure search result Positive structure search result
Figure 24. Structures, showing equivalent and non-equivalent results of structure searches

Spectra Manager
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

Spectra Manager
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.
Figure 26. Structures page of the Spectra Manager

Spectra Manager
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.

Spectra Manager
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 time-
consuming 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.

Spectra Manager
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

Spectra Manager
'JOOJHBO9DBMJCVS ________________________________ Mass Spectral Data Exchange Between Modules
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

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.
• 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

Library Utilities
NIST/EPA/NIH Mass Spectral Database________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS _________________________________________________________Library Installation
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 CD-
ROM, 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

Library Utilities
Library Installation _________________________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS _____________________________________________________Creating User Libraries
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

Library Utilities
Creating User Libraries _____________________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS __________________________________________________________ Uninstall Library
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

Library Utilities
Adding Records to a User Library _____________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS ____________________________________________ 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.

Library Utilities
Deleting Library Entries _____________________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS ____________________________________________________ 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

Library Utilities
Changing Structures in Libraries ______________________________________________ 'JOOJHBO9DBMJCVS
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.

Library Utilities
'JOOJHBO9DBMJCVS __________________ Opening and Saving Spectra, Structures, and Spectra References
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.

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

• 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

Features_________________________________________________________________ 'JOOJHBO9DBMJCVS
5.1 Features
The Fragments & Mechanisms module is based on an expert system, which
uses a mathematical approach for the simulation of unimolecular ion-
decomposition 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.

'JOOJHBO9DBMJCVS ________________________________________________________________ Features
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.

Fragmentation, Rearrangement and Resonance Reactions _________________________ 'JOOJHBO9DBMJCVS
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.

'JOOJHBO9DBMJCVS @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ Fragmentation, Rearrangement and Resonance Reactions
Table 1. Fragmentation, rearrangement, and resonance reactions
_____________________ Finnigan Xcalibur HighChem: Mass Frontier Software __________________________________ 77

Fragmentation, Rearrangement and Resonance Reactions _________________________ 'JOOJHBO9DBMJCVS
Table 2. Reaction formalism used in Mass Frontier
α Alpha cleavage
π Pi electron ionization
σ Sigma electron ionization
cr Charge stabilization
-e- Non-bonding electron ionization
es Electron sharing
i Inductive cleavage
+H+ Protonation
-H• Hydrogen radical loss
-H: Hydride abstraction
rHA Radical site rearrangement
rHB Charge site rearrangement (α, β)
rHC Charge site rearrangement (γ)
rHR Charge-remote rearrangement
rH1,2 Hydrogen shift to adjacent position
rr Radical resonance

'JOOJHBO9DBMJCVS ________________________________________________________Starting Generation
5.3 Starting Generation

Fragmentation and rearrangement pathways can be generated from any user-
supplied 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.

Starting Generation ________________________________________________________ 'JOOJHBO9DBMJCVS
Figure 35. Generation of Fragments & Mechanisms dialog box
A combo box in the Already Generated m/z group box stores mass-to-
charge 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.

'JOOJHBO9DBMJCVS ________________________________________________________Starting Generation
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 Window ____________________________________________ 'JOOJHBO9DBMJCVS
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
Display Fragments Only
Copy Mechanism
Display Mechanisms
Copy Selected Fragment
Copy List of Fragments Ionization Method
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

'JOOJHBO9DBMJCVS ___________________________________________ Fragments & Mechanisms Window
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.
Note. Only a selected fragment can be copied to the Clipboard.
Several possible isobaric fragments can be generated for a particular mass-

to-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.

Reaction Restrictions_______________________________________________________ 'JOOJHBO9DBMJCVS
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

'JOOJHBO9DBMJCVS ______________________________________________________ Reaction Restrictions
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).
• •
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

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 H-

Rearrangement page

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 by-
products, 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

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.

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

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.

Figure 43. Generation of Fragments & Mechanisms dialog

box, showing the Reactions Limit box

Generated Fragments Linked with Spectrum ____________________________________ 'JOOJHBO9DBMJCVS
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.

'JOOJHBO9DBMJCVS ______________________ 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-to-
charge ratio values will again be listed.

Simulation of MSn Experiments ______________________________________________ 'JOOJHBO9DBMJCVS
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

'JOOJHBO9DBMJCVS ________________________________________________________Unexplained Peaks
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 compound-
specific 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.

Unexplained Peaks ________________________________________________________ 'JOOJHBO9DBMJCVS
100 149
O
75
O
OH O
50
O
O
O 263
25 57 O
41 77 104 133
65 207
0
40 60 80 100 120 140 160 180 200 220 240 260 280
100 149
O
75
O
OH
50
57
279 307 O
43
O O
71
25
85
167 O
113
141
97
0
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320
100 323
43 O O O
75
O OH O O
50 O O
O O O O
25 57
95
425
O O O
611
29 69 252 341 509 694
149
0
50 100 150 200 250 300 350 400 450 500 550 600 650 700
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.

'JOOJHBO9DBMJCVS _____________________________________ Too Many Proposed Fragments for a Peak
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-10-
methyl-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 N
99 NH
25
39 65 79 91 105
51 182
128 154 211
0
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
Figure 46. Spectrum of 2,3,3a,4,5,6-Hexahydro-10- methyl-1H-

pyrazino[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.

Too Many Proposed Fragments for a Peak______________________________________ 'JOOJHBO9DBMJCVS
1.
•
π • α α
N N N • N
NH NH NH NH
m/z 226 m/z 226 m/z 198
2.
π • α α
N N N N
NH NH • 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 75
N
N N
50 NH 50
274
65
39 M-28
25 25 51 167
40 63 227 302
51 89 100 44 115 127 154
77 115 145 77 211
127 154 102
0 0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Figure 48. Spectra from a substructure search

'JOOJHBO9DBMJCVS _____________________________________ Too Many Proposed Fragments for a Peak
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 2
. . N
NH NH
100 143 100 121
M-28 Cl
75 75
231
50
NH 50 N
NH
25 25
115 36 197
77 84 89 128 167 92 98 167
28 39 51 63 102 140 154 51 67 75 111 202
0 0
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
100 177 100 193 264
M-28
75 Cl 75 72
N
50 205 50
NH 220
N 165
25 25 179
115
41 51 63
75 84 89 142 132 249
57 71 154 85 115 152
0 0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
100 211 100 43

O
183
75 75 N F
M-28 O
N
50 50
168
H2N N
70
N 254
25 25
196
77 84 90 154
41 115 183
51 63 106 128 83 109 137 153
0 0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 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

Too Many Proposed Fragments for a Peak______________________________________ 'JOOJHBO9DBMJCVS
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).
100 ___________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

'JOOJHBO9DBMJCVS _________________________________________________________ Bar Code Spectra
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.
100 70
75
50 216
160
92 119
25 41 187
64 132
146
76
0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
100 43 55 56 68 77 91 98 105 118 124 145 159 171 175 199 201 216
75
50
25
0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
Figure 50. Experimental and Bar Code spectrum of Pyrrolidine[2,1-c]-2H,5H-1,4-

benzodiazepin-2,5-dione,1,3-dihydro- (Electron Ionization mode)
_________________Finnigan Xcalibur HighChem: Mass Frontier Software __________ 101

Bar Code Spectra _________________________________________________________ 'JOOJHBO9DBMJCVS
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 in any Fragments &

Mechanisms window.
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 spectra-
structure relationships. The primary purpose of generating bar code spectra
is that they allow the possibility to identify spectral differences in
structurally similar compound classes, for which mass spectra are not
available. To study fragmentation dissimilarities between structurally
related compounds, it is far easier and quicker to compare two or more bar
code in Spectra Manager than manually compare fragments and their mass-
to-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.

'JOOJHBO9DBMJCVS _________________________________________________________ Bar Code Spectra
The following example demonstrates how to identify the correct structure

for an unknown spectrum. Let us assume two structures are suggested: 1-
Butanone, 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.
100 43 71 77 105 107 133 148
75
O
50
25
0
50 60 70 80 90 100 110 120 130 140
100 43 70 71 77 78 102 107 119 120 133 148
75
O
50
25
0
50 60 70 80 90 100 110 120 130 140
100 105
75
77
50 Unknown Spectrum
51
25
120
43 55
0
50 60 70 80 90 100 110 120 130 140
O • OH OH
rHA α
• •
m/z 148 m/z 148 m/z 120
Figure 51. Postulating structures from isobaric possibilities using Bar Code spectra

Marking Fragments ________________________________________________________ 'JOOJHBO9DBMJCVS
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.

Chapter 6
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.

________________________________________________________________________ 'JOOJHBO9DBMJCVS
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.

'JOOJHBO9DBMJCVS _______________________________________________________________________
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.
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.

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 computer-
oriented 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.
• Spectra Classification
• Principal Component Analysis (PCA)
• Neural Networks (Self-Organizing Maps)
• Spectra Transformation

Spectra Classification ______________________________________________________ 'JOOJHBO9DBMJCVS
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 spectra-
structure 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.
100 59
166
75 151
73
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43 123
25 83 109 137
97
45 55 69 91
27 31 81 87
0
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
PCA or Neural
Networks
Method
Class Class
A B
Class
X
Figure 53. Classification of mass spectra in Mass Frontier

'JOOJHBO9DBMJCVS _________________________________________________ Spectra Classification
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 n-
dimensional 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.
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

Principal Component Analysis (PCA) __________________________________________ 'JOOJHBO9DBMJCVS
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

'JOOJHBO9DBMJCVS ______________________________________ Principal Component Analysis (PCA)
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.

Neural Networks (Self-Organizing Maps) _______________________________________ 'JOOJHBO9DBMJCVS
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 n-

dimensional 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.
Activated Neuron
by Spectrum 1
1
100 44
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25
31 2 43
13 24
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15 20 25 30 35 40 45
Activated Neuron
by Spectrum 2
100 29
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15
44
50
25
14 1 42 43
13 16
25 26 27 28
0
15 20 25 30 35 40 45
Figure 56. SOM Architecture used in Mass Frontier. Each spectrum

activates a neuron in the map

'JOOJHBO9DBMJCVS ________________________________________________Spectra Transformation
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) =
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.

Spectra Transformation _____________________________________________________ 'JOOJHBO9DBMJCVS
Mass Frontier offers the following spectra transformations:
• Modulo-14 spectra (normalized) A1

• Modulo-14 spectra (not normalized) A2
• Auto-correlation spectra (normalized) B1
• Auto-correlation spectra (not normalized) B2
• Original spectra (normalized) 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.

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).
• Spectra Classifier Window

• Classifying Mass Spectra
• Maintaining Groups of Spectra

Spectra Classifier
Spectra Classifier Window___________________________________________________ 'JOOJHBO9DBMJCVS
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 user-
selected 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:
• Click the Spectra Classifier button on the tool bar in the

main window, or
• Choose the Tools | Spectra Classifier menu item to open
an empty Spectra Classifier window

Spectra Classifier
'JOOJHBO9DBMJCVS __________________________________________________ Spectra Classifier Window
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
Spectra Classifier Window___________________________________________________ 'JOOJHBO9DBMJCVS
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

Spectra Classifier
'JOOJHBO9DBMJCVS __________________________________________________ Spectra Classifier Window
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.
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.

Spectra Classifier
Classifying Mass Spectra ___________________________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Classifier
'JOOJHBO9DBMJCVS ___________________________________________________ Classifying Mass Spectra
Add selected records to

Spectra Classifier
Substructure search
Select all records
Figure 58. Retrieving data for a classification task

Spectra Classifier
Maintaining Groups of Spectra _______________________________________________ 'JOOJHBO9DBMJCVS
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.

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.
• 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

Spectra Projector
Generating Spectra Projector Module __________________________________________ 'JOOJHBO9DBMJCVS
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

Spectra Projector
'JOOJHBO9DBMJCVS ____________________________________________________ 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.

Spectra Projector
Spectra Projector Window ___________________________________________________ 'JOOJHBO9DBMJCVS
Open Projections
Save All Projections
Print Projection Legend of Group Members
Copy Projection
3D Projection mode
Show Axes
Select Spectra and
show their origin
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.

Spectra Projector
'JOOJHBO9DBMJCVS _________________________________________________________ 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 well-
recognized 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.

Spectra Projector
Opening and Saving of Classification Results____________________________________ 'JOOJHBO9DBMJCVS
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.

Spectra Projector
'JOOJHBO9DBMJCVS ________________________________________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.

Spectra Projector
Adding External Spectrum ___________________________________________________ 'JOOJHBO9DBMJCVS
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

Chapter 10
Neural Networks
The Neural Networks module displays classification results from Self-

Organizing 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 two-
dimensional 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.

Neural Networks
________________________________________________________________________ 'JOOJHBO9DBMJCVS
• Generating Neural Networks Module

• Neural Networks Window
• Working with Neural Networks

Neural Networks
'JOOJHBO9DBMJCVS ____________________________________________ 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

Neural Networks
Neural Networks Window ____________________________________________________ 'JOOJHBO9DBMJCVS
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 Show Legend
Open External Spectrum
Paste External Spectrum
Show Distances between Neurons
Select Spectra and
show their origin
Spectrum
Neuron activated by
Distance between neurons external spectrum
Neuron
Figure 63. Neural Networks window

Neural Networks
'JOOJHBO9DBMJCVS _____________________________________________________ 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.

Neural Networks
Working with Neural Networks ________________________________________________ 'JOOJHBO9DBMJCVS
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

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.
• GC/LC/MS Processor Window

• Data File Formats

GC/LC/MS Processor
________________________________________________________________________ 'JOOJHBO9DBMJCVS
• 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

GC/LC/MS Processor
'JOOJHBO9DBMJCVS _________________________________________________ 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 Components Detection and Spectra Deconv.
Save Spectrum Selected Ion Chromatogram (SIC)
Print Window Set Background Subtraction Scans
Copy Window and Sel. Spectra Cancel Background Subtraction
Chromatogram Layout Spectra Average
Spectrum Layout Select Scans or Comp.
Search Spectrum Clear Chromatogr.
Spectra Show Scan
Classifier Points
Figure 64. GC/LC/MS Processor window

GC/LC/MS Processor
Data File Formats __________________________________________________________ 'JOOJHBO9DBMJCVS
11.2 Data File Formats

Mass Frontier supports various data file formats for importing GC/MS and
LC/MS files, including the following:
• 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.

GC/LC/MS Processor
'JOOJHBO9DBMJCVS _____________________________________________________ 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.

GC/LC/MS Processor
TIC Page _________________________________________________________________ 'JOOJHBO9DBMJCVS
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.

GC/LC/MS Processor
'JOOJHBO9DBMJCVS _________________________________________________________________ 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

GC/LC/MS Processor
Spectra Averaging __________________________________________________________ 'JOOJHBO9DBMJCVS
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

GC/LC/MS Processor
'JOOJHBO9DBMJCVS ______________________________________________________ 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.

GC/LC/MS Processor
Processing Extracted Spectra _________________________________________________ 'JOOJHBO9DBMJCVS
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.

GC/LC/MS Processor
'JOOJHBO9DBMJCVS ___________________________________________________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 multi-
component 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.

GC/LC/MS Processor
Selected Ion Chromatogram __________________________________________________ 'JOOJHBO9DBMJCVS
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

GC/LC/MS Processor
'JOOJHBO9DBMJCVS ________________________ 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.

GC/LC/MS Processor
Automated Component Detection and Spectra Deconvolution ________________________ 'JOOJHBO9DBMJCVS
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.

GC/LC/MS Processor
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 time-
consuming 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.

GC/LC/MS Processor
Automated Component Detection and Spectra Deconvolution ________________________ 'JOOJHBO9DBMJCVS
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.

GC/LC/MS Processor
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.

GC/LC/MS Processor
Processing Xcalibur MSn Data ________________________________________________ 'JOOJHBO9DBMJCVS
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.

GC/LC/MS Processor
'JOOJHBO9DBMJCVS _________________________________________________Processing Xcalibur MSn Data
Figure 70. GC/LC/MS Processor window, showing MS/MS Tree

and Spectra Selector tabs

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-mass-
unit, 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

Index
Finnigan Xcalibur ________________________________________________________________
Index
A G
Already Generated Mass To Charge Ratio GC/LC/MS Processor, 139
group box, 80 background subtraction, 147
AutoPlay feature, 4 data file formats, 142
Info page, 145
opening chromatograms, 143
B processing extracted spectra, 148
selected ion chromatogram, 149
bar code spectra, 101
spectra averaging, 146
Total Ion Current page, 144
C GC/LC/MS Processor Window, 141
Check Structures option, 6

Compare Spectra page, 37 I
importing data from Microsoft Excel, 41
D
data exchange between Microsoft Excel and L
Spectra Manager, 39
Library Utilities, 59
add records to user library, 66
E changing structures, 70
creating user libraries, 63
exporting data to Microsoft Excel, 40 deleting entries, 68
installation, 61
opening and saving spectra, structures, and
F spectra references, 71
uninstall, 65
fragment comparison, 105
limitations, 6
Fragments & Mechanisms, 6, 73
bond cleavages, 75
charge localization concept, 74
eliminating fragments, 93
M
even-electron rule, 75 marking fragments, 104
features, 74 Mass Differences page, 36
formally possible solutions, 75 Mass Frontier
fragmentation reactions, 76 Full Activation Access Codes, 5
fragments linked with spectra, 92 installation, 4
general fragmentation and software, 1
rearrangement rules, 74 system requirements, 3
H-Rearrangement page, 86 mass spectra classification, 109, 110
ionization methods, 75 mathematical transformation, 115
reaction restrictions, 84 neural networks (self-organizing maps), 114
Resonance page, 87 principal component analysis (PCA), 112
simulation of mass spec experiements, 94 Spectra Classifier module, 117
Sizes page, 91 Spectra Projector module, 117
starting generation, 79 mass spectral data, 1
too many peaks, 97 MDI, 2
unexplained peaks, 95 Merge Results Into Active Spectra Manager check
unimolecular linear reaction, 74 box, 31
Fragments & Mechanisms window, 82 Microsoft Excel, 39
Fragments Comparator window, 106 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 Info page, 33

Fragments & Mechanisms, 9 mass spectral data exchange, 57
Fragments Comparator, 9 molecular formula search, 54
GC/LC/MS Processor, 9 molecular mass search, 54
Isotope Pattern, 9 name search, 54
Neural Networks, 9 records, 31, 33
Periodic Table, 9 search constraints, 55
Spectra Classifier, 9 search utilities, 47
Spectra Manager, 8 spectrum search, 49
Spectra Projector, 9 structures, 43
Structure Editor, 8 working with spreadsheets, 46
Monoisotopic dialog window, 159 Spectra Manager structures, importing, 44
Monoisotopic Mass Settings, 159 Spectra Manager window, 31
Spectra Projector, 125
3D projection mode, 129
N accessing spectra and structures, 131
adding external spectrum, 132
Neural Networks, 133
generating, 126
generating, 135
opening and saving results, 130
working with, 138
Spectra Projector window, 127
Neural Networks window, 136
Status bar, 128
Status bar, 137
spectrum editor, using Microsoft Excel, 42
NIST library icons, 59
Structure Editor, 6
NIST/EPA/NIH Mass Spectral database, 60
atom properties, 21
bond properties, 22
R checking structures, 27
cleaning structures, 26
Reactions Limit bar, 80 copying structures, 23
rearrangement reactions, 76 drawing tools, 24
resonance reactions, 76 layout, 16
mirroring structures, 25
moving structures, 25
S MS calculations, 28
pasting structures, 24
Search dialog box, 31
resizing structures, 25
software access code, 5
rotating structures, 25
Spectra Classifier
templates, atoms and bonds, 19
classifying mass spectra, 122
text, 18
maintaining groups of spectra, 124
Structure Editor module, 11
Spectra Classifier window, 118
Structure Editor window, 12
Spectra Manager, 29
data formats, 15
(sub)structure search, 51
opening and saving structures, 15
assigning a structure to a record, 45
reduction, 14
CAS number search, 55
restoring defaults, 14
cutting, copying and pasting records, 38
ID number search, 54
II ______________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

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Printing History: Revision A printed in June 2003.

Published by Technical Publications, Thermo Electron Corporation, San Jose, California.

Additional Comments: (Attach additional sheets if necessary.)

EDITOR, TECHNICAL PUBLICATIONS

Read This First............................................................................................................................ vii

Typographical Conventions .................................................................................................................. xiii

Reply Cards........................................................................................................................................... xvi

Introducing Mass Frontier............................................................................................................1

1.2 Modules Overview........................................................................................................................ 8

Structure Editor ...........................................................................................................................11

2.2 Structure Layout.......................................................................................................................... 16

2.4 Templates: Selecting Atoms and Bonds..................................................................................... 19

2.5 Atom Properties .......................................................................................................................... 21

2.6 Bond Properties........................................................................................................................... 22

2.7 Copying Structures...................................................................................................................... 23

2.8 Pasting Structures........................................................................................................................ 24

2.9 Moving, Resizing, Rotating, and Mirroring Structures .............................................................. 25

Thermo ______________ Finnigan Xcalibur HighChem: Mass Frontier Software _________________

2.10 Cleaning Structures......................................................................................................................26

2.11 Checking Structures.....................................................................................................................27

2.12 MS Calculations ..........................................................................................................................28

3.2 Mass Differences .........................................................................................................................36

3.3 Comparing Spectra ......................................................................................................................37

3.4 Cutting, Copying, and Pasting Records.......................................................................................38

3.5 Data Exchange between Microsoft Excel and Spectra Manager.................................................39

3.6 Structures in Spectra Manager.....................................................................................................43

3.7 Working with Spreadsheets .........................................................................................................46

3.8 Search Utilities ............................................................................................................................47

3.9 Mass Spectral Data Exchange Between Modules .......................................................................57

4.2 Library Installation ......................................................................................................................61

4.3 Creating User Libraries ...............................................................................................................63

4.4 Uninstall Library..........................................................................................................................65

ii _________________ Finnigan Xcalibur HighChem: Mass Frontier Software_______________

4.5 Adding Records to a User Library .............................................................................................. 66

4.6 Deleting Library Entries.............................................................................................................. 68

4.7 Changing Structures in Libraries ................................................................................................ 70

Fragments & Mechanisms ..........................................................................................................73

5.2 Fragmentation, Rearrangement and Resonance Reactions ......................................................... 76

5.3 Starting Generation ..................................................................................................................... 79

5.4 Fragments & Mechanisms Window............................................................................................ 82

5.5 Reaction Restrictions .................................................................................................................. 84

5.6 Generated Fragments Linked with Spectrum.............................................................................. 92

5.7 Eliminating Generated Fragments Not Present in a Spectrum ................................................... 93

5.8 Simulation of MSn Experiments ................................................................................................. 94

5.9 Unexplained Peaks...................................................................................................................... 95

5.10 Too Many Proposed Fragments for a Peak ................................................................................. 97

5.11 Bar Code Spectra ...................................................................................................................... 101

5.12 Marking Fragments ................................................................................................................... 104

Mass Spectra Classification ......................................................................................................109

7.2 Principal Component Analysis (PCA)....................................................................................... 112

7.3 Neural Networks (Self-Organizing Maps) ................................................................................ 114

7.4 Spectra Transformation ............................................................................................................. 115

Spectra Classifier .......................................................................................................................117

8.2 Classifying Mass Spectra...........................................................................................................122

8.3 Maintaining Groups of Spectra..................................................................................................124

Spectra Projector .......................................................................................................................125

9.2 Spectra Projector Window .........................................................................................................127

9.3 3D Projection Mode...................................................................................................................129

9.4 Opening and Saving of Classification Results ..........................................................................130

9.5 Accessing Spectra from Spectra Projector ................................................................................131

9.6 Adding External Spectrum ........................................................................................................132

Neural Networks ........................................................................................................................133

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