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Finnigan ™

Xcalibur ®

Getting Productive:
Quantitative Analysis

Revision A
XCALI-97063
Finnigan™ is a trademark of and Xcalibur® is a registered trademark of Thermo Electron Corporation. Microsoft® and Windows® are registered
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Technical information contained in this publication is for reference purposes only and is subject to change
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Thermo Electron Corporation assumes no responsibility and will not be liable for any errors, omissions,
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This publication is not part of the Agreement of Sale between Thermo Electron Corporation and the purchaser
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Printing History: Revision A printed in June 2003.


Software Version: Xcalibur 1.4

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fold
Contents
Finnigan Xcalibur ________________________________________________________________________

Contents

Read This First ..............................................................................................................................v


Changes to the Manual and Online Help ............................................................................................... vi

Abbreviations ........................................................................................................................................ vii

Typographical Conventions ................................................................................................................... xi


Data Input ............................................................................................................................. xi
Boxed Information............................................................................................................... xii
Topic Headings................................................................................................................... xiii

Reply Cards .......................................................................................................................................... xiv

Quantitative Analysis ............................................................................................................... 1-1


1.1 About Quantitative Analysis...................................................................................................... 1-2

1.2 The Quantitation Procedure ....................................................................................................... 1-3


Identification........................................................................................................................ 1-4
Detection.............................................................................................................................. 1-5
Calibration ........................................................................................................................... 1-5
Quantitation ......................................................................................................................... 1-6

1.3 Quantitation Techniques ............................................................................................................ 1-7


External Standard Quantitation ........................................................................................... 1-7
Internal Standard (ISTD) Quantitation ................................................................................ 1-7

1.4 Sequences................................................................................................................................... 1-8


Sample Types....................................................................................................................... 1-8
Brackets ............................................................................................................................... 1-9
Levels and Replicates ........................................................................................................ 1-15

1.5 Quantitation with Xcalibur....................................................................................................... 1-16

Quantitative Processing ............................................................................................................ 2-1


2.1 Processing Setup ........................................................................................................................ 2-2

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___________________________________________________________________ Finnigan Xcalibur

2.2 The Processing Setup Window.................................................................................................. 2-3


The Title Bar ....................................................................................................................... 2-4
The Toolbar ......................................................................................................................... 2-4
Quan View........................................................................................................................... 2-4
Components List ................................................................................................................. 2-5
Applying Changes to a Page ............................................................................................... 2-5
Customizing Processing Setup............................................................................................ 2-8

2.3 Using Quan View Interactively ................................................................................................. 2-9


Previewing Processing ........................................................................................................ 2-9
Setting Processing Parameters .......................................................................................... 2-10
Cursor Actions .................................................................................................................. 2-10
Using the Toolbar.............................................................................................................. 2-13
Customizing the Previews................................................................................................. 2-13

2.4 Identification............................................................................................................................ 2-14


Detector............................................................................................................................. 2-15
Filter .................................................................................................................................. 2-15
Trace.................................................................................................................................. 2-15
Mass or Wavelength.......................................................................................................... 2-17
Retention Time.................................................................................................................. 2-19

2.5 Detection.................................................................................................................................. 2-20


Peak Integration ................................................................................................................ 2-21
Peak Detection .................................................................................................................. 2-22
Advanced Detection Parameters ....................................................................................... 2-28
Data Flags ......................................................................................................................... 2-32

2.6 Calibration ............................................................................................................................... 2-34


Assigning an ISTD............................................................................................................ 2-35
Assigning a Target............................................................................................................. 2-36
Isotope Correction............................................................................................................. 2-38
Setting Calibration and Quantitation Flags....................................................................... 2-40

2.7 Levels....................................................................................................................................... 2-43


Standard Dilution .............................................................................................................. 2-44

2.8 System Suitability.................................................................................................................... 2-47


Resolution ......................................................................................................................... 2-48
Symmetry .......................................................................................................................... 2-48
Peak Classification............................................................................................................ 2-49

2.9 Peak Purity............................................................................................................................... 2-53


Enable Peak Purity ............................................................................................................ 2-54
Limit Scan Wavelength ..................................................................................................... 2-54

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Contents
Finnigan Xcalibur ________________________________________________________________________

2.10 Reports ..................................................................................................................................... 2-55


Sample Reports.................................................................................................................. 2-56
Summary Reports .............................................................................................................. 2-57

2.11 Programs .................................................................................................................................. 2-58

Automating Analysis ................................................................................................................. 3-1


3.1 Sequence Setup .......................................................................................................................... 3-2

3.2 The Sequence Setup Window .................................................................................................... 3-3

3.3 About Sequences........................................................................................................................ 3-4


Arranging the Columns ....................................................................................................... 3-5
Changing User Labels ......................................................................................................... 3-6

3.4 Creating a New Sequence .......................................................................................................... 3-8


Importing a Sequence .......................................................................................................... 3-8
Letting Xcalibur Create Your Sequence .............................................................................. 3-9
Creating a Sequence Manually.......................................................................................... 3-13

3.5 Working with a Sequence......................................................................................................... 3-15


Filling Down Columns ...................................................................................................... 3-15
Inserting a Row.................................................................................................................. 3-16
Deleting a Row .................................................................................................................. 3-16
Going to a Sequence Row ................................................................................................. 3-16
Transferring Row Information........................................................................................... 3-17
Printing a Sequence ........................................................................................................... 3-18
Checking Disk Space......................................................................................................... 3-20
Exporting a Sequence ........................................................................................................ 3-21

3.6 Running Samples ..................................................................................................................... 3-23


Running a Single Sample .................................................................................................. 3-23
Running a Sequence .......................................................................................................... 3-23
Setting up the Run ............................................................................................................. 3-23

3.7 Reprocessing Samples.............................................................................................................. 3-28

3.8 The Acquisition Queue ........................................................................................................... 3-30


Sample Information Dialog Box ....................................................................................... 3-31
Managing Tasks................................................................................................................. 3-31

Reviewing Quantitation ............................................................................................................ 4-1


4.1 Results Review........................................................................................................................... 4-2

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4.2 About Quan Browser ................................................................................................................. 4-3


How Quan Browser Works ................................................................................................. 4-3
Getting Started in Quan Browser ........................................................................................ 4-6

4.3 The Quan Browser Window ...................................................................................................... 4-8


The Title Bar ....................................................................................................................... 4-9
The Toolbar and Menu Bar ................................................................................................. 4-9
Component List................................................................................................................. 4-10
Results Grid....................................................................................................................... 4-10
Chromatogram View ......................................................................................................... 4-10
Companion View................................................................................................................4-11

4.4 The Results Grid...................................................................................................................... 4-13


Bracket/Group in Use........................................................................................................ 4-14
Calibration File ................................................................................................................. 4-14
Results Grid Columns ....................................................................................................... 4-14
Working Directly With The Grid ...................................................................................... 4-15

4.5 Chromatogram View................................................................................................................ 4-19


Chromatogram View Shortcut Menu ................................................................................ 4-19
Viewing Peak Information ................................................................................................ 4-20
Qualifier Peak Information ............................................................................................... 4-26
Spectrum Candidate Information ...................................................................................... 4-27
Setting User Peak Detection Parameters........................................................................... 4-28
Changing Display Options ................................................................................................ 4-35

4.6 Calibration Companion View .................................................................................................. 4-36


Calibration Companion View Shortcut Menu................................................................... 4-36
Adjusting Calibration Settings .......................................................................................... 4-37

4.7 Spectrum Companion View..................................................................................................... 4-45


Changing the Spectrum..................................................................................................... 4-45

4.8 Reports..................................................................................................................................... 4-46


Selecting Samples for Reports .......................................................................................... 4-47

4.9 Quan Browser Procedures ....................................................................................................... 4-48


Editing a Sequence............................................................................................................ 4-48
Reviewing Samples........................................................................................................... 4-49
Reviewing a Chromatogram ............................................................................................. 4-50
Modifying Detection and Identification............................................................................ 4-51
Integrating Chromatogram Peaks Manually ..................................................................... 4-52
Modifying Calibration Parameters.................................................................................... 4-52

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Read This First

Welcome to Xcalibur®, the Thermo Electron Finnigan™ mass spectrometry


data system!
This Getting Productive: Quantitative Analysis manual describes how to
use your Finnigan system and Xcalibur for quantitative analysis.
It describes how to:
• Set up a method for automatic quantitative processing.
• Create a sequence or batch of samples for analysis and processing under
full software control.
• Review and rework your data using Xcalibur’s quantitative reviewing
utility, Quan Browser.
It is assumed that you have read your instrument’s Getting Started and are
familiar with the basic features of Xcalibur such as Home Page and
Instrument Setup.
The Getting Productive: Quantitative Analysis manual includes the
following chapters:
Chapter 1: Quantitative Analysis explains some of the basic principles and
terminology of quantitation.
Chapter 2: Quantitative Processing describes Processing Setup and
explains how you can use it to create a method for automated batch analysis.
It leads you through the parameters required for data processing, calibration,
quantitation, reporting and running additional programs.
Chapter 3: Automating Analysis describes Sequence Setup and explains
how to set up and use a sequence for automated quantitative analysis.
Chapter 4: Reviewing Quantitation describes the underlying principles of
Quan Browser and explains how to use it for reviewing and reworking
sequences, calibration curves and quantitation.

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Read This First
Changes to the Manual and Online Help _____________________________________ Finnigan Xcalibur

Changes to the Manual and Online Help


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

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Read This First
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

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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

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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

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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)

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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”.

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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.

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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

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Read This First
Reply Cards __________________________________________________________ Finnigan Xcalibur

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

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Chapter 1
Quantitative Analysis

You perform Quantitative analysis in Xcalibur®, the Finnigan™ mass


spectrometry data system.
This chapter explains some of the basic principles and terminology of
quantitation.
The topics in this chapter are as follows:
• About Quantitative Analysis
• The Quantitation Procedure
• Quantitation Techniques
• Sequences
• Quantitation with Xcalibur

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Quantitative Analysis
About Quantitative Analysis ______________________________________________ Finnigan Xcalibur

1.1 About Quantitative Analysis


Quantitative analysis is the process of measuring the amount of a particular
component in a sample.
In some cases, such as in trace analysis, you may only want to estimate the
quantity of a component. It may be sufficient to know that the component is
present at a level significantly higher or lower than a defined threshold. For
example, it is not generally important to know whether a patient has
overdosed 15 or 20 times above a prescribed limit, but simply that the limit
has been exceeded. In such cases, a rapid measurement is required rather than
a precise one. This form of measurement is generally called semi-quantitative
analysis.
In other applications, a clinical trial for example, you may be seeking the
maximum possible accuracy from your measurements. Time and cost of
analysis are less important than achieving the highest possible standards in
precision and accuracy.
Quantitative analysis consists of the following steps:
• Preparing samples
• Developing a suitable chromatographic method
• Calibrating the mass spectrometer’s response
• Analyzing the samples
• Reviewing the results
It is beyond the scope of this manual to describe sample preparation and
chromatographic procedures. This manual assumes that you have achieved
these important prerequisites to high quality quantitation. Refer to your
Getting Started and Hardware manuals for guidance in these areas.

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Quantitative Analysis
Finnigan Xcalibur __________________________________________________ The Quantitation Procedure

1.2 The Quantitation Procedure


The first step in quantitation is the development of a chromatographic method
using an appropriate combination of column, flow rate and operating
conditions. The analysis should provide chromatographic separation of the
target component from the solvent and other components that may interfere
with Xcalibur’s detection process.
A typical chromatogram with good component separation and sharp,
symmetrical, peaks is shown in Figure 1-1. The position of a peak in the
chromatogram is called its Retention time and is determined by the total time
that the compound remains on the chromatograph column.

Figure 1-1. A typical chromatogram

Xcalibur’s quantitation procedure consists of four steps:


Identification Optimizing the chromatogram and targeting a
component.
Detection Assigning a chromatogram peak to the
component.
Calibration Calculating the instrument’s response to the
component using one or more samples containing
known amounts of it.
Quantitation Applying the calibration to samples containing
unknown amounts of the component.

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Quantitative Analysis
The Quantitation Procedure ______________________________________________ Finnigan Xcalibur

Identification
So that Xcalibur can correctly identify each component peak in a
chromatogram, you need to:
• Optimize the chromatogram trace for the component(s) of interest using a
scan filter, mass (or wavelength) range, or trace combination.
• Provide Xcalibur with the expected retention time of each component
together with a time ‘window’ to account for run-to-run variations.

Scan Filters and Traces


You can direct Xcalibur to create a chromatogram tailored for the component
of interest, effectively screening out other components. This is particularly
important in multi-component analyses, or when solvent effects or column
bleed could interfere with a more general chromatogram (for example, TIC).
There are three methods, which can be used in combination:
• You can specify a scan filter. A scan filter allows you to specify that
processing is applied to a subset of the scans in a raw file. Xcalibur
creates scan filters from Instrument Method settings.
• You can specify a quantitation mass or mass range directly in the
processing method. This minimizes the chance of interference and
maximizes the quantitative accuracy.

In the case of UV detectors, you would use a quantitation wavelength or


wavelength range.
• You can specify a trace, or trace combination. For example, you could
subtract one mass range from another to remove the effects of an
impurity.

Retention Time
For each component, you supply Xcalibur with an expected retention time and
a time range window describing an allowable deviation from the expected
time. Xcalibur uses this information to determine the retention time window,
within which to look for components within a chromatogram.
In some analyses, there is sufficient variation in retention times to make it
difficult to provide a reliable retention time window. In such cases, you can
compensate for any retention time drift by assigning a retention time
reference. This is a component whose actual retention time is used to
dynamically adjust the expected retention time of other components.

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Quantitative Analysis
Finnigan Xcalibur __________________________________________________ The Quantitation Procedure

Detection
In many cases, the defined retention time window may contain two or more
peaks. Often, it is not possible to define a sufficiently small retention time
window, or unique quantitation mass that will unambiguously identify a target
component.
Xcalibur uses one of three methods to assess chromatogram peaks and hence
confirm the identify of a component (detection):
Spectrum Using a reference spectrum of the component, Xcalibur
examines the spectrum of each peak within the reten-
tion time window. The peak with the spectrum most
closely matching the reference (within user-specified
tolerances) is assigned to the component.
Highest peak Xcalibur assigns the largest peak (in terms of peak
height) within the retention time window to the compo-
nent.
Nearest RT Xcalibur assigns the peak closest to the expected reten-
tion time to the component.
With Highest Peak and Nearest RT modes, Xcalibur offers an optional
procedure called Ion Ratio Confirmation. This allows you to provide details
for up to five qualifier ions. Xcalibur ensures that these are present within the
assigned chromatogram peak (utilizing user-specified tolerances) before
finally confirming it as the component.

Calibration
To carry out quantitation, you need to evaluate the instrument’s response to
known amounts of the target component.
Response is based on either the height of the chromatogram peak, or more
commonly, the area under the peak’s profile; in both cases taking account of
the detected peak’s baseline. Xcalibur determines the area of chromatogram
peaks by an integration calculation. Figure 1-2 (a) illustrates an integrated
chromatogram peak.
Instrument response is generally measured with several samples commonly
called standards, or calibration standards. These represent a suitably wide
range of concentrations or amounts. Responses to these standards are plotted
in a graph called the calibration curve. See Figure 1-2 (b). This usually
reveals an essentially linear relationship between amount and response,
although more complex relationships are occasionally observed.
Xcalibur fits an equation to the calibration curve by a user chosen method (for
example, a least squares regression). This provides a Response factor - a
comparative measure of the response of the mass spectrometer to a
component. It is based on the amount of sample injected and the resulting

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Quantitative Analysis
The Quantitation Procedure ______________________________________________ Finnigan Xcalibur

peak area or peak height. Thus, the response factor gives an intuitive (and
quantitative) measure of how responsive or sensitive the mass spectrometer is
to a certain component.

Figure 1-2. (a) Integrated chromatogram peak, and (b) the calibration curve.

Quantitation
Xcalibur achieves quantitation of samples containing unknown amounts of
the target component by first calculating the peak area or height and then
computing and applying the appropriate response to the equation derived
from the calibration curve. This provides an estimate of the amount of the
unknown component. The precision of the measurement depends on the
quality (and to a lesser extent the quantity) of the calibration data.

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Quantitative Analysis
Finnigan Xcalibur _____________________________________________________ Quantitation Techniques

1.3 Quantitation Techniques


There are two basic techniques:
• External standard quantitation
• Internal standard (ISTD) quantitation
The chosen method determines the manner in which response is calculated,
both for the generation of calibration curves and for subsequent quantitation.

External Standard Quantitation


The external standard quantitation technique utilizes a component’s absolute
peak area or height directly as its response factor. The response factor is then
used to refine a calibration curve (from standard samples), or to compute an
amount (for unknown samples) using the calibration curve.
The external standard quantitation approach offers time and cost effective
quantitation although it is not capable of achieving the very highest precision
and accuracy. For semi-quantitative analysis, a single external standard
sample may provide sufficient calibration information to achieve the required
accuracy.

Internal Standard (ISTD) Quantitation


An ISTD is a component added to a sample to act as a response reference for
one or more non-ISTD components in the sample. The concentration or
amount of an ISTD in any standard or unknown sample remains constant and
the ISTD component is added as the last step of sample preparation prior to
the sample’s use.
Since the ISTD and non-ISTD components are analyzed together, the internal
standard quantitation approach has the advantage that it corrects for injection
and other sample handling errors.
Ideally, an ISTD should be closely related to the target component in terms of
both its physical and chemical properties. Typically, ISTD components are
analogs, homologues or isomers of the target non-ISTD component. An ideal
ISTD is a structural or isotopically labeled analog of one of the target
components.
The internal standard quantitation technique computes response values based
on the ratio of a component’s absolute peak area or height to that of the ISTD.
This provides a peak area ratio. The peak area ratio value is then used to form
a calibration curve (from standard samples), or to calculate an amount (for
unknown samples) using the calibration curve.
There can be any number of ISTD components in a sample but each
non-ISTD component can only be calibrated against one ISTD.

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ELECTRON CORPORATION 1-7
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur

1.4 Sequences
Each quantitative analysis consists of a number or sequence of samples. The
sequence represents the order of sample analysis, or data acquisition. Xcalibur
processes the sample data, generating calibration curves and performing
quantitation according to the sequence’s bracket type.

Sample Types
A quantitation sequence will contain:
• One or more standards
• One or more unknowns
For more demanding applications, you can also use additional, optional,
sample types called Blanks and QCs.

Standards
A Standard is a sample containing a known amount of all target components.
The purpose of a standard is to measure the response of the instrument to the
target component(s), such that a calibration curve can be computed for each
component.
For the purpose of generating a calibration curve, Xcalibur recognizes and
utilizes several different standard types:
• Standard Clear
• Standard Update
• Standard Bracket
• Start Bracket
• End Bracket
These definitions are a consequence of the various bracket types (see
Brackets) and determine how Xcalibur processes the standards.

Unknowns
An Unknown sample is one containing an unknown amount of the target
components. Xcalibur performs quantitative analysis of any sample defined as
an Unknown.

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ELECTRON CORPORATION
Quantitative Analysis
Finnigan Xcalibur _______________________________________________________________ Sequences

Blanks
A Blank sample contains no target components, but may well contain ISTD
components when the internal standard quantitation technique is being used.
Xcalibur performs quantitative analysis of any sample defined as a blank
using the same calibration settings as it uses on Unknown samples.
The analysis of a Blank sample:
• Effectively purges all residual components prior to the analysis of the next
sample or sequence.
• Allows you to confirm that there are no residual components (often called
carryover) in the solvent system that can cause erroneous results.
If you use the New Sequence Template dialog box in Sequence Setup, Blank
samples can be associated with Calibration samples (which they both precede
and succeed) and/or QC samples (which they succeed).

QCs
A QC (quality control) sample contains known amounts of one or more
specific target components. QC samples are placed in a sequence so that
quantitation results can be tested for quality assurance purposes. Xcalibur
performs quantitation on QC samples using the same calibration settings as it
uses on Unknown samples. The measured quantity is then compared with the
expected value and an acceptability range. The quantitation of a QC is
classified as PASSED if the difference between the observed and expected
quantities is within the defined tolerance. A QC sample is classified as
FAILED if the difference between the observed and expected quantities is
outside the defined tolerance.

Brackets
Xcalibur organizes quantitation sequences into brackets. Brackets are simply
a way of ordering, grouping and processing standard, unknown, QC and blank
samples. This helps to ensure accurate quantitation by creating and applying
the most up-to-date calibration curve to each unknown sample.
Bracketing basically involves organizing standard samples into two groups,
encompassing Unknown, Blank and QC Samples. The standard samples are
therefore said to bracket (surround) the other sample types. This approach
helps to compensate for any long term systematic variations that may occur
during an analysis.
Calibration and quantitation are performed for each defined bracket in the
sequence. All the standards within a bracket are used to create a calibration
curve, which is then used to quantitate the bracketed samples (Unknowns,
QCs and Blanks). It is important to understand that a sequence represents the
order of sample analysis but not necessarily the processing order. Within a

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ELECTRON CORPORATION 1-9
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur

bracket, Xcalibur processes the raw files for all the standards before
quantifying any other sample types, irrespective of the order of samples
within the sequence.
Xcalibur recognizes four bracketing options:
• None
• Open
• Overlapped
• Non-overlapped

None
With this option, bracketing is not applied. Samples are processed in the order
in which they are submitted in the sequence. For non-bracketed sequences,
Xcalibur uses a named calibration file (.XCAL) to store data representing
each component’s calibration curve. Although in theory it is possible to have a
different calibration file name for every sample, in practice it is usual to have
only one named calibration file per sequence.
Xcalibur uses two classifications for non-bracketed standards: Standard Clear
and Standard Update. Whenever Xcalibur encounters a Standard Clear in a
sequence, it discards the previous calibration data and starts a new calibration
with this standard. Standard Updates are simply appended to the calibration
file. Effectively, a named calibration file accumulates calibration data from
Standard Update samples until a Standard Clear is encountered in the
sequence.
An illustration of the method used by Xcalibur to process an unordered
non-bracketed sequence is shown in Figure 1-3.

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ELECTRON CORPORATION
Quantitative Analysis
Finnigan Xcalibur _______________________________________________________________ Sequences

Blank
Standard Clear 1
Standard Update 2 1
5
Standard Update 3
2
Standard Update 4 3
Standard Update 5 4

Blank
Unknown 1
Blank
Standard Update 6
Standard Update 7
1
Unknown 2 5 6
Standard Update 8 7 2
3
Standard Update 9 4
Standard Update 10
Blank
QC 1
QC 2
Blank
Unknown 3 1
Unknown 4 5 6
7 2
Blank 3 9
Standard Clear 11 4
8
Standard Update 12
10
Standard Update 13
Standard Update 14
Standard Update 15
Unknown 5

14

12 11
13

15

Figure 1-3. Processing of an unordered non-bracketed sequence

If you allow Xcalibur to order your sequence (using the New Sequence
Template dialog box), the samples are arranged as follows:
1. Blank (associated with standards)
2. Standard Clear
3. Standard Updates
4. Blank (associated with standards)
5. QCs

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 1-11
ELECTRON CORPORATION
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur

6. Blank (associated with QCs)


7. Unknowns

Open
In an open bracket, samples do not need to be ordered in the sequence. There
is only one bracket no matter how many sets of standard samples. All
Standard samples are processed before Blank, QC and Unknown sample
types.
Within Xcalibur, the standards in Open (and Overlapped) brackets are
classified as samples of type: Standard Bracket.
An example of the way Xcalibur arranges an Open sequence is shown in
Figure 1-4.

Blank
Standard Bracket 1
Standard Bracket 2 1
Standard Bracket 3 5 6
7 2
Standard Bracket 4
3 9
Standard Bracket 5 4
Blank 8
10
QC 1
QC 2
Blank
Unknown 1
Unknown 2
Blank
Standard Bracket 6
Standard Bracket 7
Standard Bracket 8
Standard Bracket 9
Standard Bracket 10
Blank

Figure 1-4. Processing of an open bracket

Note that a sequence lists samples in their acquisition order. This is not the
same order used by Xcalibur during the processing of a bracket. Irrespective
of the acquisition sequence order, the processing order is:
1. Standard Bracket samples (in acquisition order)
2. Blank, QC and Unknown samples (in acquisition order)

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Quantitative Analysis
Finnigan Xcalibur _______________________________________________________________ Sequences

Overlapped
In overlapped brackets, standard samples (classified as Standard Bracket
samples) are arranged before and after Unknown, Blank and QC samples in
the acquisition sequence. However, standard samples from the end of one
bracket are used as the standard samples for the beginning of the next bracket.
Adjacent brackets therefore overlap because they share standard samples. An
example of a two-bracket, overlapped sequence is shown in Figure 1-5.

Blank
Standard Bracket 1
Standard Bracket 2
1
Standard Bracket 3 7 6
Standard Bracket 4 5 2
10
Standard Bracket 5 9
Blank
Bracket 1 8
3
4
QC 1
QC 2
Blank
Unknown 1
Unknown 2
Blank
Standard Bracket 6
Standard Bracket 7 12 11
Standard Bracket 8 7 6
15
Standard Bracket 9
10
Standard Bracket 10 9
Blank 8 14

QC 3 13
QC 4
Blank
Bracket 2 Unknown 3
Unknown 4
Blank
Standard Bracket 11
Standard Bracket 12
Standard Bracket 13
Standard Bracket 14
Standard Bracket 15
Blank

Figure 1-5. Processing of two overlapping brackets

The first sample in a bracket clears the stored calibration curve data and starts
a new set of calibration data.
As in other bracket types, the Standard samples within each bracket are
processed before Blank, QC and Unknown sample types.

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ELECTRON CORPORATION
Quantitative Analysis
Sequences __________________________________________________________ Finnigan Xcalibur

Non-Overlapped
In non-overlapped brackets, Standard samples from one bracket are not
shared with another bracket. The first sample in a bracket clears the stored
calibration curve data and starts a new set of calibration data. As in other
bracket types, Standard samples within each bracket are processed before
Blank, QC and Unknown sample types. Standards are classified as Start
Bracket or End Bracket types to denote the bracket boundaries. An example
of a two bracket non-overlapped sequence is shown in Figure 1-6.

Blank
Start Bracket 1
Start Bracket 2 1
Start Bracket 3 7 6
5 2
Start Bracket 4
10
Start Bracket 5 9
Blank 8 4
3
QC 1
QC 2
Bracket 1 Blank
Levels
Unknown 1
Unknown 2
Blank
End Bracket 6
End Bracket 7 This figure also illustrates
End Bracket 8
the use of calibration
End Bracket 9
levels to define sample
End Bracket 10
amounts. See Levels.
Blank
Blank
Start Bracket 11
Start Bracket 12
12 11
Start Bracket 13 17 16
Start Bracket 14 15
19
Start Bracket 15 20
Blank 18 14
QC 3
Bracket 2 QC 4
13

Blank
Unknown 3 Levels
Unknown 4
Blank
End Bracket 16
End Bracket 17
End Bracket 18
End Bracket 19
End Bracket 20
Blank

Figure 1-6. Processing of non-overlapped brackets

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Quantitative Analysis
Finnigan Xcalibur _______________________________________________________________ Sequences

Levels and Replicates


Xcalibur uses the following terms to describe sequence samples:
• Levels
• Replicates

Levels
A Level is a user-specified name associating component amounts with
particular Standard or QC samples in a sequence. Calibration and QC levels
are defined in a Processing Method. The individual amounts do not need to be
the same. However, a calibration standard or QC sample injected for a
particular level needs to contain the amounts specified for that level.
In the non-overlapped bracket shown in Figure 1-6, for example, there are
five calibration levels. Also, for example, samples 1, 6, 11 and 16 all belong
to the same calibration level.

Replicates
When Xcalibur orders a bracket for acquisition, it analyzes each standard
sample both before and after the QC and Unknown samples. You can also
specify multiple injections of standards within each bracket. A particular
standard mixture may therefore be analyzed several times within a sequence.
These repeated analyses are called replicates.
In the non-overlapped bracket shown in Figure 1-6, for example, samples 1, 6,
11 and 16 are replicates at the same calibration level.
For non-bracketed sequences, Xcalibur stores information about replicates in
a calibration file. This allows you to choose, during review, which replicates
to include or exclude from a calibration.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 1-15
ELECTRON CORPORATION
Quantitative Analysis
Quantitation with Xcalibur ________________________________________________ Finnigan Xcalibur

1.5 Quantitation with Xcalibur


With Xcalibur, quantitative analysis involves:
1. Developing an Instrument Setup Method for data acquisition in
Instrument Setup.
If you require high selectivity and sensitivity for a target component, use a
SIM analysis method. If these factors are not important, you could
alternatively use a Full Scan analysis method and would certainly do so, if
you also need qualitative information from your samples. These
procedures are described in detail in your Getting Started manual.
2. Creating a Processing Method in Processing Setup.
Xcalibur uses a processing method to identify, detect and integrate
components in a chromatogram, generate calibration curves, quantify
unknowns, and produce reports. Processing Setup is described in
Chapter 2.
3. Building a sequence of samples in Sequence Setup.
A sequence defines each sample as a standard, unknown, QC or blank,
and identifies its position on an autosampler tray (if appropriate). A
sequence also identifies the Instrument Method to be used for data
acquisition, and the Processing Method to be applied during automatic
processing. Sequence Setup is described in Chapter 3.
4. Reviewing calibration and quantitation in Quan Browser.
Quan Browser allows you to examine the results of automatic processing.
You can adjust all processing parameters, generate new calibration data
and recalculate quantitation. Quan Browser is described in Chapter 4.

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ELECTRON CORPORATION
Chapter 2
Quantitative Processing

This chapter describes Processing Setup and explains how you can use it to
create a method for automated batch analysis. It leads you through the
parameters required for data processing, calibration, quantitation, reporting
and running additional programs.
The topics in this chapter are as follows:
• Processing Setup
• The Processing Setup Window
• Using Quan View Interactively
• Identification
• Detection
• Levels
• System Suitability
• Reports
• Programs

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ELECTRON CORPORATION 2-1
Quantitative Processing
Processing Setup ______________________________________________________ Finnigan Xcalibur

2.1 Processing Setup


Processing Setup allows you to create a method for automated batch analysis.
It leads you through the parameters required for data processing, reporting
and running additional programs (such as file copying procedures).
Sequence Setup uses the Processing Method to initiate qualitative and
quantitative processing, reporting and additional programs or macros.
A single processing method consists of four views:
Quan Quantitative processing setup
Qual Qualitative processing setup
Reports Reporting setup
Programs Program and macro selection

This chapter concentrates on the functionality of the Quan Reports and


Programs views.
You can find a full description of the Qual view in Finnigan Xcalibur
Getting Productive: Qualitative Analysis.

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Quantitative Processing
Finnigan Xcalibur ________________________________________________ The Processing Setup Window

2.2 The Processing Setup Window


The Processing Setup window (Figure 2-1) consists of:
• A title bar containing a description about the current method
• A menu bar
• A toolbar
• A view bar containing graphical buttons leading to the four views of
Processing Setup: Quan, Qual, Reports and Programs
• The selected view – Quan and Qual views are multi-paged
• The Components list
• A status bar showing information about your activities within Processing
Setup

Figure 2-1. The Quan view in the Processing Setup window

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ELECTRON CORPORATION 2-3
Quantitative Processing
The Processing Setup Window ____________________________________________ Finnigan Xcalibur

You can display or hide the View Bar, Components list, Toolbar, and Status
Bar by choosing the appropriate View menu command:
• Choose View > View Bar to display or hide the View Bar
• Choose View > Components list to display or hide the Components list
• Choose View > Toolbar to display or hide the Toolbar
• Choose View > Status Bar to display or hide the Status Bar
If you want to maximize the display of a Processing Setup view, hide all four
of these features.

The Title Bar


The Title Bar lists:
• The application name – Processing Setup
• The active view (Quan, Qual, Reports or Programs)
• The active page (for example, Identification)
• The name of the opened method, or Untitled if a new method has not yet
been saved
• The selected type of calibration, internal or external standard (abbreviated
to Int Std and Ext Std respectively)

The Toolbar
The Toolbar contains shortcuts for frequently used menu commands. For
specific information about Processing Setup’s menu commands or Toolbar
buttons refer to Xcalibur’s online Help.

Quan View
Quan view consists of six tabbed pages:
Identification Name components and specify retention time and peak
identification criteria.
Detection Control peak detection and integration in the
chromatogram plot.
Calibration Determine the type of calibration applied to your data.
Levels Define calibration and QC levels and amounts.
System Carry out a sequence of automated chromatographic
Suitability checks that assign a pass or fail qualification to target
peaks.

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Quantitative Processing
Finnigan Xcalibur ________________________________________________ The Processing Setup Window

Peak Purity Specify parameters for peak purity determination:


percent of peak to cover, minimum intensity of PDA
scan to accept, and wavelength range of the PDA to
use.

Processing Setup displays the Chromatogram and Spectrum previews with the
first two pages. You can use these to preview the results of peak identification,
detection and integration in the chromatogram preview. A secondary use of
these previews is to set some of the Quan parameters interactively using an
existing raw file.

Components List
Xcalibur displays the Components list (if enabled) at the far right of the
Processing Method window within a Quan view. It lists all of the component
names defined in the active Processing Method. A processing method may
contain different Identification, Detection, Calibration, Levels and System
Suitability page parameters for each listed component.
To view the parameters for a particular component, click on its name in the
Components list.
To add a new component to the list, select <new> in the Name combo box on
the Identification page. Type the name of the new component and then click
on OK to apply the change. The new component name appears in the Name
list and Components list.
To delete a component from the list, first click on its name in the Components
list. Then, choose Options > Delete component (for example,
Options > Delete Anthracene) and confirm the deletion.

Applying Changes to a Page


Each Processing Setup page features OK and Cancel buttons. These are
enabled only if you change one or more parameters on the page, otherwise
they are grayed and disabled. When you have changed or edited a parameter:
Click on OK to apply the changes to the current processing method. Xcalibur
reports any validation errors.
Click on Cancel to undo all changes made to the page, and revert to the
previously applied values.

Note. These actions do not affect the saved version of the processing
method. This can only be modified by using the File > Save command.

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ELECTRON CORPORATION 2-5
Quantitative Processing
The Processing Setup Window ____________________________________________ Finnigan Xcalibur

Xcalibur displays the Apply Changes dialog box (Figure 2-2). In the Apply
Changes dialog box, choose:
• Yes to apply changes.
• No to discard any changes and proceed with the selected action.
• Cancel to abort the intended action and return to the current page
without applying or discarding changes.
Select the Don’t Tell Me About This Again check box to suppress the display
of the Apply Changes dialog box. In future cases where it would normally be
displayed, Xcalibur treats changes according to your final selection in the
dialog box:
• If you clicked on the Yes button, Xcalibur applies changes if validation is
successful and continues with your selected action. If validation fails,
your intended action is aborted and you are returned to Processing Setup
to correct or discard changes made to the page.
• If you clicked on the No button, Xcalibur automatically discards all
changes and continues with your selected action. In such cases, you must
apply changes explicitly, by clicking on the OK button, before you initiate
the action.
Choose Options > Enable Warnings to re-enable this and all other
warning dialog boxes.
If you attempt one of the following actions without applying or discarding
changes:
• Switch to another page
• Switch to another component
• Switch to another View, using either the buttons in the View Bar or the
options on the View menu
• Change chromatography type in the Chromatography Options dialog box
(Options > Chromatography By)
• Change calibration type in the Calibration Options dialog box
(Options > Calibration By)
• Click on the Close button on the title bar
• Choose one of the following menu commands:
• File > Open
• File > <most recently used file list>
• File > Save
• File > Save As
• File > Exit
• File > Import Method

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ELECTRON CORPORATION
Quantitative Processing
Finnigan Xcalibur ________________________________________________ The Processing Setup Window

• File > New


• Options > Standard Dilution
Xcalibur will not allow you to proceed with any of these actions until you
apply or undo the page modifications.
In the Apply Changes dialog box choose:
• Yes to apply changes.
• No to discard any changes and proceed with the selected action.
• Cancel to abort the intended action and return to the current page
without applying or discarding changes.
Select the Don't Tell Me About This Again check box to suppress the display
of the Apply Changes dialog box. In future cases where it would normally be
displayed, Xcalibur treats changes according to your final selection in the
dialog box:
• If you clicked on the Yes button, Xcalibur applies changes if validation is
successful and continues with your selected action. If validation fails,
your intended action is aborted and you are returned to Processing Setup
to correct or discard changes made to the page.
• If you clicked on the No button, Xcalibur automatically discards all
changes and continues with your selected action. In such cases, you must
apply changes explicitly, by clicking on the OK button, before you initiate
the action.
Choose Options > Enable Warnings to re-enable this and all other
warning dialog boxes.

Figure 2-2. Apply Changes dialog box

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ELECTRON CORPORATION 2-7
Quantitative Processing
The Processing Setup Window ____________________________________________ Finnigan Xcalibur

Customizing Processing Setup


By default, Xcalibur loads the most recently used method into Processing
Setup at startup. You can change this option, and configure Xcalibur to open a
raw file into the Chromatogram and Spectrum previews when a processing
method is opened.
To adjust these options choose the Options > Settings menu command.
The Settings dialog (Figure 2-3) is then displayed.

Figure 2-3. Settings dialog box

In the Startup mode group box select:


• Load last processing method, or
• Create new processing method
In the Auto-open raw file group box, select:
• On, or
• Off

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Quantitative Processing
Finnigan Xcalibur ________________________________________________ Using Quan View Interactively

2.3 Using Quan View Interactively


Processing Setup displays the Chromatogram and Spectrum previews with the
Identification and Detection pages. Using a representative raw file, you can
use these to:
• Preview the results of peak detection and integration in the
Chromatogram Preview.
• Set some of the Identification and Detection parameters interactively.
To use Quan view interactively, choose File > Open Raw File or click on
the Open Raw File toolbar button. Select a relevant raw file and click on
Open.

Note. If a processing method is saved when a raw file is present, the raw file
name is saved in the processing method. The associated raw file will be
opened automatically whenever the processing method is opened if you have
selected the On option button in the Auto-Open Raw File group box in the
Settings dialog box.

Previewing Processing
Using a suitable raw file, you can use the Chromatogram and Spectrum
previews to assess processing parameters for the following:
• Peak identification
• Peak detection and integration
For MS data, Xcalibur processes the raw file using the parameters of the
Identification and Detection pages. The chromatogram preview is centered on
the component’s Expected Retention Time value and its display width is
based on its View Width value. It shades all detected peaks and indicates the
start and end of each peak with a blue baseline. Initially, the spectrum shown
in the Spectrum preview will be the one corresponding to the apex scan of the
first detected peak in the chromatogram. If no peak has been detected in the
chromatogram, the chromatogram preview will show the whole raw file and
the spectrum preview will show the spectrum for the first scan in the raw file.
You can re-scale the chromatogram or spectrum previews by using:
• Cursor actions (refer to Cursor Actions)
• Toolbar buttons
• Zoom menu commands, either from the top-level menu, or from the
shortcut menu (refer to Using the Toolbar)

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ELECTRON CORPORATION 2-9
Quantitative Processing
Using Quan View Interactively _____________________________________________ Finnigan Xcalibur

If you manually edit values on the Identification or Detection pages, or


perform some interaction on the chromatogram and spectrum previews, which
changes one or more parameters, the shading and baselines are removed from
all detected peaks in the chromatogram. This indicates that the previews do
not match the information currently shown on the page. To proceed, either:
• Click on OK to perform the peak detection processing again using the
current parameters, or
• Click on Cancel to discard all changes made to the page.
Xcalibur shades all detected peaks and adds their baselines to indicate the
peak start and end positions.

Setting Processing Parameters


You can generate a quantitative processing method simply by typing values
for all the required Quan view parameters. In the Identification and Detection
pages, you may want to take advantage of the interactive features of
Processing Setup. This involves the use of the chromatogram and spectrum
previews together with a raw file representative of your analysis
requirements.
You can use the previews to set:
• Expected retention time range for the component (refer to Retention
Time)
• Mass or Wavelength Ranges (refer to Mass or Wavelength)
• Spectrum Qualifier table (refer to Spectrum Detection)

Cursor Actions
Within the chromatogram and spectrum previews, you can use the cursor in
three ways:
• A click picks a point on the preview
• A line dragged parallel to any axis picks a range
• A line dragged in any diagonal direction selects an area
The effect of these actions depends on the state of the preview:
• Inactive
• Active and unpinned (each preview has a pin icon in its top right corner)
• Active and pinned
Only one of the previews can be active at any one time. The active preview is
highlighted with a gray border. In the method shown in Figure 2-1, for
example, the spectrum preview is active, but not pinned.

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ELECTRON CORPORATION
Quantitative Processing
Finnigan Xcalibur ________________________________________________ Using Quan View Interactively

Pinning fixes the active status of a preview. To make a preview active:


1. Make sure the currently active preview is not pinned. If it is, click on the
pin icon to unpin it.
2. Click anywhere within the preview you want to be active. Xcalibur
highlights it with a gray border. Click on its pin icon if you want to fix it
as the active preview.
Cursor actions in an active preview cause the preview to be scaled according
to the dimensions of the dragged line or area (refer to Table 2-1).
The same actions in the unpinned or inactive preview have a very different
effect. In this case, the cursor actions affect the active preview (refer to
Table 2-2)
Important points to note are:
• The cursor action is always applied to the pinned preview.
• Within an active preview, cursor actions rescale the plot.

Note. Right click on the active preview to display a shortcut menu with
Display Options (see Customizing the Previews) and Zoom commands.

Table 2-1. Cursor action in active, unpinned, preview

Cursor Action Effect


Drag parallel to X-axis Rescale graph showing selected X
range only, same Y range
Drag parallel to Y-axis Rescale graph showing selected Y
range only, same X range
Dragged area Rescale graph showing both the
selected X and Y ranges

Table 2-2. Cursor action in inactive or unpinned preview

Quan page Pinned preview Cursor action Effect


Identification Spectrum Click on Detector type: All
Chromatogram The retention time selected is entered into
preview. the Expected (min) text box. Spectrum
preview displays the mass spectrum of that
retention time.
Identification Spectrum Drag across a Detector type: All
time range in The retention time of the highest point of the
Chromatogram dragged range is entered into the Expected
preview. (min) text box. Spectrum preview displays
the mass spectrum of that retention time.

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Table 2-2. Cursor action in inactive or unpinned preview, continued

Quan page Pinned preview Cursor action Effect


Identification Chromatogram Click on Detector type: MS
Spectrum The selected mass is entered into the Mass
preview. (m/z) text box as an addition to any existing
value(s). If the Trace type is TIC, it is
changed to Mass Range.
Detector type: PDA
The selected wavelength is entered into the
Wavelength (nm) text box as an addition to
any existing value(s). If the Trace type is
Total Scan, it is changed to Wavelength
Range.
Identification Chromatogram Drag across an Detector type: MS
m/z range in The selected mass range is entered into the
Spectrum Mass (m/z) text box as an addition to any
preview. existing value(s). If the Trace type is TIC, it is
changed to Mass Range.
Detector type: PDA
The selected wavelength range is entered
into the Wavelength (nm) text box as an
addition to any existing value(s). If the Trace
type is Total Scan, it is changed to
Wavelength Range.
Detection Spectrum Click on Detector type: MS
Chromatogram Spectrum preview displays the mass
preview. spectrum of the selected retention time.
If Peak Detection is set to Spectrum, the
spectrum table is populated with all ions in
the displayed spectrum (replacing any
existing values); subject to the Low Intensity
Cutoff threshold set in the Spectrum Options
dialog box (Options > Spectrum command).
Detection Spectrum Drag across a Detector type: MS
time range in Spectrum preview displays the mass
Chromatogram spectrum of the retention time of the highest
preview. point in the dragged range.
If Peak Detection is set to Spectrum, the
spectrum table is populated with all ions in
the displayed spectrum (replacing any
existing values); subject to the Low Intensity
Cutoff threshold set in the Spectrum Options
dialog box (Options > Spectrum command).

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Table 2-2. Cursor action in inactive or unpinned preview, continued

Quan page Pinned preview Cursor action Effect


Detection Chromatogram Drag across an Detector type: MS
m/z range in If Peak Detection is set to Spectrum, the
Spectrum spectrum table is populated with all ions in
preview. the dragged range (replacing any existing
values); subject to the Low Intensity Cutoff
threshold set in the Spectrum Options dialog
box (Options > Spectrum command).

Using the Toolbar


Use the toolbar buttons to re-scale a chromatogram or spectrum preview. The
toolbar buttons are:
• Normalize Y
• Zoom out Y
• Zoom in Y
• Auto Range Y
• Zoom in X
• Zoom out X
• Display all data on X axis
• Reset scaling to full scale for both X and Y axes
The Zoom menu contains equivalent commands. This can also be displayed as
a shortcut menu by right clicking on the appropriate preview. You can also
re-scale the chromatogram using the cursor.

Customizing the Previews


You can customize the display of a chromatogram or spectrum:
1. Click anywhere within the preview to make it active.
2. Choose Options > Display Options.
Alternatively, right-click on the appropriate preview and choose Display
Options from the shortcut menu.
The Display Options dialog box contains five tabbed pages for changing the
plotting style, colors, axes, labels and normalization method. For more
information about display options, refer to Xcalibur’s online Help.

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2.4 Identification
Figure 2-4 shows the Identification page of the Quan view. Xcalibur uses the
parameters on this page to:
• Generate a chromatogram from a raw file
• Identify each component peak within the chromatogram

Figure 2-4. Identification page of Quan view

The parameters on the Identification page are as follows:


Name This combo box lists all the component names for the
active Processing Method. To display the component
identification settings for a component on the list, click
on the name of the component in the Name combo box.
You can also do this by clicking on a component name
in the Components pane. To add a new component:

1. Select the <new> entry in the combo box list.


2. Replace it with the new name.
3. Press <Enter> or click on OK

The new component appears in the Components pane.


Keys Use this text field for comments about the component’s
analysis. The text field holds up to 30 characters and is
case sensitive for alphabetic characters (for example
“abc” is recognized as being different from “Abc”).

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Detector
Detector Type Select the type of detector: MS, Analog, A/D card,
PDA, or UV.
Peak Detection Select the type of peak detection algorithm: Genesis,
Method ICIS, or Avalon.

Filter
The Filter field is enabled if you select an MS detector type. Use this combo
box to specify a scan filter. A scan filter causes processing to be applied to a
subset of the scans in a raw file.
When you load a raw file, Xcalibur lists the scan filters associated with it in
the Filter combo box (Xcalibur creates scan filters from the Instrument
Method during data acquisition). Select a scan filter from the list. Xcalibur
applies the scan filter to the data in the raw file and displays the resulting
filtered chromatogram data in the Chromatogram preview if you click on OK.
For advanced uses, you can type your own filter although you must write it in
the scan filter format. For information about scan filter formats consult the
online Help.

Trace
Use these fields to specify the type of chromatogram you want to use for
qualitative processing. The Trace options depend on your selection of
Detector Type:
• For MS scans, select Mass Range, TIC or Base Peak.
• For Analog data, select from four channels (labeled Analog 1-4).
• For data from an A/D Card, select from four channels (labeled A/D Card
Ch. 1-4).
• For PDA data, select Wavelength Range, Total Scan, or Spectrum
Maximum.
The three Trace list boxes allow you to choose:
• A basic chromatogram type, for example, TIC.
• A logical operator: - and, according to the trace, +. Your selection of an
operator then enables:
• A second chromatogram type to add to, or subtract from, the first trace.
For example, Mass Range. The list contains valid traces which may be
subtracted from, or added to, the trace specified in the first list box.

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In most cases, you will probably use a single trace such as TIC. A second
trace is useful for subtracting contributions to a chromatogram from a solvent
or other noise.
Table 2-3 lists the various MS traces and gives examples of their use.
Table 2-4 lists traces for non-MS detectors.
Table 2-3. MS Traces and combinations

Trace Use
TIC Compiles a chromatogram from all the ions in each MS scan.
Mass Range Compiles a chromatogram from a single mass, or a range of masses in each scan.
This can be a list of masses or ranges separated by commas and summed.
Base Peak Compiles a chromatogram from the most abundant ion within the specified mass
range.
TIC - Mass Allows you to ‘clean up’ a TIC by subtracting a range of background contamination
Range and thereby allowing less abundant masses to have a more significant effect on the
chromatogram. For example, consider data acquired from 50 to 1000 with dominant
solvent or contaminant peaks in the range 50 to 150. Use this trace combination with
Mass Range = 50 to 150.
TIC - Base Useful in situations where the most intense spectral peak throughout the run is due to
Peak a contaminant. Subtracting the base peak from the TIC would then remove this. You
could also use the TIC-mass range combination.
Mass Range - Can be used to remove a variety of background, solvent or contaminant peaks from a
Mass Range chromatogram. Consider an example in which data has been acquired from
m/z 50 to 900: solvent contamination is evident below m/z 150 and there are intense
contaminant peaks in the intermediate range m/z 500 to 600. Use Mass Range
1 = 150 to 900; Mass Range 2 = 500 to 600.
Mass Range + Similar uses to Mass Range – Mass Range above. Considering the same example
Mass Range as above, identical results could be obtained using this trace combination with:
Mass Range 1 = 150 to 499; Mass Range 2 = 601 to 900.
Base Peak - Rarely used. Consider an example in which the most intense peaks in the spectrum
Mass Range are, say, m/z 130 at one point in the chromatogram and m/z 140 at another. If there
are no sample masses in this range BPI– (125 to 145) could remove the effect of
these peaks.
Base Peak + Useful if the Base Peak trace does not show up every chromatogram peak of
Mass Range interest. The mass range of interest can then be added to enhance the spectrum.

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Table 2-4. Other traces and combinations

Trace Use
Analog x For monitoring any external detector, such as an FID detector, that
provides an analog signal.
Analog x - Analog y For some external detectors that give out an analog signal, such as UV
detectors, it is possible to monitor more than one channel (typically two)
and to set channels to a range, for example, 220 to 500 nm. These
outputs are simple analog voltages (typically 0 to 1V). You could acquire
two channels from the same detector, one a range and one a single
wavelength or smaller range (for example, at a contaminants' specific
wavelength). Then subtract one from the other (for example,
(220 to 500) – (260 to 280) nm).
Analog x + Analog y As Analog x - Analog y above. You could add two channels
corresponding to the wavelengths of two compounds of interest (ranges
cannot be set on some detectors, only single channels).
A/D Card Channel For monitoring any external detector that provides a digital signal. You
can also specify channel combinations.
Wavelength Range PDA detector wavelength range
Wavelength Range + You could add two channels corresponding to the wavelengths of two
Wavelength Range compounds of interest (ranges cannot be set on some detectors, only
single channels).
Wavelength Range – You could acquire two channels from the same detector, one a range
Wavelength Range and one a single wavelength or smaller range (for example, at a
contaminants' specific wavelength) then subtract one from the other, for
example, (220 to 500) – (260 to 280) nm.
Total Scan PDA detector total scan
Total Scan – Wavelength Use this option to subtract a single wavelength or small range (for
Range example, at a contaminants' specific wavelength) from the total scan.
Spectrum Maximum PDA spectrum maximum

Mass or Wavelength
This parameter is available only if you select an MS or PDA detector type in
the Detector Type text box (Figure 2-4).
For an MS detector type, use the Mass text box to specify the mass or mass
range for trace combinations featuring Mass Range or Base Peak traces (for
example, Mass Range, TIC - Base Peak, TIC - Mass Range). If you use Base
Peak ± Mass Range or Mass Range ± Mass Range trace combinations, an
additional Mass (m/z) text box is displayed for you to specify the second mass
range.
For the PDA detector type, use the Wavelength text box in the cases where the
specified Trace combination features Spectrum Maximum or Wavelength
Range to specify the wavelength or wavelength range for the chromatogram.

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If you use a trace combination such as Wavelength Range + Wavelength


Range (see Table 2-4), an additional Wavelength text box is displayed for you
to specify the second wavelength range.
To change the range or to add a new range, either:
• Type the range in the text box. The valid range is dependent upon the
configured detector. The format is [Low Mass/Wavelength] - [High
Mass/Wavelength]. For example, for the range m/z 123 through 456, type
the following: 123 – 456.
• Open a representative raw file in the Chromatogram and Spectrum
previews.
Then:
1. Pin the Chromatogram preview
2. Drag the required mass range on the Spectrum preview (or click to select
a single mass-to-charge ratio value).
The mass range is added to the Mass text box.
You can have up to 50 ranges in the Mass/Wavelength text boxes. These
should be separated using the List separator character, normally a comma.
This can be found on the Number tab of Regional Settings in the Control
Panel of Microsoft® Windows® XP Professional.

Note. You must provide a mass or wavelength range for each enabled Mass
Range or Wavelength Range text box. If a Mass Range or Wavelength Range
text box is blank, Xcalibur will not allow you to save the parameters or
change to another page until you have provided a range (or switched to a
different trace combination which does not involve Mass/Wavelength
Ranges).

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Retention Time
These settings define the expected retention time in minutes and the error
window in seconds for component peak elution and detection.
Expected (min) Enter the anticipated retention time for the detection of
the selected component by Xcalibur. The valid range is
dependent upon the configured hardware.

Xcalibur identifies the selected component as the


highest peak with an apex within the expected retention
time range. The valid, and default, range is 0.0 to 999.0
minutes. To specify a time range, either:
• Type the time range directly (for example,
0.3–1.6), or
• Use a representative raw file and allow Xcalibur to
calculate an effective window. To do this:
1. Pin the spectrum preview.
2. Drag the cursor horizontally across the component
peak in the Chromatogram preview. Xcalibur
updates the Expected (min) field with the time of
the apex scan. If you click on the chromatogram,
the ‘clicked on’ time is transferred to the Expected
text box whether it is the apex scan or not.
Window (sec) Enter a retention time window for the elution of the
selected component. The value should be equal to, or in
excess of, the peak width. The valid range is 1.0 to
999.0 seconds.
Use as RT Enable this check box if you want Xcalibur to use the
reference actual retention time (RT) of the active component to
adjust the expected retention time of one or more of the
remaining components.
View width (min) Use this text box to enter the current view width (in
minutes) for the chromatogram preview. The valid
range is 0.1 to 999 minutes.
Adjust using Enable this check box if you want the expected
retention time (RT) of the active component to be
adjusted using the actual retention time of an RT
Reference (see Use as RT reference). Then, select an
RT Reference from the adjacent list box. At least one
RT Reference must be available for the check box to be
active.

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2.5 Detection
The Detection page (Figure 2-5) consists of two groups of parameters:
• Peak integration
• Peak detection
Peak Detection modes are determined by your selection in the
Chromatography Options dialog box. Xcalibur detects the type of instrument
(LC or GC) connected (when it is run for the first time) and makes this the
default type. Peak Integration parameters are common to both GC and LC
Chromatography modes.

Figure 2-5. Detection page of Quan view

To change the chromatography mode, choose Options >


Chromatography By and select GC or LC as required in the
Chromatography Options dialog box (Figure 2-6).

Figure 2-6. Chromatography Options dialog box

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Peak Integration
Xcalibur provides three peak detection algorithms. The ICIS peak detection
algorithm has been designed for MS data and has superior peak detection
efficiency at low MS signal levels. This is the Xcalibur default peak detection
algorithm. The Genesis peak detection algorithm is the original Xcalibur peak
detection algorithm. This algorithm has been provided for backward
compatibility with Xcalibur 1.0 studies. The Avalon peak detection algorithm
supports detectors other than MS, detects negative chromatographic peaks,
and shoulders more accurately than Genesis or ICIS.
For new methods only, you can change the default between the ICIS, the
Genesis, and the Avalon Xcalibur peak detection algorithms at any time for
each type of detector. From the Roadmap view of the Home Page, choose
Tools > Configuration and select the appropriate option on the Detection
page.
For example, the ICIS Peak Integration group box contains the following
options for peak integration:
Smoothing Use this text box to enter the amount of smoothing that
Points Xcalibur applies before integration. The value must be
odd and in the range 1 (minimum smoothing) to 15
(maximum smoothing).
Baseline Use this text box to enter the number of scans over
Window which to look for a local minima. The valid range is 1.0
to 500. The default value is 40 scans.
Area Noise Use this text box to specify the noise level multiplier
Factor used to determine the peak edge after the location of a
peak candidate. The valid range is 1 through 500. The
default multiplier is 5.
Peak Noise Use this text box to specify the noise level multiplier
Factor used to determine the potential peak signal threshold.
The valid multiplier range is 1 through 1000. The
default multiplier is 1.
Enable or disable the Constrain Peak Width check box to adjust integration
parameters for tailing peaks:
Peak height Enter the percentage of the total peak height that a
signal needs to be above the baseline before integration
is turned on or off. The valid range is 0 to 100.0%
Tailing factor Enter the maximum ratio of the trailing edge to the
leading side of a constrained peak. The valid range is
0.5 to 9.0.

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The two graphical display boxes (entitled Min and Max) at the right of the
ICIS Peak Integration group box depict the effect of small and large values for
the selected option as a visual reminder of how the option operates on data.
For example, the boxes in the margin above show the large and small values
for the peak integration parameters, and illustrate their effects on a simple
data representation, not the actual data.

Peak Detection
The ICIS, Genesis and Avalon Peak Detection group boxes contain options
for determining how Xcalibur detects peaks within the retention time window.
There are three options:
Spectrum This option is only available in GC chromatography
mode. It allows you to use a reference spectrum for
component identification. Xcalibur attempts to match
the reference spectrum with a series of unknown
spectra and calculates a score for each comparison.
Highest Peak This option allows you to identify the active component
with the highest peak in the retention time window.
This is the default LC chromatography mode option.
Nearest RT This option allows you to identify the selected
component with the peak having a retention time
nearest to the Expected value.
The following parameter is applicable to all peaks, irrespective of the
detection mode:
Minimum peak This is a signal-to-noise threshold for peak integration.
height Peaks with signal-to-noise less than this value are not
integrated. Peaks with signal-to-noise greater than this
value are integrated. The valid range is 0.0 to 999.0.

In GC Chromatography mode, Ion Ratio Confirmation is available with the


Highest Peak and Nearest RT options (refer to Ion Ratio Confirmation).

Spectrum Detection
The spectrum detection mode is designed specifically for use in gas
chromatography (GC), where peak widths are typically about 6 seconds and
often significantly less. It can therefore be difficult to define a precise
retention time window for a specific peak. If you use a large retention time
window, it is possible that several peaks will be identified within it. In LC,
peak widths are significantly greater and the definition of a retention time
window is generally a simple matter.

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Spectrum detection relies on you providing a reference spectrum for the


component peak. If this is available, spectrum detection is preferable to using
Highest Peak or Nearest RT modes for GC users.
The reference spectrum may consist of up to 50 ions and there are three
match-criteria thresholds (Figure 2-7). Xcalibur uses the reference spectrum
to locate the target component within the chromatogram. It then uses the
thresholds to filter potential candidates.
When using this mode, the Identification page would normally be set for an
MS detector type with a single Mass value specified.

Figure 2-7. ICIS Peak Detection group box with spectrum detection
enabled

How it Works
The full spectrum detection procedure is as follows:
1. Xcalibur calculates the component’s predicted retention time range. This
consists of using the parameters you specified on the Identification page
to calculate the expected retention time and window.
2. Xcalibur compares the reference spectrum with the chromatogram. This
consists of comparing each raw file spectrum across the component’s
retention time range with the reference spectrum and calculating Forward
and Reverse match factors.
3. Xcalibur computes the peak detection function. This consists of utilizing
the Forward and Reverse match values, together with the intensity of the
component’s mass, for each spectrum within the retention time range.
4. Xcalibur carries out peak detection and integration on the chromatogram
plot and peak detection function. Using the parameters on the Detection
page, Xcalibur detects peaks in both the component’s mass chromatogram

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plot and the peak detection function. If enabled, smoothing is applied


before detection.
5. Peaks in the two plots are compared and Match values are calculated.
Xcalibur selects potential candidates for the component peak. If a peak
apex in the peak detection function is within two scans of a peak apex of
the chromatogram plot, the peak is identified as a candidate. For each
candidate its Match value is computed. This takes into account how close
a candidate is to the component’s predicted retention time and the width
of the component’s retention time range.
6. Xcalibur filters the results, discarding any candidate with one or more of
its Forward, Reverse or Match values below the specified threshold
values.
7. Xcalibur selects the top three candidates. It chooses the highest Match
value candidate as the ‘found’ peak and stores information about any
second- and any third-best candidate. You can view this information in
Quan Browser or in printed reports.

Using Spectrum Detection


The following procedure describes how to use the Spectrum Detection mode:
1. Open the Chromatography Options dialog box:
Choose Options > Chromatography By.
2. Select GC detection mode:
Select the GC option button. Click on OK.
3. Back on the Detection page, select spectrum detection:
In the Peak Detection group box, select the Spectrum option button.
Spectrum detection options are then displayed.
4. For semi-automated mass spectral peak entry, Xcalibur discards any ions
with intensities below the Low Intensity Cutoff percentage parameter in
the Spectrum Options dialog box (Figure 2-8). To adjust this parameter,
select Options > Spectrum.
5. Enter mass/charge [m/z] and intensity data for up to 50 mass spectral
peaks in the Spectrum peak identification table. You can do this manually
or semi-automatically using a raw file containing good quality spectral
data of the component.
To enter data manually:
a. Select an m/z table text box and enter the value for an ion
characteristic of the component.
b. Select the Intensity percentage table text box and enter a value for the
relative intensity of the ion.
c. Repeat this procedure for all the ions in the reference spectrum (up to
a maximum of 50).

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To enter data using a raw file:


a. Pin the Spectrum preview.
b. Drag the cursor across the appropriate component peak in the
Chromatogram preview. Xcalibur displays the spectrum from the
scan at the peak apex in the Spectrum preview.
c. Pin the Chromatogram preview and drag the cursor across the
required Spectrum range. The ion m/z and Intensity values are copied
to the peak identification table, overwriting any existing values.
d. Perform manual adjustments to the peak identification table values, as
described above for manual data input.
5. Select Thresholds for spectrum matching:
a. Use the Forward text box to enter a Forward comparison threshold
b. Use the Reverse text box to enter a Reverse comparison threshold
c. Use the Match text box to enter a Match comparison threshold
6. Click on OK to save your settings.

Figure 2-8. Spectrum Options dialog box

Editing the Peak Identification Table


A shortcut menu is available for you to insert, delete, clear or move rows in
the table.
To insert a row:
1. Click on the row number above the position.
2. Right click and select Insert Row from the shortcut menu.
To delete a row or range of rows:
1. Click on the row number of the row you want to delete. If you want to
delete a range of rows, drag the cursor to the final row in the range.
2. Right click and select Delete Rows from the shortcut menu, or press
<Delete>.

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Ion Ratio Confirmation


Ion Ratio Confirmation is for use only with Highest Peak and Nearest RT
peak detection in Xcalibur’s GC Chromatography mode.
Using Ion Ratio Confirmation, Xcalibur can confirm the identity of a target
peak using qualifier ions. This can be useful when several peaks are present in
the retention time window. This often occurs in gas chromatography where
narrow peak widths and large numbers of peaks make it difficult to target a
component using a retention window alone.
It is particularly useful in applications using SIM acquisition (rather than full
scan) to achieve high sensitivity for high accuracy quantitative results. In this
case, Spectrum peak detection is not appropriate because limited mass
spectral peak data are available.
The qualifier ion table (Figure 2-9) allows you to enter mass/charge [m/z]
values for up to 5 qualifier ions. For each qualifier ion, you must also provide
a Target Ratio and target ratio tolerance [Window ±%].

Figure 2-9. Qualifier ion table in the Ion Ratio Confirmation group box

How it Works
The Ion Ratio Confirmation procedure is as follows:
1. Xcalibur generates a mass chromatogram for the quantitation mass(es).

Using the parameters you specified in the Trace, Filter and Mass (m/z)
fields, Xcalibur generates a mass chromatogram for the quantitation
mass(es).
2. Xcalibur carries out peak detection.

Using the parameters you specified on the Identification and Detection


pages, Xcalibur carries out peak detection. If no peak is found, the
component is flagged as “not found” and no ion ratio confirmation is
carried out.

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3. Xcalibur generates a mass chromatogram for each specified qualifier ion.

Using the same Identification and Detection parameters, Xcalibur


generates a mass chromatogram for each qualifier ion and detects peaks.
If these chromatograms do not feature a peak or if the retention time of
the qualifier ion peak apex lies outside of the Qualifier Ion Coelution
window (centered on the quantitation peak), Xcalibur rejects the
quantitation peak and terminates the ion ratio confirmation procedure.
4. Xcalibur calculates the ratio of each qualifier ion peak to the quantitation
peak.

If you are using area response, Xcalibur integrates each qualifier ion peak
and ratios it with the quantitation peak area. Xcalibur then compares this
ratio with your specified target ratio. If the calculated ratio is outside of
the target ratio by more than your specified tolerance (Window ±%), the
quantitation peak is rejected and the IRC Flag for the quantitation peak is
set to false.
If you are using Height response, Xcalibur ratios the qualifier ion peak
height with that of quantitation peak. Xcalibur then compares this ratio
with your specified target ratio. If the calculated ratio is outside of the
target ratio by more than your specified tolerance (Window ±%), the
quantitation peak is rejected and the IRC Flag for the quantitation peak is
set to false.
These four steps are repeated for each qualifier ion. All qualifier ratios must
be within the target ratio tolerances for the IRC Flag to be set to true.

Using Ion Ratio Confirmation


1. Open the Chromatography Options dialog box:
Choose Options > Chromatography By.
2. Select the GC option button and click on OK.
3. Back on the Detection page, select the Highest Peak or Nearest RT option
button as required.
4. Select the Ion Ratio Confirmation check box.
5. Enter details of the qualifier ions for the current component:
a. Select an m/z text box and enter the value for an ion characteristic of
the component.
b. Select the Target Ratio % text box and enter a value for the Target
ratio.
c. Select the Window ±% text box and enter a value for the relative
intensity tolerance applied to the Target Ratio percentage.
d. Repeat this procedure for all the ions (up to a maximum of 5).

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6. Select a Window% calculation option:


• Select the Absolute option button if you want Xcalibur to use the
target ratio tolerances in the Window ±% column as absolute
percentages of the target ratio.
• Select the Relative option button if you want Xcalibur to use the
target ratio tolerances in the Window ±% column as relative
percentages of the target ratio.
7. Enter a value for the Qualifier Ion Coelution window in minutes. If the
retention time of any qualifier ion peak apex lies outside of the Qualifier
Ion Coelution window (centered on the quantitation peak), Xcalibur
rejects the quantitation peak.

Quantitation peaks with matching qualifier ion peaks (within the


Coelution window) are tested by Xcalibur for ion ratio confirmation
according to the selected method.
8. Click on OK to save your settings.

Editing the Qualifier Ion Table


A shortcut menu is available for you to insert or delete rows in the table.
To insert a row:
1. Click on the row number above the position.
2. Right click and select Insert Row from the shortcut menu.
To delete a row or range of rows:
3. Click on the row number of the row you want to delete. If you want to
delete a range of rows, drag the cursor to the final row in the range.
4. Right click and select Delete Row from the shortcut menu, or press
<Delete>.

Advanced Detection Parameters


Xcalibur’s default options provide suitable chromatographic peak detection
for most applications. In certain circumstances, you might need to change
some of these parameters. Advanced options are available from the Advanced
button of the Detection page:
Identification Use this dialog box to adjust the parameters for
Options baseline noise analysis and retention time correction.
ICIS Advanced This dialog box is displayed if you are using the ICIS
Parameters peak detection algorithm.

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Genesis This dialog box is displayed if you are using the


Advanced Genesis peak detection algorithm.
Detection
Options
Avalon Event This dialog box is displayed if you are using the Avalon
List peak detection algorithm

For more information about ICIS, Genesis, and Avalon advanced detection
options, refer to Xcalibur online Help.

Peak Identification and Baseline


Choose the Options > Identification menu command to open the
Identification Options dialog box (Figure 2-10). This contains the parameters
used by Xcalibur to estimate baseline noise and to correct retention time
assignments for void time.

Figure 2-10. Identification Options dialog box

Void Time
The Void Time parameter allows you to obtain correct retention times for each
peak. Void time is the time taken by a non-retained compound to elute from
the column. To obtain the correct retention time for each peak either:
• Select the Value (min) option button and enter a value for the void time
(this is subtracted from the elution time for all recorded peaks), or

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• Select the First Peak option button and set the void time to that of the first
detected peak. Xcalibur subtracts this time from the elution time for all
remaining peaks.

Baseline
Xcalibur calculates baseline noise (Genesis Detection only) in an iterative
process using the filtered and smoothed mass chromatogram. The objective of
the noise calculation process is to draw a line through the baseline composed
of a number of points with a noise ratio that is less than a specified tolerance.
Xcalibur uses the calculated baseline noise value throughout the peak
characterization process to determine whether or not baseline adjusted
intensities or heights of measurements are significant. The value is judged
significant if it is greater than the product of noise and S/N threshold
(Genesis Detection page only). Likewise, when values are less than this
product, they are considered baseline values.
The parameters defining this process are:
Baseline and Xcalibur applies this parameter to each peak and
noise window calculates the baseline and baseline noise within this
(min) window (valid range 0.1 to 1000). To ensure an
accurate noise calculation, enter a value that includes
the base width of the peak and an appreciable amount
of baseline. If the window is too small, the baseline will
be positioned up the sides of the peak.
Baseline noise This parameter controls how the baseline is drawn in
tolerance (%) the noise data. The higher the baseline noise tolerance
value, the higher the baseline is drawn through the
noise data. The valid range is 0.0 to 100.0.
Minimum This parameter defines the minimum number of scans
number of that Xcalibur uses in the baseline calculation. A larger
scans in number includes more data in determining an averaged
baseline baseline. The valid range is 2 to 100.0.

ICIS Advanced Parameters


For an example of Advanced Parameters, click on the Advanced button on the
Detection page to open the ICIS Advanced Parameters dialog box
(Figure 2-11). This allows you to select ICIS advanced chromatogram peak
detection criteria.

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Figure 2-11. ICIS Advanced Parameters dialog box

Note. The default values are suitable for most analysis requirements. Change
these settings only if standard chromatogram detection and integration
options do not provide the desired result.

Noise Method
Xcalibur uses this advanced parameter to determine how the noise level of the
data is determined by the ICIS peak detection algorithm.
INCOS Noise This option allows you to use a single pass algorithm to
determine the noise level. This is the default noise
method.
Repetitive Noise This option allows you to use a multiple pass algorithm
to determine the noise level. In general, this algorithm
is more accurate in analyzing the noise than the INCOS
Noise method.
RMS This option allows you to use a root mean squared
(RMS) algorithm to determine the noise level. By
default, Xcalibur uses Peak To Peak for the noise
calculation. RMS is automatically selected if you
determine the noise region manually.

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Peak Parameters
The Xcalibur ICIS peak detection algorithm uses the following advanced
peak detection parameters:
Min Peak Width This text box allows you to enter the minimum number
of scans in a peak. The valid range is 0 to 100 scans.
The default value is 3 scans.
Multiplet This text box allows you to enter the minimum
Resolution separation in scans between the apexes of two potential
peaks. This is a criterion to determine if two peaks are
resolved. The valid range is 1 to 500 scans. The default
value is 10 scans.
Area Tail This text box allows you to enter the number of scans
Extension past the peak endpoint to use in averaging the intensity.
The valid range is 0 to 100 scans. The default value is 5
scans.
Area Scan This text box allows you to enter the number of scans
Window on each side of the peak apex to be allowed. The valid
range is 0 to 100 scans. The default value of 0 scans
specifies that all scans from peak start to peak end are
to be included in the area integration.

Data Flags
The Data Flags dialog box (Figure 2-12) allows you to set flags for peak area
and height thresholds. Flags are recorded as true or false in the result file. If
you set a value to zero, the flag will always be false. Flags are reported in
Quan Browser and in printed or exported Reports.

Figure 2-12. Data Flags dialog box

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To open the Data Flags dialog box, click on Flags on the Detection page.
Flags are reported as true if they exceed these thresholds:
Area threshold Enter a value for the Area Threshold Data flag. This is
an absolute value of peak area (counts).
Height Enter a value for the Height Threshold Data flag. This
threshold is an absolute value of peak height (counts).

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2.6 Calibration
The Calibration page allows you to assign one of the following types to each
of the components defined on the Identification page:
• Target compound
• ISTD
If you select Target compound, the Target Compounds group box is enabled.
This allows you to:
• Assign an ISTD to the compound
• Carry out an Isotope Contribution Correction
• Select a calibration curve type
• Select the response type
• Specify units
If you select ISTD:
• The ISTD group box becomes active
• The Target Compounds group box is grayed
• The Levels page becomes unavailable
• In the ISTD group box, you provide the amount and units for the ISTD
component.
ISTD options are available only if you have selected the Internal Standard
option button in the Calibration Options dialog box (Figure 2-13). This is the
default option for Xcalibur.

Figure 2-13. Calibration Options dialog box

To change the calibration mode:


1. Choose Options > Calibration By.
2. In the Calibration Options dialog box, select Internal Standard or
External Standard as required.

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3. Click on OK to save the setting.

Note. If you select External Standard, any ISTD components in the active
method will be converted to Target Compounds. The ISTD option button
and group box will be grayed. The Amount and Unit information will be
lost. If you select Internal Standard, Xcalibur will prompt you to assign at
least one of the components as an ISTD.

Assigning an ISTD
The following procedure describes how to define a component as an ISTD:
1. Click on a component in the Components list located at the far right of the
Processing Setup window. If this is not visible, choose View >
Components list.
2. Select the ISTD option button in the Component type group box
(Figure 2-14). The ISTD group box is enabled.
3. Use the Amount text box to specify the amount of the internal standard
injected into each sample.
4. Use the Units text box to specify the units of the internal standard injected
into each sample.

Figure 2-14. Calibration page for an ISTD component type

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Assigning a Target
The following procedure describes how to define a Target Compound:

Note. When creating an internal standard method, you need to define at least
one component to be an ISTD before you can define any other components
as target compounds.

1. Click on a component in the Components list located at the far right of the
Processing Setup window. If this is not visible, choose View >
Components list.
2. Select the Target Compound option button in the Component type group
box (Figure 2-15). The Target Compounds group box is enabled.

Figure 2-15. Calibration page for a Target Compound type.

3. Select an Internal Standard (ISTD) for the Target from those listed in the
ISTD combo box.
4. If you want to make calibration corrections for isotope contributions of
the internal standard to the target compound or the target compound to the
internal standard click on the Isotope percent button. This opens the
Correction For Isotope Contribution dialog box (Figure 2-16).

Note. Check that the values in the Correction For Isotope Contribution
dialog box are set to zero if you do not require isotope contribution
correction.

5. Select a Calibration Curve type from those listed in the Calibration Curve
combo box. The options are:
Linear A linear polynomial curve of the following mathematical
form: Y = mX + B, where m is the slope of the curve and B
is the intercept point on the Y-axis.

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Quadratic A quadratic polynomial curve of the following


mathematical form: Y = AX2 + BX + C, where A, B, and C
are the polynomial coefficients.
Linear A linear polynomial curve of the following mathematical
Log-Log form: log10[Y] = m log10[X] + B, where m is the slope of
the curve and B is the intercept point on the Y-axis.
Quadratic A quadratic polynomial curve of the following
Log-Log mathematical form: log [Y] = A log [X2] + B log [X] + C,
where A, B, and C are the polynomial coefficients.
Average RF A calibration curve in which the slope of the calibration
curve is constructed from the average response factor of all
levels. This calibration curve always passes through the
origin.
Point-to- A curve in which straight lines are drawn between averaged
Point replicate data at each calibration level.
Cubic Spline A calibration curve in which a cubic polynomial curve is
fitted between each pair of calibration levels such that the
slopes of the separate cubic polynomial curves match at
common calibration curve points.
Locally A calibration curve that is constructed from individual line
Weighted segments. At multiple points across the calibration region, a
weighted linear regression is performed. The point slopes
are then connected to form a continuous line. At any point
on the curve, the calculated amount is based on the nearest
weighted linear regression.

6. If you have selected a Linear or Quadratic calibration curve, select one of


the calibration point Weighting option buttons: Equal, 1/X, 1/X2, 1/Y, 1/Y2,
or 1/s2. Xcalibur applies the weighting when it calculates the
least-squares regression calibration curve.
7. If you have selected a Linear, Quadratic, Point-to-Point or Cubic Spline
curve type, select how to treat the origin in the calibration curve
calculation:
Ignore Do not include the origin in the calibration curve
calculation.
Force Require that the calibration curve passes through the origin.
Include Include the origin as an extra data point (not available for
Point-to-Point or Cubic Spline curve types).

8. Use the Units text box to enter the units to be displayed on graphs and
reports.

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9. Select the peak response method:


Area If you want Xcalibur to quantitate based upon the
integrated area of component peaks.
Height If you want Xcalibur to quantitate based upon the
calculated height of component peaks.

Repeat this procedure for all the components in the active method that you
wish to define as target compounds.

Isotope Correction
The Correction For Isotope Contribution dialog box (Figure 2-16) allows you
to correct for an impurity in the internal standard compound that elutes at the
same time as the target compound and/or correct for an impurity in the target
compound that elutes at the same time as the internal standard.
Access the dialog box by clicking on Isotope % in the Target Compounds
group box on the Detection page. This is only available for components
defined as Target Compounds.

Figure 2-16. Correction For Isotope Contribution dialog box

The dialog box displays two text boxes:


• Contribution of ISTD to Target Compound
• Contribution of Target Compound to ISTD

Note. Check that the values in the Correction For Isotope Contribution
dialog box are set to zero if you do not require isotope contribution
correction.

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Contribution of ISTD to Target Compound


To make a correction to the target compound arising from a contribution due
to the ISTD:
1. Analyze the ISTD reagent using the Processing Method to be used for
quantitation of the target compound. Use the respective peak areas or
heights to determine the following ratio:
ISTDimpurity/ISTDpure
Where:
ISTDimpurity is the response due to the impurity compound in the internal
standard reagent that elutes at the same time as the target compound.
ISTDpure is the response of the pure internal standard compound.
2. Enter this value in the Contribution of ISTD to Target Compound (%) text
box.
Xcalibur uses this ratio as the value x in the following impurity correction
expressions:
ISTDcorr = [ISTDobs - y TCobs]/[1-yx]
TCcorr = [TCobs - x ISTDobs]/[1-yx]
Where:
ISTDcorr is the corrected amount of internal standard.
ISTDobs is the apparent amount of ISTD, as measured by Xcalibur at the
retention time for the ISTD. This peak consists of ISTDcorr + TCimpurity.
TCcorr is the corrected amount of the target compound.
TCobs is the apparent amount of target compound, as measured by
Xcalibur at the retention time for the target compound. This amount
consists of
TCcorr + ISTDimpurity.

Contribution of Target Compound to ISTD


To make a correction to the Target Compound arising from a contribution due
to the ISTD:
1. Analyze the target compound using the Processing Method to be used for
quantitation of the target compound without the ISTD present. Use the
respective peak areas or heights to determine the following ratio:
TCimpurity/TCpure
Where:
TCimpurity is the response due to the impurity compound in the target
compound that elutes at the same time as the ISTD.

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TCpure is the response of the pure target compound.


2. Enter this value in the Contribution of Target Compound to ISTD (%) text
box.
Xcalibur uses this ratio as the value y in the following impurity correction
expressions:
ISTDcorr = [ISTDobs - y TCobs]/[1-yx]
TCcorr = [TCobs - x ISTDobs]/[1-yx]
Where:
ISTDcorr is the corrected amount of internal standard.
ISTDobs is the apparent amount of ISTD, as measured by Xcalibur at the
retention time for the ISTD. This peak consists of ISTDcorr + TCimpurity.
TCcorr is the corrected amount of the target compound.
TCobs is the apparent amount of target compound, as measured by
Xcalibur at the retention time for the target compound. This amount
consists of TCcorr + ISTD impurity.

Setting Calibration and Quantitation Flags


You can set limits for the calibration and quantitation flags from the
Calibration page. Xcalibur reports these flags in result files, in printed reports,
and in Quan Browser. To set limits for the flags, click on the Flags button. The
Calibration And Quantitation Flags dialog box is then displayed
(Figure 2-17).

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Figure 2-17. Calibration And Quantitation Flags dialog box

Calibration Flag
R-squared Enter a threshold to test the goodness of fit of the
calibration curve. Xcalibur calculates a coefficient of
determination (R-squared) whenever it computes a
calibration curve. If the value is less than the R-squared
threshold, the R-squared flag in the result file is set to
true; otherwise it is set to false.

Quantitation Flags
Detection limit Enter a threshold for the limit of detection. If the
quantified component concentration is less than the
Detection Limit threshold, the Detection Limit flag in
the result file is set to true; otherwise it is set to false.
Linearity limit Enter a threshold for the linearity limit. If the quantified
component concentration is greater than the Linearity
Limit threshold, the Linearity Limit flag in the result
file is set to true; otherwise it is set to false.

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Quantitation This setting allows you to specify a flag threshold for


limit the limit of quantitation. If the quantified component
concentration is less than the Quantitation Limit
threshold, the Quantitation Limit flag in the result file
is set to true; otherwise it is set to false.
Carryover limit Enter a threshold for the carryover limit. If the
quantified component concentration is greater than the
Carryover Limit threshold, the Carryover Limit flag in
the result file is set to true; otherwise it is set to false.

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2.7 Levels
The Levels page (Figure 2-18) allows you to define the concentrations of
target compounds in calibration standard samples. A Level is a text ‘label’ for
each of the defined amounts. Cal Levels are associated with calibration
standard samples when you set up a sequence. QC Levels are associated with
QC samples when you set up a sequence.
The Levels page is not available for components defined as ISTDs in the
active method (since they are spiked into samples at a fixed amount as
specified by the Amount text box of the ISTD group box on the Calibration
page).

Figure 2-18. Levels page

The page consists of two tables:


• Calibration levels
• QC (Quality Control) levels
To set levels:
1. Click on a target component in the Components list located at the far right
of the Processing Setup window. The Levels page is not available for
ISTD components.
2. Enter Calibration Level data into the Calibration Levels table:
• Use the Cal Level text boxes to enter calibration level labels.
• Use the Amount text boxes to enter the amount of the component
added at each level (in a calibration standard sample).
You can also use the Standard Dilution dialog box (Figure 2-19) to set
calibration levels for all the target components (refer to Standard Dilution).

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3. Enter QC Level data. Type the following information in the QC Level


table:
• Use the QC Level text boxes to enter QC level labels.
• Use the Amount text boxes to enter the amount of the component
added at each level (in a QC sample).
• Use the QC % Test text boxes to enter the percent tested at each QC
level. Xcalibur measures the quantity of the QC component in the
same manner as unknown components. The measured quantity is then
compared with a user defined expected quantity and a user defined
percent test.
4. Repeat the procedure for all target components.
5. Click on the OK button to save your settings.

Standard Dilution
The Standard Dilution dialog box (Figure 2-19) allows you to enter
calibration level information for all target components simultaneously. To
open the dialog box, choose Options > Standard Dilution from the Levels
page.
At the top of the dialog, the Target Compound Components readback line
displays the total number of target components defined in the processing
method out of the total number of all components in the method.
The Selected Components readback line displays the selected number of
non-ISTD components for Standard Dilution out of the total number of all
components in the method.

Entering Standard Dilution Information


To use the Standard Dilution dialog box to enter Calibration Levels
information for all the target components:
1. Use the Amount text boxes in the Base Amounts table to enter the base
amount for each component. You must provide a value for each listed
component.
2. Now enter information in the Dilution Factors table:
• Use the Cal Level text boxes to enter up to 50 calibration levels. To
enter a calibration level, type the new name in the appropriate Cal
Level text box (32 characters maximum). To delete a Cal Level row,
click on the numbered tile to the left of the row. Xcalibur highlights
the row. Then, press <Delete>.

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• Use the Dilution text boxes to enter the stock dilution factors for each
calibration level. To enter a dilution factor, type the value in the
appropriate Dilution text box. The value must be greater than
0.00000001 and less than or equal to 1.
3. Click on OK to save the new settings and close the dialog box.

Figure 2-19. Standard Dilution dialog box

How Xcalibur Uses the Information


Xcalibur calculates the calibration levels for all the target compound
components defined in the method using the parameters you have provided in
the Standard Dilution dialog box. It does this by:
• Transferring the Cal Level values to the Cal Level column of the
Calibration Levels table for each component
• Multiplying the Dilution factor with the Base Amount value. The result is
transferred to the corresponding Amount text box in the Calibration
Levels table for the component.

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Xcalibur repeats this procedure for all Calibration Levels, and all
components.

Note. The Standard Dilution dialog box is not updated with any changes
made directly on the Levels page. If you use the dialog box to set up a
method, you should ensure that you use it to modify the method
subsequently. Any manual changes made to the Levels page will be lost if
you subsequently use the Standard Dilution dialog box.

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2.8 System Suitability


The System Suitability page (Figure 2-20) allows you to enable a number of
automated chromatographic checks that assign a pass or fail qualification to a
target peak. These checks are based on an analysis of the quantitation peak
and, if ion ratio confirmation is enabled, all qualifier ion peaks within the
retention time window. Warning flags are reported in Sample and Summary
report, and in Quan Browser.

Figure 2-20. System Suitability page in Quan view

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The tests are divided into 3 groups:


• Resolution parameters
• Symmetry parameters
• Peak classification parameters
Flagging is used to:
• Fail a compound and therefore a sample
• Monitor trends occurring in successive sample injections
You can use any of the three System Suitability test groups by selecting the
Enable check box associated with it.

Resolution
The resolution test uses a single parameter - the resolution threshold.
This enables Xcalibur to check the resolution of quantitation peaks against a
threshold value. Resolution testing is based on a comparison of the peak
height with the adjacent valley ‘height’ within the quantitation window.
Resolution threshold is defined as the ratio:
100 × V/P
where:
V is the horizontal asymptote extended from the target peak’s apex to the
lowest point in the valley between the target peak and a neighboring peak
P is the height of the target peak
The default value for the resolution threshold is 50% and the valid range is 0
to 100%.
Compounds are flagged (R) if any of the target peaks fail to meet the
resolution threshold.

Symmetry
These settings allow you to specify system suitability checks for the
symmetry of quantitation peaks. Symmetry is determined at a specified peak
height and is a measure of how even-sided (symmetrical) a peak is about a
perpendicular dropped from its apex.
The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures the symmetry of target peaks. You
can enter any value within the range 1% to 100%.

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Symmetry This text box specifies the Symmetry Threshold value.


threshold You can enter any value within the range 0% to 100%.
The default value is 90% at 50% peak height. A
realistic practical tolerance for capillary GC data might
be 70% at 50% peak height.

Xcalibur determines symmetry at the peak height specified in the Peak Height
% text box. For the purposes of the test, a peak is considered symmetrical if:
(Lesser of L and R) × 100 / (Greater of L and R) > Symmetry Threshold %
where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
R is the distance from the right side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (S) if any of the target peaks fail to meet the
symmetry threshold.

Peak Classification
This group consists of 5 classification checks:
• Detect peak width
• Detect tailing
• Detect column overload
• Detect baseline clipping
• Detect minimum signal-to-noise ratio

Peak Width
These settings allow you to specify system suitability checks for the width of
quantitation peaks. The test uses three parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur tests the width of target peaks. You can enter
any value within the range 0% to 100%. The default
value is 50%.

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Min peak width This text box specifies the minimum peak width, at the
(sec) specified peak height, for the peak width suitability
test. The default value is 1.8. You can set any value in
the range 0 to 30 seconds.
Compounds are flagged (W) if the peak width is less
than the minimum peak width.
Max peak width This text box specifies the maximum peak width, at the
(sec) specified peak height, for the peak width suitability
test. The default value is 3.6. You can set any value in
the range 0 to 30 seconds.

Compounds are flagged (W) if the peak width is greater


than the maximum peak width.

Tailing
These settings allow you to specify system suitability checks for the tailing of
peaks. The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures the tailing of target peaks. You can
enter any value within the range 1% to 100%.
Failure This text box specifies the failure threshold for the
threshold tailing test. The valid range is 1 to 100.

Tailing is calculated at the value defined in the Peak Height % text box. For
the purposes of the test, a peak is considered to be excessively tailed if:
R / L > Failure Threshold %
where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
R is the distance from the right side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (T) if any of the target peaks exceed the tailing failure
threshold.

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Column Overload
These settings allow you to specify system suitability checks for column
overloading. The test uses two parameters:
Peak height This text box specifies the Peak Height % at which
Xcalibur measures column overloading. You can enter
any value within the range 1% to 100%.
Failure This text box specifies the failure threshold value for
threshold the column overload test. The valid range is 1 to 100.

A peak is considered to be overloaded if:


L / R > Failure Threshold %
where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
R is the distance from the right side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (O) if any of the target peaks exceed the failure
threshold.

Baseline Clipping
This test uses a single parameter to check quantitation peaks for baseline
clipping - Number of peak widths for noise detection.
A peak is considered to be baseline clipped if there is no signal (zero
intensity) on either side of the peak within the number of peak widths
specified in the Number Of Peak Widths For Noise Detection text box. The
default value is 1.0 and the permitted range is 0 to 100. The range is truncated
to the quantitation window if the specified number of peak widths extends
beyond the window’s edge.
Compounds are flagged (B) if any of the target peaks fail the
baseline-clipping test. Baseline clipping is often indicative of problems with
the MS detector and associated electronics.

Minimum Signal to Noise Ratio


This test (Genesis Detection only) checks for a low signal-to-noise ratio
within the quantitation window. It uses a single parameter - Signal-to-noise
ratio threshold.

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ELECTRON CORPORATION
Quantitative Processing
System Suitability ______________________________________________________ Finnigan Xcalibur

This is the threshold for system suitability testing of the signal-to-noise ratio.
The default value is 3 and the permitted range is 0 to 100. Xcalibur calculates
the signal-to-noise ratio within the quantitation window using only baseline
signal. Any extraneous, minor, detected peaks are excluded from the
calculation.
Compounds are flagged (N) if any of the target peaks fail the signal-to-noise
test.

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Quantitative Processing
Finnigan Xcalibur _______________________________________________________________ Peak Purity

2.9 Peak Purity


The Peak Purity page (Figure 2-21) allows you to enable or disable peak
purity parameters for PDA chromatograms in the active cell. Unresolved
peaks in data generated from a PDA (UV) detector can indicate the presence
of analyte impurities. The Peak Purity computation detects changes in peak
shape during a run, assesses peak purity from the changes in the shape of a
peak, and then computes a correlation factor from the collected data. The
correlation factor is a measure of the purity of the scan at the apex of a single
chromatogram peak, when compared with the scans at other times within the
same peak.

Figure 2-21. Peak Purity page in the Quan view

This feature is active in Processing Setup or Qual Browser only when both of
the following conditions are true:
• A raw data file for a PDA analysis is open
• PDA is selected from the Detector Type list box in the Quan view of
Processing Setup or PDA is selected from the Detector list box in the
Chromatogram Ranges dialog box of Qual Browser
You can determine suitable Peak Purity parameters for raw data by processing
the raw file in Qual Browser; Xcalibur displays the correlation factor in the
active chromatogram view of Qual Browser. Then, you can include this
correlation factor in a Processing Method by using the Quan view and/or the
Qual view of Processing Setup. Finally, you can produce a Peak Purity report
by using the Reports view of Processing Setup when you include the
correlation factor in a Processing Method.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 2-53
ELECTRON CORPORATION
Quantitative Processing
Peak Purity __________________________________________________________ Finnigan Xcalibur

Enable Peak Purity


The Enable check box allows you to enable or disable Peak Purity parameters
for the PDA chromatograms in the active cell. To enable Peak Purity
parameters and calculate Peak Purity results, select the Enable check box.
Peak detection occurs automatically prior to the peak purity calculation.

Scan Threshold
The Scan Threshold text box allows you to specify a minimum value of
intensity for wavelength scans in milliabsorbance units (mAU). A Peak Purity
computation using scan threshold starts with a scan at the apex of the peak
and collects wavelength data from scans on both sides of the apex until the
scan threshold is reached.
Use scan threshold for either symmetrical or asymmetrical peaks. The default
value for scan threshold is 3 mAU. The range of possible values is 0 to
1000 mAU (or 1 AU). In a sample with high background or noise, you might
start with a value for scan threshold of 40 mAU.

Peak Coverage
The Peak Coverage text box allows you to specify a maximum percent value
of the width of the integrated peak. A Peak Purity computation using peak
coverage starts with the scan at the apex of the peak and collects wavelength
data from scans on both sides of the apex until the percent peak coverage is
reached. Use peak coverage for symmetrical peaks. The default value for peak
coverage is 95% of the integrated peak width.

Limit Scan Wavelength


The Limit Scan Wavelength check box allows you to enable or disable the
wavelength range text box. Select the check box if you want to limit the
number of wavelengths to include in the Peak Purity computation. Then enter
a range in the Wavelength Range text box.
The Wavelength Range text box allows you to specify a range of UV scans (in
nanometers) that includes the wavelengths of your peak(s) of interest. A Peak
Purity computation using wavelength range starts with the scan at the apex of
a peak and collects wavelength data from scans on both sides of the apex until
all the wavelengths in the range are included. Use wavelength range for either
symmetrical or asymmetrical peaks. The default wavelength range is the full
width of the scan.

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Quantitative Processing
Finnigan Xcalibur __________________________________________________________________ Reports

2.10 Reports
Xcalibur’s automated reporting creates comprehensive, high quality, printed
documentation. Xcalibur’s Merlin reporting package uses Microsoft Word
to create report templates, utilizing a palette of Report Objects for insertion at
any point in a page. You can customize reports to suit your own requirements
using Merlin. Reports are specified in the Reports view. (See Figure 2-22.)
The Reports view of Processing Setup lists:
Sample reports The reports issued for each sample.
Summary The summary reports issued after processing of a
reports sequence quantitation bracket (or non-bracketed
sequence).

Xcalibur is equipped with a number of standard templates.

Figure 2-22. The Reports view in Processing Setup

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ELECTRON CORPORATION
Quantitative Processing
Reports _____________________________________________________________ Finnigan Xcalibur

Sample Reports
The Sample Reports list consists of seven columns:
Enable Enable or disable report.
Std Enable to produce report for a Standard sample type.
QC Enable to produce report for a QC sample type.
Unk Enable to produce report for Unknown sample type.
Other Enable to produce report for all other sample types.
Save As Select a report export option. Xcalibur saves the
exported file with the sample file name and the
appropriate extension in the Data folder where result
files are stored. Valid export file types are:
Noneprint only, no exported file
TextASCII text file (*.txt), no printed copy
DocWord XP file (*.doc), no printed copy
HTMLHTML file, (*.html), no printed copy
Report Template Enter the full pathname of the template to be used by
Name Xcalibur in the generation of the report.

When you have specified a Report Template Name you


can invoke Merlin to edit the report template. To do
this:
1. Click on the cell
2. Right-click to display the shortcut menu
3. Choose Open from the shortcut menu.

You can specify a Report Template Name in three ways:


• Click on the cell and type the full path and filename.
• Double click on the cell and browse to the file.
• Click on the cell first, then right click on the cell and select Browse from
the shortcut menu.
To change any of the report ‘sample type’ fields (Enable, Std, QC, Unk, or
Other) click on the appropriate cell to display a check box. Enable or disable
the option as required.
A shortcut menu is available within the grid. Right click within a row to
access additional commands to:
• Open Merlin to edit a report template
• Delete the selected row or rows
• Insert a row above the selected row or rows

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Finnigan Xcalibur __________________________________________________________________ Reports

Summary Reports
The summary reports list contains three fields:
Enable Enable or disable report.
Save As Select a report export option. Xcalibur saves the
exported file with the file name and the appropriate
extension in the Data folder where result files are
stored. Valid export file types are:
None print only, no exported file
Text ASCII text file (*.txt), no printed copy
Doc Word XP file (*.doc), no printed copy
HTML HTML file, (*.html), no printed copy
Report Template Enter the full pathname of the template to be used by
Name Xcalibur in the generation of the report.
When you have specified a Report Template Name you
can invoke the GRD to edit the report template. To do
this:
1. Click on the cell
2. Right-click to display the shortcut menu
3. Choose Open from the shortcut menu.

You can edit cells and rows in the same manner as described above, in the
topic Sample Reports.

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ELECTRON CORPORATION
Quantitative Processing
Programs ____________________________________________________________ Finnigan Xcalibur

2.11 Programs
The Programs view of Processing Setup (Figure 2-23) allows you to list
programs or macros to be run by Xcalibur after the analysis of a sample and
the processing of the resulting data. Xcalibur runs the programs in the listed
order.

Figure 2-23. The Programs view in Processing Setup

Programs are defined by nine headings:


Enable Determines whether Xcalibur runs the specified
program during post-processing.
Std Determines whether Xcalibur runs a program after a
Standard sample analysis.
QC Determines whether Xcalibur runs a program after a
QC sample analysis.
Unk Determines whether Xcalibur runs the program after an
Unknown sample analysis.
Other Determines whether Xcalibur runs the program after
any other type of sample analysis.
Action Displays Run Program or Run Microsoft Excel Macro
options.
Program or Lists the full pathname of the program or Excel macro
Macro Name

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Finnigan Xcalibur _________________________________________________________________Programs

Sync Determines whether the selected program is to be run


synchronously. If you select Yes, Xcalibur waits for the
program to terminate before starting any other
processing task. If you select No, Xcalibur continues
with other processing tasks without waiting for the
program to terminate.
Parameters Specifies any command parameters for the selected
program. You can use the following macro parameters
in the Parameters column (see Table 2-5).

Table 2-5. Example Program Parameters

Macro Macro Parameter Replacement


Parameters
%R Provides the current raw file
%F Provides the current result file
%% Provides a single % character in the run line
%X Provides the result file name with extension .xls.

To specify a Program or Macro:


1. Click on the Enable field of the appropriate row. Then:
• Click on the Program or Macro Name cell and type the full path
name, or
• Double click on the cell to identify the program using a standard
Browse dialog box, or
• Click on the cell first, then right click on it and select Browse from
the shortcut menu
2. To change any of the program sample type fields (Std, QC, Unk, or Other)
click on the appropriate cell to access a checkbox: enable or disable the
option as required.
A shortcut menu is available within the grid. Right click within a row to
access additional commands to:
• Browse to a program or macro file (enabled only when a Program or
Macro Name cell has been selected)
• Delete the selected row or rows
• Insert a row above the selected row or rows

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ELECTRON CORPORATION
Chapter 3
Automating Analysis

This chapter describes Sequence Setup and explains how to set up and use a
sequence for automated quantitative analysis.
The topics in this chapter are as follows:
• Sequence Setup
• The Sequence Setup Window
• About Sequences
• Creating a New Sequence
• Working with a Sequence
• Running Samples
• Reprocessing Samples
• The Acquisition Queue

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Automating Analysis
Sequence Setup_______________________________________________________ Finnigan Xcalibur

3.1 Sequence Setup


You can automate data acquisition or reprocessing using Sequence Setup.
Sequence Setup allows you to compile a sequential list containing a variety of
sample types (Figure 3-1). Each row of the list corresponds to one sample
injection. Sequences can be bracketed (open, non-overlapped, or overlapped)
to accommodate calibration standards. From within Sequence Setup you can
run a single sample, or complete sequence, or reprocess a batch of previously
acquired raw data files. During acquisition Xcalibur controls your
instrumentation using information from the sequence.
The Acquisition Queue page allows you to control and prioritize sequences.
Each time you select Processing options in Sequence Setup Xcalibur also
starts a process queue service in the background. When Xcalibur finishes an
acquisition, it sends the data to the process queue for processing. Xcalibur
processes samples and sequences using a first-in first-out queue priority. You
can:
• Pause and resume processing
• Delete acquisitions or purge the entire queue
• Re-order and prioritize sequences and samples
• Obtain information about processing
This chapter specifically describes how to set up and use a sequence for
automated quantitative analysis. The companion manual Finnigan Xcalibur
Getting Productive: Qualitative Analysis describes how to set up and use a
sequence solely for automated qualitative analysis. Xcalibur permits both to
be performed simultaneously and, if you want to do this, you should consult
both documents.

Figure 3-1. A simple sequence for the quantitative analysis of five samples

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur _________________________________________________The Sequence Setup Window

3.2 The Sequence Setup Window


Sequence Setup is one of the view options on Xcalibur’s Home Page. To
select Sequence Setup, display the Home Page and then:
Choose View > Sequence Setup View, or
Click on the Sequence View button on the View toolbar. If this is not
displayed, choose View > View Toolbar.
Home Page has four toolbars:
View Select Home Page view options.
Road Map Tools for controlling acquisitions and for accessing
Instrument Setup and Processing Setup.
Sequence Tools for editing sequences in Sequence Setup View.
Editor
Plot Tools for controlling real time plots during data
acquisition.

Within Sequence Setup, you are recommended to display the View and
Sequence Editor toolbars. Display or hide a toolbar by choosing the
appropriate View menu command.
For details about Road Map and Plot toolbars and more information about the
use of Home Page, refer to your Getting Started manual.
For specific information about Home Page’s toolbar buttons or menu
commands refer to Xcalibur’s online Help.

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Automating Analysis
About Sequences ______________________________________________________ Finnigan Xcalibur

3.3 About Sequences


Each row in the sequence table describes a single sample acquisition. In
quantitation, a sequence can contain standards, QC samples, blanks and
unknowns in a number of formats.
Sequence rows may contain many parameters described by the column
headings. You can choose which columns are displayed or hidden (refer to
Arranging the Columns).
The following list describes the parameters that define a sample acquisition.
Sample Type Type of sample, selected from the following:
Unknown
Blank
QC (quality control)
Standard Clear
Standard Update
Start Bracket
End Bracket
Standard Bracket

File Name Name of the file to contain the sample data.


Sample ID An identifier unique to the sample. This field can also
be used to import a barcode identifier.
Path The path to the raw file that Xcalibur creates for the
sample data. Xcalibur creates this file with extension
.raw.
Inst Meth The path and file name of the Instrument Method to be
used for acquisition.
Proc Meth The path and file name of the processing method to be
used to process the acquired data.
Position The sample’s vial number. The format of the entry
depends on the configured autosampler, for example,
Surveyor AS could have C:C2 or A:B5.
Inj Vol The volume of sample to be injected in microliters.
Dil Factor Dilution factor used to prepare the sample.
Level The level, if defined, for sequence rows corresponding
to Calibration or QC samples.
ISTD Corr Amt The amount of internal standard present in the sample.

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Finnigan Xcalibur __________________________________________________________ About Sequences

Study User-defined topic with a default heading of Study.


Client User-defined topic with a default heading of Client.
Laboratory User-defined topic with a default heading of
Laboratory.
Company User-defined topic with a default heading of Company.
Phone User-defined topic with a default heading of Phone.
Comment An additional field for any other information about the
sample or analysis procedure.
Sample Name Text description of the sample.
Sample Wt A reporting feature (not used in quantitation
calculations).
Sample Vol A reporting feature (not used in quantitation
calculations).

To open an existing sequence, click on the Open toolbar button or choose


File > Open.
To start a new sequence, click on the New toolbar button or choose
File > New. The New Sequence Template dialog box is opened.
To save the current sequence, click on the Save toolbar button or choose
File > Save.

Arranging the Columns


To change the arrangement of columns in Sequence Setup:
1. Choose Change > Columns or click on the Column Arrangement
toolbar button to open the Column Arrangement dialog box
(Figure 3-2). The columns currently displayed are listed in the Displayed
Columns pane in the order that they appear.
2. If you want to display a column that is currently hidden:
a. Select the column heading in the Available Columns list.
b. Click on the Add button.
The column title is moved from the Available Columns list to the
Displayed Columns list.
3. If you want to hide a column that is currently displayed:
a. Select the column heading in the Displayed Columns list.
b. Click on the Remove button.
The column title is moved from the Displayed Columns list to the
Available Columns list.

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About Sequences ______________________________________________________ Finnigan Xcalibur

4. If you want to change the order of the Displayed Columns:


a. Select the column heading you want to move.
b. Click on the Move Up button or the Move Down button.
Repeat the procedure with other columns.
Click on OK to save the changes and close the dialog box. Xcalibur displays
the columns in Sequence Setup in the new arrangement.

Figure 3-2. The Column Arrangement dialog box

Changing User Labels


You can define the caption labels of the five user defined columns.
To change a heading caption in the User Labels dialog box (Figure 3-3):
1. Select Change > User Labels or click on the User Labels button on the
toolbar.
2. Enter the new heading caption in the heading text box to replace the
current heading caption. If you do not want to use a heading, delete the
text and leave the text box blank.
Repeat for each of the five heading captions that you want to change.
Click on OK to save your new captions.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur __________________________________________________________ About Sequences

Figure 3-3. User Labels dialog box

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ELECTRON CORPORATION 3-7
Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur

3.4 Creating a New Sequence


There are three ways to create a new sequence. You can:
• Import a sequence from a text file
• Provide Xcalibur with some basic details and allow it to create the
sequence
• Type the sequence manually

Importing a Sequence
Xcalibur allows you to import data into some or all of the columns in a
sequence.
Xcalibur reads comma separated text files with file extension .csv. This file
format can be created by a text editor, such as Microsoft Notepad, or a
spreadsheet program, such as Microsoft Excel.
To import a sequence:
1. Choose File > Import Sequence to open the Import Sequence dialog
box (Figure 3-4).

Figure 3-4. Import Sequence dialog box

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ___________________________________________________ Creating a New Sequence

2. Use the Browse button to select the file for importing. Alternatively, type
the path and file name directly into the Import from File field.
3. In the Select Columns to Import group box, select the sequence columns
to be included in the imported file.
4. Click on All to select all the column options
5. Click on Clear to deselect all the column options
6. Click on OK to import the selected columns of the sequence you have
specified. Xcalibur displays the imported file in Sequence Setup.
Xcalibur generates an ‘invalid file’ message if you attempt to import a file:
• With an incorrect extension or file type, or
• In which the separator character is different from the character currently
set in the International dialog box (see Changing the List Separator
Character).

Letting Xcalibur Create Your Sequence


This method is particularly useful when you are running large numbers of
similar samples or when you are running bracketed, calibration, or QC
samples.
To let Xcalibur create your sequence, click on the New Sequence toolbar
button or select File > New. The New Sequence Template dialog box is
displayed (Figure 3-5).

Entering General Information


In the General group box of the New Sequence Template dialog box enter:
Base File Name Enter a base file name for the raw files. Xcalibur
applies this name to all of the raw files that it creates
using the new sequence. Xcalibur then determines an
incremental numeric suffix for the base name starting at
001. If you want to have Xcalibur start with a different
number, enter the number in the Starting Number text
box.
Path Type a path to the directory where you want Xcalibur to
store the raw files. Alternatively, use the Browse button
to locate the drive and directory.
Instrument Select an existing instrument method. Use the Browse
Method button to locate the file.
Processing Select an existing processing method. Use the Browse
Method button to locate the file.

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ELECTRON CORPORATION 3-9
Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur

Calibration File • For first time processing, enter a new name for
the calibration file. Xcalibur will create this during
processing.
• For batch reprocessing, enter a new name for the
calibration file or select an existing calibration file.
Use the Browse button to locate the file.

Figure 3-5. The New Sequence Template dialog box

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ___________________________________________________ Creating a New Sequence

Specifying Samples
In the Samples group box, you enter details about the number of samples in
the sequence and configure the autosampler.
Enter details about your samples:
Number of Enter the number of samples to be analyzed, for
Samples example, 96 for a single analysis of each sample in a
Surveyor AS tray.
Injections per Enter the number of times each sample is to be
Sample analyzed.
Base Sample ID Enter the identifier code for the sequence. The Base
Sample ID is an alphanumeric prefix to the Sample ID
that Xcalibur applies to each sample in a new sequence.
Xcalibur adds a suffix to the base sample ID starting
with 001. For example, if you enter a base sample ID of
AB12, Xcalibur numbers the first five samples as
follows:
AB12001
AB12002
AB12003
AB12004
AB12005
Select the autosampler configuration:
Tray Type Select the autosampler tray type from the drop-down
list.
Initial Vial Enter the first vial number in the tray.
Number
Re-Use Vial Select this option if you want Xcalibur to use the same
Numbers vial number for replicate samples.

The Select Vials button displays the Vial Selection dialog box (Figure 3-6).
This allows you to create a sequence of samples from individually selected
vials on any of the configured trays. Select or deselect a vial simply by
clicking on it. You can deselect all selected vials (highlighted in blue) by
clicking on the Cancel Selection button in the New Sequence Template dialog
box.

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ELECTRON CORPORATION
Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur

Figure 3-6. The Vial Selection dialog box

Note. The Select Vials feature is only available with suitable autosamplers
such as Surveyor Autosampler.

Choosing a Bracket Type


In the Bracket Type group box, select one of the calibration bracket options:
None, Open, Non-Overlapped, Overlapped.

Specifying Standards and Blanks


In the Calibration group box, select Add Standards to add calibration
samples. Then enter:
Injections per Enter the number of injections (replicates) for each QC
Level level.
Add Blanks Enable this check box to add blanks to the brackets.

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ELECTRON CORPORATION
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Finnigan Xcalibur ___________________________________________________ Creating a New Sequence

Specifying QC Samples
In the QC group box, select Add QCs if you want to add quality control
samples. Choose between the following QC options:
• After First Calibration Only
• After Every Calibration
Add Blanks Enable this option to add blanks immediately after the
QC samples.

Completing the Sequence


Click on OK to save the changes and close the New Sequence Template
dialog box. Xcalibur now generates a Sequence based on the information you
have provided.

Creating a Sequence Manually


To create a sequence manually you must define the following parameters for
each sample/row in the sequence:
Sample Type Click on the Sample Type cell and select: Unknown,
Blank, QC, Std Clear, or Std Update from the list.
File Name Enter a file name for storing the sample data.
Sample ID Enter a Sample identification number.
Path Enter the directory path where the sample’s raw file
will be stored. Alternatively, double-click in the text
box and browse to the appropriate directory using the
Select Directory dialog box.
Inst. Meth Enter the path and filename of an instrument method
file. Alternatively, double-click in the text box and
browse to the appropriate file using the Select
Directory dialog box. Instrument methods have file
extension .meth.
Proc Meth Enter the path and filename of the processing method
file. Alternatively, double-click in the text box and
browse to the appropriate file using the Select
Directory dialog box. A process method is required if
the sample type is QC, Std Clear or Std Update or if
you want to run a process on the raw file. Processing
methods have file extension .pmd.

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ELECTRON CORPORATION
Automating Analysis
Creating a New Sequence _______________________________________________ Finnigan Xcalibur

Cal File Enter the path and filename of the calibration file.
Alternatively, double-click in the text box and browse
to the appropriate file using the Select Directory dialog
box.
Position Specify the position of the vial on the autosampler tray.
Inj. Vol. Enter the injection volume (in microliters). Xcalibur
sends this volume to the syringe pump. If you do not
enter an injection volume, Xcalibur uses the default
injection volume set in the experiment method.
Level Specify a level if the sample type is QC, Std clear, or
Std Update (you need to create and select a processing
method with Calibration or QC levels before you can
select a level). Double-click in the Level text box to
open the Select Level dialog box. Select a level and
click on OK.
ISTD Corr Amt Enter the correct amount of internal standard used, if
the internal standard amount injected into the sample is
different from the amount specified in the active
Processing Setup. The internal standard response will
then be corrected by the ratio of specified/actual.
Dil Factor Enter the sample dilution factor.

The remaining columns:


• User Labels 1-5 (On installation these have defaults: Study, Client,
Laboratory, Company, Phone)
• Comments
• Sample Name
• Sample Weight
• Sample Volume
are simple text fields used for reporting purposes. The columns may be
hidden, refer to Arranging the Columns. They are not essential for the
running of a sample or sequence.
To enter text information under any of these column headings:
1. Click on the relevant grid cell.
2. Type the required information.
3. Click on any other cell.
Repeat these procedures for all samples/rows in the sequence. To save time in
duplicating column entries use the Fill Down command or toolbar button
(refer to Filling Down Columns).

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ____________________________________________________ Working with a Sequence

3.5 Working with a Sequence


The Sequence Setup is equipped with a number of tools and commands to
assist you in compiling a sequence.

Filling Down Columns


The Fill Down command allows you to copy information from one row to any
number of rows immediately below it in the Sequence table. You can copy
information from a single cell or a complete row.
To fill down sample settings:
1. Select the cells in the row you want to copy.
2. Drag downwards to select the range of columns to be filled (you can edit
the selection in step 4). You must select at least one row to enable the
command.
3. Choose Edit > Fill Down or click on the Fill Down button in the toolbar.
The Fill Down dialog box is displayed (Figure 3-7).

Figure 3-7. Fill Down dialog box

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ELECTRON CORPORATION
Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur

4. Xcalibur identifies the first selected row as the one to copied, and all
subsequent selected rows as targets for the Fill Down operation. Check
the extent of the range to be filled:
Fill Rows Y to If required, type in a new value for Row Z, the last row
Z using to be filled with Row X duplicates. If X is incorrect,
Row X click on the Cancel button to close the dialog box and
repeat the procedure from step 1.

5. Choose the columns to be copied down by checking the relevant boxes.


• Click on All to select all the column check boxes
• Click on Clear to deselect all the column check boxes
6. Click on OK to close the dialog box and execute the Fill Down command.
Xcalibur copies appropriate information from the first row into the
selected range.

Inserting a Row
To insert a row:
1. Select the row immediately below where you want to insert a row.
2. Choose Edit > Insert Row. A dialog box now asks for confirmation of
the action Insert above line x?.
3. Click on the Yes button.
4. The inserted row is a copy of the row immediately prior to the row
selected in step 1.

Deleting a Row
To delete a row:
1. Select the row you want to delete
2. Choose Edit > Delete Row. A dialog box now asks for confirmation of
the action Delete line x?.
3. Click on the Yes button.

Going to a Sequence Row


To go to a specified row in the current sequence:
1. Select Edit > Go To Row.
2. Enter a valid row number in the Go To Line Number dialog box
(Figure 3-8).

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ____________________________________________________ Working with a Sequence

3. Click on OK.
4. Xcalibur closes the dialog box and highlights the selected row.

Figure 3-8. Go To Line Number dialog box

Transferring Row Information


This procedure allows you to ensure that all occurrences of a particular
Sample ID or Position have the same parameters. Xcalibur copies the
parameters from the first row featuring a Sample ID or Position to all other
rows in the sequence with the same Sample ID or Position.
1. Choose Change > Transfer Row Information or click on the
Transfer Row Info toolbar button.
2. Select from the following options in the Transfer Row Information dialog
box (Figure 3-9):
Match by Select this option if you want to copy the parameters
Sample ID from the first sequence row with a particular Sample ID
to all other sample rows with the same Sample ID.
Match by Select this option if you want to copy the parameters
Position from the first sequence row with a particular Position to
all other sample rows with the same Position.

3. Click on OK to close the dialog box.


Xcalibur performs the selected copy operation.
To undo the copy operation, immediately choose Edit > Undo or click on the
Undo button in the toolbar.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-17
ELECTRON CORPORATION
Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur

Figure 3-9. Transfer Row Information dialog box

Printing a Sequence
You can print a full sequence or a vial list compiled from the active sequence.
To preview the appearance of the Sequence before printing:
1. Choose File > Print Preview. The Print Selection dialog box is
displayed (Figure 3-10).
2. Select either:
Vial Position List To review the vial list from the active sequence
Full Sequence To preview the active sequence.

3. Click on OK to open the Print Preview dialog box (Figure 3-11).


4. Use Next Page, Previous Page, Two Page, Zoom In or Zoom Out
to preview the active list pages.
5. Select:
Close To return to Sequence Setup
Print To print the displayed list.

To print the Sequence without preview:


1. Choose File > Print or click on the Print toolbar button The Print
Selection dialog box is displayed (Figure 3-10).
2. Select one of the following print output options:
Vial Position List To print the vial list from the active sequence.
Full Sequence To print the active sequence.

3. Click on OK to open the Print dialog box.


4. Complete the printer settings and click on OK to print the selected list.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ____________________________________________________ Working with a Sequence

Refer to the Xcalibur online Help for a complete description of all controls
contained in the Print dialog box.

Figure 3-10. Print Selection dialog box

Figure 3-11. Print Preview dialog box

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-19
ELECTRON CORPORATION
Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur

Checking Disk Space


A Sequence can generate a large number of raw files. Sequence Setup
provides a simple utility for you to check the amount of available disk space
on system drive(s):
1. Choose Actions > Check Disk Space or click on the Disk Space
button on the toolbar. The Disk Space dialog box is displayed
(Figure 3-12). This shows:
• The current drive and directory. For example:
C:\Xcalibur\SYSTEM\PROGRAMS
• The number of MB that are available (free) on the current drive and
the percentage of the total capacity of the drive that is available. For
example: 214 MBytes (17.6%) Free
• A pie-chart in which the available space is shown in the color green
and the used space is shown in the color red
• The total capacity of the current drive, for example: 1220 MBytes
Total.
2. Use the Directory button to check disk space on another disk.
3. Click on OK to close the dialog box.

Figure 3-12. Disk Space dialog box

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ____________________________________________________ Working with a Sequence

Exporting a Sequence
You can export a Sequence as a separator delimited text file with a file
extension .csv. This file format can be read by a text editor, such as Microsoft
Notepad, or a spreadsheet program, such as Microsoft Excel.
The exported sequence file contains the current list separator character
(normally a comma) that is set in the Microsoft Windows dialog box (refer to
Changing the List Separator Character).
To export a sequence:
1. Choose File > Export Sequence. The Export Sequence dialog box is
displayed (Figure 3-13).
2. Enter the path and file name of the exported sequence file in the Export
To File text box. The file extension should be .csv Alternatively, you may
use the Browse button to select a path for the exported sequence file.
Xcalibur assigns extension .csv to the exported file.
3. Use the options in the Export Sequence group box to select the sequence
columns to be included in the exported file.
• Click on All to select all the column options
• Click on Clear to deselect all the column options
4. Click on OK to export the selected columns of the active sequence to the
specified file and location.

Figure 3-13. Export Sequence dialog box

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-21
ELECTRON CORPORATION
Automating Analysis
Working with a Sequence ________________________________________________ Finnigan Xcalibur

Changing the List Separator Character


When you export a sequence, Xcalibur creates a text file with file extension
.csv and inserts a list separator character between each field of each column of
the sequence. This file format can be read by a text editor, such as Microsoft
Notepad, or a spreadsheet program, such as Microsoft Excel.
The list separator may be any alphanumeric character. However, characters
that cannot be distinguished from the characters used in the sequence text
fields (such as alphabetic characters) should be avoided because they result in
unreadable (invalid) files. The most common list separators are the comma (,)
and the semicolon (;). Each country has a default list separator. For example,
the default list separator for United States is the comma.
When you import a sequence, the list separator character used in a sequence
file to be imported must be the same as that specified in the Microsoft
Windows XP Professional operating system.
To change the list separator character:
1. In the Windows Taskbar, click on the Start button and choose Settings >
Control Panel.
2. Double-click on the Regional and Language Options icon.
3. Click on the Regional Options tab.
4. Click on Customize to open the Customize Regional Options
dialog box.
5. Enter the new list separator character into the List Separator combo box.
6. Click on OK to store the new list separator and close the dialog box.
7. Click on OK to close the Customize Regional Options dialog box.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur __________________________________________________________ Running Samples

3.6 Running Samples


You can run a single sample from a sequence, a range of samples or the full
sequence.

Running a Single Sample


To run a single sample from the current sequence:
1. Select the sample you want to run by clicking on its row number. Xcalibur
highlights the row. If you do not select a sequence row, Xcalibur will
assume that you want to run Sample 1, by default.
2. Choose Actions > Run This Sample or click on the Run Sample
toolbar button.
The Run Sequence dialog box is then displayed. Refer to Setting up the Run.

Running a Sequence
To run a sequence:
1. Highlight the samples you want to run. Click on the left-most column of
the first sample and drag to the last sample you wish to run.
2. Choose Actions > Run Sequence or click on the Run Sequence
toolbar button.
3. The Run Sequence dialog box is then displayed. Refer to Setting up the
Run below.

Setting up the Run


The Run Sequence dialog box (Figure 3-14) allows you to:
• Identify the range of samples for analysis from the current list
• Configure instruments to be used in the run
• Run instrument start up methods before the sequence is initiated
• Run instrument shutdown methods when the sequence is complete
• Execute programs before and/or after each sample acquisition
• Prioritize the sequence so that it is positioned at the head of the
Acquisition Queue
• Select processing and reporting options

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-23
ELECTRON CORPORATION
Automating Analysis
Running Samples ______________________________________________________ Finnigan Xcalibur

Figure 3-14. Run Sequence dialog box

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur __________________________________________________________ Running Samples

Setting General Run Options


Run Rows Check the Run Rows information. If it is incorrect,
either:
• Type in the correct range, or
• Click on Cancel to close the dialog box. Select a
different sample or range of samples and repeat the
procedure.
User Enter the name of the operator (up to 10 characters).
Priority Choose this option if you wish to position the sequence
Sequence or sample ahead of all others in the Acquisition Queue.
If Xcalibur is running a quantitation bracket it will
queue the priority sequence immediately after the
bracket.
Start When Choose this option if you want Xcalibur to perform an
Ready autosampler injection as soon as the system is ready. If
you want to initiate autosampler activation using the
Start Analysis command from the Home Page, ensure
that Start When Ready is disabled.

Choosing Acquisition Options


The Acquisition Options window at the top of the dialog box lists the
Instruments assigned to process the sequence:
Instrument Names of the instruments assigned to sequence
analysis. If you want to add or remove an instrument,
click on Change Instruments (refer to Changing
Instruments, below).
Start Instrument Identifies the instrument used by Xcalibur to start the
acquisition. Xcalibur assumes that the Start Instrument
controls all other active instruments, for example, via
contact closure. If no instrument is flagged as the start
device, Xcalibur expects an unlisted instrument to
provide an appropriate signal to start the acquisition.

To change the Start Instrument, click on Change


Instruments.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-25
ELECTRON CORPORATION
Automating Analysis
Running Samples ______________________________________________________ Finnigan Xcalibur

Changing Instruments
The Change Instruments button on the Run Sequence dialog box opens the
Change Instruments in Use dialog box (Figure 3-15).

Figure 3-15. Change Instruments in Use dialog box

To change the status of any instrument in the current configuration, toggle the
In Use field by clicking on it.
To change the Start Instrument assignment, toggle the Start Instrument fields
as appropriate. Only one instrument can be designated as the Start Instrument.

Selecting a Startup or Shutdown Method


Xcalibur allows you to specify instrument methods to be run before and after
the sequence (for example, for tuning or calibration).
Start Up Select an existing method to start up the instrument.
This will be run through the instrument before the first
sample is queued. Click on Browse to select the drive
and directory where the file is located.
Shut Down Select an existing method to shut down the instrument.
This will be run through the instrument after the last
sample has been analyzed. Click on Browse to select
the drive and directory where the file is located.

No data are acquired during the execution of a start up or shut down method.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur __________________________________________________________ Running Samples

Specifying Pre- and Post-Run Acquisition


Programs
Xcalibur allows you to specify programs or macros to be run before and/or
after each acquisition. They might be used, for example, to issue commands to
prepare an instrument for acquisition. This will be of particular use for
instruments that are not controlled directly by Xcalibur.
Pre Acquisition Select an existing program to run before each
acquisition. Click on Browse to select the drive and
directory where the file is located.
Post Acquisition Select an existing program to run after each acquisition.
Click on Browse to select the drive and directory where
the file is located.
Run Select Synchronous if you want Xcalibur to wait for a
Synchronously program to be completed before continuing with its
next action

Select Asynchronous if you want Xcalibur to continue


with its next action immediately after initiating the
program.

Choosing Processing Actions


Choose from the following processing and reporting options:
Quan Select this check box to carry out quantitative
processing.
Qual This check box would be selected if you also wanted to
carry out qualitative processing.
Reports Select this check box if you want to print the reports
specified in the processing method.
Programs Select this check box if you want to run the programs
and macros specified in the processing method.
Print Methods Select this check box if you want to print the methods
used to process the sample(s).
Create Select this option if you want to print a summary report
Summary for the sample(s).

Click on OK to save the settings and close the dialog box. Xcalibur then
places the selected sample(s) in the run queue or starts processing
immediately.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-27
ELECTRON CORPORATION
Automating Analysis
Reprocessing Samples __________________________________________________ Finnigan Xcalibur

3.7 Reprocessing Samples


To reprocess a batch of samples:
1. Select the rows to be reprocessed from the current sequence.
(Alternatively, you can specify the row numbers using the Process Rows
text box in the Batch Reprocess Setup dialog box – see below). Xcalibur
highlights the selected rows.
2. Choose Actions > Batch Reprocess or click on the Batch Reprocess
toolbar button to display the Batch Reprocess Setup dialog box
(Figure 3-16).

Figure 3-16. Batch Process Setup dialog box

1. Check the Process Rows information. If it is incorrect:


Click on Cancel to close the dialog box. Select a different sample or
range of samples and repeat the procedure.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ______________________________________________________ Reprocessing Samples

Alternatively, type in the correct range. The format is either [Row] for one
sample or [First Row - Last Row] for multiple samples.
2. Select the Quan check box to reprocess quantitative data. Select the
following quantitative processing options:
Peak Select this check box if you want to generate new peak
Detection & detection and integration data.
Integration
Calibration Select this check box if you want to carry out new
calibration calculations using the sequence’s standards.
Quantitation Select this check box if you want to re-calculate the
quantitation data for unknown samples in the sequence.

3. Select the Reports check box if you want to print new reports.
Print Sample Select this check box if you want to generate new
Reports sample reports, based on those listed in the processing
method(s).
Print Select this check box if you want to generate new
Summary summary reports, based on those listed in the
Reports processing method(s).

4. Select the Programs check box if you want to run the post-processing
programs or macros, based on those listed in the processing method(s).
5. Select the Print Methods check box if you want to print the Experiment
and Processing Methods used during batch reprocessing.
6. Select the Create Quan Summary Spreadsheet option if you want
Xcalibur to generate a summary spreadsheet for the reprocessed
sequence.
7. Select the Advanced Options – Replace Sample Info check box if you
want to replace the sample information generated during data acquisition
in the sample headers with new information generated during
reprocessing.
8. Click on OK. Xcalibur initiates batch reprocessing of the selected
samples.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-29
ELECTRON CORPORATION
Automating Analysis
The Acquisition Queue __________________________________________________ Finnigan Xcalibur

3.8 The Acquisition Queue


The Acquisition Queue (Figure 3-17) shows all the sequences and samples
submitted for analysis. The Explorer style tree view shows two levels of
detail: the sequence names and, within each branch, the raw sample
filenames.
You can use the Acquisition Queue to:
• Delete sequences (unless they are currently being run).
• Delete samples within a sequence (unless they have already been
acquired, are currently undergoing acquisition, or are part of the
quantitation bracket currently being acquired).

Figure 3-17. The Acquisition Queue with the Sample Information window displayed

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ______________________________________________________ The Acquisition Queue

You can manipulate entries in the acquisition queue:


• Right-click on the name of the sequence or sample to view the shortcut
menu. This contains a single command: Properties. When selected, this
displays the Sample Information window.
• Double-click on a sequence to load it into Sequence Setup.
• Double-click on a sample to open the Sample Information window.
A check box is displayed alongside each sequence and sample. These allow
you to select one or more items for deletion.
To delete a sample or sequence from the queue, select its check box and then
press the Delete key.
Deleted samples are identified by a large cross in the check box. Xcalibur also
appends the word ‘DELETED’ to the sample or sequence identifier.

Sample Information Dialog Box


The Sample Information dialog box shows the parameters for all the sequence
fields (refer to About Sequences for descriptions of all the fields).
The Sample Information dialog box closes if you click anywhere outside it.
Click on the pin icon if you want to keep it open. You must then click on the
close icon to close the dialog box, or unpin the dialog box (by clicking on the
pin icon again) and click anywhere outside the dialog box.
A pinned dialog box is updated with the details of any selected sequence.

Managing Tasks
Queue Manager (Figure 3-18) provides additional functions for managing
queued tasks. It is active whenever samples or sequences are queued for
reprocessing. If it is not visible, it may be minimized to the Windows toolbar.

Figure 3-18. Queue Manager window

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-31
ELECTRON CORPORATION
Automating Analysis
The Acquisition Queue __________________________________________________ Finnigan Xcalibur

Use the following procedures to manage the Xcalibur Processing Queue:

Temporarily Pause the Processing Queue


Click on the Pause button in the toolbar.
Alternatively, choose Queue > Pause.

Resume the Processing Queue (when it is in Pause mode)


Click on the Resume button in the toolbar.
Alternatively, choose Queue > Resume.

Update the Display with the Latest Information


Choose View > Refresh.

Remove a Task from the Queue


1. Select the task to be removed.
2. Click on the Remove Job button in the toolbar. Alternatively choose
Analysis > Remove From Queue from Queue.

Remove all Tasks from the Queue


Choose Queue > Purge Queue.

View Details of Selected Analysis


1. Select the required analysis in Queue Manager
2. Click on the Details button in the toolbar.
3. Alternatively, choose Analysis > Details.
The Details of Selected Analysis dialog box is then displayed
(Figure 3-19). This shows:
File The sample’s filename.
Status The current queue status.
Submitted The time and date the job was submitted.
From The source of the job.
Actions Lists the tasks required to complete the selected job and
their current status.

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ELECTRON CORPORATION
Automating Analysis
Finnigan Xcalibur ______________________________________________________ The Acquisition Queue

Figure 3-19. Details of Selected Analysis dialog box

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ___________ 3-33
ELECTRON CORPORATION
Chapter 4
Reviewing Quantitation

This chapter describes the underlying principles of Quan Browser and


explains how to use it for reviewing and reworking sequences, calibration
curves and quantitation.
The topics in this chapter are as follows:
• Results Review
• About Quan Browser
• The Quan Browser Window
• The Results Grid
• Chromatogram View
• Calibration Companion View
• Spectrum Companion View
• Reports
• Quan Browser Procedures

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ____________


ELECTRON CORPORATION 4-1
Reviewing Quantitation
Results Review _______________________________________________________ Finnigan Xcalibur

4.1 Results Review


Xcalibur’s data reviewing component is called Results Review (Figure 4-1).
This is organized into three core Browsers.

Figure 4-1. Results Review section of Xcalibur’s home page

The core Browsers are:


Qual Browser Allows you to display and manipulate chromatograms
and spectra, activate library searches and view
qualitative processing results. Qual Browser is
described in Finnigan Xcalibur Getting Productive:
Qualitative Analysis.
Quan Browser Allows the peak integration and calibration curve
results of a Processing Method’s Quantitative
processing to be displayed and manipulated.
Library Browser Activates the NIST library utility to match spectra to
library entries. Also used to generate and manipulate
user libraries Library Browser is described in Finnigan
Xcalibur Getting Productive: Qualitative Analysis.

This chapter describes Quan Browser and its use for analyzing processed
quantitation sequences. The chapter explains the properties and uses of each
component within the Quan Browser window. It also describes how you can
use Quan Browser to achieve calibration and quantitation reviewing tasks.

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ELECTRON CORPORATION
Reviewing Quantitation
Finnigan Xcalibur ________________________________________________________About Quan Browser

4.2 About Quan Browser


Quan Browser is a powerful and versatile utility for reviewing and reworking:
• Component peak identification and integration criteria
• Standards, QCs, blanks and unknowns
• Calibration curves for quantitation standards
If you make any changes, you can save the new results with an audit trail
describing the reason for the change.
Quan Browser incorporates a calibration curve display, peak integration, and
results view that allows you to:
• Reprocess quantitation sequences
• Interactively edit processing parameters and audit the changes for XQN.
• Create new files that keep track of processing results for individual raw
files and include a copy of the method used to generate the results.
Result files changed using Quan Browser do not affect the original processing
method.

How Quan Browser Works


Quan Browser helps you step through a sequence and review the results for
each component in each file. The Quan Browser assists you to quickly review
component peak identification and integration criteria. If you make any
changes, you can save the new results with an audit trail describing the reason
for the change. Result files changed using the Quan Browser do not affect the
original processing method. Only Processing Setup can edit processing
methods and only the Quan Browser can edit result files.

Calibration Replicates
Calibration replicates are multiple injections of the calibration mixture at the
same calibration level or amount. These standard samples all contain the same
amount of target compound and therefore correspond to the same calibration
level. Xcalibur allows you to choose which replicates to include or exclude
from the calibration curve by using the Calibration Companion View.

Named Calibration File


When you create a sequence with Bracket Type set to None you can specify a
calibration file name in the Cal File field. Although in theory it is possible to
have a different calibration file name for every sample, in practice it is usual
to have only one name per sequence.
Named calibration files are not available with bracketed sequences.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ____________


ELECTRON CORPORATION 4-3
Reviewing Quantitation
About Quan Browser ___________________________________________________ Finnigan Xcalibur

Non-Bracketed Sequence
Xcalibur processes a non-bracketed sequence using a procedure known as the
continuing calibration method. Each time it processes a non-bracketed
sequence it creates or updates the calibration file(s) named in the sequence.
In this way and by avoiding the use of Std Clear, a calibration file can have
replicate data incrementally added to it over any desired period of time,
without the existing, ‘historical’ replicate data being discarded.
Quan Browser breaks down non-bracketed sequences into logical groups,
which are somewhat analogous to brackets. It does this by first ordering the
samples chronologically (with respect to acquisition date and time) and then it
examines the sequence and starts a new group whenever it encounters a
standard. After a non-standard sample has then subsequently been found, the
group ends at the non-standard immediately preceding the next standard
found.
Note that, the first group will always start with the first sample, even if it is
not a standard, and the last group will always end with the last sample.
Further, a Std Clear always starts a new group, even if no intervening
non-standard has been found following one or more Std Updates.

Note. Additional logical groups will be formed, for the situations where
different named calibration files have been specified in the Cal File entries
of the sequence; where each change of name causes a new group to be
formed As the use of multiple named calibration files is not the normal
operation, its usage will not be considered any further in this document, but
should be deducible from the discussions on groups.

As each group is processed, its samples are quantitated against the current
calibration curve. As each standard is encountered, it is processed and either
replaces (sample type set to Std Clear) or adds to (sample type set to Std
Update) the calibration replicate list and a new, calibration curve is generated.
Quan Browser processing closely emulates that of batch processing (either
that directly after acquisition, or subsequently as a batch re-process
operation). However, it has the additional capability that, if a cal file is
specified but cannot be found nor opened by Quan Browser, the message Cal
File Unavailable - Using Embedded Calibration will appear in the
Calibration File edit box and replicate data will be taken from that stored in
the result file. In most cases this will be identical to that contained within the
original calibration file anyway.
Once Quan Browser has set up the groups, they are independent and are
effectively treated as brackets. In other words, changes in one group do not
affect any other group, unlike in batch processing where subsequent groups
may well be affected.

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ELECTRON CORPORATION
Reviewing Quantitation
Finnigan Xcalibur ________________________________________________________About Quan Browser

The following list illustrates the procedure (for a single named calibration
file):

Sample 1 Unknown Group 1 start


Sample 2 Unknown Group 1 end
Sample 3 Std Clear Group 2 start
Sample 4 Unknown Group 2 end
Sample 5 Std Update Group 3 start
Sample 6 Std Update
Sample 7 Unknown
Sample 8 Unknown
Sample 9 Blank
Sample 10 QC Group 3 end
Sample 11 Std Update Group 4 start/end
Sample 12 Std Clear Group 5 start
Sample 13 Blank Group 5 end

Open Bracket Sequence


The replicate list is created directly from all standard samples in the sequence;
that is, without utilizing any calibration data embedded in result files.

Note. If you open Quan Browser with a single Result file it is treated as a
sequence with only one entry and is brought in as an unknown. In order to
show the calibration curve used to quantitate the sample, the replicate list is
created from the embedded information.

Non-Overlapping Bracket Sequence


Quan Browser creates a separate replicate list for each bracket. Each replicate
list is created directly from all standard samples in the bracket; that is, without
utilizing any calibration data embedded in result files.

Overlapping Bracket Sequence


Generally, Quan Browser creates a separate replicate list for each bracket.
Each replicate list is created directly from all standard samples in the bracket;
that is, without utilizing any calibration data embedded in result files.

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ____________


ELECTRON CORPORATION 4-5
Reviewing Quantitation
About Quan Browser ___________________________________________________ Finnigan Xcalibur

Exceptions occur for shared standard samples between brackets. If a standard


that is shared undergoes a change, that change is reflected in all brackets that
contain that sample. If a shared standard is deleted, it is deleted in all brackets
that contain that sample and the replicate lists for all brackets are adjusted.
You may add a sample to any bracket. If it is added as a standard, it will be
added to the replicate list automatically. To add a sample as a shared sample
you must add it separately to each bracket.
The exclusion status of the replicates is independent for each bracket. Even
shared samples may be excluded in one bracket but not another. This is the
only exception to a shared sample having identical settings.

Getting Started in Quan Browser


To start Quan Browser:
• Click on the Quan Browser icon on the Home Page
• Choose GoTo > Quan Browser from Processing Setup or Qual
Browser.
At startup, Quan Browser displays an Open dialog for you to select an
existing file. Supported file types are:
• Sequence files (.SLD)
• Result files (.RST)
• Quan Browser files (.XQN)
Quan Browser closes if you click on the Cancel button.
Quan Browser handles result files as single entry sequences.
If you select a sequence file, Xcalibur checks that all the associated raw and
result files are available. If it encounters a problem with the sequence file,
Xcalibur informs you of the likely cause in a warning dialog box. You will
then be asked to exit the application or select a different file.
After verifying that the files exist and can be opened, Xcalibur displays the
View Sample Types dialog box (Figure 4-2). This offers the following
Viewing Options:
• Show standard and QC sample types
• Show all sample types
These options determine how the Result Grid is configured at startup. The
dialog box includes a Don’t Ask Again check box. If you select this check
box, the dialog box is not displayed when you start subsequent sessions in
Quan Browser and the current selection is used by default.

Note. You can re-enable this, and all other Don’t Ask Again type dialog
boxes by choosing Options > Enable Warnings.

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Figure 4-2. View Sample Types dialog box

Click on OK to start the session. Quan Browser now loads the specified
sequence or file and configures the Results Grid using your selected viewing
option.

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4.3 The Quan Browser Window


The Quan Browser window (Figure 4-3) incorporates the following:
1. Title Bar
2. Toolbar
3. Menu Bar
4. Component list
5. Results Grid
6. Chromatogram View
7. Companion View

1.Title Bar 3. Menu Bar 2. Toolbar 4. Component List

5. Results Grid

7. Companion View

6. Chromatogram
View

Figure 4-3. Quan Browser in action

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The Title Bar


The title bar lists:
• The application name – Quan Browser
• The active view (Browser or Report)
• The name of the opened sequence, result or Quan Browser file
• Additional information: the Bracket in use and the viewing preference.
The bracket information will be labeled as Bracket x. The viewing
preference will be labeled as View Stds and QCs or View All.
In Figure 4-3, the title bar display is:
Quan Browser – Browser – L1908_q3.sld (Bracket 1, View All)

The Toolbar and Menu Bar


Toolbar buttons are available on the dockable toolbar. The toolbar can be
customized so that you can design a toolbar with buttons for your preferred
commands.
The default layout is shown below:

Open Display the Open dialog box to select a different file.


You will be prompted to save all changes to the current
document. The supported file types are Sequence list
(*.SLD), Result (*.RST) and QuanBrowser (*.XQN)
files.
Save Create an Xcalibur Quan File (*.XQN) from the current
Quan Browser data. This file contains all the necessary
information needed to recreate the current session.
Calibration Set the companion view to display the calibration curve
Companion for the currently selected bracket.
View
Spectrum Set the companion view to display the spectrum plot.
Companion Xcalibur initially displays the spectrum at the apex of
View the current peak in the chromatogram view.
Reports Opens the Reports dialog box. This allows you to
generate sample or summary reports for selected
samples or the entire sequence.

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Manual Noise Click on the Manual Noise Region button and then drag
Region the cursor horizontally across the region of the
chromatogram that you want to select as the noise
region. Xcalibur marks the region with a red baseline.
Xcalibur calculates noise based on the data points you
select. Xcalibur uses all selected data points as noise
points and calculates noise based on those points. You
can select the noise region from an individual trace or
different noise regions from multiple traces.
Note. A raw file must be open and a chromatogram
selected for this button to be active.
Delete Manual Click on the Delete Manual Noise Region button and
Noise Region then drag the cursor over the region that was previously
selected as the noise region. Release the mouse button
to delete the noise region.

The next group of buttons are Zoom controls used to adjust the display of the
component chromatogram and companion views. These include the zoom in
and out, for both the vertical and horizontal axes as well as the ‘display all’
button to expand the plot to its limits.
The last button is the application Help button.
Many of Quan Browser’s functions are accessed from shortcut menus from
within the Results Grid, Chromatogram View, or Companion View.
For specific information about Quan Browser’s menu commands or toolbar
buttons refer to Xcalibur’s online Help.

Component List
The Component list displays all the components within the current bracket
sorted by retention time. Click on the name of a component to update the
Chromatogram View and Companion View with data for the selected
component.

Results Grid
The results grid is made up of sequence entries. Each row defines a result file
and associated parameters.

Chromatogram View
The Chromatogram View displays the chromatogram for the currently
selected component from the currently selected result file.

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If a filter is stored within the embedded processing method for the current
compound this will be applied to the chromatogram. You can adjust the
chromatogram plot using the Zoom menu commands or toolbar buttons.
The type of integration used is displayed in the results grid but can be
overridden. The three types are Method Settings, User Settings and Manual
Integration. You can change the Integration method by using commands on
the shortcut menu within the Chromatogram View.

Companion View
The Companion View has two display modes:
Calibration Displays the calibration curve for the current bracket
Curve
Spectrum Plot Displays the spectrum at the selected retention time.
Initially the display shows the spectrum at the apex of
the current peak in the chromatogram view.

Calibration Companion View


The Calibration Companion View displays a calibration curve for the current
bracket or group.

For a Bracketed Sequence


In a bracketed sequence, Xcalibur calculates the points on the graph from the
embedded calibration information stored in the result file.

For a Non-Bracketed Sequence


For a non-bracketed sequence, the points on the graph consist of the replicates
in the specified calibration file for the current component and any standards in
the first group. Unless a Standard Clear is encountered, each subsequent
group includes all the standards from the prior groups as well as the standards
from the calibration file. If a standard clear is encountered, the calibration
tables are cleared and only the standards from the current group are used.
All standards within a group are used to evaluate the calibration curve. This
means that the calibration curve is the same for all samples within a group At
the time of sample acquisition, non-standard samples are processed
immediately after acquisition using a calibration curve determined from the
standards acquired so far (that is, omitting any standards following the sample
in the sequence).

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Spectrum Companion View


The spectrum companion view displays a spectrum from the current
chromatogram in the chromatogram view. You can view spectra from the
apex, left peak edge or right peak edge using commands from the shortcut
menu. When the View is pinned, you can also view scans from any part of the
chromatogram by clicking on the chromatogram.

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4.4 The Results Grid


The Results Grid (Figure 4-4) is made up of sequence entries. Each row
defines a result file and associated parameters.

Figure 4-4. The Results Grid

There are two viewing configurations. These are determined by your choice in
the Viewing Sample Types dialog box at startup, or by your choice in the
Options menu.
If the Options menu viewing preference is set to View Stds and QCs, the
results grid has 3 pages:
All All Standard and QC samples
Stds Standard samples only
QCs QC samples only

If the viewing preference is set to View All, the results grid will have 5 pages:
All All Standard, QC, Blank and Unknown samples
Stds Standard samples only
QCs QC samples only
Blanks Blank samples only
Unknowns Unknown samples only

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Above the Results Grid, Xcalibur displays the following:


• Bracket/Group in Use combo box
• Calibration File text box

Bracket/Group in Use
For bracketed sequences, this combo box lists the available brackets in
sequential order. Xcalibur selects the first bracket in the list when the file is
first loaded into Quan Browser, and displays the samples within this bracket
in the results grid.
If you load a non-bracketed sequence, the samples are broken into logical
groups (refer to How Quan Browser Works). The combo box lists the
available groups.
Selecting a new bracket or group from the combo list refills the results grid
with the samples from the selected bracket or group. Xcalibur updates all the
other Views and dialog boxes automatically.

Calibration File
This read only text box shows the calibration method applied to the current
bracket or group.
If the calibration information for the current bracket was obtained from the
embedded processing method and not from a separate cal file, the text box
displays Embedded Calibration.
For non-bracketed sequences, the text box displays the name of the calibration
file associated with the current Group in the sequence. To change the
calibration file, choose File > Replace Calibration. This option is not
available for bracketed sequences.

Results Grid Columns


Xcalibur lists samples under some or all of the following headings:
File Name The raw file containing the acquisition data for this run
Sample Type Standard, QC, Blank or Unknown.
Sample Name Sample name given to this sample when the sequence
was prepared in Sequence Setup.
Integration Type The method applied to integrating the peak. The
choices are Method, User and Manual.
Area (or Height) Integrated area (or height) under the detected peak
(count secs or counts)

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ISTD Area Integrated area (or height) under the Internal Standard
(or ISTD Height) peak (count secs or counts)
Area Ratio The area (or height) ratio between the selected peak and
(or Height Ratio) the Internal standard
Specified The amount of the component at the Cal or QC level
Amount
Calculated Amount of component as determined by the response
Amount ratio and calibration curve
% Diff Percentage difference between the calculated amount
and the specified amount
% RSD Relative Standard Deviation of the difference between
the calculated amount and the specified amount as
expressed as a percentage of the specified value
Peak Status Low is displayed if the %Difference is < 0, High is
displayed if the %Difference is > 0 and Fail is
displayed if the %Difference is greater than the QC fail
percentage test value.
Level The name of the calibration or QC level of the sample.
Units The units defined in the processing method for quantity
or concentration.
RT Retention time in minutes at the peak apex.
Sample ID Unique sample identification string given to this
sample when the sequence was prepared in Sequence
Setup.
Exclude Indicates if the sample point is to be included or
excluded from the calibration curve for the current
bracket or group.

Working Directly With The Grid


You can change any of the following by clicking on the appropriate grid cell:
Sample Type Select from Standard, QC, Blank or Unknown.
Integration Type Select from Method Settings, User Settings or Manual
Integration.
Levels Select another defined level from the list.
Exclude Select or clear the check box to exclude or include the
sample in the bracket calibration. Selecting excludes
the data and is indicated in the grid by Yes.

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If a sample is shared between two brackets you cannot change its Sample
Type. Xcalibur will also notify you when a sample is part of two overlapping
brackets if you attempt to change its Integration Type, Level or Exclude state.
You can then either confirm or cancel the change.

Results Grid Shortcut Menu


Most of the commands for manipulating the Results Grid are available on a
shortcut menu. You can display this by right-clicking anywhere within the
grid. The menu displays the following commands:
Columns Displays the Result List Column Hiding dialog box
(see Figure 4-5). This allows you to select the columns
to be displayed in the results grid.
Delete Selected Removes the currently selected sample(s) from the
Samples results grid. Samples are selected by dragging a range
across one or more columns in the rows to be deleted. If
you delete Standards or QCs the RSD% parameters are
recalculated for the bracket.
Add Sample Displays the Open Result Data File dialog box for you
to select a new file. Xcalibur adds this to the current
bracket and displays it in the results grid according to
the new sort order. If the sample(s) added are standards,
Xcalibur recalibrates the bracket and recalculates the
RSD % parameters. RSD% is also recalculated if the
new sample is a QC sample.
Copy Row Duplicates the selected row. If the copied sample is a
standard, Xcalibur recalibrates the bracket and
recalculates the RSD % parameters. RSD% is also
recalculated if the copied sample is a QC.
Set Sorting Brings up the Quantitation Results Sorting Order dialog
Order box. This allows you to set the sorting criteria for
samples in the results grid.
Send to Qual Launches Qual Browser with the results file for the
Browser currently selected sample.

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Hiding or Displaying Columns


Quan Browser’s Results Grid contains many columns. You can choose to
display some or all of these columns using the Result List Column Heading
dialog box (Figure 4-5). To open this dialog box, right click within the Result
Grid and choose Columns form the shortcut menu.

Figure 4-5. Result List Column Hiding dialog box

Select the check box for a column heading to display it; clear the check box to
hide the column.

Changing the Sort Order


To change the sort order for entries in the results grid, right click on the grid,
and choose Set Sorting Order from the shortcut menu. The Quantitation
Results Sorting Order dialog box is displayed (Figure 4-6).

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Figure 4-6. Quantitation Results Sorting Order dialog box

The first sorting criteria is fixed to be Sample type. The second and third sort
criteria can be any of the remaining columns, even if the column is currently
hidden.
Click on the Save As Default button if you want to replace the default sorting
criteria with your new selections.

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4.5 Chromatogram View


The Chromatogram View displays the chromatogram for the currently
selected component from the currently selected result file. Most of the
commands for manipulating the Chromatogram View are available on a
shortcut menu.

Chromatogram View Shortcut Menu


You can display a shortcut menu by right-clicking anywhere within the
chromatogram view. The menu displays the following commands:
Method Settings Quan Browser uses the integration parameters
embedded within the processing method for the
selected component. The method for a given bracket
derives from the first result file.
User Settings Quan Browser uses your settings, provided in the User
Identification Settings dialog box (refer to Setting User
Peak Detection Parameters).
Manual Quan Browser integrates the chromatogram according
Integration to the manually dragged baseline markers.
Show Peak Info Displays the Peak Information dialog box. This shows
information about the peak or one of the peaks used by
the Spectrum search or Ion Ratio Confirmation routines
in a read only format (refer to Viewing Peak
Information).
User Peak Displays the User Identification Settings dialog box.
Detection This allows you to adjust the peak identification,
Settings detection and integration parameters for the selected
component.
Display Options Displays the Display Options dialog box. This allows
you to change chromatogram peak labeling.
Manually Add Allows you to place a baseline on the chromatogram
Peak plot. When you use this option, Quan Browser changes
the Integration Type to Manual Integration. This option
is only available if Xcalibur has failed to detect a peak
using the existing method or user settings. You can
disable the existing detection using the Set Peak To Not
Found Status command.
Update This command updates the retention time specified in
Expected the processing method with the retention time that
Retention Time Xcalibur detected.

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Set Peak to Not Allows you to override the identification method and
Found Status classify the current component as Not Found. When
used, the chromatogram view re-displays over the
whole acquisition range.
Reset Scaling Allows you to reset the chromatogram scaling to show
the full peak in a normalized window.

Viewing Peak Information


Quan Browser displays information about the currently displayed component
peak, qualifier ion or spectrum candidate in the Peak Information dialog box.
The title bar contains the component name. To open this dialog box, right
click within the Chromatogram View and choose Show Peak Info from the
shortcut menu.
If the peak is a qualifier ion the title bar contains the text Qual Ion Mass xxx.x
where the xxx.x represents the mass of the qualifier ion.
If the peak is for a Spectrum candidate the title bar contains the text Spectrum
Candidate.
For a component peak, the Peak Information dialog box has 5 tabbed pages:
Info Shows statistics about the chromatogram peak
Flags Displays integration and detection flag results
More Flags Shows flags for detection, calibration and quantitation
thresholds
Suitability Shows system suitability test results
Spectrum Displays the spectrum at the apex retention time
If the component peak is the leading Spectrum detection candidate the dialog
box features an additional page labeled More Info. This shows information
about ion coelution and ion ratio testing.
For a qualifier ion, the dialog box has 7 tabbed pages. These include the Info,
Flags, More Flags, Suitability and Spectrum pages described above. The two
additional pages are:
More Info Displays the results of the Ion Coelution and Ion Ratio
tests.
Chro Displays the chromatogram for the qualifier ion.

For a spectrum candidate, the dialog box has 3 tabbed pages:

Info Shows information about the chromatogram peak and


spectrum matching.
Chro Displays the TIC for the spectrum candidate
Spectrum Displays the spectrum at the apex of the spectrum
candidate chromatogram peak.

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If the component peak is not found then the dialog box consists of a single tab
labeled No Peak. This displays the text No Peak Found: Cannot show Peak
Info.
The Peak Information dialog box is read only. If you select other components
or samples, the dialog box is updated with peak information for the displayed
component chromatogram peak.

Info Page
The peak Info page (Figure 4-7) displays the following properties:
Left (min) Left point in minutes of integration baseline
Apex (min) Location of apex in minutes
Right (min) Right point in minutes of integration baseline
Height Height at peak apex
Area (cts-secs) Area measured in count seconds
Baseline Baseline height directly beneath the apex
Base Peak (m/z) Mass to charge ratio of ion with largest response
Signal to Noise Measured signal-to-noise
Expected RT Expected retention time in minutes of peak
(min)
ISTD Response Area (or Height) of internal standard peak
Response Ratio Ratio of this peak’s area (or height) to the ISTD peak’s
area (or height)
Calculated Amount in sample as calculated with response ratio and
Amount calibration curve

Figure 4-7. Info Page of Peak Information dialog box

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Flags Page
The Flags page (Figure 4-8) displays information about peak detection.

Figure 4-8. Flags page of Peak Information dialog box

The displayed parameters are:


Detected By A read only edit box which will contain the description
of the detection method used. The available types are
Spectrum, Highest Peak and Nearest RT. In the case
where an LC method is used, only the Highest Peak and
Nearest RT are available.
Valid A flag indicating whether or not the peak was
successfully detected.
Left Edge Type Displays one of the following, based on the detection
method used during peak detection. Baseline, Valley,
Manual, Stripe, Tail, Tilt or Unknown.
Right Edge Type Displays one of the following, based on the detection
method used during peak detection. Baseline, Valley,
Manual, Stripe, Tail, Tilt or Unknown.
The page also displays the state of the following calibration flags. If the flag
is true its box is checked.
Saturated Indicates that one or more of the scans within the peak
were saturated.
Calculated Indicates that a quantitation calculation was performed.
Amount
Valley Detect Indicates that valley detection was enabled in the
processing method.

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QC Failed Indicates whether or not the sample failed a QC check.


RT Ref OK Indicates whether or not the Retention Time Reference
component was found.
Response OK Indicates that a Response Factor was calculated.
Response Low Indicates if the calculated amount was less than the
lowest level of the component and therefore determined
by extrapolation from the lowest level.
Response High Indicates if the calculated amount was greater than the
highest level of the component and therefore
determined by extrapolation from the highest level.

More Flags Page


The More Flags page (Figure 4-9) displays the state of flags for detection,
calibration and quantitation thresholds. If the flag is true, its box is checked.

Figure 4-9. More Flags page of Peak Information dialog box

The detection threshold flags (defined in Processing Setup in the Data Flags
dialog box accessed from the method’s Detection page) are:
Area True if the peak area exceeds the defined absolute peak
area.
Height True if the peak height exceeds the defined absolute
peak height.

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The calibration and quantitation threshold flags (defined in Processing Setup


in the Calibration and Quantitation Flags dialog box accessed from the
method’s Calibration page) are:
R-squared True if the R-squared threshold value (a test of the
goodness of fit of the calibration curve) is greater than
the threshold value, otherwise false.
Detection Limit True if the quantified component concentration is less
than the Detection Limit threshold, otherwise false.
Linearity Limit True if the quantified component concentration is less
than the Linearity Limit threshold, otherwise false.
Quantitation True if the quantified component concentration is less
Limit than the Quantitation Limit threshold, otherwise false.
Carryover Limit True if the quantified component concentration is less
than the Carryover Limit threshold; otherwise false.

Suitability Page
The Suitability page (Figure 4-10) displays the results of specific tests that
may have been performed (as determined by the System Suitability
parameters in the processing method) on the component peak to determine its
suitability to be considered a valid peak.

Figure 4-10. Suitability page of Peak Information dialog box

There are 3 possible responses for each test:


• Passed
• Failed
• Not Tested

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The tests reviewed on the Suitability page are:


Symmetrical Determines if the peak is symmetrical about the apex
Resolution Determines if peaks are well resolved into individual
peaks
Peak Width Determines if peak width is within specified limits
Tailing Determines if tailing has occurred
Column Determines if the column was overloaded during
Overload acquisition
Baseline Determines if the baseline is clipped (no noise) outside
Clipping the peak
Signal-To- Determines if minimum Signal-to-Noise ratio is met.
Noise Ratio
Concave Determines if peak exhibits a concave depression (local
minimum) due to noise
Saturation Determines if detector was saturated during acquisition

Spectrum Page
The Spectrum page (Figure 4-11) displays the current spectrum at the Apex
retention time. No adjustments can be made to the plot.

Figure 4-11. Spectrum page of Peak Information dialog box

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Qualifier Peak Information


When you select a qualifier ion, the Peak Information dialog box also features
More Info and Chro pages.

More Info Page


The More Info page (Figure 4-12) displays the results of Ion Coelution and
Ion Ratio tests. In Processing Setup, these are defined in the Ion Ratio
Confirmation group box on the method’s Detection page.

Figure 4-12. More Info page of Peak Information dialog box for a
qualifier ion

Ion Coelution Indicates whether the selected qualifier ion passes the
Test Passed Coelution test. To do this, its mass chromatogram must
have a peak apex within the Qualifier Coelution
Window specified on the processing method’s
Detection page (see Chapter 2).

If the qualifier ion fails the Coelution test, the Ion Ratio
test is not performed and the Ion Ratio Test group box
is not displayed.
Ion Ratio Test Indicates whether the selected qualifier ion passes the
Passed Ion Ratio test. The Target ratio % and Absolute
Window % parameters display the results of the test.
Target Ratio The calculated Target Ratio Percentage. See Chapter 2
for more details.
Absolute The calculated Absolute window Percentage. See
Window Chapter 2 for more details.

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Chro Page
The Chro page (Figure 4-13) displays the qualifier ion mass chromatogram
within the component peak window. No adjustments can be made to the plot.

Figure 4-13. Chro page of Peak Information dialog box for a qualifier ion

Spectrum Candidate Information


When a Spectrum candidate is selected the Peak Information dialog box
features three tabbed pages: Info, Chro and Spectrum.
Spectrum candidates are only displayed if Spectrum detection was specified
in the processing method. Spectrum detection is only available if the GC
chromatography option is selected. See Chapter 2 for more details.

Info Page
The parameters in the Peak Info group box (Figure 4-14) are the same as those
described for the standard peak and qualifier ion Info page.
See Chapter 2 or the online Help for a more detailed description of these
parameters and Spectrum detection.

Note. If the main component peak was detected using Spectrum detection
Xcalibur displays the standard Info page with the Spectrum Results group
box on an additional tabbed page called More Info.

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Figure 4-14. Info page of Peak Information dialog box for a Spectrum
candidate

The Spectrum results group parameters are:


Forward Calculated forward match factor for the spectrum
candidate and the reference spectrum.
Reverse Calculated reverse matching factor for the spectrum
candidate and the reference spectrum.
Match Calculated probability matching factor for the spectrum
candidate and the reference spectrum.

Chro Page
The Chro page displays a TIC plot for the Spectrum candidate. The plot has
the width used by the component peak display.

Spectrum Page
The Spectrum page is effectively the same as that described for a standard
peak. It displays the spectrum corresponding to the apex of the spectrum
candidate chromatogram.

Setting User Peak Detection Parameters


When you first open a Sequence in Quan Browser component identification,
peak detection, calibration and quantitation information is obtained from the
Result file.

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Within Quan Browser, Xcalibur allows you to apply unique peak detection
parameters to the chromatogram using the User Identification Settings dialog
box. This duplicates the parameters available in the Identification and
Detection pages in Quan View of Processing Setup, allowing you to adjust
and test the effect of different values. You can:
• Save the settings in a Quan Browser file (*.XQN). Choose File > Save or
File > Save As.
• Export the User Settings as a full Processing Method using the File >
Export Method menu command.
To open the User Identification Settings dialog box, right click on the
Chromatogram View and select User Peak Detection Settings from the
shortcut menu.
The User Identification Settings dialog box for the ICIS peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
ICIS Integration ICIS peak detection algorithm parameters applied to
the component peak.
ICIS Advanced ICIS advanced parameters used by Xcalibur during
peak identification and integration.
Flags Detection flagging thresholds applied to the selected
component to validate detection.

The User Identification Settings dialog box for the Genesis peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
Genesis Genesis peak detection algorithm parameters applied to
Integration the component peak.
Genesis Genesis advanced parameters used by Xcalibur during
Advanced peak identification and integration.
Flags Detection flagging thresholds applied to the selected
component to validate detection.

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The User Identification Settings dialog box for the Avalon peak detection
algorithm consists of the following tabbed pages:
Identification Parameters used by Xcalibur to identify the selected
component in the chromatogram.
Detection Settings used by Xcalibur to confirm peak detection.
Avalon Avalon peak detection algorithm parameters
Integration applied to the component peak.
Flags Detection flagging thresholds applied to the selected
component to validate detection.

Identification Page
Xcalibur uses the parameters on the Identification page (Figure 4-15) to:
• Generate a chromatogram from raw data
• Identify the component within the chromatogram
The parameters are identical to those on the Identification page of Quan View
in Processing Setup. These are described in Chapter 2, in the topic
Identification.

Figure 4-15. Identification page of User Identification Settings dialog


box

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Detection Page
Xcalibur uses the parameters on the Detection page (Figure 4-16) to confirm
the identity of the component within the retention time window defined by the
Identification settings. The options available on this page depend on the
Chromatography mode selected in the original processing method used to
generate the raw data (refer to Chapter 2).
The parameters are identical to those in the Peak Detection group box on the
Detection page of Quan View in Processing Setup. These are described in
Chapter 2, in the topic Peak Detection.

Figure 4-16. Detection page of User Identification Settings dialog box


with Spectrum detection mode selected

The controls on the Detection page vary depending upon whether you are
using a GC or LC and also whether the detection method is Spectrum, Highest
Peak, or Nearest RT. For more information, refer to Xcalibur online Help.

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ICIS Integration Page


Xcalibur applies the settings on the Integration page (Figure 4-17) during
peak integration.
The parameters are identical to those in the ICIS Peak Integration group box
on the Detection page of Quan View in Processing Setup. These are described
in Chapter 2, in the topic Peak Integration.

Figure 4-17. ICIS Integration page of User Identification Settings dialog


box

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ICIS Advanced Page


Xcalibur applies the ICIS Advanced page (Figure 4-18) parameters during
ICIS peak detection and integration.
The parameters are identical to those in the Advanced Detection Options
dialog box accessed from the Advanced button on the Detection page of Quan
View in Processing Setup. These are described in Chapter 2, in the topic
Advanced Detection Parameters.

Figure 4-18. ICIS Advanced page of User Identification Settings dialog


box

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Flags Page
Xcalibur applies the parameters on the Flags page (Figure 4-19) to test the
validity of detected peaks.
The parameters are identical to those in the Data Flags dialog box accessed
from the Detection page of Quan View in Processing Setup. These are
described in Chapter 2, in the topic Data Flags.

Figure 4-19. Flags page of User Identification Settings dialog box

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Changing Display Options


The Display Options dialog box (Figure 4-20) allows you to change the way
Quan Browser displays the Chromatogram View. To open this dialog box,
right click on the Chromatogram View and choose Display Options from
the shortcut menu.

Figure 4-20. Display Options dialog box

For more information about these settings, refer to Xcalibur online Help.

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4.6 Calibration Companion View


To display the Calibration Companion View choose View >
Set Companion View > Show Calibration Curve.
Alternatively, you can use the shortcut menu from within the Companion
View. If the companion View is currently displaying a spectrum plot, right
click within it and choose Show Calibration Curve.

Calibration Companion View Shortcut Menu


Right click on the calibration curve plot to display the Calibration Companion
View shortcut menu. The following menu commands are available:
Calibration Displays the Calibration Settings dialog box. This
Settings allows you to change ISTDs, apply a new calibration
curve, adjust levels, and change flag thresholds.
Normally, Xcalibur uses the settings from the
embedded processing method.
Save Calibration Displays the Save As dialog box. Use the Save As
File dialog box to save the calibration settings in a
calibration file with extention .xcal.
Exclusion List Displays the Cal Exclusion List dialog box. This allows
you to exclude levels and all associated samples from
the calibration.
Show Spectrum Changes the Companion View to the Spectrum
Plot Companion View.
Reset Scaling Resets the scaling of the calibration curve.
Copy Graph Copies the calibration graph to the Microsoft Windows
Clipboard. It can then be pasted into other applications
for presentation purposes.

Excluding a Data Point


To exclude a data point from the calibration curve, right click on it and choose
Exclude from the shortcut menu. If the data point is currently included in the
calibration, Xcalibur:
• Recalculates the calibration curve without it.
• Updates the corresponding Peak Status and Exclude field in the Results
Grid and Exclusion List to show that it is excluded.
• Redraws the excluded data point as an unfilled square.

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Restoring an Excluded Data Point


To restore a data point that you have previously excluded, right click on the
data point and choose Include from the shortcut menu. Xcalibur:
• Incorporates the data point into the calibration and recalculates the curve.
• Updates the corresponding Peak Status and Exclude field in the Results
Grid and Exclusion List to show that the point is now included.
• Redraws the included data point as filled square.
You may include or exclude samples that are shared between brackets. Their
status will be unique to the bracket. For example, excluding a shared sample
in bracket 1 will have no effect on it’s inclusion status in bracket 2.

Adjusting Calibration Settings


When you first open a sequence in Quan Browser component identification,
peak detection, calibration and quantitation are carried out according to the
settings of the associated processing method.
Within Quan Browser, Xcalibur allows you to apply unique calibration
parameters and level definitions to the chromatogram using the Calibration
Settings dialog box. This duplicates most of the parameters available in the
Calibration and Levels pages in the Quan View of Processing Setup, allowing
you to adjust and test the effect of different calibration and quantitation
parameters. You can:
• Save the settings in a Quan Browser file (*.XQN). Choose File > Save or
File > Save As.
• Export the Calibration Settings as a full processing method using the
File > Export Method menu command.
To open the Calibration Settings dialog box, right click on the Calibration
Companion View and select Calibration Settings from the shortcut menu. The
dialog box consists of 5 tabbed pages:
Type Allows you to change the sample type: Target or ISTD.
Curve Allows you to change the calibration curve calculation
and plotting methods.
Levels Allows you to change level definitions for a target
compound.
Isotope % Allows you to adjust the isotope contributions of ISTD
and Target compounds.
Flags Allows you to adjust the threshold values for
calibration and quantitation flags.

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Type Page
The Type page (Figure 4-21) displays the component type, target compound
or ISTD. For a target compound, you can change the ISTD to be used with it.
For ISTDs, you can change the Amount and Units.
The parameters are identical to those in the Component Type and ISTD group
boxes on the Calibration page of Quan View in Processing Setup. These are
described in Chapter 2, Calibration.

Figure 4-21. Type page of Calibration Settings dialog box

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Curve Page
The Curve page (Figure 4-22) allows you to change the way Xcalibur
calculates and plots the calibration curve from the data points.
Calibration Use this list box to change the type of algorithm applied
Curve to fit the data points. The available types are Linear,
Quadratic, Linear Log-Log, Quadratic Log-Log,
Average RF, Point-to-Point, Cubic Spline and Locally
Weighted
Weighting Use these option buttons to change the weighting
applied to the individual data points. The available
types include Equal, 1/x, 1/x^2, 1/y, 1/y^2 and 1/s^2
Response Use these option buttons to change the component
response used in the calibration curve: area or height.
Units Use this text box to change the units label used in the
Calibration Companion View, on the Levels page, and
in reports.

These parameters are identical to those in the Target Compounds group box
on the Calibration page of Quan View in Processing Setup. These are
described in more detail in Chapter 2, Calibration.

Figure 4-22. Curve page of Calibration Settings dialog box

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Levels Page
The Levels page (Figure 4-23) allows you to change Calibration and QC level
names and their associated amounts. It is not available for ISTD components
(the page displays the message This component does not use levels).
These parameters are identical to those on the Levels page of Quan View in
Processing Setup. These are described in more detail in Chapter 2, Levels.

Figure 4-23. Levels page of Calibration Settings dialog box

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Isotope % Page
The Isotope% page (Figure 4-24) allows you to correct data for:
• An impurity in the internal standard compound that elutes at the same
time as the target compound.
• An impurity in the target compound that elutes at the same time as the
internal standard.
These parameters are identical to those in the Correction For Isotope
Contribution dialog box, accessed from the Calibration page of Quan View in
Processing Setup. These are described in more detail in Chapter 2,
Calibration.

Figure 4-24. Isotope% page of Calibration Settings dialog box

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Flags Page
The Flags page (Figure 4-25) allows you to change the threshold values for
calibration and quantitation flags for the selected compound. An entered value
of 0 will force the flag to be false.
These parameters are identical to those in the Data Flags dialog box, accessed
from the Calibration page of Quan View in Processing Setup. These are
described in more detail in Chapter 2, Calibration.

Figure 4-25. Flags page of Calibration Settings dialog box

If you edit any of the values in the Quantitation Flags group, Xcalibur checks
that the relationships between the four fields are maintained. If an entry in one
parameter forces a change to occur in another, Xcalibur displays the
Automatic Adjustment warning dialog box (Figure 4-26).

Figure 4-26. Automatic Adjustment warning dialog box

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Excluding Calibration Levels


You can use the Cal Exclusion List dialog box (Figure 4-27) to exclude levels
from the calibration (see How Quan Browser Works for a description of the
procedures used to generate this list). This is particularly useful when you
cannot use the Include and Exclude commands because of overlapping points
on the calibration curve. If you are using a named calibration file, levels may
not be represented in the Results Grid but will always be listed in the Cal
Exclusion List dialog box.

Figure 4-27. Cal Exclusion List dialog box

To open the dialog box, right click on the Calibration Companion View and
choose Exclusion List from the shortcut menu.
The dialog box lists all the replicates used in the current bracket or group and
their exclusion status. Levels are listed under the following headings:
Level Shows the name for the Level
Expected Displays the expected amount for Level
% Diff Shows the percentage difference between measured
and expected amounts
Exclude Denotes excluded levels by the word Yes

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To exclude a level, click in the Exclude column adjacent to the level to be


excluded.
Xcalibur then:
• Recalculates the calibration curve without any samples using the level.
• Updates the corresponding Peak Status and Exclude fields in the Results
Grid to show that the samples are excluded.
• Redraws excluded data points as unfilled squares.
To restore an excluded level, click in the Exclude column adjacent to the level
(on the word Yes) to be restored. Xcalibur then:
• Incorporates all samples using the level into the calibration and
recalculates the curve.
• Updates corresponding Peak Status and Exclude fields in the Results Grid
to show that the points are now included.
• Redraws the included data point as filled square.

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4.7 Spectrum Companion View


To display the Spectrum Companion View choose View >
Set Companion View > Spectrum Plot.
Alternatively, you can use the shortcut menu from within the Companion
View. If the view is currently displaying the calibration curve, right-click on it
and choose View Spectrum Plot.
You can use the Spectrum Companion View to examine the identity of peaks
and other features (such as the background) in the chromatogram. For further
analysis, including library matching of spectra, you can export data to Qual
Browser using the Send to Qual Browser option in the Result List shortcut
menu.

Changing the Spectrum


Initially, Xcalibur displays the spectrum corresponding to the scan at the
current chromatogram’s apex retention time. If no peak was detected,
Xcalibur displays the expected retention time as defined by the processing
method.
You can change the spectrum companion view by:
• Selecting options on the shortcut menu.
• Pinning the cell and selecting a scan in the chromatogram view.
Access the shortcut menu by a right click within the Spectrum Companion
View. The menu contains four viewing options:
Spectrum at Peak Displays the spectrum at the current
Apex chromatogram’s apex retention time.
Spectrum at Peak Displays the spectrum at the current integration
Left Edge baseline’s left edge retention time.
Spectrum at Peak Displays the spectrum at the current integration
Right Edge baseline’s right edge retention time.
Show Calibration Changes the Companion view to display the
Curve calibration curve.
Reset Scaling Resets the view to display the full spectrum in a
normalized window.

Pin the spectrum companion view if you want to display spectra from other
regions of the chromatogram. Any click within the Chromatogram View will
then result in the Spectrum Companion View being updated with a spectrum
corresponding to the scan at the ‘clicked’ retention time.
In a pinned Spectrum View you can also use the cursor to rescale the plot. The
Zoom menu commands and toolbar buttons are also effective.

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4.8 Reports
To generate reports for the current sequence either:
• Click on the Reports toolbar button.
• Choose View > Reports dialog.
The Reports dialog box (Figure 4-28) duplicates the Reports view in
Processing Setup. When opened, it displays the reports specified in the
processing method associated with the active sequence. The displayed
parameters may change as you select different brackets.

Figure 4-28. Reports dialog box

For a description of the Sample Reports and Summary Reports tables see
Chapter 2, Reports view. The Reports dialog box features the following
additional parameters:
Include Sample Select this check box if you want to include
Reports sample reports in any print run.
Include Summary Select this check box if you want to include
Reports summary reports in any print run.
Select Samples Click on this button to open the Select Report
Samples dialog box. This allows you to choose
samples in the sequence for report generation and
printing.
Print Reports Click on this button to initiate report generation
and printing as defined in the dialog box.

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Selecting Samples for Reports


The Select Samples button in the Reports dialog box opens the Select Report
Samples dialog box (Figure 4-29). This allows you to choose the samples to
be processed during report generation.

Figure 4-29. Select Report Samples dialog box

To include a sample in report processing:


1. Click on its name in the Sample Choices list box.
2. Click on the Add button.
You can select multiple files using the <Shift> and <Ctrl> keys:
• Hold the <Shift> key down to select a range of samples.
• Hold the <Ctrl> key down to select multiple samples.
3. Click on the Add All button to add all the samples listed in the Sample
Choices list box to the Selected Samples list box.
To exclude a sample from report processing:
1. Click on its name in the Selected Samples list box.
2. Click on the Remove button.
3. Click on the Remove All button to remove all samples listed in the
Selected Samples list box to the Selected Samples list box.

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4.9 Quan Browser Procedures


This section describes procedures for the common tasks associated with
reviewing calibration and quantitation results.

Editing a Sequence
To review and edit an existing Sequence, do the following:
Inspect the Sequence. Ensure that the correct raw files are listed in the Results
Grid. Make sure that each raw file in the Sequence is properly associated with
a calibration level, QC level, blank, or unknown.
1. To remove raw files from the sequence:
a. Select the row(s) in the Sequence that you want to delete.
b. Right-click on the mouse in the Sequence to display a menu.
c. Finally, left-click on the Delete Selected Samples command to delete
the selected row(s) in the Sequence.
2. To add raw files to the Sequence:
a. Select the row in the Sequence above where you want the new row
(sample) to be located.
b. Right-click on the mouse in the Results Grid to display a menu.
c. Select the Add Sample command to open the Open Result File dialog
box.
d. Locate the raw file you want to add to the Sequence, and then click on
the Open button to open the Add Sample dialog box.
e. Specify sample information in the Add Sample dialog box, and then
click on the OK button.
3. To change the sample type:
a. Click on the Sample Type list box arrow to display a list of sample
type options.
b. Select the new sample type. Quan Browser displays the new sample
type in the Sample Type list box.
4. To save the sequence with all current detection and calibration settings,
choose File > Save or File > Save As. The resulting Xcalibur Quan
file (extension .XQN) contains all the necessary information required to
recreate the current Quan Browser session.

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Reviewing Samples
Use the following procedure to review and rework samples:
1. Select a component. Xcalibur automatically updates the Result List,
Chromatogram and Companion Views.
2. Click on the Standards tab to display calibration standards results.
3. Inspect the calibration curve in the companion view. If it is not currently
displayed either:
• Choose View > Set Companion View >
Show Calibration Curve, or
• Right click in the Chromatogram View and choose
Set Companion View > Show Calibration Curve from the
shortcut menu.
4. Inspect the calibration curve according to the criteria used in your
laboratory.
5. Select the first row in the Results Grid.
6. Check the peak detection and integration fields in the Result Grid for peak
detection and integration problems. Make sure that the selected data file
corresponds to the correct level and sample type.
7. Inspect the plot in the Chromatogram View.
• Make sure that Xcalibur found the peak. Xcalibur shades found peaks
gray and marks the starting and ending points with square integration
markers.
• Make sure that Xcalibur integrated the peak properly. Check that the
shaded area accurately represents the contribution of the component
to the chromatogram.
8. If necessary, modify the peak detection and integration settings:
• Right-click on the mouse in the Chromatogram View and select User
Peak Detection Settings. This opens the User Identification Settings
dialog box.
• If you want to change the detection method, modify the settings in the
Detection tab.
• If you have problems with noise in the peak, unresolved peaks, or
peak tailing, modify the settings in the Integration tab.
• If baseline noise is interfering with peak identification or integration,
modify the settings in the Advanced tab. Advanced options should
only be used if the standard options do not provide sufficiently
selective detection criteria.

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• Alternatively, perform manual integration. You can manually change


the starting and ending points and baseline of the peak by clicking on
and dragging the square integration markers to the desired location.
9. Repeat the procedure (from step 5) for the next data file.
10. Repeat the procedure for QC, Blank and Unknown samples.
11. Repeat the procedure for the remaining components.

Reviewing a Chromatogram
Use the following procedure to review a chromatogram:
1. Right click in the Chromatogram View and choose Show Peak Info.
This opens the Peak Information dialog box. Review the chromatogram
peak data:
• The properties of the detected peak on the Peak Info page.
• Integration information and flags on the Peak Flags page.
• System Suitability test results on the Peak Suitability page.
• The spectrum for the peak apex scan on the Peak Spectrum page.
2. You can adjust the chromatogram in the following ways:
• To change detection or integration parameters refer to Modifying
Detection and Identification.
• To manually integrate peaks refer to Integrating Chromatogram
Peaks Manually.
• To change chromatogram peak labeling, right click in the
Chromatogram View and choose Peak Labeling. Select the labels
and label styles you require in the Display Options dialog box.
3. If you want to view spectra for the peak, display the Spectrum
Companion View. Right click in the Chromatogram View and choose
Set Companion View > Show Spectrum Plot from the shortcut
menu.
4. View spectra across the chromatogram. Pin the Spectrum Companion
View. Click on points of interest in the chromatogram to view the
corresponding spectrum.
5. To carry out a detailed qualitative analysis of the chromatogram, export
the results file to Qual Browser. Right click on the Results Grid and
choose Send to Qual Browser.

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Modifying Detection and Identification


Use the following procedure to modify and test component peak detection
criteria:
1. Review the displayed data for the selected component to determine if the
results are consistent with your expectations:
• Are there peaks that were not found?
• Are neighboring peaks resolved?
• Are tailing peaks detected properly?
2. To modify detection criteria, right click in the Chromatogram View and
choose User Peak Detection Settings. This opens the User
Identification Settings dialog box.
3. If you want to change the chromatogram trace, or adjust the retention time
window modify the settings in the Integration tab.
4. If you want to change the detection method modify the settings in the
Detection tab.
5. If you have identified problems with noise in the peak, unresolved peaks,
or peak tailing, modify parameters on the Integration page.
6. If baseline noise is interfering with peak identification or integration,
modify the settings in the Advanced tab. Advanced options should only
be used if the standard options do not provide sufficiently selective
detection criteria.
7. Save your settings as a new processing method. Choose File >
Export Method.

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Integrating Chromatogram Peaks Manually


You can integrate peaks manually in either of two ways. The first way is to
use the cursor to drag the baseline endpoints to new positions.
See Figure 4-30 (a) and (b).

Figure 4-30. Chromatogram (a) shows a partially integrated peak and chromatogram (b) shows a
manual integration of the peak achieved by dragging the baseline endpoint to a new
location.

You use the second way when Xcalibur has not detected the peak of choice, as
follows:
1. Right click on the Chromatogram View again and select Manually Add
Peak. Xcalibur changes the cursor shape to denote the mode.
2. In the chromatogram, click on one side of the peak you want to manually
integrate, and holding down the mouse button, drag across the peak to
define the point on the other side.
Repeat the procedure for other samples and components as required. If you
want to restore automatic peak detection and integration, right click in the
Chromatogram View and choose Method Settings or User Settings.

Modifying Calibration Parameters


Use the following procedure to modify the sequence calibration:
1. Select a target component. Xcalibur automatically updates the Result
Grid, Chromatogram and Companion Views.
2. Click on the Standards tab to display calibration standards results.
3. Inspect the calibration curve in the Companion View. If it is not currently
displayed either:

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• Choose View > Set Companion View >


Show Calibration Curve, or
• Right click in the Chromatogram View and choose
Set Companion View > Show Calibration Curve from the
shortcut menu.
4. Inspect the calibration curve according to the criteria used in your
laboratory. The Calibration Companion View displays the calibration
equation, the goodness of fit parameter, R2, and the weighting, W.
To view calibration and quantitation flags:
1. Right click on the Calibration Companion View and choose
Calibration Settings from the shortcut menu.
2. Select the Flags tab.
3. To exclude a point or sample from the calibration curve, right click on it
and choose Exclude from the shortcut menu. To include a previously
excluded point, right click on it and select Include from the shortcut
menu.
4. To exclude a level, right click on the Calibration Companion View and
choose Exclusion List from the shortcut menu. This opens the Cal
Exclusion List dialog box for the selected component.
• To exclude a level, click in the Exclude column adjacent to the level
to be excluded.
• To restore an excluded level, click in the Exclude column adjacent to
the level (on the word Yes) to be restored.
5. To adjust the calibration settings, right click on the Calibration
Companion View and choose Calibration Settings from the shortcut
menu. This opens the Calibration Settings dialog box.
• To adjust the ISTD associated with the component, select a new ISTD
on the Type page.
• To adjust the calibration equation, weighting or units, change the
Curve page parameters.
• To adjust calibration or QC levels, change use the Levels page.
• To make corrections for isotope contributions to ISTD or Target
components, adjust the parameters on the Isotope % page.
• To change calibration and quantitation flag thresholds, adjust settings
on the Flags page.
• To apply any changes to the sequence, click on the Apply button.
6. To export the calibration settings with peak integration and detection
parameters as a new method, choose File > Export Method.
Repeat the procedure for all brackets.

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Index
Finnigan Xcalibur ________________________________________________________________________

Index

A calculated amount, 4-15


Calculated Amount flag, 4-22
calibration, 1-5
Absolute Window text box, 4-26
file, 1-10, 3-10, 3-14, 4-3
Acquisition Queue page, 3-2, 3-30
flag, 2-41
pausing, 3-32
levels, 1-15
purging, 3-32
modifying parameters, 4-52
resuming, 3-32
replicates, 1-15, 4-3
Add Samples command, 4-16
Set Companion View list, 4-11
adding
Calibration And Quantitation Flags dialog box, 2-40
QCs to a sequence, 3-13
Calibration Companion view, 4-9, 4-36
standards to a sequence, 3-12
calibration curve, 1-5, 2-36, 4-11
Adjust Using Setting check box, 2-19
editing samples, 4-49
All (samples) page, 4-13
excluding a point, 4-36
All (stds/QC) page, 4-13
restoring a point, 4-37
Amount (Cal Level) text box, 2-43
units, 2-37
Amount (ISTD) text box, 2-35
Calibration File text box, 4-14
Amount (QC Level) text box, 2-44
Calibration Options dialog box, 2-34
Apply Changes dialog box, 2-5
Calibration page, 2-34
area, 2-38, 4-14
calibration settings
ratio, 4-15
Curve page, 4-39
Area Noise Factor text box, 2-21
Flags page, 4-42
Area Scan Window text box, 2-32
Levels page, 4-40, 4-41
Area Tail Extension text box, 2-32
Type page, 4-38
Area Threshold flag, 2-33, 4-23
Calibration Settings dialog box, 4-36, 4-37, 4-39
automating analysis, 3-2
carryover, 1-9
autosampler configuration, 3-11
Carryover Limit flag, 4-24
Avalon Event List dialog box, 2-28
Carryover Limit text box, 2-42
Avalon peak detection algorithm, 2-21
Change Instrument In Use dialog box, 3-26
User Identification Settings dialog box, 4-30
Chro page, 4-27, 4-28
average RF, 2-37
chromatogram
cursor actions, 2-10
B preview, 2-9
trace, 2-15
Base File Name text box, 3-9 Chromatogram view, 4-10
Base Sample ID text box, 3-11 Context menu, 4-19
baseline, 2-30 editing samples, 4-49
and noise window, 2-30 reviewing, 4-50
minimum number of scans in, 2-30 working in, 4-19
noise tolerance, 2-30 Chromatography Options dialog box, 2-20
baseline clipping, 2-51 Column Arrangement dialog box, 3-6
baseline clipping suitability test, 4-25 column overload, 2-51
Baseline Window text box, 2-21 suitability test, 4-25
Batch Reprocess Setup dialog box, 3-28 Columns command, 4-16, 4-17
blanks, 1-9, 3-12, 3-13 columns in sequences
Blanks page, 4-13 arranging, 3-5
Bracket Type group box, 3-12 changing user labels, 3-6
brackets, 1-8, 1-9, 4-11 Component List text box, 4-10
none, 1-10 component name, 2-14
non-overlapped, 1-14 Components list
open, 1-12 Processing Setup, 2-4
overlapped, 1-13 Components list text box, 2-5
Brackets/Groups In Use combo box, 4-14 concave suitability test, 4-25
browsers, 4-2 configuring the autosampler, 3-11
constrain peak width, 2-21
C continuing calibration method, 4-4
Contribution Of ISTD To Target Compound text box, 2-39
Cal Exclusion List dialog box, 4-43 Contribution Of Target Compound To ISTD text box, 2-39
Cal Level text box, 2-43 Copy Graph command, 4-36

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis _______________ I


ELECTRON CORPORATION
Index
___________________________________________________________________ Finnigan Xcalibur

Copy Row command, 4-16 linearity limit, 2-41


Correction For Isotope Contribution dialog box, 2-38 minimum signal-to-noise ratio, 2-51
cubic spline, 2-37 peak width, 2-49
cursor actions in Processing Setup, 2-10 quantitation limit, 2-42
resolution threshold, 2-48
D R-squared, 2-41
symmetry threshold, 2-48
Data Flags dialog box, 2-32 tailing, 2-50
Delete Selected Samples command, 4-16 Flags button, 2-33, 2-40
Details Of Selected Analysis dialog box, 3-32 Flags page, 4-22
Detected By flag, 4-22 Calibration Settings dialog box, 4-42
detecting components in a chromatogram, 2-20 User Identification Settings dialog box, 4-34
detection, 1-5 force origin, 2-37
limit, 2-41 Forward Matching factor, 4-28
Detection Limit flag, 4-24
Detection page, 2-20 G
peak detection, 2-22
User Identification Settings dialog box, 4-31 Genesis
detector type, 2-15 Advanced Detection Options dialog box, 2-28
Diff%, 4-15 peak detection algorithm, 2-21
Dilution Factor text box, 3-4, 3-14 User Identification Settings dialog box, 4-29
Disk Space dialog box, 3-20 Go To Line Number dialog box, 3-16
Display Options dialog box groups, 4-4
Processing Setup, 2-13
Quan Browser, 4-35 H
E height, 2-38, 4-14
ratio, 4-15
editing Height Threshold flag, 4-23
a sample, 4-49 highest peak, 1-5, 2-22
a sequence, 3-15, 4-48 Home Page
Enable Warnings command, 2-5 Information view, 3-30
end bracket, 1-14
Exclude command, 4-15
Exclusion List command, 4-36, 4-43
I
Expected text box, 2-19 ICIS
Export Sequence dialog box, 3-21 Advanced page
exporting User Identification Settings dialog box, 4-33
report, 2-56, 2-57 Advanced Parameters dialog box, 2-28, 2-30
sequence, 3-21 Detection page
external calibration file, 4-4 peak integration, 2-21
external standard, 1-7 Integration page
User Identification Settings dialog box, 4-32
F peak detection, 2-22
peak detection algorithm, 2-21
Failure Threshold text box, 2-50 User Identification Settings dialog box, 4-29
file name, 4-14 identification of components in a chromatogram, 1-4,
base, 3-9 2-14
File Name text box, 3-4, 3-13 Identification Options dialog box, 2-28, 2-29
File Path text box, 3-4, 3-13 Identification page, 2-14
Fill Down dialog box, 3-15 detector, 2-15
First Peak option button, 2-30 Expected text box, 2-19
flags mass range, 2-17
area threshold, 2-33 name, 2-14
baseline clipping, 2-51 peak detection, 2-15
calibration and quantitation, 2-40 Retention Time Window text box, 2-19
carryover limit, 2-42 scan filter, 2-15
column overload, 2-51 trace, 2-15
data, 2-33 User Identification Settings dialog box, 4-30
detection limit, 2-41 wavelength range, 2-17
height threshold, 2-33 ignore origin, 2-37

II_______________ Finnigan Xcalibur Getting Productive: Quantitative Analysis_____________


Thermo
ELECTRON CORPORATION
Index
Finnigan Xcalibur ________________________________________________________________________

Import Sequence dialog box, 3-8 signal-to-noise ratio, 2-51


Include command, 4-37 Minimum Peak Height text box, 2-22
include origin, 2-37 Minimum Peak Width text box, 2-32
Include Sample Reports check box, 4-46 More Flags page, 4-23
Include Summary Reports check box, 4-46 More Info page, 4-26
INCOS Noise method, 2-31 Multiplet Resolution text box, 2-32
INCOS Noise option, 2-31
Info page, 4-21, 4-27
Information view
N
Acquisition Queue page, 3-30 nearest RT, 1-5, 2-22
Injection Volume text box, 3-4, 3-14 New Sequence Template dialog box, 3-9
Instrument Method option box, 3-9 non-bracketed sequence, 4-4, 4-11
Instrument Method text box, 3-4, 3-13 none, bracket type, 1-10
Integration Type option box, 4-14 non-overlapped, bracket type, 1-14, 4-5
internal standard, 1-7 number of peak widths for noise detection, 2-51
International dialog box, 3-9
interpreting data, 4-2
Ion Coelution test, 4-26 O
ion ratio confirmation, 1-5, 2-26
Ion Ratio test, 4-26 open, bracket type, 1-12, 4-5
Isotope button, 2-38 opening
ISTD, 1-7, 2-35 files in Quan Browser, 4-9
area, 4-15 raw file in Processing Setup, 2-9
assigning to a target, 2-36 sequence in Sequence Setup, 3-5
correcting for contribution to target, 2-39 origin, 2-37
height, 4-15 overlapped, bracket type, 1-13, 4-5
ISTD Correction Amount text box, 3-4, 3-14
P
L
path, 3-13
labeling peaks, 4-35 Path text box, 3-9
Left Edge Type flag, 4-22 pausing the acquisition queue, 3-32
level, 4-15 peak
Level column, 1-15 area ratio, 1-7
Level text box, 3-4, 3-14 classification parameters, 2-49
Levels page detection, 2-22
Calibration Settings dialog box, 4-40, 4-41 detection method, 2-15
Processing Setup, 2-43 height
Limit Scan Wavelength check box column overload testing, 2-51
peak purity, 2-54 constrain peak width, 2-21
linear, 2-36 peak width testing, 2-49
linear log-log, 2-37 symmetry threshold, 2-48
Linearity Limit flag, 4-24 integration, 2-21
Linearity Limit text box, 2-41 labeling, 4-19, 4-35
list separator character, 3-22 parameters, 2-32
locally weighted, 2-37 status, 4-15
width, 2-49
M suitability test, 4-25
Peak Coverage text box
peak purity, 2-54
macros, 2-58 Peak Detection Settings command
Manual Integration command, 4-19 User Identification Settings dialog box, 4-28
Manually Add Peak command, 4-19 Peak Height text box
mass, 2-17 peak tailing, 2-50
Match by Position option, 3-17 Peak Information dialog box, 4-20
Match by Sample ID option, 3-17 Chro page, 4-27, 4-28
Match Probability factor, 4-28 Flags page, 4-22
Max Peak Width text box, 2-50 Info page, 4-21, 4-27
Method Settings command, 4-19 More Flags page, 4-23
Min Peak Width text box, 2-50 More Info page, 4-26
minimum qualifier ions, 4-26
number of scans in baseline, 2-30 spectrum candidates, 4-27

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ______________ III
ELECTRON CORPORATION
Index
___________________________________________________________________ Finnigan Xcalibur

Spectrum page, 4-28 Standard Dilution dialog box, 2-44


Suitability page, 4-24, 4-25 System Suitability page, 2-47
Peak Noise Factor text box, 2-21 zoom commands, 2-13
peak purity Reports view, 2-55
Enable check box, 2-54 Settings dialog box, 2-8
Limit Scan Wavelength check box, 2-54 Spectrum Options dialog box, 2-24
PDA chromatograms, 2-54 Standard Dilution dialog box, 2-44
Peak Coverage text box, 2-54 view bar, 2-4
Scan Threshold text box, 2-54 window, 2-3
Wavelength Range text box, 2-54 programs, 3-27
Peak Purity page, 2-53 Programs view, 2-58
Percent Test (QC Level) text box, 2-44
pinning, 2-11
point-to-point, 2-37
Q
previewing processing, 2-9 QC, 1-9
previews QC Failed flag, 4-23
active, 2-10 QC level, 1-15
pinning, 2-10 QC Level table, 2-44
print QC Level text boxes, 2-44
methods, 3-27 QC page, 4-13
sequence, 3-18 quadratic
Print Preview dialog box, 3-19 log-log, 2-37
Print Reports button, 4-46 Qual check box, 3-27
Print Selection dialog box, 3-19 qualifier ions, 4-26
Process Method text box, 3-13 qualitative processing, 3-27
processing actions, 3-27 quality control, 1-9
Processing Method option box, 3-9 Quan
Processing Method text box, 3-4 quantitative reprocessing option, 3-29
Processing Setup, 2-2 view, 2-9, 2-10
Apply Changes dialog box, 2-5 Quan Browser, 4-2
auto open raw file, 2-8 Cal Exclusion List dialog box, 4-43
buttons, 2-5 Calibration Settings dialog box, 4-37
Calibration And Quantitation Flags, 2-40 Chromatogram view, 4-10
Calibration Options dialog box, 2-34 component list, 4-10
Calibration page, 2-34 Display Options dialog box, 4-35
Cancel button, 2-5 getting started, 4-6
Chromatography Options dialog box, 2-20 menu bar, 4-9
Components list, 2-5 opening files, 4-6
Correction For Isotope Contribution dialog box, 2-38 Peak Information dialog box, 4-20
customizing setup, 2-8 Quantitation Results Sorting Order dialog box, 4-17
Data Flags dialog box, 2-32 quantitative processing option, 3-27
Display Options dialog box, 2-13 Reports dialog box, 4-46
Identification Options dialog box, 2-29 Result List Column Hiding dialog box, 4-17
Load Last Processing Method option button, 2-8 results grid, 4-10
OK button, 2-5 Select Report Samples dialog box, 4-47
Open Raw File command, 2-9 Set Companion View list, 4-11
Programs view, 2-58 title bar format, 4-9
Quan view toolbar, 4-9
Calibration And Quantitation Flags dialog box, 2-40 User Identification Settings dialog box, 4-28
Calibration Options dialog box, 2-34 View Sample Types dialog box, 4-6
Calibration page, 2-34 window features, 4-8
changing display options, 2-13 Quan view, 2-3, 2-4
chromatogram preview, 2-3 Calibration page, 2-34
Correction For Isotope Contribution dialog box, 2-38 Detection page, 2-20
Data Flags dialog box, 2-32 Identification page, 2-14
Detection page, 2-20 Levels page, 2-43
Identification Options dialog box, 2-29 Peak Purity page, 2-53
Identification page, 2-3, 2-14 previewing processing, 2-9
Levels page, 2-43 System Suitability page, 2-47
pages, 2-4 title bar format, 2-4
Peak Purity page, 2-53 toolbar, 2-4
spectrum preview, 2-3 using interactively, 2-10

IV ______________ Finnigan Xcalibur Getting Productive: Quantitative Analysis_____________


Thermo
ELECTRON CORPORATION
Index
Finnigan Xcalibur ________________________________________________________________________

using the toolbar, 2-13 adjust using, 2-19


zoom commands, 2-13 expected, 2-19
quantitation, 1-3, 1-6 reference, 1-4
flags RT reference, 2-19
carryover limit, 2-42 View Width text box, 2-19
detection limit, 2-41 window (sec), 2-19
linearity limit, 2-41 Window text box, 2-19
quantitation limit, 2-42 Reverse Matching factor, 4-28
mass, 1-4 Right Edge Type flag, 4-22
techniques, 1-7 rows
Quantitation Flags dialog box, 4-42 deleting, 3-16
Quantitation Limit flag, 4-24 inserting, 3-16
Quantitation Limit text box, 2-42 RSD%, 4-15
Quantitation Results Sorting Order dialog box, 4-17 R-squared, 2-41
quantitative analysis, 1-2 R-squared flag, 4-24
initiating, 3-27 RT, 4-15
reprocessing data, 3-29 RT Ref OK flag, 4-23
queue manager Run Sequence dialog box, 3-23
Details Of Selected Analysis dialog box, 3-32 running
updating the display, 3-32 programs or macros, 2-58
Queue Manager window, 3-31 sample or sequence, 3-23

R S
removing jobs from the acquisition queue, 3-32 S/N threshold, 2-30
Repetitive Noise option, 2-31 sample
Replace Calibration command, 4-14 ID, 4-15
replicate samples, 3-11 name, 4-14
replicates, 1-15, 4-3 position, 3-17
reports type, 4-14
exporting, 2-56, 2-57 Sample ID text box, 3-4, 3-13
from processing method, 3-27 Sample Information window, 3-31
sample, 2-56 Sample Type option box, 3-4, 3-13
template, 2-56, 2-57 samples
Reports command, 4-9, 4-10 editing in Quan Browser, 4-49
Reports dialog box, 4-46 ID, 3-13, 3-17
Select Samples button, 4-47 name, 3-5
Reports view, 2-55 QC, 3-13
reprocessing, 3-28 removing from the acquisition queue, 3-32
rescaling a preview display, 2-13 replicate, 3-11
Reset Scaling command, 4-20, 4-36, 4-45 reports, 2-56
resolution suitability test, 4-25 running, 3-23
resolution threshold, 2-48 type, 3-4, 3-13
response, 2-38 unknowns, 3-11
response factor, 1-5 volume, 3-5
Response High flag, 4-23 weight, 3-5
Response Low flag, 4-23 Saturated flag, 4-22
Response OK flag, 4-23 saturation suitability test, 4-25
Result List Column Hiding dialog box, 4-17 Save command, 4-9
results grid, 4-10 saving
changing the sort order, 4-17 page parameters in Processing Setup, 2-5
column headings, 4-14 sequence in Sequence Setup, 3-5
context menu, 4-16 scan filters, 1-4, 2-15
displaying columns, 4-17 Scan Threshold text box
editing a sequence, 4-48 peak purity, 2-54
editing samples, 4-49 Select Directory dialog box, 3-13
hiding columns, 4-17 Select Report Samples dialog box, 4-47
working in, 4-13 Select Samples button, 4-46
results review, 4-2 semi-quantitative analysis, 1-2
resuming the acquisition queue, 3-32 Send To Qual Browser command, 4-16
retention time, 1-3, 1-4 separator character, 3-9

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis ______________ V


ELECTRON CORPORATION
Index
___________________________________________________________________ Finnigan Xcalibur

Sequence Setup, 3-2 Spectrum At Peak Right Edge command, 4-45


Batch Reprocess Setup dialog box, 3-28 Spectrum Companion view, 4-9, 4-10, 4-12, 4-45
Change Instrument In Use dialog box, 3-26 Spectrum Options dialog box, 2-24
Column Arrangement dialog box, 3-6 Spectrum page, 4-25, 4-28
Disk Space dialog box, 3-20 Spectrum plot, 4-11, 4-45
Export Sequence dialog box, 3-21 standard
Fill Down dialog box, 3-15 bracket, 1-12
Go To Line Number dialog box, 3-16 clear, 1-10, 4-4
Import Sequence dialog box, 3-8 update, 1-10, 4-4
International dialog box, 3-9 Standard Dilution dialog box, 2-44
New Sequence Template dialog box, 3-9 standards, 1-5, 1-8, 3-12
Print Preview dialog box, 3-19 start bracket, 1-14
Print Selection dialog box, 3-19 start instrument, 3-25
Run Sequence dialog box, 3-23 startup, 3-26
Select Directory dialog box, 3-13 Startup Mode group box, 2-8
Transfer Row Information dialog box, 3-17 Status Bar command
User Labels dialog box, 3-6 Processing Setup, 2-4
sequences, 1-8 Stds page, 4-13
base sample ID, 3-11 Suitability page, 4-24
brackets, 3-12 baseline clipping test, 4-25
columns, 3-4 column overload test, 4-25
creating automatically, 3-9 concave test, 4-25
creating manually, 3-13 peak width test, 4-25
editing in Quan Browser, 4-48 resolution test, 4-25
editing in Sequence Setup, 3-15 saturation test, 4-25
exporting, 3-21 signal-to-noise ratio test, 4-25
importing, 3-8 symmetrical test, 4-25
new, 3-8 tailing test, 4-25
New command, 3-9 summary reports, 2-57, 3-27
pausing, 3-32 symmetrical suitability test, 4-25
printing, 3-18 symmetry threshold, 2-48
priority, 3-25 System Suitability page, 2-47
QC samples, 3-13
removing from the acquisition queue, 3-32
resuming, 3-32
T
running, 3-23 tailing, 2-50
samples, 3-11 tailing factor, 2-21
standards, 3-12 tailing suitability test, 4-25
starting number, 3-9 target, 2-36
Set Companion View list, 4-11 correcting for contribution to ISTD, 2-39
Set Peak To Not Found Status command, 4-20 target ratio %, 4-26
Set Sorting Order command, 4-16, 4-17 title bar, Quan Browser, 4-9
Settings dialog box, 2-8 toolbar
show all sample types, 4-6 Processing Setup, 2-4
Show Calibration Curve command, 4-36, 4-45 Quan Browser, 4-9
Show Peak Info command, 4-19, 4-20 trace, 1-4, 2-15
Show Spectrum View command, 4-36 combinations, 2-15
Show Standards And QC commands, 4-6 Transfer Row Information dialog box, 3-17
shutdown, 3-26 tray type, 3-11
signal-to-noise ratio threshold, 2-51 Type page
signal-to-noise suitability test, 4-25 Calibration Settings dialog box, 4-38
smoothing in processing method, 2-21
Smoothing Points text box, 2-21
sort order, 4-17 U
specified amount, 4-15
spectrum Units
candidates, 4-27 ISTD, 2-35
detection, 1-5, 2-22 units, 2-37, 4-15
preview, 2-9 unknowns, 1-8
cursor actions, 2-10 Unknowns page, 4-13
Spectrum At Peak Apex command, 4-45 updating queue manager, 3-32
Spectrum At Peak Left Edge command, 4-45 use as RT reference, 2-19

VI ______________ Finnigan Xcalibur Getting Productive: Quantitative Analysis_____________


Thermo
ELECTRON CORPORATION
Index
Finnigan Xcalibur ________________________________________________________________________

User Identification Settings dialog box


Detection page, 4-31
W
Flags page, 4-34
Warning
ICIS Advanced page, 4-33
automatic adjustment, 4-42
ICIS Integration page, 4-32
Enable Warning command, 2-5
Identification page, 4-30
flags, 2-47
User Labels dialog box, 3-6
Warning dialog box, 4-6
User Peak Detection Settings command, 4-19, 4-28
wavelength, 2-17
User Settings command, 4-19
Wavelength Range text box
user-defined columns, 3-5, 3-6, 3-14
peak purity, 2-54
weighting, 2-37
V window (sec), 2-19
windows
Valid flag, 4-22 Processing Setup, 2-3
Valley Detect flag, 4-22 Quan Browser, 4-2
Value option button, 2-29 queue manager, 3-31
Vial list, 3-18 Sequence Setup, 3-2, 3-3
vial number, 3-4, 3-11, 3-14 working in the results grid, 4-13
Vial Position text box, 3-4
view all, 4-13
View Sample Types dialog box, 4-6
X
View Spectrum Plot command, 4-45
xcal files, 4-4
View Stds and QCs option, 4-13
View Width text box, 2-19
Void Time group box, 2-29 Z
Zoom commands, 4-10

Thermo ____________ Finnigan Xcalibur Getting Productive: Quantitative Analysis _____________ VII
ELECTRON CORPORATION