Xcalibur Processing Setup and Analysis

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Printing History: Revision A printed in May 2003.

Software Revision: Xcalibur 1.4
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fold
Contents
'JOOJHBO9DBMJCVS ________________________________________________________________________
Contents
Read This First ............................................................................................................................ iii

Changes to the Manual and Online Help ............................................................................................... iv
Abbreviations .......................................................................................................................................... v
Typographical Conventions ................................................................................................................... ix

Data Input ............................................................................................................................. ix
Boxed Information................................................................................................................. x
Topic Headings..................................................................................................................... xi
Reply Cards ........................................................................................................................................... xii
Processing Setup and the Analysis of Quantitation Data ..........................................................1

Creating a Processing Method ................................................................................................................. 6
Opening the Processing Setup Window ................................................................................. 7
Specifying Chromatography by LC........................................................................................ 8
Specifying Calibration by Internal Standard .......................................................................... 9
Opening a Raw Data File ..................................................................................................... 10
Specifying Component Identification Settings for the Internal Standard ............................ 12
Specifying Component Identification Settings for the Target Compound ........................... 19
Entering Peak Integration and Detection Parameters........................................................... 21
Selecting Calibration Settings .............................................................................................. 25
Specifying Calibration and QC Levels................................................................................. 29
Checking System Suitability Options................................................................................... 32
Specifying Report Templates ............................................................................................... 33
Saving the Processing Method ............................................................................................. 36
Adding the Processing Method to a Sequence ....................................................................................... 39
Processing the Raw Files and Reviewing Results.................................................................................. 47
Creating Reports..................................................................................................................................... 52
Sample Reports ...................................................................................................................................... 54
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Welcome to Xcalibur®, the Thermo Electron Finnigan™ mass spectrometry

data system!
Xcalibur uses a Processing Method to automatically detect and determine the
concentration (amount) of the sample being analyzed. This Getting
Productive: Processing Setup and the Analysis of Quantitation Data
manual steps you through a procedure for creating a Processing Method to
analyze a sample data set provided with the Xcalibur data system. This
manual also describes how to review your results and to present your results
in reports.
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Read This First
Changes to the Manual and Online Help @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
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
'JOOJHBO9DBMJCVS _____________________________________________________________ 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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Abbreviations @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
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
'JOOJHBO9DBMJCVS _____________________________________________________________ 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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Abbreviations @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
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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'JOOJHBO9DBMJCVS ___________________________________________________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 plus 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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Typographical Conventions @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
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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'JOOJHBO9DBMJCVS ___________________________________________________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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Reply Cards @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
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,
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Processing Setup and the
Analysis of Quantitation Data
Processing Setup is part of Xcalibur®, the Finnigan™ mass spectrometry

data system.
You use the Processing Setup window to build a Processing Method. The
Processing Method provides instructions to Xcalibur on how to perform
qualitative analysis on a raw file. Xcalibur uses a Processing Method to
automatically detect and determine the concentration (amount) of the sample
being analyzed. The Processing Method is a defined set of parameters that
provide Xcalibur a recipe for automatic quantitation.
In a typical quantitation experiment, Xcalibur measures the response of the
detection system to the compounds in your sample. The response
measurement is taken from the area under each peak and is determined by an
integration calculation. See Figure 1.
In a quantitation experiment involving internal standards, Xcalibur calculates
the ratio of the peak area of the target compound to the peak area of the
internal standard to provide an area ratio. When peak area ratios of several
known standards are measured, a plot of amount versus area ratio can be
drawn to provide a calibration curve, as shown in Figure 2. Xcalibur
accomplishes quantitative analysis by comparing the measured peak area ratio
of a sample to the calibration curve and reading the amount of compound
(according to the curve) that gives rise to that peak area ratio. This is the
calculated amount.
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Processing Setup and the Analysis of Quantitation Data
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Figure 1. Integrated chromatogram peak
Figure 2. Calibration curve
Xcalibur detected and integrated the peak shown in Figure 1, and constructed
the calibration curve shown in Figure 2, by using parameters contained in a
Processing Method.
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Xcalibur can fit the calibration data to the following curve types:
• Linear
• Quadratic
• Linear log-log
• Quadratic log-log
• Average RF
• Point-to-point
• Cubic spline
• Locally weighted
When performing the least squares fit to the calibration data, Xcalibur can
weight the calibration data with the following weighting functions:
• Equal
• 1/X
• 1/X2
• 1/Y
• 1/Y2
• 1/S2, where S2 = X2 + Y2
You can have Xcalibur ignore the origin, use the origin as a data point, or
force the calibration curve to include the origin.
In addition, the Processing Method can define single or multiple target
compounds and single or multiple internal standards. You can define multiple
internal calibration levels or quality control (QC) levels for selected target
compounds. Processing Methods are saved as file type .pmd.
This manual gives a stepwise procedure for analyzing quantitation data. A
flow diagram for analyzing quantitation data is shown in Figure 3. The third
step in Figure 3, running a sequence, is grayed because data acquisition is not
discussed in this manual.
In the example contained in this manual you analyze an example data set
provided in the C:\Xcalibur\examples\data directory of your Xcalibur data
system. This data was collected on a proprietary pharmaceutical product in
the applications laboratory at Thermo Finnigan using LC/MS/MS techniques
in the electrospray ionization mode.
The pharmaceutical product has the code name drugx. An isotopically labeled
internal standard (ISTD) was used to quantitate the pharmaceutical. The
internal standard is a deuterated analogue of drugx that has four deuterium
atoms exchanged for hydrogen atoms in the compound. In this example, the
internal standard has the code name D4. The concentration of the internal
standard in the calibration standards and quality control (QC) samples in this
example is 100 pg/mL.
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Calibration standards were prepared by spiking human plasma with drugx to

give nine calibration levels with concentrations of 10, 25, 50, 100, 200, 400,
600, 800, and 1000 pg/mL. Triplicate samples were run at the high
(1000 pg/mL) and low (10 pg/mL) ends of the curve with single samples run
in between.
QC samples were prepared similarly by spiking human plasma with drugx to
give three QC levels with concentrations of 10, 400, and 1000 pg/mL. Six
replicates per QC level were run.
In this manual you create a new Processing Method and add it to an existing
Sequence. You then process the raw data files, review calibration standards,
and QCs, and create and review reports.
Analyzing quantitation data involves the following steps:
• Creating a Processing Method
• Adding the Processing Method to a Sequence
• Processing the raw files and reviewing the results
• Creating reports
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PROCESSING SETUP:
CREATE PROCESSING
METHOD
SEQUENCE SETUP:
ADD PROCESSING
METHOD TO SEQUENCE
QUAN BROWSER:
PROCESS RAW FILES
USING SEQUENCE
PROCESSING SETUP OR
ALL PEAKS FOUND NO QUAN BROWSER: MODIFY
AND INTEGRATED PEAK DETECTION OR
CORRECTLY? INTEGRATION PARAMETERS
YES
PROCESSING SETUP
NO CALIBRATION
OR QUAN BROWSER:
MODIFY CALIBRATION CURVE OK?
CURVE PARAMETERS
YES
SEQUENCE SETUP:
CREATE REPORTS
Figure 3. Flow diagram for analyzing quantitation data with Processing Setup
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Creating a Processing Method

You use the Processing Setup window to create a Processing Method.
Creating a Processing Method for this example involves the following steps:
• Open the Processing Setup window. You open the Processing Setup
window from the Xcalibur Home Page.
• Specify chromatography by LC. You specify that the drugx data set was
obtained by an LC/MS/MS experiment.
• Specify calibration by internal standard. You specify that an internal
standard was used in the quantitative analysis.
• Open a raw data file. You need to open a raw file from your data set to test
peak detection and integration parameters. Processing Setup also obtains
scan filter information from the raw file.
• Specify component identification settings for the internal standard.
Processing Setup needs this information to associate the internal standard
with a chromatographic peak.
• Specify component identification settings for the target compound. The
component identification settings for the target compound are slightly
different than those for the internal standard.
• Enter peak integration and detection parameters. You test peak integration
and detection settings using the data in the raw file you opened earlier.
During batch processing, Xcalibur uses this information to detect all
component peaks in all raw files in your data set.
• Specify calibration settings. You identify the target compounds and
internal standards and specify what type of calibration curve to fit the
calibration data to.
• Specify calibration and QC levels. You need to specify the amount of
analyte in the calibration standards and quality control (QC) standards for
Xcalibur to construct and test the calibration curve.
• Check system suitability options (optional). The system suitability
options allow you to carry out a sequence of automated chromatographic
checks that assign a pass or fail qualification to a target peak.
• Specify report templates. Xcalibur uses report templates to create reports
of your quantitative analysis experiment.
• Save the Processing Method. When you save the Processing Method it
becomes available to other Xcalibur windows such as Sequence Setup.
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'JOOJHBO9DBMJCVS ________________________________________________ Creating a Processing Method
Opening the Processing Setup Window

You open the Processing Setup window from the Xcalibur Home Page. See
Figure 4.
Open the Processing Setup window – Quan view – Identification page as
follows:
1. In the Xcalibur Home Page, click on the Processing Setup button in the
Road Map or choose Goto > Processing Setup.
2. Click on the Quan button to display the Quan view (if it is not already
displayed).
3. Click on the Identification tab to display the Identification page (if it is
not already displayed). The Identification page is shown in Figure 5.
The Identification page of the Quan view of the Processing Setup window
allows you to name the components of your sample, to display the spectrum,
and to specify retention time and peak identification criteria. If a method is
automatically loaded, press File > New to start with a fresh display.
Figure 4. Xcalibur Home Page
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Figure 5. Processing Setup window – Quan view – Identification page
Specifying Chromatography by LC
The data in this example was obtained by using liquid chromatography (LC).
Specify chromatography by LC as follows:
1. In the Processing Setup window, choose Options > Chromatography
By to open the Chromatography Options dialog box. See Figure 6.
2. In the Chromatography Options dialog box, select the LC option.
3. Click on OK to specify chromatography by LC and to dismiss the dialog
box.
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Figure 6. Chromatography Options dialog box
Specifying Calibration by Internal Standard

This example uses an isotopically labeled internal standard to quantitate the
pharmaceutical drugx. Specify that you are calibrating by internal standard as
follows:
1. In the Processing Setup window, choose Options > Calibration By to
open the Calibration Options dialog box. See Figure 7.
2. In the Calibration Options dialog box, select the Internal Standard option.
3. Click on OK to specify that you are calibrating by internal standard and to
dismiss the dialog box.
Figure 7. Calibration Options dialog box
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Opening a Raw Data File

You need to open a raw file from your data set to determine peak detection
and integration parameters. In this example you use the drugx_03.raw file. In
general, you open a raw file corresponding to a low-concentration calibration
standard.
To open drugx_03.raw using the Open Raw File dialog box, proceed as
follows:
1. Click on the open raw file button or choose File > Open Raw File to
open the Open Raw File dialog box. See Figure 8.
2. Browse through the directories to find the file, for example:
C:\Xcalibur\examples\data\drugx_03.raw.
3. Click on drugx_03.raw, and then click on Open. Processing Setup selects
the drugx_03.raw file and displays the unfiltered total ion chromatogram.
See Figure 9.
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Figure 8. Open Raw File dialog box, showing the selected directory
path and raw file
Note. If you save a Processing Method 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 you open the Processing Method if the
Auto-open raw file On option button has been selected in the Settings dialog
box.
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Figure 9. Identification page, showing the total ion current (TIC) chromatogram of
drugx_03.raw
Specifying Component Identification

Settings for the Internal Standard
Processing Setup needs component identification information to associate the
internal standard with a chromatographic peak. The following topics describe
how to specify the identification settings for the internal standard, D4:
• Naming components
• Selecting detector type
• Specifying trace type
• Matching scan filters with components
• Displaying the spectrum and determining the retention time
12 5IFSNP
Naming Components
You use You use the Name combo box in the Identification page to name the
components of your sample. When you change settings, Processing Setup
changes the settings for the named component only.
Enter the name of the target compound in the Name combo box, as follows:
1. In the Name combo box, select <New>. Then, enter D4 to specify the
name of the internal standard.
2. Click on OK to save the new name. See Figure 10.
Figure 10. Identification page, after the name D4 has been entered
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Selecting the Detector Type

You use the Detector list box on the Identification page to specify the type of
detector you used to obtain the raw file.
In the Detector Type list box, select MS.
Selecting the Peak Detection Type

You use the Peak Detect list box on the Identification page to specify the type
of peak detection algorithm (ICIS, Genesis, or Avalon) you want to use to
analyze raw data. These algorithms also apply smoothing, construct a
chromatogram using the scan and/or mass filters, assign peak numbers,
generate a peak list, and determine the peak start and peak end points. All
algorithms provide component peak detection and chromatographic peak
detection.
In the Peak Detect list box, select ICIS, Genesis or Avalon.
Matching Scan Filters with Components

Xcalibur creates unique scan filters to acquire data according to the type of
experiment you specify in the Instrument Method. When you load a raw file,
Xcalibur lists in the Filter combo box the scan filters associated with the raw
file. In this example, selected reaction monitoring (SRM) data were acquired
on a proprietary drug of molecular weight 465 u1 and a deuterated internal
standard of molecular weight 469 u2 (drugx and D4, respectively) using
alternating product ion scans. To calibrate and quantitate drugx it is necessary
to filter the total ion current chromatogram.
Match D4 with its scan filter as follows:
1. In the Filter combo box, click on the down arrow to display the scan
filters in the file drugx_03.raw.
2. Click on + c SRM ms2 [email protected][423.30-425.30] to select the scan
filter for D4.
3. Click on OK to display the mass chromatogram for D4. See Figure 11.
Selecting the Trace Type

You use the Trace list box on the Identification page to specify the type of
chromatogram you want to use for processing.
In the Trace list box, select TIC (total ion current).
1
Parent ion m/z 465; product ion m/z 420
2Parent ion m/z 469; product ion m/z 424
14 5IFSNP
Figure 11. Identification page, showing the mass chromatogram for D4.
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Displaying the Spectrum and Determining the

Retention Time
To display the mass spectrum and automatically enter the retention time, do
the following:
1. Display the mass spectrum of the currently active component and
automatically enter the retention time of the LC peak as follows:
a. Click on the pin button (currently grayed) in the upper right corner of
the Spectrum pane (see arrow in Figure 11) to activate the Spectrum
pane. The pin background turns green and the pin appears stuck into
the screen. ( ).
b. Click and drag the cursor in the Chromatogram pane from left to right
across the chromatogram peak as shown in Figure 12.
Figure 12. Identification page, showing the click-and-drag procedure for selecting the scan
corresponding to the peak maximum in the chromatogram pane
16 5IFSNP
c. Release the mouse button, and the following events occur

automatically:
• Processing Setup selects the retention time of the peak maximum
and highlights the selected scan in the LC peak with a red marker
in the Chromatogram pane.
• Processing Setup enters the retention time corresponding to the
selected scan in the Expected text box in the Retention Time
group box.
• Processing Setup displays the mass spectrum of the product
ion(s) in the Spectrum pane. See Figure 13.
Figure 13. Identification page, showing a marker (vertical line) indicating the retention time
corresponding to the peak maximum
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2. Select the Use as RT Reference check box to use the retention time of
D4 as the retention time reference.
3. Enter 2.00 into the View Width text box.
4. Click on OK to save the component identification information for D4.
Processing Setup automatically shades the integrated portion of the peak
gray and displays blue integration markers at the starting and ending
points of the peak integration. The baseline is indicated by a blue line that
connects the integration markers. See Figure 14.
Figure 14. Identification page, showing the area of peak integration (grayed) and the integration
markers (blue circles indicated by arrows)
5. Inspect the integrated peak and ensure the following:

• Ensure that the retention time on the peak agrees with that in the
Expected text box in the Retention Time group box.
• Ensure that the scan filter in the Filter text box is matched to the
correct component in the Components list.
If the peak has been identified properly, you are ready to specify the peak
identification parameters for the target compound. Go to the next topic:
Specifying Component Identification Settings for the Target Compound.
18 5IFSNP
If the peak has not been identified, repeat the procedure. Make sure the
Spectrum pane (the pane on the right of the Chromatogram pane) is pinned
before performing step 1b.
Specifying Component Identification

Settings for the Target Compound
You also use the Identification page to specify component identification
settings for the target compound, drugx. The settings for the target compound
are slightly different from those for the internal standard.
Specify the settings for the target compound, as follows:
1. In the Name combo box, select <New>. The Apply Changes dialog box
appears if you have warnings enabled. Click on Yes to apply your
changes and proceed.
2. Then, enter drugx to specify the name of the target compound. Click on
OK to save the new name.
3. In the Detector Type list box, select MS (if it is not already selected).
4. In the Peak Algorithm combo box, select ICIS.
5. Match the target compound with its scan filter as follows:
a. Click on the down arrow in the Filter list box to display the scan
filters in the file drugx_03.raw.
b. Click on + c SRM ms2 [email protected][419.30-421.30] to select the
scan filter for drugx.
c. Click on OK to apply the scan filter to the total ion current.
Processing Setup automatically displays the mass chromatogram
corresponding to the target compound.
6. In the Trace list box, select TIC (if it is not already selected).
7. Display the mass spectrum of the currently active component and
automatically enter the retention time of the LC peak, as follows:
a. Click on the pin button (currently grayed) in the upper right corner of
the Spectrum pane to activate the Spectrum pane. The pin background
turns green and the pin appears stuck into the screen.
b. Click and drag the cursor in the Chromatogram pane from left to right
across the chromatogram peak.
c. Release the mouse button, and the following events occur
automatically:
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• Processing Setup selects the retention time of the peak maximum

and highlights the selected scan in the LC peak with a red marker
in the Chromatogram pane.
• Processing Setup enters the retention time corresponding to the
selected scan in the Expected text box in the Retention Time
group box.
8. Specify to use the retention time of D4 to adjust the retention time of
drugx, as follows:
a. Select the Adjust Using check box. The Adjust Using list box
becomes active.
b. In the Adjust Using list box, select D4.
c. Enter 2.00 into the View Width text box.
9. Click on OK to accept the peak identification settings for drugx. See
Figure 15.
Figure 15. Identification page, showing the peak identification settings for drugx
20 5IFSNP
Entering Peak Integration and Detection

Parameters
You use the Detection page of the Quan view of the Processing Setup window
to enter peak integration and detection parameters. You test the peak
integration and detection parameters on the raw file you opened earlier.
Note. Xcalibur provides the ICIS, Genesis, and Avalon peak detection
algorithms. All algorithms analyze raw data, apply smoothing, construct a
chromatogram using the scan and/or mass filters, assign peak numbers,
generate a peak list, and determine the peak start and peak end points. The
ICIS peak detection algorithm has been designed for MS data and has
superior peak detection efficiency at low MS signal levels. The Genesis peak
detection algorithm has been provided for backwards compatibility with
Xcalibur 1.0 studies. The Avalon peak detection algorithm has been
designed for chromatographic data and supports detectors other than MS
detectors.
All new Processing Setup methods are created using the Xcalibur default
peak detection algorithms. To change the default peak detection algorithm,
choose Tools > Configuration from the Xcalibur Home Page to open the
Xcalibur Configuration dialog box. Then, click on the Peak Detection tab
and change the default peak detection algorithm for each data type.
Entering peak integration and detection parameters involves the following:

• Displaying the Detection page
• Entering peak integration parameters
• Entering peak detection parameters
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Displaying the Detection Page

Click on the Detection tab of the Processing Setup window to display the
Detection page. The Detection page is shown in Figure 16.
Figure 16. Processing Setup window – Quan view – Detection page
Entering Peak Integration Parameters

You enter the peak integration parameters to specify how Xcalibur determines
the area of each peak in the chromatogram. The peak integration parameters
are contained in the Peak Integration group box on the Detection page.
Note. When you are entering many components with similar peak
integration parameters, first enter all of the identification parameters for one
of the components. Then, click on Save As Default. These parameters
then become the default values for new components.
Enter the ICIS peak integration parameters as follows:

1. In the Components list, click on D4 to select the internal standard, D4.
22 5IFSNP
The Smoothing Points text box allows you to enter the number of points
used in the moving average used to smooth data. The valid range is 1 (no
smoothing) to 15 (maximum smoothing).
2. In the ICIS Peak Integration group box, in the Smoothing Points text box,
enter 5 to specify five smoothing points.
3. In the Baseline Window text box, enter 40 to set to 40 scans the maximum
number of scans that Xcalibur looks for a local minimum.
The area noise factor is a noise level multiplier used to determine the
location of a peak edge after the location of the possible peak. The valid
range is 1 to 500.
4. In the Area Noise Factor text box, enter 5 to specify an area noise factor
of 5.
The peak noise factor is a noise level multiplier used to determine the
potential peak signal threshold. The valid range is 1 to 1000.
5. In the Peak Noise Factor text box, enter 10 to specify a peak noise factor
of 10.
The constrain peak width option allows you to control how much of the
peak is integrated by specifying a peak height threshold and a tailing
factor.
6. Leave the Constrain Peak Width check box empty ( ).
7. Click on OK to save the peak integration parameters.
8. Select drugx in the Components list and repeat steps 2 through 7 for the
target compound.
Entering Peak Detection Parameters

You enter the peak detection parameters to specify how Xcalibur determines
whether a component has been found. The peak detection parameters are
contained in the Peak Detection group box.
Enter the peak detection parameters as follows:
1. In the Components list, select D4 to select the internal standard, D4.
2. In the ICIS Peak Detection group box, select the Highest Peak option
button to associate D4 with the highest peak in the chromatogram.
3. In the Minimum Peak Height (S/N) text box, enter 3 to have Xcalibur
ignore all peaks that do not have a signal-to-noise ratio of 3 or greater.
The ICIS Advanced Parameters dialog box allows you to specify
advanced component detection criteria. These additional criteria can be
used if the standard detection criteria do not provide the desired results.
Refer to the Xcalibur online Help for information on the parameters in the
ICIS Advanced Parameters dialog box.
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4. Ensure that the advanced peak detection parameters are set to their default
values:
a. Click on the Advanced button to open the ICIS Advanced Parameters
dialog box.
b. Inspect the ICIS Advanced Parameters dialog box. Make sure the
settings are the same as those in Figure 17.
c. Click on OK to close the dialog box
5. If it is needed, click on OK to save the peak detection parameters.
6. Select drugx in the Components list and repeat steps 2 through 5 for the
target compound. The Detection page should look like the one shown in
Figure 18.
Figure 17. ICIS Advanced Parameters dialog box,

showing default settings
24 5IFSNP
Figure 18. Detection page, showing settings for drugx and D4
Selecting Calibration Settings

You use the Calibration page of the Quan view of the Processing Setup
window to identify the target compound and internal standard and to specify
settings used by Xcalibur to create a calibration curve.
Select the calibration settings, as follows:
1. Click on the Calibration tab to display the Calibration page. See
Figure 19.
2. Enter the calibration settings for the internal standard, D4, as follows:
a. In the Components list, select D4.
b. In the Component Type group box, select the ISTD option button to
select D4 as the internal standard.
c. In the Amount text box in the ISTD group box, enter 100 to specify
an internal standard amount of 100 pg/mL.
d. In the Units text box, enter pg/mL to specify pg/mL as the units of
concentration.
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Figure 19. Processing Setup window – Quan view – Calibration page
e. Click on OK to save the settings for D4. The Calibration page should
look like Figure 20.
3. Enter the calibration settings for the target compound, drugx:
a. In the Components list, select drugx.
b. In the Component Type group box, select the Target Compound
option button to specify drugx as the target compound.
c. In the Target Compounds group box, in the ISTD list box select D4 as
the internal standard.
d. Click on OK to save the settings for drugx.
26 5IFSNP
Figure 20. Calibration page, showing the settings for the internal standard, D4
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4. Enter the calibration curve settings as follows:

a. In the Calibration Curve list box, select Quadratic to specify a
quadratic calibration curve.
b. In the Units text box, enter pg/mL to specify the units of
concentration.
c. In the Weighting group box, select the 1/X option button to specify a
weighting of 1/X.
d. In the Origin group box, select the Ignore option button to have
Xcalibur not include the origin as a data point when fitting the
calibration curve.
e. In the Response group box, select the Area option button to have
Xcalibur use the area of the peak to determine response.
f. Click on OK to save the settings. The Calibration page should look
like the one shown in Figure 21.
Figure 21. Calibration page, showing the settings for the target compound, drugx
28 5IFSNP
Specifying Calibration and QC Levels

You use the Levels page of the Quan view of the Processing Setup window to
specify the amount of analyte in the calibration standards and quality control
(QC) standards. Xcalibur uses this information to construct and test the
calibration curve.
Specify calibration and QC levels, as follows:
1. From the Quan view of the Processing Setup window, select drugx in the
Components list. Then select the Levels tab to display the Levels page.
See Figure 22.
2. The Levels page is not available for ISTD components and a warning box
will appear if you have selected the ISTD and try to open the Levels page.
Figure 22. Processing Setup window – Quan view – Levels page
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3. Enter Calibration Level data as follows:

a. In the first Cal Level text box, enter cal 1 as the name of the first
calibration level.
b. Press <Tab> to advance the cursor to the first Amount text box.
c. Enter 10 in the text box to specify an injection amount of 10 pg.
d. Press <Tab> twice to create a new row and to advance the cursor to
the second Cal Level text box.
e. Repeat this procedure until you fill in the nine calibration levels as
shown in Table 1.
Table 1. Calibration Level Table, showing the amount of drugx (in
picograms) injected in 10 µL of the corresponding
calibration solution
Cal Level Amount
cal 1 10
cal 2 25
cal 3 50
cal 4 100
cal 5 200
cal 6 400
cal 7 600
cal 8 800
cal 9 1000
4. Enter QC Level data as follows:

a. In the first QC Level text box, enter QC 1 as the name of the first QC
level.
b. Press <Tab> to advance the cursor to the first Amount text box. Then,
enter 10 in the text box to specify an injection amount of 10 pg.
c. Press <Tab> to advance the cursor to the first % Test text box. Then,
enter 20 in the text box to specify a 20% test value.
Note. The % Test values for QCs in this example are shown in Table 2.
These are the criteria used in this example to determine whether QCs pass.
You can use any % Test parameters that are appropriate for your particular
application.
d. Press <Tab> twice to create a new row and to advance the cursor to
the second QC Level text box.
30 5IFSNP
e. Repeat this procedure until you fill in the three QC levels as shown in
Table 2.
Table 2. QC Level Table, showing QC levels, amounts
(in pg/injection), and % Test for drugx
QC Level Amount % Test

QC 1 10 20
QC 2 400 15
QC 3 1000 15
5. Click on OK to save the calibration and QC level settings. The Levels

page should look like the one shown in Figure 23.
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Figure 23. Levels page, showing the completed Calibration Level and QC Level Tables
Checking System Suitability Options

The System Suitability page of the Quan view of the Processing Setup
window allows you to set up a sequence 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 (chromatography by GC only), all qualifier ion peaks within the
retention time window. In this example, you do not use the system suitability
checks.
Ensure the system suitability options are turned off, as follows:
1. Click on the System Suitability tab to display the System Suitability page.
See Figure 24.
2. Ensure that the Resolution Parameters, Peak Classification Parameters,
and Symmetry Parameters check boxes are unchecked ( ).
3. If you made any changes, click on OK to save the changes.
32 5IFSNP
Figure 24. Processing Setup window – Quan view – System Suitability page
Specifying Report Templates

Xcalibur contains sample report templates that allow you to create reports of
your results. You specify report templates in the Reports view of the
Processing Setup window. In addition, you can use the Merlin Report Wizard
to create your own custom report templates. Refer to the Xcalibur Getting
Productive: Merlin, the Custom Report Wizard manual for procedures for
creating report templates.
Specify report templates as follows:
1. In the view bar, click on the Reports button to display the Reports view.
See Figure 25.
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Figure 25. Processing Setup window - Reports view
2. In the Sample Reports table, click in the Enable text box to display the
enable check box for the first row. Then, select the Enable check box.
3. Double-click in the Save As text box in the first row of the Sample
Reports table to display the drop down list. Then, select Doc to save the
report as a .doc file.
4. Still in the first row of the Sample Reports table, double click in the
Report Template Name text box. Xcalibur displays the Browse for
Sample Report Templates dialog box. See Figure 26.
34 5IFSNP
Figure 26. Browse for Sample Report Template dialog box
5. Select PeakIntegration2.doc and click on Open to choose the sample

peak integration report template.
6. Click in the Enable text box in the second row of the Sample Reports
table to display the enable check box for the second row. Then, select
the Enable check box.
7. Click in the Save As text box in the second row of the table to display the
drop down list. Then, select Doc to save the report as a .doc file.
8. Double-click in the Report Template Name text box in the second row of
the table. Xcalibur displays the Browse for Sample Report Templates
dialog box. See Figure 26.
9. Select CompCalReport2_ICIS.doc and click on Open to choose the
sample component calibration report template.
10. Click on OK to save the settings. The Report view should look like the
one shown in Figure 27.
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Figure 27. Reports view, showing the sample peak integration report and component
calibration report templates selected
Saving the Processing Method

You need to save the Processing Method to use it in other windows. Save the
Processing Method as drugx_example.pmd as follows:
1. Choose File > Save As to display the File Summary Information dialog
box.
2. Type Processing Method for drugx example in the description text box.
See Figure 28.
3. Click on OK to open the Save As dialog box. See Figure 29.
4. Find the C:\Xcalibur\examples\methods directory or the directory where
you saved the Xcalibur examples and name the Processing Method
drugx_example.pmd, as follows:
a. Browse through the directory tree to find the
C:\Xcalibur\examples\methods directory.
b. In the File Name text box, enter drugx_example.pmd.
36 5IFSNP
5. Click on Save to save the Processing Method and close the dialog box.
Figure 28. File Summary Information dialog box
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Figure 29. Save As dialog box, showing drugx_example.pmd
38 5IFSNP
'JOOJHBO9DBMJCVS ____________________________________ Adding the Processing Method to a Sequence
Adding the Processing Method to a

Sequence
A Sequence is a list containing sample information. You use the Sequence
Setup view to create or modify a Sequence. For this example, you add the
Processing Method you just created, drugx_example.pmd, to an existing
Sequence, drugx.sld.
Note. For additional information about setting up a Sequence for

Quantitative Analysis refer to Xcalibur Getting Productive: Quantitative
Analysis and/or the online Help.
Use the Sequence Setup view to add the Processing Method to the Sequence
as follows:
1. in the Xcalibur Home Page window (See Figure 4), click on the Sequence
Setup button to open the Sequence Setup view.
2. Click on the Open (Sequence) toolbar button (or choose File > Open) to
display the Open dialog box. See Figure 30.
3. Browse through the directories to find the Sequence file
C:\Xcalibur\examples\methods\drugx.sld.
4. Select drugx.sld, and then click on Open to select the Sequence file
drugx.sld. Sequence Setup displays the Sequence drugx.sld. See
Figure 31.
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Adding the Processing Method to a Sequence @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
Figure 30. Open dialog box, showing the selected Sequence,

drugx.sld
40 5IFSNP
Figure 31. Sequence Setup view, showing the Sequence drugx.sld saved on the C:\ drive.
5. Change the Processing Method to

C:\Xcalibur\examples\methods\drugx_example.pmd in the Sequence as
follows:
a. Double-click on the top cell (cell number 1) in the Proc Meth column.
Xcalibur displays the Select Processing Method dialog box.
b. Browse through the directories to find the Processing Method
C:\Xcalibur\examples\methods\drugx._example.pmd. See Figure 32.
c. Select drugx_example.pmd, and then click on Open to enter the
Processing Method
C:\Xcalibur\examples\methods\drugx._example.pmd in the first cell.
d. Single-click on the first cell in the Proc Meth column to select it.
Then, click on Proc Meth above the column to select the entire
column.
5IFSNP
e. Click on the Fill Down button in the toolbar. Xcalibur opens the Fill
Down dialog box. See Figure 33.
f. Ensure that the parameters are set to fill rows 2 to 31 of the
Processing Method column with row 1. (Figure 33)
g. Click on OK to change the Processing Method to
C:\Xcalibur\examples\methods\drugx_example.pmd in all rows of the
Sequence. The Sequence should now look like the one shown in
Figure 34.
Figure 32. Select Processing Method dialog box, showing the

Processing Method drugx_example.pmd selected
42 5IFSNP
Figure 33. Fill Down dialog box, with settings to fill rows 2 to 31 of
the Processing Method column with row 1.
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Figure 34. Drugx Sequence, with drugx_example.pmd selected as the Processing Method
6. Save the Sequence as follows:

a. Choose File > Save As. Xcalibur opens the File Summary dialog
box.
b. In the Description text box, enter drugx example Sequence. See
Figure 35.
c. Click on OK to display the Save As dialog box.
d. In the File Name text box, enter drugx_example. See Figure 36.
e. Click on Save to save the Sequence as
C:\Xcalibur\examples\methods\drugx_example.sld.
44 5IFSNP
Figure 35. File Summary Information dialog box
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Figure 36. Save As dialog box for saving the Sequence as

drugx_example.sld
46 5IFSNP
'JOOJHBO9DBMJCVS _________________________________ Processing the Raw Files and Reviewing Results
Processing the Raw Files and

Reviewing Results
Xcalibur processes the raw files when you open a Sequence in the Quan
Browser window. You then use the Quan Browser window to step through the
Sequence and review the results for each component in each raw file.
Note. For additional information about Quan Browser, refer to Xcalibur

Getting Productive: Quantitative Analysis and/or the online Help.
1. Use the following procedure to process the raw files and review results:
Process the raw files and open the Quan Browser window as follows:
a. Click on the Quan Browser button in the Xcalibur Home Page or
choose GoTo > Quan Browser in the Sequence Setup view.
Xcalibur displays the Open dialog box.
b. Browse through the directories to find the Sequence
C:\Xcalibur\examples\methods\drugx._example.sld. See Figure 37.
c. Select drugx_example.sld, and then click on Open to open the View
Sample Types dialog box. See Figure 38.
d. Select the Show Standard and QC Sample Types option button and
click on OK. Xcalibur processes the raw files in the Sequence
drugx._example.sld and opens the Quan Browser window. The Quan
Browser window is shown in Figure 39.
5IFSNP
Processing the Raw Files and Reviewing Results @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
Figure 37. Open dialog box, showing the Sequence

drugx_example.sld selected
Figure 38. View Sample Types dialog box
48 5IFSNP
Figure 39. Quan Browser window, with the internal standard D4 selected, showing the
All tab (arrow)
2. Click on the Results Grid view to make it active, then click on the All tab
(see arrow in Figure 39) to display all of the data files.
3. In the Results Grid view, click on the first row to select the first data file.
4. Check the entries in the selected Result Grid row for peak detection and
integration problems. Make sure that the selected data file corresponds to
the correct level and sample type. If necessary, modify the peak
identification, detection or integration parameters in the Processing
Method.
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Processing the Raw Files and Reviewing Results @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
Note. You can modify the Processing Method in either Processing Setup or
Quan Browser. In Quan Browser you modify the component identification,
detection, and integration settings of the Processing Method in the User Peak
Detection Settings dialog box. To open the User Peak Detection Settings
dialog box, right click on the Chromatogram Plot view and choose User
Peak Detection Settings from the dropdown menu. To save the modified
Processing Method, choose File > Export Method in the Quan Browser
window.
5. Inspect the component peak in the Chromatogram Plot view. Ensure that
Xcalibur found the peak. Xcalibur shades found peaks gray and marks the
starting and ending points with square integration markers. Ensure that
Xcalibur integrated the peak properly. Ensure that the shaded area
accurately represents the contribution of the component to the
chromatogram. If necessary, modify the peak detection or integration
parameters of the Processing Method.
6. Click on the next row in the Results Grid view to select the next data file.
Perform steps 4 and 5 for each data file.
7. Select drugx in the Components list to display the results for the target
compound. Perform steps 3 through 6 for the target compound.
8. Inspect the calibration curve in the Calibration Curve Plot view. Evaluate
the calibration curve according to the criteria used in your laboratory. If
necessary, modify the calibration curve parameters of the Processing
Method. See Figure 40.
Note. You can modify the Processing Method in either Processing Setup or
Quan Browser. In Quan Browser you modify the calibration curve and
calibration levels settings of the Processing Method in the Calibration
Settings dialog box. To open the Calibration Settings dialog box, right click
on the Calibration Curve view and choose Calibration Settings from the
dropdown menu. To save the modified Processing Method, choose File >
Export Method in the Quan Browser window.
50 5IFSNP
Figure 40. Quan Browser window, with the target compound drugx selected
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Creating Reports @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
Creating Reports
Sequence Setup uses the report templates that you specified in the Reports
view of Processing Setup to create reports in Xcalibur.
To create reports, proceed as follows:
1. Click on the Sequence Setup button in the Xcalibur Home Page to open
the Sequence Setup view.
2. Open the Sequence drugx_example.sld (if it is not already open):
a. Click on the Open Sequence toolbar button (or choose File > Open)
to display the Open dialog box.
b. Browse through the directories to find the Sequence
C:\Xcalibur\examples\methods\drugx_example.sld.
c. Select drugx_example.sld, and then click on Open to open the
Sequence drug_example.sld. See Figure 31.
3. Choose Actions > Batch Reprocess to open the Batch Reprocess
Setup dialog box. See Figure 41.
4. Set the batch reprocess options as shown in Figure 41:
a. Select the Quan, Peak Detection & Integration, Quantitation, Reports,
and Print Sample Reports options.
b. Enter 1-31 in the Process Rows text box.
5. Click on OK to start batch reprocessing and report generation. Xcalibur
exports the reports for each raw file to the C:\Xcalibur\examples\data\
folder. See Figure 42.
Note. Xcalibur exports the reports to the folder where the raw files are
located. In this example the reports and raw files are in the
C:\Xcalibur\examples\data\ folder.
Several sample peak identification and component calibration reports are

shown in the next topic: Sample Reports.
52 5IFSNP
'JOOJHBO9DBMJCVS ___________________________________________________________ Creating Reports
Figure 41. Batch Reprocess Setup dialogbox
Figure 42. Microsoft® Windows® Explorer, showing the contents of C:\Xcalibur\examples\data\
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Sample Reports @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 'JOOJHBO9DBMJCVS
Sample Reports
This topic contains sample peak integration and component calibration
reports for the drugx_31.raw file. The sample peak integration report is
drugx_31_PeakIntegration2.doc, and the sample component calibration
report is drugx_31_CompCalReport2_ICIS.doc. You specify the report
templates in the Reports view of the Processing Setup window. Refer to the
topic Specifying Report Templates.
54 5IFSNP
Peak Integration Report
drugx
Sample Name: Sample Type: QC Sample ID: 44

Acquisition Date: 05/05/96 05:10:30 Revision: 1.1 Operator: linda
AM
High Mass (m/z): 425.30 Low Mass (m/z): 419.30 Scans: 323
Run Time (min): 6.97 Inj Vol (uL): 50.00 Vial: 144
Cal Level: QC 3 Samp Wt: 0.00 Samp Vol: 0.00
ISTD Amt: 0.000 Dil Factor: 1.00
Comments: QC=1000 pg/ml,IS=100 pg/ml
Inst Method: C:\LCQ\Methods\
Data Path: C:\Xcalibur\examples\data\
Proc Method: C:\Xcalibur\examples\methods\drugx_example
Study:
Client:
Laboratory:
Company:
Phone:
Component: drugx ISTD: D4

ERT: 4.89 R T : 3.87 - 5.87 S M: 5G ERT: 4.89 R T : 3.88 - 5.88 S M: 5G
R T : 4.87 NL: R T : 4.88 NL:
RTW: 30.00 100 RTW: 30.00 100 2.59E5
2.29E6
VW: 2.00 90 T IC F : + c
VW: 2.00 90 T IC F : + c
RTR: no S R M ms 2 RTR: no 80
S R M ms 2
80 469.40 [
ID: Nearest RT 465.30 [ ID: Nearest RT
Relative Abundance 70 423.30-
Relative Abundance
70 419.30-
MPH: 3.00 421.30] MS
MPH: 3.00 425.30] MS
60 60
SM: 5 drugx_ 31 SM: 5 drugx_ 31
PDAU: Genesis 50 PDAU: Genesis 50
VD: yes 40 VD: yes 40
EPW: 20.00 30 EPW: 20.00 30
CP: no 20 CP:: no 20
ISTD: 10
ISTD: 10
D4 D4
0 0
4 5 4 5
T ime (min) T ime (min)
Actual Calculated Base Signal Area Area Specified

Component Name RT Amount Response Line To Noise % Diff Ratio ISTD Amount
drugx 4.87 1019.273 22277136 BB 10217.38 1.927 9.103 24471 1000.000
98
Peak Integration Report
D4
Sample Name: Sample Type: QC Sample ID: 44

Acquisition Date: 05/05/96 05:10:30 Revision: 1.1 Operator: linda
AM
High Mass (m/z): 425.30 Low Mass (m/z): 419.30 Scans: 323
Run Time (min): 6.97 Inj Vol (uL): 50.00 Vial: 144
Cal Level: QC 3 Samp Wt: 0.00 Samp Vol: 0.00
ISTD Amt: 0.000 Dil Factor: 1.00
Comments: QC=1000 pg/ml,IS=100 pg/ml
Inst Method: C:\LCQ\Methods\
Data Path: C:\Xcalibur\examples\data\
Proc Method: C:\Xcalibur\examples\methods\drugx_example
Study:
Client:
Laboratory:
Company:
Phone:
Component: D4 ISTD: D4
ERT: 4.87 R T : 3.88 - 5.88 S M: 5G ERT: 4.87 R T : 3.88 - 5.88 S M: 5G
R T : 4.88 NL: R T : 4.88 NL:
RTW: 30.00 100 RTW: 30.00 100 2.59E5
2.59E 5
VW: 2.00 90 T IC F : + c
VW: 2.00 90 T IC F : + c
RTR: yes S R M ms 2 RTR: yes 80
S R M ms 2
80
ID: Nearest RT 469.40 [ ID: Nearest RT Relative Abundance 469.40 [
Relative Abundance
70 423.30- 70 423.30-
MPH: 3.00 425.30] MS
MPH: 3.00 425.30] MS
SM: 5 60 SM: 5 60
drugx_ 31 drugx_ 31
PDAU: Genesis 50 PDAU: Genesis 50
VD: yes 40 VD: yes 40
EPW: 20.00 30 EPW: 20.00 30
CP: no 20 CP:: no 20
ISTD: 10
ISTD:
10
- 0
-
0
4 5 4 5
T ime (min) T ime (min)
Actual Calculated Base Signal Area Area Specified

Component Name RT Amount Response Line To Noise % Diff Ratio ISTD Amount
D4 4.88 0.000 2447198 BV 469.24 0.000 0.000 0 0.000
Component Calibration Report
drugx
Identification drugx
Detector Type MS
Filter: + c SRM ms2 [email protected] [ 419.30-421.30]
8
Trace TIC
Quan Masses N/A 7
Retention Time 6
Area Ratio
Expected RT (min) 4.89 5
Window (sec) 30.0
4
View Width (min) 2.00
RT Reference N/A 3
Adjust Expected RT Yes 2

Adjust Using D4 1
Detection 0
Smoothing Points 5 0 200 400 600 800 1000
S/N Threshold N/A pg/ml
Valley Detection N/A
Expected Peak Width (sec) N/A
Cal Level Amount
Constrain Peak Width N/A
cal 1 10.000
Peak Height (%) N/A
cal 2 25.000
Tailing Factor N/A
cal 3 50.000
Identify By Nearest RT
cal 4 100.000
Minimum Peak Height (S/N) N/A
cal 5 200.000
Calibration cal 6 400.000
Component Type Target Component cal 7 600.000
Internal Standard D4 cal 8 800.000
Weighting 1/Xˆ cal 9 1000.000
Origin Ignore
Response Area
Calibration Curve Quadratic QC Level Amount
Units pg/ml QC 1 10.000
Number of Cal. Levels 9 QC 2 400.000
Number of QC. Levels 3 QC 3 1000.000
Sample Area Area Area ISTD Specified Calculated % Diff % RSD

Ratio Amount Amount
Component Calibration Report
D4
Identification D4
Average R es pons e F actor = 24692.7
Detector Type MS
Filter: + c SRM ms2 [email protected] [ 423.30-425.30] 4500000
Trace TIC
4000000
Quan Masses N/A
3500000
Retention Time
3000000
Expected RT (min) 4.89
Area
Window (sec) 30.0 2500000
View Width (min) 2.00 2000000

RT Reference Yes 1500000
Adjust Expected RT N/A 1000000
Adjust Using N/A
500000
Detection 0
Smoothing Points 5 0 50 100 150 200
S/N Threshold N/A pg/mL
Valley Detection N/A
There are no Cal. levels to report.
Expected Peak Width (sec) N/A
Constrain Peak Width N/A
Peak Height (%) N/A
Tailing Factor N/A There are no QC levels to report.
Identify By Nearest RT
Minimum Peak Height (S/N) N/A
Calibration
Component Type Internal Standard
Internal Standard N/A
Weighting N/A
Origin N/A
Response N/A
Calibration Curve N/A
Units N/A
Number of Cal. Levels N/A
Number of QC. Levels N/A
Sample Area Area Area ISTD Specified Calculated % Diff % RSD

Ratio Amount Amount
Index
'JOOJHBO9DBMJCVS ________________________________________________________________________
Index
detectors
A selecting type, 1-14
drugx
Advanced Parameters dialog box
example, description, 1-3
displaying, 1-24
figure, 1-24
note, 1-23 F
Figures
B Advanced Parameters dialog box, 1-24
calibration curve, 1-2
Batch Reprocess Setup dialog box
Calibration Options dialog box, 1-9
displaying, 1-52
Calibration page, 1-26
figure, 1-53
Chromatography Options dialog box, 1-9
Detection page, 1-22, 1-25
C File Summary Information dialog box, 1-37, 1-45
Fill Down dialog box, 1-43
calibration curve ICIS Advanced Parameters dialog box, 1-24
creating, 1-47 Identification page, 1-8, 1-12, 1-13, 1-15, 1-16, 1-17,
curve types, 1-3 1-18, 1-20
figure, 1-2 integrated chromatogram peak, 1-2
modifying in Quan Browser (Note), 1-50 Levels page, 1-29, 1-32
weighting, 1-3 Open Raw File dialog box, 1-11
calibration levels Quan Browser window, 1-49, 1-51
modifying in Quan Browser (Note), 1-50 quantitation flow diagram, 1-5
specifying, 1-29 Reports view, 1-34, 1-36
table, 1-30 Sample Report Template dialog box, 1-35
Calibration Options dialog box Select Processing Method dialog box, 1-42
displaying, 1-9 Sequence Setup view, 1-41, 1-44
figure, 1-9 System Suitability page, 1-33
Calibration page Xcalibur Home Page, 1-7
displaying, 1-25 File Summary Information dialog box, 1-45
figure, 1-26 displaying, 1-36
chromatograms figure, 1-37
integrated peaks (Figure), 1-2 Fill Down dialog box
selecting trace type, 1-14 displaying, 1-42
chromatography figure, 1-43
specifying LC or GC, 1-8 flow diagram
Chromatography Options dialog box analyzing quantitation data, 1-5
displaying, 1-8
figure, 1-9
component calibration
G
Calibration page, 1-25
GC
entering parameters, 1-25
specifying chromatography by GC, 1-8
components
Genesis peak detection
identification settings, 1-12
note, 1-21
identifying, 1-12
selecting, 1-21
naming, 1-13
D I
ICIS Advanced Parameters dialog box
data files
figure, 1-24
opening, 1-10
note, 1-23
processing, 1-47
ICIS peak detection
Detection page
note, 1-21
displaying, 1-22
selecting, 1-21
figure, 1-22, 1-25
Identification page
detection, peak
displaying, 1-7
entering parameters for, 1-23
figure, 1-8, 1-12, 1-13, 1-15, 1-16, 1-17, 1-18, 1-20
5IFSNP
&-&$530/$03103"5*0/ ____________ Finnigan Xcalibur: Getting Productive with Processing Setup ______________ I
Index
________________________________________________________________________ 'JOOJHBO9DBMJCVS
integrated chromatogram peak (Figure), 1-2 opening a raw file, 1-10

integration, peak processing raw files, 1-47
Detection page, 1-22 saving the Processing Method, 1-36
entering parameters for, 1-22 selecting chromatogram trace type, 1-14
internal standards selecting detector type, 1-14
specifying calibration by, 1-9 specifying calibration levels, 1-29
specifying component identification settings for target
L compound, 1-19
specifying QC levels, 1-29
LC specifying report templates, 1-33
specifying chromatography by LC, 1-8 Processing Methods
Levels page adding to Sequence, 1-39
displaying, 1-29 creating, 1-6
figure, 1-29, 1-32 description, 1-1
modifying in Quan Browser, 1-50
modifying in Quan Browser (Note), 1-50
N saving, 1-36
Processing Setup window
Notes displaying, 1-7
Advanced Parameters dialog box, 1-23 figure, 1-8
creating Sequences, 1-39
default peak identification parameters, 1-22
Genesis peak detection, 1-21 Q
ICIS Advanced Parameters dialog box, 1-23
ICIS peak detection, 1-21 QC levels
percent test, 1-30 modifying in Quan Browser (Note), 1-50
Quan Browser, 1-47 specifying, 1-29
raw files in Processing Methods, 1-11 table, 1-31
Quan Browser window
displaying, 1-47
O figure, 1-49, 1-51
modifying Processing Methods, 1-50
Open Raw File dialog box modifying Processing Methods (Note), 1-50
displaying, 1-10 reviewing results, 1-47
figure, 1-11 quantitation
flow chart, 1-5
P introduction, 1-1
peak detection R
Detection page, 1-22
entering parameters, 1-21 raw files
modifying parameters in Quan Browser (Note), 1-50 Open Raw File dialog box, 1-11
peak integration opening, 1-10
Detection page, 1-22 processing, 1-47
entering parameters, 1-21 report templates
modifying parameters in Quan Browser (Note), 1-50 description, 1-33
peaks specifying, 1-33
detection parameters, entering, 1-23 reports
integration parameters, entering, 1-22 drugx sample report listing (Figure), 1-53
setting default identification parameters (Note), 1-22 sample reports, 1-54
unresolved, 1-23 specifying templates, 1-33
percent test (Note), 1-30 Reports view
procedures displaying, 1-33
checking system suitability options, 1-32 figure, 1-34, 1-36
choosing the Create Method tab, 1-7 retention time
creating a Processing Method, 1-6 determining, 1-16
determining retention time, 1-16
displaying a spectrum, 1-16
entering component calibration parameters, 1-25 S
entering peak detection parameters, 1-21, 1-23
entering peak integration parameters, 1-21, 1-22 Sample Report Template dialog box
matching scan filters with components, 1-14 figure, 1-35
naming components, 1-13 sample reports, 1-54
II_______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________ 5IFSNP
&-&$530/$03103"5*0/
Index
'JOOJHBO9DBMJCVS ________________________________________________________________________
scan filters System Suitability page

matching with components, 1-14 displaying, 1-32
Select Processing Method dialog box, 1-42 figure, 1-33
Sequence Setup view
displaying, 1-39
figure, 1-41, 1-44
T
Sequences Tables
adding a Processing Method to, 1-39 Calibration Level, 1-30
creating (Note), 1-39 QC Level, 1-31
drugx (figure), 1-44 target compound
spectrum specifying component identification settings, 1-19
displaying, 1-16
system suitability options
checking, 1-32 X
description, 1-32
Xcalibur
Home Page (Figure), 1-7
5IFSNP
&-&$530/$03103"5*0/ ____________ Finnigan Xcalibur: Getting Productive with Processing Setup _____________ III

Xcalibur Processing Setup and Analysis

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Printing History: Revision A printed in May 2003.

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

Additional Comments: (Attach additional sheets if necessary.)

EDITOR, TECHNICAL PUBLICATIONS

Read This First ............................................................................................................................ iii

Typographical Conventions ................................................................................................................... ix

Reply Cards ........................................................................................................................................... xii

Processing Setup and the Analysis of Quantitation Data ..........................................................1

Adding the Processing Method to a Sequence ....................................................................................... 39

Processing the Raw Files and Reviewing Results.................................................................................. 47

Sample Reports ...................................................................................................................................... 54

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

Changes to the Manual and Online Help

vi ______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________5IFSNP

ppm parts per million

Note. Exponents are written as superscripts. In the corresponding online

Note. Boxes such as this are used to display information.

Boxed information can be of the following types:

1.2 Second Level Topics

Third Level Topics

Fourth Level Topics

Fifth Level Topics

Processing Setup is part of Xcalibur®, the Finnigan™ mass spectrometry

Figure 1. Integrated chromatogram peak

Figure 2. Calibration curve

Calibration standards were prepared by spiking human plasma with drugx to

Creating a Processing Method

Opening the Processing Setup Window

Figure 4. Xcalibur Home Page

Figure 5. Processing Setup window – Quan view – Identification page

Figure 6. Chromatography Options dialog box

Specifying Calibration by Internal Standard

Figure 7. Calibration Options dialog box

Opening a Raw Data File

Specifying Component Identification

Selecting the Detector Type

Selecting the Peak Detection Type

Matching Scan Filters with Components

Selecting the Trace Type

Displaying the Spectrum and Determining the

c. Release the mouse button, and the following events occur

5. Inspect the integrated peak and ensure the following:

Specifying Component Identification

• Processing Setup selects the retention time of the peak maximum

Entering Peak Integration and Detection

Entering peak integration and detection parameters involves the following:

Displaying the Detection Page

Figure 16. Processing Setup window – Quan view – Detection page

Entering Peak Integration Parameters

Enter the ICIS peak integration parameters as follows:

Entering Peak Detection Parameters

Figure 17. ICIS Advanced Parameters dialog box,

Figure 18. Detection page, showing settings for drugx and D4

Selecting Calibration Settings

Figure 19. Processing Setup window – Quan view – Calibration page

'JOOJHBO¥

vi __ Finnigan Xcalibur: Getting Productive with Processing Setup 5IFSNP