xcalibur processing setup and analysis

Technical information contained in this publication is for reference purposes only and is subject to change without notice. Every effort has been made to supply complete and accurate information; however, Thermo Electron Corporation assumes no responsibility and will not be liable for any errors, omissions, damage, or loss that might result from any use of this manual or the information contained therein (even if this information is properly followed and problems still arise).

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System Configurations and Specifications supersede all previous information and are subject to changewithout notice.

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Printing History: Revision A printed in May 2003.Software Revision: Xcalibur 1.4

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Contents________________________________________________________________________

Contents

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

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

Abbreviations .......................................................................................................................................... v

Typographical Conventions ................................................................................................................... ixData Input ............................................................................................................................. ixBoxed Information................................................................................................................. xTopic Headings..................................................................................................................... xi

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

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

Creating a Processing Method ................................................................................................................. 6Opening the Processing Setup Window ................................................................................. 7Specifying Chromatography by LC........................................................................................ 8Specifying Calibration by Internal Standard .......................................................................... 9Opening a Raw Data File ..................................................................................................... 10Specifying Component Identification Settings for the Internal Standard ............................ 12Specifying Component Identification Settings for the Target Compound ........................... 19Entering Peak Integration and Detection Parameters........................................................... 21Selecting Calibration Settings .............................................................................................. 25Specifying Calibration and QC Levels................................................................................. 29Checking System Suitability Options................................................................................... 32Specifying Report Templates ............................................................................................... 33Saving 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

____________Finnigan Xcalibur: Getting Productive with Processing Setup ______________ i

Read This First

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.

______________Finnigan Xcalibur: Getting Productive with Processing Setup ___________ iii

Read This FirstChanges to the Manual and Online Help

Changes to the Manual and Online HelpTo suggest changes to this manual or the online Help, please send your comments to:

Editor, Technical PublicationsThermo Electron San Jose355 River Oaks ParkwaySan Jose, CA 95134-1991U.S.A.

You are encouraged to report errors or omissions in the text or index. Thank you.

iv ______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Read This First_____________________________________________________________ Abbreviations

AbbreviationsThe 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

______________Finnigan Xcalibur: Getting Productive with Processing Setup ___________ v

Read This FirstAbbreviations

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

vi ______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Read This First_____________________________________________________________ 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

______________Finnigan Xcalibur: Getting Productive with Processing Setup __________ vii

Read This FirstAbbreviations

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)

viii _____________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Read This First___________________________________________________Typographical Conventions

Typographical ConventionsTypographical 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”.

______________Finnigan Xcalibur: Getting Productive with Processing Setup __________ ix

Read This FirstTypographical Conventions

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.

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.

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

x_______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Read This First___________________________________________________Typographical Conventions

Topic Headings

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

Chapter 1Chapter Name

1.2 Second Level Topics

Third Level Topics

Fourth Level Topics

Fifth Level Topics

______________Finnigan Xcalibur: Getting Productive with Processing Setup __________ xi

Read This FirstReply Cards

Reply CardsThermo 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.

xii______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Processing Setup and theAnalysis 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.

____________Finnigan Xcalibur: Getting Productive with Processing Setup ______________ 1

Processing Setup and the Analysis of Quantitation Data

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.

Figure 1. Integrated chromatogram peak

Figure 2. Calibration curve

2_______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Processing Setup and the Analysis of Quantitation Data________________________________________________________________________

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.


Processing Setup and the Analysis of Quantitation Data

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


Processing Setup and the Analysis of Quantitation Data________________________________________________________________________

Figure 3. Flow diagram for analyzing quantitation data with Processing Setup

PROCESSING SETUP:

CREATE PROCESSING

METHOD

SEQUENCE SETUP:

ADD PROCESSING

METHOD TO SEQUENCE

QUAN BROWSER:

PROCESS RAW FILES

USING SEQUENCE

ALL PEAKS FOUND

AND INTEGRATED

CORRECTLY?

NO

PROCESSING SETUP OR

QUAN BROWSER: MODIFY

PEAK DETECTION OR

INTEGRATION PARAMETERS

CALIBRATION

CURVE OK?

SEQUENCE SETUP:

CREATE REPORTS

YES

YES

PROCESSING SETUP

OR QUAN BROWSER:

MODIFY CALIBRATION

CURVE PARAMETERS

NO


Processing Setup and the Analysis of Quantitation DataCreating a Processing Method

Creating a Processing MethodYou 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.


Processing Setup and the Analysis of Quantitation Data________________________________________________ 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



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.

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



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 6. Chromatography Options dialog box

Figure 7. Calibration Options dialog box



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.

10 _____________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________


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.

____________Finnigan Xcalibur: Getting Productive with Processing Setup ____________ 11


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

Figure 9. Identification page, showing the total ion current (TIC) chromatogram of drugx_03.raw



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



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).

1Parent ion m/z 465; product ion m/z 420

2Parent ion m/z 469; product ion m/z 424



Figure 11. Identification page, showing the mass chromatogram for D4.



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



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



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.

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.

Figure 14. Identification page, showing the area of peak integration (grayed) and the integration markers (blue circles indicated by arrows)



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:



• 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



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.

Entering peak integration and detection parameters involves the following:

• Displaying the Detection page

• Entering peak integration parameters

• Entering peak detection parameters

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.



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.

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.

Enter the ICIS peak integration parameters as follows:

1. In the Components list, click on D4 to select the internal standard, D4.

Figure 16. Processing Setup window – Quan view – 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.



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.



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



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.

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



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.

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



Figure 20. Calibration page, showing the settings for the internal standard, D4



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



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



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.

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.

d. Press <Tab> twice to create a new row and to advance the cursor to the second QC Level text box.

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

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.



e. Repeat this procedure until you fill in the three QC levels as shown in Table 2.

5. Click on OK to save the calibration and QC level settings. The Levels page should look like the one shown in Figure 23.

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



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.

Figure 23. Levels page, showing the completed Calibration Level and QC Level Tables



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.

Figure 24. Processing Setup window – Quan view – System Suitability page

p



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.

Figure 25. Processing Setup window - Reports view



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.

Figure 26. Browse for Sample Report Template dialog box



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.

Figure 27. Reports view, showing the sample peak integration report and component calibration report templates selected



5. Click on Save to save the Processing Method and close the dialog box.

Figure 28. File Summary Information dialog box



Figure 29. Save As dialog box, showing drugx_example.pmd


Processing Setup and the Analysis of Quantitation Data____________________________________ 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.

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.

Note. For additional information about setting up a Sequence for Quantitative Analysis refer to Xcalibur Getting Productive: Quantitative Analysis and/or the online Help.


Processing Setup and the Analysis of Quantitation DataAdding the Processing Method to a Sequence

Figure 30. Open dialog box, showing the selected Sequence, drugx.sld



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.

Figure 31. Sequence Setup view, showing the Sequence drugx.sld saved on the C:\ drive.



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



Figure 33. Fill Down dialog box, with settings to fill rows 2 to 31 of the Processing Method column with row 1.



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.

Figure 34. Drugx Sequence, with drugx_example.pmd selected as the Processing Method



Figure 35. File Summary Information dialog box



Figure 36. Save As dialog box for saving the Sequence as drugx_example.sld


Processing Setup and the Analysis of Quantitation Data_________________________________ Processing the Raw Files and Reviewing Results

Processing the Raw Files and Reviewing ResultsXcalibur 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.

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.

Note. For additional information about Quan Browser, refer to Xcalibur Getting Productive: Quantitative Analysis and/or the online Help.


Processing Setup and the Analysis of Quantitation DataProcessing the Raw Files and Reviewing Results

Figure 37. Open dialog box, showing the Sequence drugx_example.sld selected

Figure 38. View Sample Types dialog box



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.

Figure 39. Quan Browser window, with the internal standard D4 selected, showing the All tab (arrow)


Processing Setup and the Analysis of Quantitation DataProcessing the Raw Files and Reviewing Results

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 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.

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.



Figure 40. Quan Browser window, with the target compound drugx selected


Processing Setup and the Analysis of Quantitation DataCreating Reports

Creating ReportsSequence 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.

Several sample peak identification and component calibration reports are shown in the next topic: Sample Reports.

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.


Processing Setup and the Analysis of Quantitation Data___________________________________________________________ Creating Reports

Figure 41. Batch Reprocess Setup dialogbox

Figure 42. Microsoft® Windows® Explorer, showing the contents of C:\Xcalibur\examples\data\


Processing Setup and the Analysis of Quantitation DataSample Reports

Sample ReportsThis 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.


Cdrug

Peak Integration Report drugx

Sample Name: Sample Type: QC Sample ID: 44 Acquisition Date: 05/05/96 05:10:30

AM Revision: 1.1 Operator: linda

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:

ERT: 4.89 RTW: 30.00 VW: 2.00 RTR: no ID: Nearest RT MPH: 3.00 SM: 5 PDAU: Genesis VD: yes EPW: 20.00 CP: no ISTD: D4

Component: drugx R T : 3.87 - 5.87 SM: 5G

4 5T ime (min)

0

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R T : 4.87 NL:2.29E6T IC F : + c S R M ms2 465.30 [ 419.30-421.30] MS drugx_31

ERT: 4.89 RTW: 30.00 VW: 2.00 RTR: no ID: Nearest RT MPH: 3.00 SM: 5 PDAU: Genesis VD: yes EPW: 20.00 CP:: no ISTD: D4

ISTD: D4 R T : 3.88 - 5.88 SM: 5G

4 5T ime (min)

0

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R T : 4.88 NL:2.59E5T IC F : + c SR M ms2 469.40 [ 423.30-425.30] MS drugx_31

omponent Name Actual

RT Calculated

Amount Response Base Line

Signal To Noise % Diff

Area Ratio

Area ISTD

Specified Amount

x 4.87 1019.273 22277136 BB 10217.38 1.927 9.103 2447198

1000.000

S

ed nt

D

Peak Integration Report D4

Sample Name: Sample Type: QC Sample ID: 44 Acquisition Date: 05/05/96 05:10:30

AM Revision: 1.1 Operator: linda

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:

ERT: 4.87 RTW: 30.00 VW: 2.00 RTR: yes ID: Nearest RT MPH: 3.00 SM: 5 PDAU: Genesis VD: yes EPW: 20.00 CP: no ISTD: -

Component: D4 R T : 3.88 - 5.88 SM: 5G

4 5T ime (min)

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R T : 4.88 NL:2.59E5T IC F : + c S R M ms2 469.40 [ 423.30-425.30] MS drugx_31

ERT: 4.87 RTW: 30.00 VW: 2.00 RTR: yes ID: Nearest RT MPH: 3.00 SM: 5 PDAU: Genesis VD: yes EPW: 20.00 CP:: no ISTD: -

ISTD: D4 R T : 3.88 - 5.88 SM: 5G

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R T : 4.88 NL:2.59E5T IC F : + c SR M ms2 469.40 [ 423.30-425.30] Mdrugx_31

Component Name Actual

RT Calculated

Amount Response Base Line

Signal To Noise % Diff

Area Ratio

Area ISTD

SpecifiAmou

4 4.88 0.000 2447198 BV 469.24 0.000 0.000 0 0.000

DetecFilterTraceQuan

ExpecWindViewRT RAdjusAdjus

SmooS/N TValleExpecConstPeak TailinIdentiMinim

CompInternWeigOrigiRespoCalibUnitsNumbNumb

Component Calibration Report drugx

Identification tor Type MS : + c SRM ms2 [email protected] [ 419.30-421.30] TIC Masses N/A

Retention Time ted RT (min) 4.89

ow (sec) 30.0 Width (min) 2.00 eference N/A t Expected RT Yes t Using D4

Detection thing Points 5 hreshold N/A

y Detection N/A ted Peak Width (sec) N/A rain Peak Width N/A Height (%) N/A g Factor N/A fy By Nearest RT um Peak Height (S/N) N/A

Calibration onent Type Target Component al Standard D4 hting 1/Xˆ n Ignore nse Area

ration Curve Quadratic pg/ml er of Cal. Levels 9 er of QC. Levels 3

drugx

0 200 400 600 800 1000pg/ml

0

1

2

3

4

5

6

7

8

Are

a R

atio

Cal Level Amount cal 1 10.000 cal 2 25.000 cal 3 50.000 cal 4 100.000 cal 5 200.000 cal 6 400.000 cal 7 600.000 cal 8 800.000 cal 9 1000.000

QC Level Amount QC 1 10.000 QC 2 400.000 QC 3 1000.000

Sample Area Area

Ratio Area ISTD Specified

Amount Calculated

Amount % Diff % RSD

DFTQ

EWVRAA

SSVECPTIdM

CInWORCUNN

SD

Component Calibration Report D4

Identification etector Type MS ilter: + c SRM ms2 [email protected] [ 423.30-425.30] race TIC uan Masses N/A

Retention Time xpected RT (min) 4.89 indow (sec) 30.0 iew Width (min) 2.00 T Reference Yes djust Expected RT N/A djust Using N/A

Detection moothing Points 5 /N Threshold N/A alley Detection N/A xpected Peak Width (sec) N/A onstrain Peak Width N/A eak Height (%) N/A ailing Factor N/A entify By Nearest RT inimum Peak Height (S/N) N/A

Calibration omponent Type Internal Standard ternal Standard N/A eighting N/A rigin N/A esponse N/A alibration Curve N/A nits N/A umber of Cal. Levels N/A umber of QC. Levels N/A

D4Average R esponse F actor = 24692.7

0 50 100 150 200pg/mL

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

Are

aThere are no Cal. levels to report. There are no QC levels to report.

Sample Area Area

Ratio Area ISTD Specified

Amount Calculated

Amount % Diff % R

Index

________________________________________________________________________

Index

AAdvanced Parameters dialog box

displaying, 1-24figure, 1-24note, 1-23

BBatch Reprocess Setup dialog box

displaying, 1-52figure, 1-53

Ccalibration curve

creating, 1-47curve types, 1-3figure, 1-2modifying in Quan Browser (Note), 1-50weighting, 1-3

calibration levelsmodifying in Quan Browser (Note), 1-50specifying, 1-29table, 1-30

Calibration Options dialog boxdisplaying, 1-9figure, 1-9

Calibration pagedisplaying, 1-25figure, 1-26

chromatogramsintegrated peaks (Figure), 1-2selecting trace type, 1-14

chromatographyspecifying LC or GC, 1-8

Chromatography Options dialog boxdisplaying, 1-8figure, 1-9

component calibrationCalibration page, 1-25entering parameters, 1-25

componentsidentification settings, 1-12identifying, 1-12naming, 1-13

Ddata files

opening, 1-10processing, 1-47

Detection pagedisplaying, 1-22figure, 1-22, 1-25

detection, peakentering parameters for, 1-23

detectorsselecting type, 1-14

drugxexample, description, 1-3

FFigures

Advanced Parameters dialog box, 1-24calibration curve, 1-2Calibration Options dialog box, 1-9Calibration page, 1-26Chromatography Options dialog box, 1-9Detection page, 1-22, 1-25File Summary Information dialog box, 1-37, 1-45Fill Down dialog box, 1-43ICIS Advanced Parameters dialog box, 1-24Identification page, 1-8, 1-12, 1-13, 1-15, 1-16, 1-17,

1-18, 1-20integrated chromatogram peak, 1-2Levels page, 1-29, 1-32Open Raw File dialog box, 1-11Quan Browser window, 1-49, 1-51quantitation flow diagram, 1-5Reports view, 1-34, 1-36Sample Report Template dialog box, 1-35Select Processing Method dialog box, 1-42Sequence Setup view, 1-41, 1-44System Suitability page, 1-33Xcalibur Home Page, 1-7

File Summary Information dialog box, 1-45displaying, 1-36figure, 1-37

Fill Down dialog boxdisplaying, 1-42figure, 1-43

flow diagramanalyzing quantitation data, 1-5

GGC

specifying chromatography by GC, 1-8Genesis peak detection

note, 1-21selecting, 1-21

IICIS Advanced Parameters dialog box

figure, 1-24note, 1-23

ICIS peak detectionnote, 1-21selecting, 1-21

Identification pagedisplaying, 1-7figure, 1-8, 1-12, 1-13, 1-15, 1-16, 1-17, 1-18, 1-20

____________Finnigan Xcalibur: Getting Productive with Processing Setup ______________ I

Index

________________________________________________________________________

integrated chromatogram peak (Figure), 1-2integration, peak

Detection page, 1-22entering parameters for, 1-22

internal standardsspecifying calibration by, 1-9

LLC

specifying chromatography by LC, 1-8Levels page

displaying, 1-29figure, 1-29, 1-32

NNotes

Advanced Parameters dialog box, 1-23creating Sequences, 1-39default peak identification parameters, 1-22Genesis peak detection, 1-21ICIS Advanced Parameters dialog box, 1-23ICIS peak detection, 1-21percent test, 1-30Quan Browser, 1-47raw files in Processing Methods, 1-11

OOpen Raw File dialog box

displaying, 1-10figure, 1-11

Ppeak detection

Detection page, 1-22entering parameters, 1-21modifying parameters in Quan Browser (Note), 1-50

peak integrationDetection page, 1-22entering parameters, 1-21modifying parameters in Quan Browser (Note), 1-50

peaksdetection parameters, entering, 1-23integration parameters, entering, 1-22setting default identification parameters (Note), 1-22unresolved, 1-23

percent test (Note), 1-30procedures

checking system suitability options, 1-32choosing the Create Method tab, 1-7creating a Processing Method, 1-6determining retention time, 1-16displaying a spectrum, 1-16entering component calibration parameters, 1-25entering peak detection parameters, 1-21, 1-23entering peak integration parameters, 1-21, 1-22matching scan filters with components, 1-14naming components, 1-13

opening a raw file, 1-10processing raw files, 1-47saving the Processing Method, 1-36selecting chromatogram trace type, 1-14selecting detector type, 1-14specifying calibration levels, 1-29specifying component identification settings for target

compound, 1-19specifying QC levels, 1-29specifying report templates, 1-33

Processing Methodsadding to Sequence, 1-39creating, 1-6description, 1-1modifying in Quan Browser, 1-50modifying in Quan Browser (Note), 1-50saving, 1-36

Processing Setup windowdisplaying, 1-7figure, 1-8

QQC levels

modifying in Quan Browser (Note), 1-50specifying, 1-29table, 1-31

Quan Browser windowdisplaying, 1-47figure, 1-49, 1-51modifying Processing Methods, 1-50modifying Processing Methods (Note), 1-50reviewing results, 1-47

quantitationflow chart, 1-5introduction, 1-1

Rraw files

Open Raw File dialog box, 1-11opening, 1-10processing, 1-47

report templatesdescription, 1-33specifying, 1-33

reportsdrugx sample report listing (Figure), 1-53sample reports, 1-54specifying templates, 1-33

Reports viewdisplaying, 1-33figure, 1-34, 1-36

retention timedetermining, 1-16

SSample Report Template dialog box

figure, 1-35sample reports, 1-54

II_______________ Finnigan Xcalibur: Getting Productive with Processing Setup ____________

Index

________________________________________________________________________

scan filtersmatching with components, 1-14

Select Processing Method dialog box, 1-42Sequence Setup view

displaying, 1-39figure, 1-41, 1-44

Sequencesadding a Processing Method to, 1-39creating (Note), 1-39drugx (figure), 1-44

spectrumdisplaying, 1-16

system suitability optionschecking, 1-32description, 1-32

System Suitability pagedisplaying, 1-32figure, 1-33

TTables

Calibration Level, 1-30QC Level, 1-31

target compoundspecifying component identification settings, 1-19

XXcalibur

Home Page (Figure), 1-7

____________Finnigan Xcalibur: Getting Productive with Processing Setup _____________ III

xcalibur processing setup and analysis

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