You are on page 1of 78

Finnigan™ is a trademark of and Xcalibur ® is a registered trademark of Thermo Electron Corporation. Microsoft ® , Windows ® , and ActiveX ® are registered trademarks of Microsoft Corporation.

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

This publication is not part of the Agreement of Sale between Thermo Electron Corporation and the purchaser of a GC/MS or LC/MS system. In the event of any conflict between the provisions of this document and those contained in Thermo Electron Corporation’s Terms and Conditions, the provisions of the Terms and Conditions shall govern.

System Configurations and Specifications supersede all previous information and are subject to change without notice.

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

A printed in May 2003. Software Revision: Xcalibur 1.4 The products of Thermo Electron San Jose

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

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

Customer Registration Register now and receive all the privile ges associated with being a Thermo
Customer Registration Register now and receive all the privile ges associated with being a Thermo
Customer Registration Register now and receive all the privile ges associated with being a Thermo

Customer Registration

Register now and receive all the privileges associated with being a Thermo

Electron, Finnigan product user, including application reports and technical reports.

 

Name

Title

Company

 

Address

City/State/Postal Code

 

Country

Telephone

System Type

Serial

#

Date Purchased

 

Tell us more Let us know more about how you use this product:

 

My Organization Is: (Check one only)

 

My Primary Application Is: (Check one only) Analytical

 

Commercial (for profit) lab Government lab Hospital / Clinic Research Institute University / College Veterinary Other

Biomedical

Clinical / Toxicology Energy Food / Agriculture Forensic / Toxicology

 

Pharmaceutical

 

Research / Education

Job Function: (Check one only) Administration

 

Other

Lab Management

Operator

 

Other

Reader Survey

Help us to improve the quality of our documentation by answering a few questions:

Finnigan Xcalibur Getting Productive:

 

Revision A

Processing Setup and the Analysis of Quantitation Data

 

XCALI-97044

 

Strongly

Strongly

Agree

Agree

Disagree

Disagree

The manual is well organized.

 

1

2

3

4

The manual is clearly written.

 

1

2

3

4

The manual contains all of the information I need.

 

1

2

3

4

The instructions are easy to follow.

 

1

2

3

4

The instructions are complete.

 

1

2

3

4

The technical information is easy to understand.

 

1

2

3

4

The figures are helpful.

 

1

2

3

4

I was able to operate the system by using this manual. (If not, please comment below.)

 

1234

Additional Comments: (Attach additional sheets if necessary.)

 

Tear this sheet from the manual, fold it closed, stamp it, and drop it in the mail.

 

From

From EDITOR, TECHNICAL PUBLICATIONS THERMO ELECTRON SAN JOSE 355 RIVER OAKS PARKWAY SAN JOSE, CA 95134-1991

EDITOR, TECHNICAL PUBLICATIONS THERMO ELECTRON SAN JOSE 355 RIVER OAKS PARKWAY SAN JOSE, CA 95134-1991 UNITED STATES OF AMERICA

PUBLICATIONS THERMO ELECTRON SAN JOSE 355 RIVER OAKS PARKWAY SAN JOSE, CA 95134-1991 UNITED STATES OF

fold

fold

Contents

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

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 First Changes to the Manual and Online Help

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.

iv

Finnigan Xcalibur: Getting Productive with Processing Setup

Abbreviations

Read This First 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

cm 3

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 First

Abbreviations

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 (10 9 )

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 (10 3 , 1000)

K

kilo (2 10 , 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 (10 6 )

M+

molecular ion

MB

Megabyte (1048576 bytes)

MH+

protonated molecular ion

min

minute

mL

milliliter

mm

millimeter

MS

mass spectrometer; mass spectrometry

MS

MS n power: where n = 1

MS/MS

MS n power: where n = 2

MS n

MS n 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

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 First

Abbreviations

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:

MS n (in this manual)

MS^n (in the online Help)

10^5 (in the online Help)

10 5 (in this manual)

viii

Finnigan Xcalibur: Getting Productive with Processing Setup

Read This First

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”.

Finnigan Xcalibur: Getting Productive with Processing Setup

ix

Read This First

Typographical 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.

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.

x

Finnigan Xcalibur: Getting Productive with Processing Setup

Topic Headings

Read This First

Typographical Conventions

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

Finnigan Xcalibur: Getting Productive with Processing Setup

xi

Read This First

Reply Cards

Reply Cards

Thermo Electron San Jose manuals contain one or two reply cards. All manuals contain a Customer Registration / Reader Survey card and some contain a Change of Location card. These cards are located at the front of each manual.

The Customer Registration / Reader Survey card has two functions. First, when you return the card, you are placed on the Thermo Electron San Jose mailing list. As a member of this list, you receive application reports and technical reports in your area of interest, and you are notified of events of interest, such as user meetings. Second, it allows you to tell us what you like and do not like about the manual.

The Change of Location card allows us to track the whereabouts of the instrument. Fill out and return the card if you move the instrument to another site within your company or if you sell the instrument. Occasionally, we need to notify owners of our products about safety or other issues.

xii Finnigan Xcalibur: Getting Productive with Processing Setup

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

1

Processing Setup and the Analysis of Quantitation Data

Processing Setup and the Analysis of Quantitation Data Figure 1. Integrated chromatogram peak Figure 2. Calibration

Figure 1.

Integrated chromatogram peak

of Quantitation Data Figure 1. Integrated chromatogram peak Figure 2. Calibration curve Xcalibur detected and integrated

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.

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/X

1/Y

1/Y

1/S 2 , where S 2 = X 2 + Y 2

2

2

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

3

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

4 Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data

PROCESSING SETUP:

CREATE PROCESSING METHOD

SEQUENCE SETUP: ADD PROCESSING METHOD TO SEQUENCE QUAN BROWSER: PROCESS RAW FILES USING SEQUENCE ALL
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
YES
PROCESSING SETUP
OR QUAN BROWSER:
NO
CALIBRATION
CURVE OK?
MODIFY CALIBRATION
CURVE PARAMETERS
YES
SEQUENCE SETUP:
CREATE REPORTS

Figure 3.

Flow diagram for analyzing quantitation data with Processing Setup

Finnigan Xcalibur: Getting Productive with Processing Setup

5

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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.

6 Finnigan Xcalibur: Getting Productive with Processing 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

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

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.

press File > New to start with a fresh display. Figure 4. Xcalibur Home Page Finnigan

Figure 4.

Xcalibur Home Page

Finnigan Xcalibur: Getting Productive with Processing Setup

7

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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.

8 Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 6. Chromatography Options dialog box Specifying

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.

by internal standard and to dismiss the dialog box. Figure 7. Calibration Options dialog box Finnigan

Figure 7.

Calibration Options dialog box

Finnigan Xcalibur: Getting Productive with Processing Setup

9

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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:

using the Open Raw File dialog box, proceed as follows: 1. Click on the open raw

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

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 8. Open Raw File dialog box, showing

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

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.

2. Click on OK to save the new name. See Figure 10. Figure 10. Identification page,

Figure 10.

Identification page, after the name D4 has been entered

Finnigan Xcalibur: Getting Productive with Processing Setup

13

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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 u 1 and a deuterated internal standard of molecular weight 469 u 2 (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 469.40@23.00[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).

14

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

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 11. Identification page, showing the mass

Figure 11.

Identification page, showing the mass chromatogram for D4.

Finnigan Xcalibur: Getting Productive with Processing Setup

15

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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:

enter the retention time of the LC peak as follows: a. Click on the pin button

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. (

turns green and the pin appears stuck into the screen. ( ). b. Click and drag

).

b. Click and drag the cursor in the Chromatogram pane from left to right across the chromatogram peak as shown in Figure 12.

to right across the chromatogram peak as shown in Figure 12. Figure 12. 16 Identification page,

Figure 12.

16

Identification page, showing the click-and-drag procedure for selecting the scan corresponding to the peak maximum in the chromatogram pane

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.

of the product ion(s) in the Spectrum pane. See Figure 13. Figure 13. Identification page, showing

Figure 13.

Identification page, showing a marker (vertical line) indicating the retention time corresponding to the peak maximum

Finnigan Xcalibur: Getting Productive with Processing Setup

17

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

2. Select

P r o c e s s i n g M e t h o d

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.

line that connects the integration markers. See Figure 14. Figure 14. Identification page, showing the area

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.has not been identified, repeat the procedure. Make sure the Specifying Component Identification Settings for the

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 465.30@23.00[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.a. Click on the pin button (currently grayed) in the upper right corner of

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:

mouse button, and the following events occur automatically: Finnigan Xcalibur: Getting Productive with Processing Setup

Finnigan Xcalibur: Getting Productive with Processing Setup

19

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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

to adjust the retention time of drugx, as follows: a. Select the Adjust Using check box.

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.

the peak identification settings for drugx. See Figure 15. Figure 15. 20 Identification page, showing the

Figure 15.

20

Identification page, showing the peak identification settings for drugx

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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

Finnigan Xcalibur: Getting Productive with Processing Setup

21

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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.

Detection page. The Detection page is shown in Figure 16. Figure 16. Processing Setup window –

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.

list and repeat steps 2 through 7 for the target compound. ). Entering Peak Detection Parameters

).

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

23

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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.

Detection page should look like the one shown in Figure 18. 24 Figure 17. ICIS Advanced

24

Figure 17.

ICIS Advanced Parameters dialog box, showing default settings

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 18. Detection page, showing settings for drugx

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

25

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 20. Calibration page, showing the settings for

Figure 20.

Calibration page, showing the settings for the internal standard, D4

Finnigan Xcalibur: Getting Productive with Processing Setup

27

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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.

page should look like the one shown in Figure 21. Figure 21. 28 Calibration page, showing

Figure 21.

28

Calibration page, showing the settings for the target compound, drugx

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.

you have selected the ISTD and try to open the Levels page. Figure 22. Processing Setup

Figure 22.

Processing Setup window – Quan view – Levels page

Finnigan Xcalibur: Getting Productive with Processing Setup

29

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

31

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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 (

and Symmetry Parameters check boxes are unchecked ( ). 3. If you made any changes, click

).

3. If you made any changes, click on OK to save the changes.

32

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

p
p

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:

report templates. Specify report templates as follows: 1. In the view bar, click on the Reports

1. In the view bar, click on the Reports button to display the Reports view. See Figure 25.

Finnigan Xcalibur: Getting Productive with Processing Setup

33

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

Figure 25.

34

Processing Setup window - Reports view

2. In the Sample Reports table, click in the Enable text box to display the

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.

enable check box for the first row. Then, select

Figure 26. enable check box for the first row. Then, select Finnigan Xcalibur: Getting Productive with

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

Analysis of Quantitation Data Creating a Processing Method Figure 26. Browse for Sample Report Template dialog

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.

The Report view should look like the one shown in Figure 27. Finnigan Xcalibur: Getting Productive

Finnigan Xcalibur: Getting Productive with Processing Setup

35

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating a Processing Method

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

to save the Processing Method and close the dialog box. Figure 28. File Summary Information dialog

Figure 28.

File Summary Information dialog box

Finnigan Xcalibur: Getting Productive with Processing Setup

37

Processing Setup and the Analysis of Quantitation Data

Creating a Processing Method

g a P r o c e s s i n g M e t h

38

Figure 29.

Save As dialog box, showing drugx_example.pmd

Finnigan Xcalibur: Getting Productive with Processing Setup

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.

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

Productive: Quantitative Analysis and/or the online Help. Use the Sequence Setup view to add the Processing

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

39

Processing Setup and the Analysis of Quantitation Data

Adding the Processing Method to a Sequence

g M e t h o d t o a S e q u e n

40

Figure 30.

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Adding the Processing Method to a Sequence

Data Adding the Processing Method to a Sequence Figure 31. Sequence Setup view, showing the Sequence

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

, and then click on Open to enter the Processing Method C:\Xcalibur\examples\methods\drugx example.pmd in the first

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

41

Processing Setup and the Analysis of Quantitation Data

Adding the Processing Method to a Sequence

g M e t h o d t o a S e q u e n

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.

Sequence should now look like the one shown in Figure 34. 42 Figure 32. Select Processing

42

Figure 32.

Select Processing Method dialog box, showing the Processing Method drugx_example.pmd selected

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Adding the Processing Method to a Sequence

Data Adding the Processing Method to a Sequence Figure 33. Fill Down dialog box, with settings

Figure 33.

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

Finnigan Xcalibur: Getting Productive with Processing Setup

43

Processing Setup and the Analysis of Quantitation Data

Adding the Processing Method to a Sequence

g M e t h o d t o a S e q u e n

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Adding the Processing Method to a Sequence

Data Adding the Processing Method to a Sequence Figure 35. File Summary Information dialog box Finnigan

Figure 35.

File Summary Information dialog box

Finnigan Xcalibur: Getting Productive with Processing Setup

45

Processing Setup and the Analysis of Quantitation Data

Adding the Processing Method to a Sequence

g M e t h o d t o a S e q u e n

46

Figure 36.

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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data 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.

Productive: Quantitative Analysis and/or the online Help. 1. Use the following procedure to process the raw

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

and opens the Quan Browser window. The Quan

drugx

example.sld

Browser window is shown in Figure 39.

Finnigan Xcalibur: Getting Productive with Processing Setup

47

Processing Setup and the Analysis of Quantitation Data

Processing the Raw Files and Reviewing Results

a n d R e v i e w i n g R e s u

Figure 37.

Open dialog box, showing the Sequence drugx_example.sld selected

box, showing the Sequence drugx_example.sld selected 48 Figure 38. View Sample Types dialog box Finnigan

48

Figure 38.

View Sample Types dialog box

Finnigan Xcalibur: Getting Productive with Processing Setup

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

Data Processing the Raw Files and Reviewing Results Figure 39. Quan Browser window, with the internal

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.

Finnigan Xcalibur: Getting Productive with Processing Setup

49

Processing Setup and the Analysis of Quantitation Data

Processing the Raw Files and Reviewing Results

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

Finnigan Xcalibur: Getting Productive with Processing Setup

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

Data Processing the Raw Files and Reviewing Results Figure 40. Quan Browser window, with the target

Figure 40.

Quan Browser window, with the target compound drugx selected

Finnigan Xcalibur: Getting Productive with Processing Setup

51

Processing Setup and the Analysis of Quantitation Data

Creating Reports

Creating Reports

r e a t i n g R e p o r t s 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

Finnigan Xcalibur: Getting Productive with Processing Setup

Processing Setup and the Analysis of Quantitation Data Creating Reports

and the Analysis of Quantitation Data Creating Reports Figure 41. Batch Reprocess Setup dialogbox Figure 42.

Figure 41.

Batch Reprocess Setup dialogbox

Creating Reports Figure 41. Batch Reprocess Setup dialogbox Figure 42. Microsoft ® Windows ® Explorer, showing

Figure 42.

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

Finnigan Xcalibur: Getting Productive with Processing Setup

53

Processing Setup and the Analysis of Quantitation Data

Sample Reports

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

Finnigan Xcalibur: Getting Productive with Processing Setup

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

RT: 3.87 - 5.87 SM: 5G

ERT:

4.89

RT: 3.88 - 5.88

SM: 5G

RTW:

30.00

   

R T : 4.87

NL:

2.29E6

T IC F : + c SRM ms2

RTW:

30.00

   

R T : 4.88

NL:

100

 

Relative Abundance

100 90 80 70 60 50 40 30 20 10 0
100
90
80
70
60
50
40
30
20
10
0

2.59E5

VW:

2.00

 

90

VW: 2.00   90 VW: 2.00 T IC F: + c SRM ms2

VW:

2.00

T IC F: + c SRM ms2

RTR:

no

80

RTR:

no

ID:

Nearest RT

465.30 [

ID:

Nearest RT

469.40 [

MPH:

SM:

3.00

5

 

Relative Abundance

70

419.30-

MPH:

3.00

5

 

423.30-

60

421.30] MS

425.30] MS

drugx_31

SM:

drugx_31

PDAU: Genesis

50

PDAU: Genesis

VD:

yes

40

VD:

yes

EPW:

20.00

30

EPW:

20.00

CP:

no

 

20

CP::

no

ISTD:

10

ISTD:

D4

 

D4

 

0

 

4

5

 

4

5

 

T ime(min)

 

T ime(min)

 
 

Actual

Calculated

 

Base

 

Signal

 

Area

Area

Specified

RT

Amount

Response

Line

To Noise

% Diff

Ratio

ISTD

Amount

Component Name drugx

4.87 1019.273 22277136 BB 10217.38 1.927 9.103 24471 1000.000 98
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

RT: 3.88 - 5.88

SM: 5G

ERT:

4.87

 

RT: 3.88 - 5.88

SM: 5G

RTW:

30.00

   

R T : 4.88

NL:

RTW:

30.00

 

R T : 4.88

NL:

100

Relative Abundance

100 90 80 70 60 50 40 30 20 10 0
100
90
80
70
60
50
40
30
20
10
0

2.59E5

VW:

2.00

 

90

2.59E5 T IC F : + c SRM ms2 469.40 [ 423.30- 425.30] MS drugx_31

2.59E5

T IC F : + c SRM ms2

469.40 [

423.30-

425.30] MS

drugx_31

VW:

2.00

T IC F: + c SRM ms2

RTR:

yes

80

RTR:

yes

ID:

Nearest RT

ID:

Nearest RT

 

469.40 [

MPH:

SM:

3.00

5

 

Relative Abundance

70

MPH:

SM:

3.00

5

 

423.30-

60

425.30] MS

drugx_31

PDAU: Genesis

50

PDAU: Genesis

VD:

yes

40

VD:

yes

EPW:

20.00

30

EPW:

20.00

CP:

no

 

20

CP::

no

ISTD:

10

ISTD:

 

-

 

-

 
 

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
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

 
Detector Type   MS    
 

Filter: + c SRM ms2 465.30@23.00 [ 419.30-421.30]

Trace

 

TIC

 

8

Quan Masses

 

N/A

7

 

Retention Time

 

6

Expected RT (min)

 

4.89

 

Area Ratio

5

Window (sec)

 

30.0

View Width (min)

 

2.00

4

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