ToxID 2.1.2 User Guide Version B - Thermo Fisher

ToxID

User Guide

XCALI-97245 Revision B February 2010

http://www.surveymonkey.com/s.aspx?sm=R7gKOvhLXn3NTkpK2BefHQ_3d_3d

© 2010 Thermo Fisher Scientific Inc. All rights reserved.

ToxID, LCquan, and Exactive are trademarks and Xcalibur is a registered trademark of Thermo Fisher Scientific Inc. in the United States.

Microsoft, Windows, and Excel are registered trademarks of Microsoft Corporation in the United States and other countries.

Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.

Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or error-free and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any use of this document, even if the information in the document is followed properly.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.

Release history: Revision A released January 2009. Revision B released February 2010

Software version: ToxID 2.1.2, Xcalibur 2.1 or earlier, LCquan 2.6 or earlier

For Research Use Only. Not for use in diagnostic procedures.

Thermo Scientific ToxID User Guide iii

C

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vContacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Chapter 1 Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Hardware and Software Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Installing ToxID Files and Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Running ToxID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Chapter 2 Working with Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Adding a Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Adding Libraries to the Search List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Archiving a Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 3 Setting Up ToxID Processing Configuration Files. . . . . . . . . . . . . . . . . . . . . . . . . .11Processing Configuration File Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Processing Configuration File Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Setting Processing Configuration File Parameters . . . . . . . . . . . . . . . . . . . . . 15

Example Processing Configuration Files and Reports . . . . . . . . . . . . . . . . . . . . 15MS/MS Spectrum and Retention Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16MS/MS and MS3 Spectra and Retention Time . . . . . . . . . . . . . . . . . . . . . . . 20MS3 Spectrum and Retention Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24MS/MS Spectrum Collected in a QED Experiment and Retention

Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27SRM Transitions and Ion Ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Exact Mass. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Exactive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Chapter 4 Running the ToxID Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Running the Application Using the ToxID User Interface. . . . . . . . . . . . . . . . . 39Running the Application Using an Xcalibur Processing Method . . . . . . . . . . . . 44Running the Application Directly After Data Acquisition for Each Sample

in the Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Troubleshooting Processing Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51

Contents

Thermo Scientific ToxID User Guide v

P

Preface

This guide describes how to install and use ToxID™ 2.1.2.

ToxID is an automated compound identification tool that dramatically simplifies toxicology screening workflows for the clinical and forensic laboratory.

To provide comments about this document, click the link below. Thank you in advance for your help.

Contacting UsThere are several ways to contact Thermo Fisher Scientific for the information you need.

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Knowledge base www.thermokb.com

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mailto:[email protected]

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PrefaceContacting Us

vi ToxID User Guide Thermo Scientific

To copy manuals from the Internet

Go to mssupport.thermo.com and click Customer Manuals in the left margin of the window.

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mailto:[email protected]

Thermo Scientific ToxID User Guide 1

1

Getting Started

ToxID is an application that provides automated compound identification from Xcalibur™ data files.

OverviewThe ToxID application identifies compounds and provides reports for the following experiments:

• Ion trap mass spectrometer experiments

– MS/MS spectra

– MS/MS and MS3 spectra

– MS3 spectra

• Triple quadruple mass spectrometer experiments

– MS/MS spectra collected in a QED experiment

– SRM transitions and ion ratios

• Hybrid mass spectrometer experiment

– Exact mass

• Orbital ion trap experiment (Exactive™)

– Exact Mass

Contents

• Overview

• Hardware and Software Requirements

• Installing ToxID Files and Components

Note The ToxID application can save retention time as an additional compound identification factor in every experiment.

1 Getting StartedHardware and Software Requirements

2 ToxID User Guide Thermo Scientific

Hardware and Software RequirementsIn order to run ToxID software, your computer must conform to the following minimum requirements:

Installing ToxID Files and ComponentsTo install ToxID software

1. Insert the ToxID installation CD and click Next.

2. To accept the license agreement terms, select the I accept the terms of the license agreement option and click Next.

3. After reviewing the hardware and software requirements, click Next.

4. To install the ToxID files:

a. Select the Program Files check box to install the ToxID application.

b. To copy the ToxID User Guide to your system, select the Manuals check box.

c. To copy the example processing configuration files to your system (recommended), select the Examples check box.

d. Click Next.

5. To start installing files, click Next.

6. Choose whether to review the saved Installation Qualification report by clicking Yes or No.

7. Click Finish.

Table 1. Hardware and software requirements

System Requirements

PC • 2.0 GHZ processor with 512 MB RAM• 75 GB hard drive (NTFS formatted)• CD-ROM drive• Display monitor resolution of 1280×1024

Software • Microsoft Windows™ XP SP2 or higher• Microsoft Office 2003 or 2007• Xcalibur 2.1 or previous versions• LCquan™ 2.6 (optional)

1 Getting StartedRunning ToxID


The ToxID installation provides the files listed in Table 2 as well as a desktop icon.

Running ToxIDIn order to run ToxID, you must configure the ToxID software, which consists of the following steps:

• Configuring spectra libraries. This step is required only for experiments implementing specific libraries in compound identification. See Chapter 2, “Working with Libraries,” on page 5.

• Setting up a processing configuration file to define experiment- and compound-specific parameters. See Chapter 3, “Setting Up ToxID Processing Configuration Files,” on page 11.

When executed, the ToxID application produces a log file. The log file contains processing information and is stored in the same directory as the raw data file.

Table 2. ToxID files and their folder locations

File type Description Default folder location

ToxID.exe (and related DLL files)

ToxID application \Xcalibur\system\programs

ToxID_User_Guide.pdf ToxID User Guide \Xcalibur\system\programs

library Folder for example library files

\Xcalibur\examples\ToxID\libraries

experiment type.csv Example ToxID processing configuration files

\Xcalibur\examples\ToxID

experiment type.raw Example raw files \Xcalibur\examples\ToxID


2

Working with Libraries

The ToxID application requires one or more spectra libraries for experiments identifying compounds based on MS/MS, MS3, or both MS/MS and MS3 spectra. This chapter provides information on adding, configuring, and archiving libraries.

To see a sample library file, go to the folder:

\Xcalibur\examples\ToxID\libraries

For more detailed information about using libraries, refer to the Creating and Searching Libraries User Guide included in the Xcalibur documentation.

Adding a LibraryTo add a library

1. Open the Xcalibur Home Page and choose Tools > Library Manager.

The Library Manager dialog box opens with the Manage libraries page shown by default (Figure 1).

Contents

• Adding a Library

• Adding Libraries to the Search List

• Archiving a Library

2 Working with LibrariesAdding a Library


Figure 1. Library Manager dialog box

2. Click Add.

The Add Library dialog box opens (Figure 2).

Figure 2. Add Library dialog box

3. Click Browse to locate the library you want to add.

The ToxID application installs example libraries in the \Xcalibur\examples\ToxID\libraries folder.

4. Click OK.

The library you selected is now shown in the Source box.

2 Working with LibrariesAdding Libraries to the Search List


5. Depending on the location of the source library, select the Copy the library to the local computer or Link to the library from either a remote location or computer check box.

6. Click OK.

The File Status message box opens and reports the progress of copying the file.

7. After you receive confirmation that the library has been added, click Dismiss.

By default, the library is copied to the folder:

\Program Files\NISTMS\MSSEARCH

8. Click Exit to close the Library Manager dialog box.

Adding Libraries to the Search ListTo add a library to the search list

1. Open the Xcalibur Home Page and click Qual Browser.

The Qual Browser window opens (Figure 3).

Figure 3. Qual Browser window

2. Choose File > Open.

The Open Raw File dialog box opens (Figure 4).

2 Working with LibrariesAdding Libraries to the Search List


Figure 4. Open Raw File dialog box

3. Select an Xcalibur raw date file.

4. Click Open.

The Qual Browser spectrum window opens (Figure 5).

Figure 5. Qual Browser spectrum window

To ensure that the spectrum window is active, look for (the pinned icon). (If you see instead, click the icon to activate the window.)

5. Right-click the activated spectrum window and choose Library > Options from the shortcut menu.

2 Working with LibrariesArchiving a Library


The Search Properties dialog box opens with the Search List page shown by default (Figure 6).

Figure 6. Search Properties dialog box

6. Select the library from the Available Libraries list.

7. Click Add to include the library in the Selected Libraries list.

8. Use the Top, Up, Down, and Bottom buttons to create a search order of multiple libraries.

9. Click Apply, and then click OK.

10. Right-click the activated spectrum window and choose Library > Search from the shortcut menu.

If you see some compound results in the library, then your library settings are valid and the NIST software is activated.

Archiving a LibraryArchiving a library creates a copy of the library as a backup or to use on another instrument.

To archive a library

1. Open the Xcalibur Home Page.

2. Choose Tools > Library Manager.

The Library Manager dialog box opens (Figure 7).

2 Working with LibrariesArchiving a Library


Figure 7. Library Manager dialog box

3. Select the library from the NIST libraries list.

4. Click Archive.

The Archive Library dialog box opens (Figure 8).

Figure 8. Archive Library dialog box

5. Click Browse and select the destination to archive the library.

6. Click OK.

7. After you receive confirmation that the library has been archived, click Dismiss.

8. Click Exit to close the Library Manager dialog box.


3

Setting Up ToxID Processing Configuration Files

The ToxID application requires experiment- and compound-specific information that you define in the processing configuration file. The processing configuration file is a .csv file that you name and save.

Processing Configuration File TemplateTwo read-only processing configuration (.csv) file templates are provided on the CD. One of the templates is for the first six experiments listed on page 1 and the other is for the Orbital ion trap experiment. As part of the installation process, these templates are put in the following folder:


In addition, the CD contains an example file for each type of experiment that you can analyze with the ToxID application, as described in “Example Processing Configuration Files and Reports” on page 15.

Use Microsoft Excel™ to edit ToxID processing configuration files. Note the following about editing the processing configuration files with Excel:

• Never add, remove, or rearrange columns or rows above row 4 in a processing configuration file. The ToxID application cannot process the analysis if any information is missing or moved.

• Changes you make to a .csv file, such as enlarging or shrinking columns or rows, are lost when you save the file.

• Always save the edited processing configuration file as a .csv file. You cannot run the ToxID application using Excel (.xls) files.

Contents

• Processing Configuration File Template

• Example Processing Configuration Files and Reports

Note If you have a processing configuration file from ToxID 1.0, open the processing configuration file template on the ToxID 2.1.2 CD and save it with a new name. Then copy the compound-related information from the ToxID 1.0 configuration file into the copy of the ToxID 2.1.2 configuration processing file you saved.

3 Setting Up ToxID Processing Configuration FilesProcessing Configuration File Template


The ToxID processing configuration file (name.csv) supplied on the ToxID CD is shown in Figure 9 and Figure 10.

Figure 9. Processing configuration file

The Exactive processing configuration file (name.csv) supplied on the ToxID CD is shown in Figure 10.

Figure 10. Exactive processing configuration file for first six experiments listed on page 1



Processing Configuration File Parameters

A ToxID processing configuration file contains compound-specific parameters located in line 4 and below. These parameters are also specific to the screening experiment.

Table 3 describes the compound-specific parameters for the six experiments and the Exactive experiment.Table 3. Compound-specific parameters (Sheet 1 of 2)

Parameter Description

Compound Name (all experiments)

Name of the compound. If a spectra library is implemented, this name must match the library compound name. Compound names are required for all screening experiments.

Parent Ion m/z (six experiments)

Parent ion mass-to-charge ratio. This parameter is required for all non-Exactive experiments.

Chemical Formula (Exactive) Chemical formula of the compound.

Polarity (Exactive) Positive or negative ionization.

Analyte Type(all experiments)

Specifies a parent, metabolite, or other analyte type, as appropriate. The first letter of an analyte type name is posted in the report. This parameter is required for all experiments.

The Code column in the reports refers to the information in the Analyte Type column: P for Parent, M for Metabolite, or I for Internal Standard.

Expected RT (all experiments)

Expected retention time. This parameter is required for all experiments.

MS2 Product Ion(six experiments)

For MS/MS, and for MS/MS and MS3experiments, specifies the most intense or most specific fragment in MS/MS spectra. The m/z value for this fragment is used to reconstruct the chromatogram.

For MS3experiments, it specifies the MS/MS fragment ion for which an MS3spectrum was collected.



Intensity Threshold(all experiments)

For MS/MS, and for MS/MS and MS3 experiments, a search hit is only reported if the signal intensity for the most intense or most specific fragment in the MS/MS spectra is equal to or above this value.

For QED MS/MS experiments, a search hit is only reported if the intensity of the fragment ion collected in an SRM scan event is equal to or above this value.

For other types of experiments, a hit is reported if the TIC signal intensity is equal to or above this value.

SI (six experiments) Specifies the minimum search index value for the reported hits. This parameter is required for the MS/MS spectra, MS/MS and MS3spectra, MS3spectra, and MS/MS QED experiments.

RSI (six experiments) Specifies the minimum reverse search index value for the reported hits. This parameter is required for the MS/MS

spectra, MS/MS and MS3 spectra, MS3 spectra, and MS/MS QED experiments.

SRM Fragment Ions(six experiments)

Specifies the fragment and, in parentheses, the relative intensity of each fragment ion. Separate each ion entry with a semicolon (;), for example: 191.3(25);245.2(100);345.9(67). This parameter is required for the SRM and QED. The SRM fragment ions input must match with the SRM scans, or else it does not return a hit.

Internal Standard(six experiments)

A reference compound name present in the data file.

Intensity Concentration Coefficient (for ion trap experiment only)

A user-defined parameter to convert the intensity ratio to concentration of analyte. See page 43 for further details.

Area Concentration Coefficient (for ion trap experiment only)

A user-defined parameter to convert the area ratio to concentration of analyte. Use intensity of most MS/MS scans.

Adduct 1, 2, 3 (Exactive) Ions attached to parent ion during ionization.

Fragment 1, 2, 3 (Exactive) Ions in the HCD scan, which the user can choose for additional information about the compound match.

Table 3. Compound-specific parameters (Sheet 2 of 2)


3 Setting Up ToxID Processing Configuration FilesExample Processing Configuration Files and Reports


Setting Processing Configuration File Parameters

To set the ToxID processing configuration file parameters

1. Do one of the following:

• Open Excel and then open the ToxID processing configuration file for the experiment you want to perform.

• Double-click the ToxID processing configuration file for the experiment you want to perform.

2. Set the compound-specific parameters as shown in Table 3 on page 13.

3. Save the file with a new name in a location of your choice.

Example Processing Configuration Files and ReportsIn addition to the processing configuration file and Exactive processing configuration file templates, the ToxID CD provides six example processing configuration files and one Exactive configuration file for each type of experiment you can analyze. These sample files are located in the following folder:


The following sections describe each example processing configuration file and provide an example of both the summary and long reports that each file generates:

• “MS/MS Spectrum and Retention Time” on page 16

• “MS/MS and MS3 Spectra and Retention Time” on page 20

• “MS3 Spectrum and Retention Time” on page 24

• “MS/MS Spectrum Collected in a QED Experiment and Retention Time” on page 27

• “SRM Transitions and Ion Ratios” on page 30

• “Exact Mass” on page 33

• “Exactive” on page 36

The ToxID application saves all reports in the raw file folder using the following format for the report file name:

raw_file_name_date_timestamp_report type

where:

raw_file_name is the name of the source file;



date is the date when the application generated the report (using the format yyyymmdd where yyyy is the year, mm is the month, and dd is the day of the month);

timestamp is the time (using the format hhmmss where hh is the hour, mm are the minutes, and ss are the seconds). The time stamp ensures that all report files are unique and never overwritten;

report type is S (Summary Report) or L (Long Report). The spreadsheet file has no report type.

Example: DrugXRawFileFolder\DrugX_20070605_100935_S

The ToxID application produces two types of reports:

• Summary Report: Lists all detected compounds and provides related chromatograms.

• Long Report: Provides one page of detailed information for each detected compound.

The CSV Report is the default report and is always generated.

MS/MS Spectrum and Retention Time

Use the ConfigFile_MS2_Test.csv file in screening experiments that identify compounds based on MS/MS spectrum and retention time. You can view the processing configuration file in Figure 11, a sample summary report in Figure 12, and a sample long report in Figure 13.



Figure 11. MS/MS spectrum and retention time processing configuration file (ConfigFile_MS2_Test.csv)



Figure 12. MS/MS spectrum and retention time sample summary report

Your Company NameSummary Report

Raw File Name: C:\Xcalibur\examples\ToxID\MS2\MS2_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS2\ConfigFile_MS2_Test.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/27/2008 6:48:33 PMScreening Conditions: Based on Full MS2 scans. m/z window (amu): 0.30, RT window (min): 0.50, MS2 Search libraries: MS2_Test,Do not use full MS scan to confirm

Peak 1

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1.30 1.81 2.254.73

4.774.704.80

4.66 4.87 5.515.40

5.455.345.495.30 5.90

4.15

4.204.114.26

3.323.28

3.353.453.25

3.853.88

3.913.814.39

4.164.10

4.214.05 4.625.295.25

5.215.355.16 5.77

NL: 3.51E4m/z= 147.80-148.40 F: ITMS + cESI d w Full ms2 [email protected][55.00-255.00]








Page 1 of 2

PeakNumber

Compound Name Code SI RSI m/z ExpectedRT

Actual RT Concentrationng/ml

Library Name

1 Albuterol P 958 959 240.10 1.40 1.40 0.00 MS2_Test2 Alprenolol P 885 887 250.20 4.80 4.73 0.00 MS2_Test3 Amitriptyline P 912 912 278.20 5.40 5.40 0.00 MS2_Test4 7-Amino-Flunitrazepam M 883 884 284.10 4.20 4.15 0.00 MS2_Test5 6-Acetylmorphine P 866 867 328.20 3.30 3.32 0.00 MS2_Test6 Acebutolol P 880 884 337.30 3.90 3.85 0.00 MS2_Test7 6-Acetylcodeine M 862 863 342.30 4.20 4.16 0.00 MS2_Test8 Haloperidol-D4 I 883 883 380.30 5.30 5.29 0.00 MS2_Test



Figure 13. MS/MS spectrum and retention time sample long report (page for hit number 1)

Your Company NameLong Report

Raw File Name: C:\Xcalibur\examples\ToxID\MS2\MS2_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS2\ConfigFile_MS2_Test.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/27/2008 6:48:33 PMScreening Conditions: Based on Full MS2 scans. m/z window (amu): 0.30, RT window (min): 0.50, MS2 Search libraries: MS2_Test,Do not use full MS scan to confirm

Page 1 of 8

PeakNumber


Actual RT Concentrationng/ml

Library Name

1 Albuterol P 958 959 240.10 1.40 1.40 0.00 MS2_Test

Peak 1

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1.36 1.43

1.471.33

1.501.531.601.30 1.81 2.351.26

NL: 3.51E4m/z= 147.80-148.40F: ITMS + c ESI d wFull [email protected][55.00-255.00]

Acquired Spectrum

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MS/MS and MS3 Spectra and Retention Time

Use the ConfigFile_MS2_MS3_Test.csv file in screening experiments that identify compounds based on MS/MS and MS3 spectra and retention time. You can view the processing configuration file in Figure 14, a sample summary report in Figure 15, and a sample long report in Figure 16 and Figure 17.

Figure 14. MS/MS and MS3 spectra and retention time processing configuration file (ConfigFile_MS2_MS3_Test.csv)



Figure 15. MS/MS and MS3 spectra and retention time sample summary report (first page)


Raw File Name: C:\Xcalibur\examples\ToxID\MS2_MS3\MS2_MS3_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS2_MS3\ConfigFile_MS2_MS3_Test.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/27/2008 6:34:32 PMScreening Conditions: Based on MS2 and MS3 scans. m/z window (amu): 0.30, RT window (min): 0.50, MS2 Search libraries: MS2_Test,MS3 Search Libraries: MS3_Test,Usefull MS scan to confirm.

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100 1.121.171.211.311.02 1.93

1.111.151.06

1.201.301.01 2.16

4.79

4.834.69 5.083.974.78

4.834.874.68 5.343.965.38

5.45

5.525.31 5.945.39

5.46

5.535.32 6.104.18

4.254.47

4.18

4.244.34

NL: 2.11E3TIC F: ITMS + c ESI d w Full [email protected]@cid37.00 [55.00-255.00]








Page 1 of 2

PeakNumber

Compound Name Code MSOrder

SI RSI m/z ExpectedRT

Actual RT Intensity Library Name

1 *Albuterol P 3 889 895 240.10 1.40 1.12 2111 MS3_Test2 *Albuterol P 2 976 976 240.10 1.40 1.11 35817 MS2_Test3 *Alprenolol P 3 830 830 250.20 4.80 4.79 18413 MS3_Test4 *Alprenolol P 2 895 897 250.20 4.80 4.78 64106 MS2_Test5 *Amitriptyline P 2 929 929 278.20 5.40 5.38 266839 MS2_Test6 *Amitriptyline P 3 877 878 278.20 5.40 5.39 62546 MS3_Test7 *7-Amino-Flunitrazepam M 3 830 833 284.10 4.20 4.18 5544 MS3_Test8 *7-Amino-Flunitrazepam M 2 868 871 284.10 4.20 4.18 25081 MS2_Test



Figure 16. MS/MS and MS3 spectra and retention time sample long report (first page of MS3 spectrum for hit number 1)



Page 1 of 16

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1 *Albuterol P 3 889 895 240.10 1.40 1.12 2111 MS3_Test

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NL: 2.11E3TIC F: ITMS + c ESId w Full [email protected]@cid37.00[55.00-255.00]

Acquired Spectrum

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Library Structure



Figure 17. MS/MS and MS3 spectra and retention time sample long report (second page of MS/MS spectrum for hit number 1)

Your Company Name Long Report


Page 2 of 16

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2 *Albuterol P 2 976 976 240.10 1.40 1.11 35817 MS2_Test

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1.251.301.441.01 1.68

NL: 3.58E4m/z= 147.80-148.40F: ITMS + c ESI d wFull [email protected][55.00-255.00]

Acquired Spectrum

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Library Structure



MS3 Spectrum and Retention Time

Use the ConfigFile_MS3_Test.csv file in screening experiments that identify compounds based on MS3 spectrum and retention time. You can view the processing configuration file in Figure 18, a sample summary report in Figure 19, and a sample long report in Figure 20.

Figure 18. MS3 spectrum and retention time processing configuration file (ConfigFile_MS3_Test.csv)



Figure 19. MS3 spectrum and retention time sample summary report


Raw File Name: C:\Xcalibur\examples\ToxID\MS3\MS3_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS3\ConfigFile_MS3_Test.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/27/2008 6:19:32 PMScreening Conditions: Based on MS3 scans. MS3_Test,Use full MS scan to confirm.

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4.764.71 4.79

4.834.64 5.285.38

5.44

5.33 5.51 5.924.15

4.204.09 4.28

3.333.29

3.373.26 3.48 4.24

3.833.88

3.923.78 4.37

4.14

4.214.07 4.655.26

5.315.21

5.395.17









Page 1 of 1

PeakNumber



1 Albuterol P 829 835 240.10 1.40 1.42 2156 MS3_Test2 Alprenolol P 837 837 250.20 4.80 4.76 21707 MS3_Test3 Amitriptyline P 860 871 278.20 5.40 5.38 51929 MS3_Test4 7-Amino-Flunitrazepam M 819 819 284.10 4.20 4.15 5435 MS3_Test5 6-Acetylmorphine P 827 827 328.20 3.30 3.33 8505 MS3_Test6 Acebutolol P 888 891 337.30 3.90 3.83 75901 MS3_Test7 6-Acetylcodeine M 841 841 342.30 4.20 4.14 29648 MS3_Test8 Haloperidol-D4 I 871 871 380.30 5.30 5.26 19816 MS3_Test



Figure 20. MS3 spectrum and retention time sample long report (page for hit number 1)


Raw File Name: C:\Xcalibur\examples\ToxID\MS3\MS3_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS3\ConfigFile_MS3_Test.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/27/2008 6:19:32 PMScreening Conditions: Based on MS3 scans. MS3_Test,Use full MS scan to confirm.

Page 1 of 8

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1 Albuterol P 829 835 240.10 1.40 1.42 2156 MS3_Test

Peak 1

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1.421.46

1.351.50

1.531.31 1.65

1.871.27 2.39

NL: 2.16E3TIC F: ITMS + c ESId w Full [email protected]@cid37.00[55.00-255.00]

Acquired Spectrum

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Library Structure



MS/MS Spectrum Collected in a QED Experiment and Retention Time

Use the ConfigFile_MS2_QED.csv file in screening experiments that identify compounds based on MS/MS spectrum collected in a QED experiment and retention time. You can view the processing configuration file in Figure 21, a sample summary report in Figure 22, and a sample long report in Figure 23.

Figure 21. MS/MS spectrum collected in a QED experiment processing configuration file (ConfigFile_MS2_QED.csv)



Figure 22. MS/MS spectrum collected in QED experiment sample summary report


Raw File Name: C:\Xcalibur\examples\ToxID\MS2_QED\MS2_QED.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS2_QED\ConfigFile_MS2_QED.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 6/18/2007 6:32:54 AMScreening Conditions: Based on SRM and MS2 scans. m/z window(amu): 0.30, RT window (min): 0.50, MS2 Search libraries: MS2_Test,Use full MS scan to confirm.

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2.922.25 3.95 4.480.223.42

3.63 4.380.63 1.584.75

5.170.53 4.571.15 6.542.37 7.454.30

7.401.15 2.05 3.66 5.58 7.785.67

6.11 6.67 7.87

NL: 5.54E5TIC F: + c ESI sid=10.00 SRM [email protected] [134.192-134.202]



NL: 3.47E5TIC F: + c ESI sid=10.00 SRM [email protected] [165.005-165.015,240.976-240.986]

NL: 8.26E5TIC F: + c ESI sid=10.00 SRM [email protected] [122.199-122.209,223.112-223.122]

Page 1 of 1

PeakNumber



1 Phenylpropanolamine p 999 999 152.000 2.50 2.65 553531 MS2_Test2 Methcathinone p 999 999 164.000 3.30 3.42 1474860 MS2_Test3 Methaqualone p 999 999 251.100 4.80 4.75 3639669 MS2_Test4 4-Hydroxynordiazepam p 999 999 287.100 4.30 4.30 347148 MS2_Test5 Ibogaine p 999 999 311.200 5.50 5.67 826397 MS2_Test



Figure 23. MS/MS spectrum collected in QED experiment sample long report (page for hit number 1)


Raw File Name: C:\Xcalibur\examples\ToxID\MS2_QED\MS2_QED.RAWConfig File Name: C:\Xcalibur\examples\ToxID\MS2_QED\ConfigFile_MS2_QED.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 6/18/2007 6:32:54 AMScreening Conditions: Based on SRM and MS2 scans. m/z window(amu): 0.30, RT window (min): 0.50, MS2 Search libraries: MS2_Test,Use full MS scan to confirm.

Page 1 of 5

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1 Phenylpropanolamine p 999 999 152.000 2.50 2.65 553531 MS2_Test

Peak 1

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1002.652.36

2.32

2.922.25 3.11 3.95 4.480.22 1.610.99

NL: 5.54E5TIC F: + c ESI sid=10.00SRM [email protected][134.192-134.202]

1 2 3 4 5 6 7 8 9 10 11 12Time (min)

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NL: 9.09E4TIC F: + c ESI sid=-10.00 dFull ms2 [email protected][30.000-157.000]

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Library Structure



SRM Transitions and Ion Ratios

Use the ConfigFile_Ion_Ratio.csv file in screening experiments that identify compounds based on single SRM transitions and retentive ion ratios and retention time. You can view the processing configuration file in Figure 24, a sample summary report in Figure 25, and a sample long report in Figure 26. The number of transitions specified in the configuration file must match the number of fragments collected in the raw file.

Figure 24. SRM transitions and ion ratios processing configuration file (ConfigFile_Ion_Ratio.csv)



Figure 25. SRM transitions and ion ratios sample summary report


Raw File Name: C:\Xcalibur\examples\ToxID\SRM_Ion_Ratio\Ion_Ratio.rawConfig File Name: C:\Xcalibur\examples\ToxID\SRM_Ion_Ratio\ConfigFile_Ion_Ratio.csvSample Name: 013108 Laboratory: Your Lab NameAcquistion Start Time: 1/31/2008 5:26:01 PMScreening Conditions: Based on SRM scans. m/z window(amu): 0.30, RT window (min): 0.50, Ion ratio calculation: Use scan at peak apex. Ion ratio window (%): 30.

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3.420.86

1.09 1.621.52

1.65

1.932.553.31

3.56 4.745.63

5.86 7.364.97

5.32 5.87

NL: 4.43E4m/z= 60.100-60.700+71.100-71.700+85.000-85.600 F: + cESI SRM ms2 130.222 [60.455-60.457, 71.414-71.416,85.313-85.315]

NL: 1.41E6m/z= 65.100-65.700+91.000-91.600+119.000-119.600 F:+ c ESI SRM ms2 136.206 [65.434-65.436,91.281-91.283, 119.251-119.253]

NL: 4.63E6m/z= 77.000-77.600+102.900-103.500+119.900-120.500F: + c ESI SRM ms2 163.000 [77.339-77.341,103.224-103.226, 120.216-120.218]





NL: 1.16E6m/z= 192.800-193.400+270.700-271.300 F: + c ESI SRMms2 314.085 [193.068-193.070, 271.041-271.043]

Page 1 of 2

PeakNumber

Compound Name Code m/z ExpectedRT

Actual RT Intensity Ion Ratio

1 1,1-Dimethylbiguanide p 130.200 0.80 0.88 44273 Fail2 Amphetamine p 136.200 3.30 3.02 1409256 Pass3 Aminorex p 163.000 3.10 3.00 4627020 Pass4 Anhydroecgonine p 168.100 0.70 0.86 667084 Fail5 AnhydroecgonineMethylE

sterp 182.100 1.60 1.52 659364 Pass

6 Antipyrine p 189.100 3.60 3.31 517562 Pass7 Amitriptyline p 278.200 6.10 5.63 3558652 Fail8 Amoxapine p 314.085 5.30 4.97 1156522 Pass



Figure 26. SRM transitions and ion ratios sample long report (page for hit number 1)


Raw File Name: C:\Xcalibur\examples\ToxID\SRM_Ion_Ratio\Ion_Ratio.rawConfig File Name: C:\Xcalibur\examples\ToxID\SRM_Ion_Ratio\ConfigFile_Ion_Ratio.csvSample Name: 013108 Laboratory: Your Lab NameAcquistion Start Time: 1/31/2008 5:26:01 PMScreening Conditions: Based on SRM scans. m/z window(amu): 0.30, RT window (min): 0.50, Ion ratio calculation: Use scan at peak apex. Ion ratio window (%): 30.

Page 1 of 10

PeakNumber

Compound Name Code m/z ExpectedRT

Actual RT Intensity ExpectedIon Ratio

Ion Ratio

1 1,1-Dimethylbiguanide p 130.200 0.80 0.88 44273 FailIon 1 60.456 77.0 49.0Ion 2 71.415 100.0 100.0Ion 3 85.314 29.0 41.6

Peak 1

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NL: 4.43E4m/z=60.100-60.700+71.100-71.700+85.000-85.600 F: + c ESI SRMms2 130.222 [60.455-60.457,71.414-71.416, 85.313-85.315]

Acquired Spectrum

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Exact Mass

Use the ConfigFile_Exact_mass.csv file in screening experiments that identify compounds based on exact mass and retention time. You can view the processing configuration file in Figure 27, a sample summary report in Figure 28, and a sample long report in Figure 29.

Figure 27. Exact mass processing configuration file (ConfigFile_Exact_mass.csv)



Figure 28. Exact mass sample summary report


Raw File Name: C:\Xcalibur\examples\ToxID\Exact_Mass\Exact_Mass_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\Exact_Mass\ConfigFile_Exact_mass.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/24/2008 4:46:43 PMScreening Conditions: Based on accurate mass scans. Exact mass window (ppm): 30, RT window(min): 0.50.

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4.18

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3.72 4.12

NL: 2.03E5m/z= 240.15580-240.16300F: FTMS + c ESI Full ms[120.00-1000.00]






Page 1 of 1

PeakNumber

Compound Name Expectedm/z

Detectedm/z

Delta(mDa)

Delta(ppm)

ExpectedRT

Actual RT Intensity

1 Albuterol 240.15940 240.15939 -0.0 -0.0 2.58 2.58 1995052 Alprenolol 250.18040 250.18039 -0.0 -0.0 4.50 4.66 126044993 Amitriptyline 278.19060 278.19061 0.0 0.0 5.00 5.19 117697554 6-Acetylmorphine 328.15430 328.15433 0.0 0.1 3.30 3.57 21120905 6-Acetylcodeine 342.17020 342.17035 0.1 0.4 4.10 4.18 45933066 Acebutolol 337.21240 337.21246 0.1 0.2 3.80 3.98 9077282



Figure 29. Exact mass sample long report (page for hit number 1)


Raw File Name: C:\Xcalibur\examples\ToxID\Exact_Mass\Exact_Mass_Test.RAWConfig File Name: C:\Xcalibur\examples\ToxID\Exact_Mass\ConfigFile_Exact_mass.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 3/24/2008 4:46:43 PMScreening Conditions: Based on accurate mass scans. Exact mass window (ppm): 30, RT window(min): 0.50.

Page 1 of 6

PeakNumber

Compound Name Expectedm/z

Detectedm/z

Delta(mDa)

Delta(ppm)

ExpectedRT

Actual RT Intensity

1 Albuterol 240.15940 240.15939 -0.0 -0.0 2.58 2.58 199505

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2.121.990.880.78 2.84 4.163.06

NL: 2.03E5m/z=240.15580-240.16300 F:FTMS + c ESI Fullms[120.00-1000.00]

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Exactive

Use the ExactiveTest_Config.csv file in screening experiments that identify compounds based on accurate mass of parent ion with adducts, retention time, or both. You can view the processing configuration file in Figure 30, a sample summary report in Figure 31, and a sample long report in Figure 32.

Figure 30. Exactive processing configuration file (ExactiveTest_Config.csv)



Figure 31. Exactive sample summary report


Raw File Name: C:\Xcalibur\examples\ToxID\Exact...\AIF_14min_KB(BCDEF)_highabund_01.rawConfig File Name: C:\Xcalibur\examples\ToxID\Exactive\ToxID211_WithHCD.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 6/27/2008 4:08:24 PMScreening Conditions: Based on Exactive screening method. Accurate mass window (ppm): 0, Adducts: H+, NH4+, Na+, No specified retention time, Use most highest peak. TheDelta, Actual RT and intensity are reported based on the adduct labeled with "*".

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7.41 9.29 9.83 10.78 13.296.97

6.93 7.00

7.27 9.32 9.81 10.97 12.064.85

5.05 7.987.200.10 12.85

NL: 5.19E7m/z= 376.14741 F:FTMS + p ESI Full ms[120.00-1800.00]





Page 1 of 1

# Comp.Index

Compound Name Formula Detectedm/z

Delta(ppm)

ExpectedRT

ActualRT

Intensity Adducts FragmentsH+ NH4+ Na+ 1 2 3

1 1 Haloperidole C21H23ClFNO2 376.14722 -0.5 6.70 6.68 51847909 Y* N N Y Y Y2 2 Buspirone C21H30N5O2 - - 5.60 - - N N N Y Y Y3 3 Nefazodone C25H32ClN5O2 470.23154 -0.4 7.20 7.17 45992184 Y* N Y Y Y Y4 4 Verapamil C27H38N2O4 455.29056 0.3 7.00 6.97 72759571 Y* Y Y Y Y Y5 5 Urapidil C20H29N5O3 388.23428 -0.1 4.90 4.85 7811124 Y* Y Y Y Y N



Figure 32. Exactive sample long report


Raw File Name: C:\Xcalibur\examples\ToxID\Exact...\AIF_14min_KB(BCDEF)_highabund_01.rawConfig File Name: C:\Xcalibur\examples\ToxID\Exactive\ToxID211_WithHCD.csvSample Name: Laboratory: Your Lab NameAcquistion Start Time: 6/27/2008 4:08:24 PMScreening Conditions: Based on Exactive screening method. Accurate mass window (ppm): 0, Adducts: H+, NH4+, Na+, No specified retention time, Use most highest peak. TheDelta, Actual RT and intensity are reported based on the adduct labeled with "*".

Page 1 of 5

Peak Number:Index in Config:

Compound Name:Formula:Polarity:

Compound Info:Expected m/z with Adduct:

Detected m/z:Delta (ppm):

Expected RT (min):Actual RT (min):

Adduct H+:Adduct NH4+:

Adduct Na+:Fragment 1:Fragment 2:Fragment 3:

11HaloperidoleC21H23ClFNO2+Parent376.14741376.14722-0.56.706.6851847909*Not foundNot foundExpected m/z: 165.07172, Detected: 165.07112Expected m/z: 358.13867, Detected: 358.13577Expected m/z: 123.02436, Detected: 123.02491

Peak 1

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9.327.00 9.84 10.884.42 12.451.23

3.85 3.883.913.81

6.66

6.979.346.195.28 9.861.06 12.113.462.45 13.86

6.646.67

6.60

7.818.03 9.34 10.79

6.66

9.336.976.16 9.964.58 10.95 12.742.730.93

NL: 5.19E7m/z= 376.14741 F:FTMS + p ESI Fullms[120.00-1800.00]

NL: 5.64E3m/z= 393.17396 F:FTMS + p ESI Fullms[120.00-1800.00]

NL: 0m/z= 398.12936 F:FTMS + p ESI Fullms[120.00-1800.00]

NL: 1.56E7m/z= 165.07172 F:FTMS + p ESI [email protected] [120.00-1800.00]



Adduct 1 H+

Adduct 2 NH4+

Adduct 3 Na+

Fragment 1

Fragment 2

Fragment 3


4

Running the ToxID Application

This chapter provides information on how to use the ToxID compound identification system. You can run this application:

• Using the ToxID user interface to process a single data file or sequence

• Performing post-acquisition processing on an entire sequence or on selected data files using an Xcalibur processing method

• During sample sequence acquisition as the acquisition of each sample is completed

A log file keeps records of any problems the ToxID application encounters during the analysis or when creating the reports.

Running the Application Using the ToxID User InterfaceUse this method to process a single data file or sequence.

To run the application on a single data file or sequence

1. Double-click the ToxID shortcut icon on your Windows desktop.

The ToxID dialog box opens (Figure 33).

Contents

• Running the Application Using the ToxID User Interface

• Running the Application Using an Xcalibur Processing Method

• Running the Application Directly After Data Acquisition for Each Sample in the Sequence

• Troubleshooting Processing Problems

4 Running the ToxID ApplicationRunning the Application Using the ToxID User Interface


Figure 33. ToxID dialog box

2. Click next to the Source File box and browse to locate the raw data file or sequence file you want to use.

3. Click next to the Processing Configuration File box and browse to locate the configuration file you want to use.

For more information about the configuration file, see “Processing Configuration File Template” on page 11.

4. Select the type of report you want to produce:

• Summary Report—Lists all detected compounds and provides related chromatograms.

• Long Report—Provides one page of detailed information for each compound.

For examples of both the summary and long reports produced by each configuration file, see “Example Processing Configuration Files and Reports” on page 15.

5. Select what type of report output you want:

• Print—Prints the report on your default printer and also saves the report as a .pdf file.

• View—Displays the report. You can view and print the report later because it is always saved as a .pdf file in the raw file folder.

Note You can choose both Summary Report and Long Report. Both are saved as .pdf files but are not displayed or printed unless you select those options as well.

Note The ToxID application still saves the reports, even if you do not select the Print or View option.



6. Click Set up. The Setup dialog box opens.

Figure 34. Setup dialog box

7. In the Setup dialog box, select a compound screening method from the Screening Method list. Select from the following:

• Auto Detect

• Based on Full MS2 scans

• Based on SRM and MS2 scans

• Based on MS2 and MS3 scans

• Based on MS3 scans

• Based on accurate mass scans



• Based on SRM scans

• Based on Exactive screening method

8. Type the information for the Company Name, Laboratory Name, Company Logo (by clicking ), m/z Window (mu), RT Window (min), MS2 Search Library, and MS3 Search Library. This information is defined in Table 4.

Table 4. Experiment configuration parameters

9. Select the Use Full MS Scan to Confirm check box if you want to confirm library search results with parent ion peak detection in the full scan event. If a peak is not detected in the full scan event, then the compound is not reported as a hit.

10. In the Semi Quantitative area, do the following:

a. Select the Report Semi-Quantitative Result check box if you wish to have the semi-quantitative result reported.

b. Type the measurement units. Be aware that the measurement units used here are for labeling purposes only.

Note Auto Detect is the screening method by default. With it, the ToxID application can identify the screening experiment implemented in the acquired data file.


Company Name Enter name of company if desired. Will be printed on the final report.

Laboratory Name Enter name of laboratory if desired. Will be printed on the final report.

Company Logo Location of the logo file for the company. For example: C:\logo.jpg The company logo will be printed on the final report if specified.

m/z Window (mu) The m/z window above and below the m/z value for the compounds.

RT Window (min) The retention time window above and below the retention time value.

MS2 Search Library The name of the library to search MS/MS spectra. If no library name is specified, the ToxID application searches all the libraries defined in Qual Browser. To specify multiple libraries, put the library name in the columns to the right. There is no limit on the number of libraries you can specify.

MS3 Search Library The name of the library to search MS3 spectra. If no library name is specified, no search takes place.



c. For the Calculation Based On, select the Scan Intensity or Peak Area option as necessary. (If you select Peak Area, the Use Full MS Scan to Confirm check box is automatically selected.) The Scan Intensity option measures the intensity of the MS/MS peak specified without performing any backgrounds subtraction. The Peak Area option measures the peak area of the reconstructed full scan chromatogram peak of the parent ion.

11. In the Ion Ratio Calculation Method (In SRM Experiment) area, do the following:

a. Select the Use Scan at Peak Apex or Use Average Scan option.

• Use Scan at Peak Apex—ion ratio is calculated based on peak apex scan spectrum.

• Use Average Scan—ion ratio is calculated based on average scan spectrum over the range of the peak’s half height.

b. Type the percentage in the Ion Ratio Window (%).

12. In the Accurate Mass Experiment area, set the parameter for the Exact Mass Window (ppm).

13. In the Exactive area, do the following:

a. Type a value for Adduct 1, Adduct 2, and Adduct 3, respectively. For example, H+, NH+, and Na+. The values entered identify the adducts listed in and applied throughout the configuration file. Adducts are polarity sensitive. For negative ionization, you can enter negative adducts.

b. Select the No Specified Retention Time check box if you do not wish to have retention time specified. Selecting this option allows the ToxID application to search the entire raw file for the peak specified.

c. If you have selected the check box in Step b, then select the First Peak or Highest Peak option. This is necessary in case more than one m/z match is found in the raw file and guides the ToxID application in selecting which peak to use for processing.

d. Select the Report All Compounds Listed in Configuration File check box if you want to have reports for all compounds in the configuration file whether or not matches are found for them. If you do not select this option, the default is to report on only those compounds where matches are found in the raw file. This option applies to the Exactive experiment only.

Note The selections in the Semi Quantitative area are only applicable to MS/MS experiments.

Note Make sure the relative intensity in the configuration file uses the selected scan method.

4 Running the ToxID ApplicationRunning the Application Using an Xcalibur Processing Method


14. Click OK to return to the ToxID dialog box.

15. Click Process in the ToxID dialog box. Clicking this button processes the source and configuration files that you selected.

Running the Application Using an Xcalibur Processing MethodTo process an entire sequence or selected rows in the sequence, create a processing method and then reprocess the sequence or selected data files using the ToxID application.

To create a processing method

1. Open the Xcalibur Home Page and choose GoTo > Processing Setup.

The Processing Setup dialog box opens with the Identification page displayed by default (Figure 35).

Figure 35. Processing Setup dialog box

2. If the left View bar is not visible, select View > View Bar.

3. In the left View bar, click .

The Programs view opens (Figure 36).



Figure 36. Programs view

4. Double-click the first available text box in the Enable column and select the check box that appears in that column.

5. In the Sample Type area (see Figure 36), select the sample type you want (only one type can be selected):

• Std: Standard

• QC: Quality Control

• Unk: Unknown

• Other

6. In the Action column, select Run Program.

7. Double-click the Program or Macro Name column.

The Browse for Program dialog box opens (Figure 37).

Sample types



Figure 37. Browse for Program dialog box

8. Select the location of the ToxID program.

The ToxID application is installed in the \Xcalibur\system\programs folder. Use the full path name for this application and select the ToxID.exe file.

9. Click Open.

The Browse for Program dialog box closes and the ToxID path is shown in the Program or Macro Name column.

10. In the Sync column, click to select the check box that appears in that column.

11. In the Parameters column, type “%R”.

For an example of how the run information appears, see Figure 38.



Figure 38. Sample list with ToxID defined as program to run

12. Click OK.

Whenever you run a sequence, the ToxID application automatically processes the sequence.

13. Save the file.

To reprocess the sequence or selected data files

1. Go to the Sequence View on the Home Page.

2. Go to the . sld file directory by choosing File > Open > Xcalibur > examples > methods.

3. Make sure to select the .sld file for the created processing method.

4. In the sequence, select the range of row numbers (samples) you want to reprocess.

5. Choose Actions > Batch Reprocess.

The Batch Reprocess Setup dialog box opens (Figure 39).

4 Running the ToxID ApplicationRunning the Application Directly After Data Acquisition for Each Sample in the Sequence


Figure 39. Batch Reprocess Setup dialog box

6. Select the Programs check box.

7. Click OK.

The ToxID application processes the samples you selected.

Running the Application Directly After Data Acquisition for Each Sample in the Sequence

Use this method to process a single data file with the ToxID application after completing data acquisition for each sample in the sequence. The data acquisition system, Xcalibur, automatically invokes the ToxID application to process the raw file.

To run the ToxID application after completing data acquisition for each sample in a sequence

1. Open the Xcalibur Home Page and choose Sequence Setup.

2. In the Sequence Setup window, choose Actions > Run Sequence.

The Run Sequence dialog box opens (Figure 40).

4 Running the ToxID ApplicationTroubleshooting Processing Problems


Figure 40. Run Sequence dialog box

3. In the Programs area, for the Post Acquisition box, click Browse and select the full path name for the ToxID program.

4. Append “%R” to the ToxID file name.

5. Under Run Synchronously, select the Post Acquisition check box.

6. Click OK.

The ToxID application processes each sample in the sequence after completing the data acquisition.

Troubleshooting Processing ProblemsThe ToxID application provides a log file to help you resolve any problems that arise if the data file appears not to have been processed correctly.

Note Never add, remove, or rearrange columns or rows above row 4 in a processing configuration file. The ToxID application cannot process the analysis if any information is missing or moved.

4 Running the ToxID ApplicationTroubleshooting Processing Problems


To troubleshoot a problem, first confirm the following:

• The processing configuration file contains the information necessary to process the data file you selected. For information about what a processing configuration file contains, see “Processing Configuration File Template” on page 11.

• If you used one of the example processing configuration files, you chose the appropriate file for the data file you are processing. For more information, see “Example Processing Configuration Files and Reports” on page 15.

• You did not add, remove, or rearrange columns or rows in the processing configuration file. For the correct structure for a processing configuration file, see “Processing Configuration File Template” on page 11.

To view the log file

Go to the folder that contains the raw data file and open the ToxID.log file.

Thermo Scientific ToxId User Guide 51

I

Aadding libraries 5adducts 14archiving libraries 9

Ccompound screening methods 41configuration files

editing 11parameters 13settings 15templates 11

Contacting Us v

Ddata file, processing 39

Eexact mass 33example, processing configuration file 15Expected RT 13experiments, types of 1

Ffragments 14

Hhardware requirements 2

Iinstalling ToxID 2

Llibraries

adding 5

adding to the search list 7archiving 9sample file 5

license agreement 2log file, viewing 50

MMS/MS and MS3 20MS2 Product Ion 13MS2 Search Library 42MS3 Search Library 42

Oopening a raw file 7

Pparameters, compound-specific 13Parent Ion m/z 13processing a sequence 39processing configuration file, setting parameters for 15processing method, creating 44

Rraw file, opening 7reports

Long 16Summary 16

reprocessing a sequence 47reprocessing, setting up for batch 47retention time 20RSI 14running Tox ID 39

Ssearch list, adding to a library 7search order, multiple libraries 9setting up batch reprocessing 47

Index

52 ToxId User Guide Thermo Scientific

Index: T

software requirements 2spectra

MS/MS and MS3 20spectrum

MS/MS 16MS3 24Qual Browser 8

SRM fragment ions 14SRM transitions 30

Ttemplate, configuration file 11ToxID

files and their folder locations 3hardware and software requirements 2installing files and components 2running 39

troubleshooting processing problems 49

ToxID 2.1.2 User Guide Version B - Thermo Fisher

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