Thermo Fisher Scientific ToxID 2.1.2 User guide

ToxID

User Guide

XCALI-97245 Revision B February 2010

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

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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

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Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v

Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Chapter 1 Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Hardware and Software Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Installing ToxID Files and Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Running ToxID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Chapter 2 Working with Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Adding a Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Adding Libraries to the Search List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Archiving a Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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

Processing Configuration File Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Processing Configuration File Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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

Example Processing Configuration Files and Reports . . . . . . . . . . . . . . . . . . . . 15

MS/MS Spectrum and Retention Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

MS/MS and MS3 Spectra and Retention Time . . . . . . . . . . . . . . . . . . . . . . . 20

MS3 Spectrum and Retention Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

MS/MS Spectrum Collected in a QED Experiment and Retention

Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

SRM Transitions and Ion Ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Exact Mass. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Exactive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Chapter 4 Running the ToxID Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Running the Application Using the ToxID User Interface. . . . . . . . . . . . . . . . . 39

Running the Application Using an Xcalibur Processing Method . . . . . . . . . . . . 44

Running the Application Directly After Data Acquisition for Each Sample

in the Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Troubleshooting 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 Us

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

YTo contact Technical Support

Find software updates and utilities to download at mssupport.thermo.com.

YTo contact Customer Service for ordering information

Phone 800-532-4752

Fax 561-688-8736

E-mail us.techsupport.analyze@thermofisher.com

Knowledge base www.thermokb.com

Phone 800-532-4752

Fax 561-688-8731

E-mail us.customer-support.analyze@thermofisher.com

Web site www.thermo.com/ms

Preface

Contacting Us

vi ToxID User Guide Thermo Scientific

YTo copy manuals from the Internet

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

window.

YTo suggest changes to documentation or to Help

• Fill out a reader survey online at

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

• Send an e-mail message to the Technical Publications Editor at

techpubs-lcms@thermofisher.com.

Thermo Scientific ToxID User Guide 1

1

Getting Started

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

data files.

Overview

The ToxID application identifies compounds and provides reports for the following

experiments:

• Ion trap mass spectrometer experiments

–MS/MS spectra

– MS/MS and MS3 spectra

–MS

3 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 Started

Hardware and Software Requirements

2ToxID User Guide Thermo Scientific

Hardware and Software Requirements

In order to run ToxID software, your computer must conform to the following minimum

requirements:

Installing ToxID Files and Components

YTo 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 ins t a l l t h e To x I D 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 Started

Running ToxID

Thermo Scientific ToxID User Guide 3

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

Running ToxID

In 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 To x I D Us er Gu i d e \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

Thermo Scientific ToxID User Guide 5

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 Library

YTo 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 Libraries

Adding a Library

6ToxID User Guide Thermo Scientific

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 Libraries

Adding Libraries to the Search List

Thermo Scientific ToxID User Guide 7

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 List

YTo 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 Libraries

Adding Libraries to the Search List

8ToxID User Guide Thermo Scientific

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 Libraries

Archiving a Library

Thermo Scientific ToxID User Guide 9

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 To p, 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 Library

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

YTo archive a library

1. Open the Xcalibur Home Page.

2. Choose To ol s > L i b r a r y Manager.

The Library Manager dialog box opens (Figure 7).

2 Working with Libraries

Archiving a Library

10 ToxID User Guide Thermo Scientific

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.

Thermo Scientific ToxID User Guide 11

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 Template

Two 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:

\Xcalibur\examples\ToxID

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 Files

Processing Configuration File Template

12 ToxID User Guide Thermo Scientific

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

3 Setting Up ToxID Processing Configuration Files

Processing Configuration File Template

Thermo Scientific ToxID User Guide 13

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.

3 Setting Up ToxID Processing Configuration Files

Processing Configuration File Template

14 ToxID User Guide Thermo Scientific

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)

Parameter Description

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Thermo Fisher Scientific ToxID 2.1.2 User guide

Thermo Fisher Scientific ToxID 2.1.2 User guide

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