MetQuest 1.2 User Guide Version A - Thermo Fisher...

Thermo

MetQuestVersion 1.2User Guide

Xcali-97468 Revision A May 2012

http://www.surveymonkey.com/s/PQM6P62

© 2012 Thermo Fisher Scientific Inc. All rights reserved.

MetQuest, Exactive, Q Exactive, Exactive Plus, Transcend, Aria, LTQ Orbitrap, Foundation, and DCMSLink are trademarks; and Accela, LTQ, UltiMate, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the United States.

The following are registered trademarks in the United States and other countries: Microsoft, Windows, and Excel are registered trademarks of Microsoft Corporation. Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated. Agilent is a registered trademark of Agilent Technologies Inc. PAL is a trademark of CTC Analytics AG.

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: MetQuest, version 1.0 December 2011; version 1.1 April 2012; version 1.2 May 2012

Software version: Thermo Foundation 2.0 SP1; Thermo LC Devices 2.5 SP2 and later

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

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiRelated Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viiiSystem Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ixGetting a License. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Getting a New License Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xInstalling a New License Code. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi

Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiContacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Chapter 1 Working with the MetQuest Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Using the Different Workflow Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2How the Application Finds Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Chapter 2 Getting Started with MetQuest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Defining Group and Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Importing a Sample List File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Typing Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Specifying Configuration Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Submitting an Experiment and Reviewing Results. . . . . . . . . . . . . . . . . . . . . . . 11

Chapter 3 Defining Sample Information for Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Defining Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Defining Group Information Using the Application . . . . . . . . . . . . . . . . . . . 18Managing Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Defining Experiment Information for Groups . . . . . . . . . . . . . . . . . . . . . . 20Identifying Elements in Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Defining Sample Information Manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Importing a File Containing Group and Sample Information . . . . . . . . . . . . 30

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Configuring Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Setting Report Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Specifying Configuration Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Specifying Instrument Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Defining Acquisition Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Specifying the Mass Tolerance Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Submitting and Reviewing an Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Working with the Sample Table Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Chapter 4 Working with Processing Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41Creating a Processing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Setting Parameters for Metabolite Searching . . . . . . . . . . . . . . . . . . . . . . . . . 42Defining Control Comparison Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Defining the Retention Time Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Specifying Expected Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Specifying Internal Standard and Elemental Composition Settings . . . . . . . . 48Defining an Internal Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Setting the Sum Area Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Defining Elemental Composition Values . . . . . . . . . . . . . . . . . . . . . . . . . . 51Changing Advanced Spectral Fit Values . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Defining Peak Acceptance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Saving Processing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Changing a Processing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Chapter 5 Processing Previously Acquired Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59Processing Data Acquired Using the MetQuest Application . . . . . . . . . . . . . . . 60Processing Data Acquired and Processed Using the MetQuest Application . . . . 61

Processing Complete Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Processing Partial Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Processing Data Acquired Using an .sld File . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Processing Data Using Raw Files Acquired in Xcalibur . . . . . . . . . . . . . . . . . . . 67

Chapter 6 Reviewing Acquisition and Processing Status . . . . . . . . . . . . . . . . . . . . . . . . . . .69Viewing Acquisition Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Viewing Processing Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Viewing Information in the Processing Display Area . . . . . . . . . . . . . . . . . . . 74Stopping Processing Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76Viewing Information in the Processing Log . . . . . . . . . . . . . . . . . . . . . . . . . . 77Understanding Error Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

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Chapter 7 Reviewing Results from the Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79Using Data Review to Review and Modify Test Results . . . . . . . . . . . . . . . . . . 80

Viewing Component Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Viewing the Component Review Table . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Viewing the Time Course Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Viewing the Extracted Ion Chromatogram (XIC). . . . . . . . . . . . . . . . . . . . 89Viewing the MS/MS or AIF Scan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Viewing Elemental Composition Information . . . . . . . . . . . . . . . . . . . . . . . . 92Viewing Elemental Composition Results . . . . . . . . . . . . . . . . . . . . . . . . . . 93Viewing the Full MS Scan Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Working with Component Results in Data Review . . . . . . . . . . . . . . . . . . . . 95Filtering Components in Data Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Sorting Elements in Data Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Changing Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Excluding a Single Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Excluding Time Points from Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Excluding Time Points from All Groups in an Experiment . . . . . . . . . . . 102Excluding a Time Course from Groups . . . . . . . . . . . . . . . . . . . . . . . . . . 103Excluding Replicate Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Manually Integrating Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Saving Changes in Data Review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Defining Report Options for Data Review . . . . . . . . . . . . . . . . . . . . . . . . . 108

Viewing Experiment Log Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Viewing Experiment Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Viewing Summary Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Viewing Detailed Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Working with an Experiment CSV File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Printing Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Chapter 8 Configuring Instruments for Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Configuring Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Adding Hardware Devices to Your System Configuration . . . . . . . . . . . . . . 124Removing Hardware Devices from Your System Configuration . . . . . . . . . 126

Working with Instrument Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Creating Instrument Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Changing an Instrument Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129Importing an Existing Instrument Method . . . . . . . . . . . . . . . . . . . . . . . . . 129

Chapter 9 Working with Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131Creating Files with Group and Sample Information . . . . . . . . . . . . . . . . . . . . 132Working with a Metabolite Modification List for Processing. . . . . . . . . . . . . . 136

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139

Thermo Scientific MetQuest User Guide v

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Preface

This guide describes how to use the Thermo MetQuest™ metabolic stability screening software to process high-resolution accurate mass data gathered from these Thermo Scientific instruments: the Exactive™, Exactive Plus™, the Q Exactive™, and the Orbitrap™ family of instruments for information on parent drug metabolism. When processing full-scan, MS/MS-scan, and AIF (All Ions Fragmentation) data, the MetQuest application provides relative quantitative and qualitative information about a parent drug and its metabolites. The application supports customers in the pharmaceutical industry who desire high-throughput screening of a large number of potential drug candidates.

To suggest changes to documentation or to Help

Complete a brief survey about this document by clicking the link below.Thank you in advance for your help.

Contents

• Related Documentation

• System Requirements

• Getting a License

• Special Notices

• Contacting Us

Thermo Scientific MetQuest User Guide vii


PrefaceRelated Documentation

viii MetQuest User Guide Thermo Scientific

Related DocumentationIn addition to the MetQuest User Guide, the MetQuest application includes the MetQuest Quick Start Guide, available as a PDF file, system Help, and a communicator bar that supplies product information specific to where you are in the product.

To view Communicator bar information

In the MetQuest window, place the cursor in any field and the Communicator bar displays information, including valid values and the default for the field.

The Communicator bar also displays information about an entire view. For more information, open Help by clicking .

To open Help

• From the MetQuest window, choose Help > MetQuest Help.

• To get help for a specific window or dialog box, click or press F1 for information about setting parameters.

To view product manuals

Go to Start > Programs > Thermo MetQuest > Manuals.

Communicator bar

PrefaceSystem Requirements

Thermo Scientific MetQuest User Guide ix

System RequirementsYour system must meet these minimum requirements. In order to install and license the MetQuest application, you must be a system administrator on the computer.

IMPORTANT The software is designed for use with the US-English version of the Microsoft™ Windows™ 7 or XP operating system. Thermo Fisher Scientific and its information partners provide no support for use with any other version of the Microsoft Windows operating system.

System Requirements

Data system computer

• 2 GHz processor with 2 GB RAM, 32-bit system or 64-bit system

• DVD/CD-ROM drive

• Video card and monitor capable of 1280×1024 resolution (XGA)

• 75 GB on the computer, with 30 GB free on the C: drive

Instrument (supported or required)

• Mass spectrometers: Exactive, the Exactive Series 2.1 (Exactive Plus and Q Exactive), LTQ™ Orbitrap™

• LC Devices supported for acquisition:

Pumps: Accela™ pump, Accela 600 Pump, Accela 1250 Pump, Agilent™ 1200 Bin Pump

Autosamplers: Accela AS, Open Accela AS, CTC Thermo PAL™ AS, Agilent 1200 AS

PDAs: Accela PDA, Agilent 1200 DAD

Combined: Transcend™ TXL-1 (Aria™ MX V.), UltiMate™ 3000 (DCMSLink™)

Software • Operating system:

Microsoft Windows XP Professional with Service Pack 3

–or–

Windows 7 Professional with Service Pack 1

• Microsoft .NET 3.5 SP1 and Microsoft .NET Framework 4 Extended

• Thermo Foundation™ 2.0 SP1

• Microsoft Office 2010

• Adobe™ Acrobat™ 10.1

• (Optional, but recommended) Thermo Xcalibur™ data system 2.2 SP1

• Hardware driver software:

Exactive driver version 1.1 SP4 or later

Exactive Series 2.1 (for Exactive Plus and Q Exactive)

For the LTQ hybrid (LTQ-Orbitrap/FT) family, install LTQ version 2.7

Thermo LC Devices version 2.5 SP2; Waters Acquity version 2.6

PrefaceGetting a License

Getting a LicenseThe MetQuest metabolic stability screening software requires a license. Follow the installation instructions on the DVD to install the software. Then, use these procedures to get a new license code from Thermo Fisher Scientific (see “Getting a New License Code” on page x) and install the new license code when you receive it (see “Installing a New License Code” on page xi).

Getting a New License Code

To get a license code, you must be a system administrator on the computer. Follow the steps to generate an inactive license and send the licensing information to the licensing group at Thermo Fisher Scientific to get an active license.

To get a license code

1. To start the MetQuest application, click or choose Start > All Programs > Thermo MetQuest > MetQuest.

Each time you start the application during the trial period, it tells you how many days you have left on your trial period and gives you the option to activate the license.

2. Choose Activate Now to apply for a license or choose Activate Later to wait.

If you have no days left in your trial period and you click Activate Later, the application closes.

After choosing Activate Later, to get a license code, choose Help > About MetQuest to display the About MetQuest dialog box. Click Activate Now.

x MetQuest User Guide Thermo Scientific

PrefaceSpecial Notices

Thermo Scientific MetQuest User Guide xi

3. Fill in all fields.

The application generates the license request based on the information in the fields.

4. Locate the bar code on the back of the MetQuest jewel case. Type the bar code in the Feature Info area.

5. Select Copy to copy the license key to the Clipboard.

6. Open an e-mail message and paste the information from the Clipboard into the body of the e-mail. Send this e-mail to [email protected].

When Thermo Fisher Scientific Customer Support sends you a new license code, see “Installing a New License Code.”

Installing a New License Code

After you receive your new license code, start the application to install it.

To install the license code

1. Click on your computer desktop.

The application displays a reminder notice if you do not have a license.

2. Click Activate Now to open the MetQuest License dialog box.

To access the MetQuest License dialog box from within the application if your trial period is still valid, choose Help > About MetQuest to display the About MetQuest dialog box. Click Activate Now.

3. Paste the new activation code in the License box.

4. Click Set to activate your MetQuest application.

5. Close the MetQuest License dialog box.

Special NoticesMake sure you follow the precautionary statements presented in this guide. The safety and other special notices appear in boxes.

Special notices include the following:

IMPORTANT Highlights information necessary to prevent damage to software, loss of data, or invalid test results; or might contain information that is critical for optimal performance of the system.

Note Highlights information of general interest.

Tip Highlights helpful information that can make a task easier.

mailto:[email protected]


PrefaceContacting Us

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

To contact Technical Support

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

To contact Customer Service for ordering information

To get local contact information for sales or service

Go to www.thermoscientific.com/wps/portal/ts/contactus.

To copy manuals from the Internet

Go to mssupport.thermo.com, agree to the Terms and Conditions, and then click Customer Manuals in the left margin of the window.

To suggest changes to documentation or to Help

• Fill out a reader survey online at www.surveymonkey.com/s/PQM6P62.

• Send an e-mail message to the Technical Publications Editor at [email protected].

Phone 800-532-4752

Fax 561-688-8736

E-mail [email protected]

Knowledge base www.thermokb.com

Phone 800-532-4752

Fax 561-688-8731

E-mail [email protected]

Web site www.thermo.com/ms

xii MetQuest User Guide Thermo Scientific


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1

Working with the MetQuest Application

The MetQuest metabolic stability screening software is a data acquisition, processing, and reporting system for users who have some experience with liquid chromatography mass spectrometry (LC/MS).

This chapter provides a basic overview of the MetQuest application.

Contents

• Using the Different Workflow Areas

• How the Application Finds Peaks

Thermo Scientific MetQuest User Guide 1

1 Working with the MetQuest ApplicationUsing the Different Workflow Areas

Using the Different Workflow AreasThis section describes how to use the main workflows of the MetQuest application. (The relative Quan/Qual experiment can use the following workflows: Acquisition Only, Processing Only, and Acquisition and Processing.) Use the Relative Quan/Qual experiment to monitor the metabolic stability of drug candidates and identify putative metabolites. Relative Quan/Qual is useful for rapidly assessing the pharmacokinetic properties of parent drugs.

To open the MetQuest application

1. Double-click to open the MetQuest application.

2. To provide more room in the application window for your data, click to hide the Thermo title bar.

3. From the Workflows view, select a workflow type from the list. Select from Acquisition Only, Processing Only, or Acquisition and Processing.

Table 1 lists the functions for each workflow.

2 MetQuest User Guide Thermo Scientific

1 Working with the MetQuest ApplicationUsing the Different Workflow Areas

Table 1. Workflow functions

Workflow Description

Opens the Acquisition Only workflow area where you can do the following:

• Instrument Setup: Create, modify, and save instrument methods. See “Working with Instrument Methods” on page 127.

• Experiment Setup: Create, modify, and save sample, group, and configuration information for acquisition. See “Defining Sample Information” on page 16.

• Real Time Display: View and modify real-time acquisition. See “Viewing Acquisition Status” on page 69.

Opens the Processing Only workflow area where you can do the following:

• Processing: Create, modify, and save processing methods. See “Creating a Processing Method” on page 42. For previously acquired experiments, see “Processing Previously Acquired Data” on page 59.

• Experiment Setup: Create, modify, and save sample and configuration information for processing. To do processing only, you must have previously acquired data files. See “Defining Sample Information” on page 16.

• Processing Status Display: View status, including processing errors and an error log, while the MetQuest application processes acquired data. See “Viewing Processing Information” on page 73.

• Reports: View reports from processed experiments. See “Viewing Experiment Reports” on page 110.

Opens the Acquisition and Processing workflow area where you can do the following:

• Instrument Setup: Create, modify, and save instrument methods. See “Working with Instrument Methods” on page 127.

• Processing: Create, modify, and save processing methods. See “Creating a Processing Method” on page 42.

• Experiment Setup: Create, modify, and save sample, group, experiment, and configuration information for acquisition.

• Real Time Display: View and modify real-time acquisition. See “Viewing Acquisition Status” on page 69.

• Processing Status Display: View status, including processing errors and an error log, while the MetQuest application processes acquired data. See “Viewing Processing Information” on page 73.

• Reports: View reports from processed experiments. See “Viewing Experiment Reports” on page 110.

Click one of the tabs at the top of the display to go from one area to another.


1 Working with the MetQuest ApplicationHow the Application Finds Peaks

How the Application Finds Peaks The MetQuest application processes relative quan/qual data for expected metabolites of a parent drug in a targeted workflow. Each experiment contains groups with a single parent and a processing method that defines a single internal standard (if defined). The application does the following:

• Creates a list of theoretical m/z values based on the expected metabolites list defined in Processing Setup > Metabolite Options > Expected Metabolites. The expected metabolite list contains names, formula changes, and mass shifts for the metabolites you want to look for. To use a template to create the expected metabolites list, see Working with a Metabolite Modification List for Processing.

• Using that list, the application proceeds to detect peaks for each theoretical m/z value and to model each peak that is found to determine the area. The application also attempts to properly split closely eluting isobaric compounds, modeling each peak separately.

• For the detected peaks, the application displays the integrated spectral peak, retention time, m/z value, peak area, elemental compositions, full-scan MS data, and appropriate MS/MS scan data in Data Review.


2

Getting Started with MetQuest

Use the MetQuest application to acquire full-scan and AIF scan high-resolution, accurate mass data to automatically generate reports. To acquire and process sample data using the procedures in this chapter, you must have already created a processing method and an instrument method.

Contents

• Defining Group and Sample Information

• Specifying Configuration Information

• Submitting an Experiment and Reviewing Results


2 Getting Started with MetQuestDefining Group and Sample Information


Defining Group and Sample InformationDefine group and sample information in the Sample Setup view.

To open the Sample Setup view

1. From the Workflows view, select a workflow type (select Acquisition and Processing to acquire and process data from samples).

2. Click the Experiment Setup tab to open the Experiment Setup views.

If you already have an experiment open, the application prompts you to save it or discard changes. The Experiment Setup tab has two sections: Sample Setup and Configuration Setup. The Sample Setup area has three main areas: Groups, Group Parameters, and Samples. Add information to all three sections to run an Acquisition and Processing workflow.

To define group and sample information, choose one of these options:

• Importing a Sample List File: Import a .csv file with group and sample information. For information about creating a .csv file for importing, see “Creating Files with Group and Sample Information” on page 132.

• Typing Sample Information: Type experiment information into the Sample Setup view.


Importing a Sample List File

To import a file containing sample information for acquisition

1. In the Sample Setup view, click Import under the Submit button, select .CSV Sample List, and browse to a file containing group and sample information.

2. Select the file and click Open.

If you import a file containing sample information, the MetQuest application overwrites all currently displayed group and sample information.

3. Review the group and sample information to make sure it is accurate.

The application validates group and sample information at the time of import, before saving, and before submitting the experiment, providing warning errors at each step. You can continue to modify an experiment with errors after importing the file. The application prevents you from submitting or saving an experiment if there are errors.

4. Select a processing method for each group.

Typing Sample Information

To define information for a group of samples

1. In the Sample Setup view, select a group name in the Groups area so that you can define group information for that group. To add a group, click Add in the Groups area and type a name for the new group.

2. In the Group Parameters area, click Import to select a processing method that defines how the MetQuest application processes data.

Processing methods define the list of expected modifications and an internal standard. To create a processing method, see “Creating a Processing Method” on page 42.

3. Type a name for the drug in the Parent Name box. Use alphanumeric characters only.



4. To add the formula for the parent, use one of the methods below.

• If you have a .mol file for the parent, select the Mol File option and click Import. Select the file and click Open to display the mass, formula, and other values in the appropriate areas from the file. To view the molecular weight and mass structure, click View Mol File to open the display box. You can resize the display.

• If you do not have a .mol file, select the Formula option and type the formula in the Formula box.

Once you add the formula, the MetQuest application adds all elements in the formula to the Elements in Use dialog box. The application calculates and displays the neutral molecular weight and other values, such as the protonated molecular weight, from the formula. See “Identifying Elements in Use” on page 23 for more information. To view the elements in use, click View Elements.

5. Type other information in the Group Parameters area as needed.

The application displays Sample ID, Replicate Number, Species, Matrix, Animal #, Route, and Dose Amount values in the summary and detailed reports.

6. Type the following required data into the Samples list before starting to acquire sample data. Provide at least one sample to begin acquiring and processing data.



To use copy or fill down, see “To copy the contents of a row” on page 38 or “To increment the contents of a cell in successive rows using Fill Down” on page 39.

a. In the Sample ID column, type a sample ID number.

b. In the File Name column, type a unique file name for results. If the experiment has replicate samples, provide a different replicate number for each replicate for a time point. Each replicate set (each row) can have its own control at time T0. The first replicate set must start with replicate number 1. Even if you delete a replicate set, replicate numbers must be consecutive. Do not define a replicate number or a time point for a blank sample.

c. In the Position column, type the sample’s vial or plate position number for the autosampler.

d. In the Inj. Volume column, specify the injection volume of sample to be injected in microliters (μL).

e. Define the time point. Do not define a time point for a blank sample.

f. Define the sample type.


2 Getting Started with MetQuestSpecifying Configuration Information


Specifying Configuration Information To specify configuration information

1. To choose the instrument method parameters, click the Experiment Setup tab and then click Configuration Setup.

2. Select report options.

3. In the Report Options area, select the Perform Data Review check box to view data results and make changes before creating reports.

4. In the Configuration Options area, select an instrument method that defines instrument acquisition parameters for the method.

5. To use a startup or shutdown method, select the appropriate check box and click Import to select and open a method file.

For the Accela, Agilent 1200, and Accela PDA autosamplers, the tray type is displayed in the Tray Name field in the Acquisition Parameters area. For autosamplers that support more than one configured tray type (Open Accela and CTC Thermo PAL autosamplers, and the UltiMate 3000 (DCMSLink) combined), the Tray Name field displays As Configured.

6. To set instrument status when processing is complete, select one of these options: On, Standby, or Off.

2 Getting Started with MetQuestSubmitting an Experiment and Reviewing Results

7. Click Instruments in Use.

8. Select instruments to use for acquisition and select a startup instrument.

Submitting an Experiment and Reviewing Results To submit an experiment and review results

1. Click Submit in the Sample Setup view to process the samples.

• To submit or save the experiment, the application prompts you to save the sequence under a unique name.

If vial or plate positions or injection volumes are incorrect, the application communicator bar displays a message specifying a range of valid values.

• To view real-time acquisition and processing status, click the Real Time Display tab. Click the Acquisition tab to see real-time acquisition data for samples to evaluate acquisition status.



To view status and log information about processing, click the Processing tab.

If you selected the Perform Data Review check box in the Report Options area, when processing is complete, the application prompts you to click the Data Review tab where you can filter or sort the data. For more information about manipulating experiment results, see “Using Data Review to Review and Modify Test Results” on page 80. If you did not select the Perform Data Review check box, the application produces reports that you can access in the Reports view.



2. To view a group report, click the Reports tab, select a group, and select from two report types, either summary or detailed.

This summary report displays an appearance/disappearance plot and the results of each time point in the table. For more information about reports, see “Viewing Experiment Reports” on page 110.


3

Defining Sample Information for Acquisition

To create an experiment for acquisition, you can use either the Acquisition Only workflow or the Acquisition and Processing workflow. For both workflows, you must define information for the groups of samples and an instrument method. For the Acquisition and Processing workflow, you need to specify a processing method as well. For the Acquisition Only workflow, once you create an experiment, you can place the samples in the autosampler and start acquisition without defining a processing method.

The samples information includes group information and a samples list for each group. A samples list can contain one control sample, one or more blank samples, and other time course samples containing a maximum of 15 different time points. Your experiment can also contain up to seven replicate samples for each time point. To use replicate samples, see “Defining Sample Information Manually” on page 26 for specific instructions.

The instrument method specifies the parameters the instrument uses to acquire samples (for more information about creating and changing instrument methods, see “Working with Instrument Methods” on page 127). The processing method specifies how the application processes the data to display in Data Review or generate reports (for more information about processing methods, see “Creating a Processing Method” on page 42).

After you have created an experiment, you can save it for later use or submit it for acquisition and processing. For more information about processing previously acquired experiments using the Processing Only workflow, see “Processing Previously Acquired Data” on page 59.

Contents

• Defining Sample Information

• Configuring Experiments

• Submitting and Reviewing an Experiment

• Working with the Sample Table Columns


3 Defining Sample Information for AcquisitionDefining Sample Information


Defining Sample InformationThe MetQuest application provides two methods of inserting group and sample information. You can use the application to insert all group and sample information manually or you can import a file containing the information.

• Defining Group Information Using the Application

• Defining Sample Information Manually

• Importing a File Containing Group and Sample Information

When creating a time course experiment, follow these requirements:

• Maximum number of time points = 15

• Maximum number of replicates = 7

• All groups within an experiment must use the same set of time points. The groups do not need to use all of the same time points, but the time point values, where present, must be identical. One group must contain all time points used by the rest of the groups in the experiment. Groups with different time points must be in separate experiments.

• Controls must be at T0 and should contain the parent compound.

To define an experiment for a group of samples

1. From the Workflows view, select a workflow:

• To generate an acquisition and processing experiment for your samples so that processing begins immediately after the raw files have been acquired, select Acquisition and Processing.

• To acquire data for later processing, select Acquisition Only.

2. Either click New Experiment to start a fresh experiment or, to continue a previously saved experiment, select an experiment from the Select an Experiment list and click Continue Experiment.

A sample experiment appears when you select Acquisition and Processing. To explore that experiment, select the Example Experiment and click Continue Experiment.



The Experiment Setup view opens, showing the Sample Setup view.

3. To provide more room on the screen for your data, click in the upper right corner to hide the Thermo banner.

4. Select a group from the Groups area to define a time course experiment for one compound. To add a group, see “Managing Groups” on page 18.

5. Define group information manually or import a file containing group and sample information.

For details about creating a file containing sample information from a template, see “Creating Files with Group and Sample Information” on page 132.



Defining Group Information Using the Application

Use the following procedures to define group information or make changes in the Groups area.

• Managing Groups

• Defining Experiment Information for Groups

• Identifying Elements in Use (for the Acquisition and Processing and Processing Only workflows)

For the Acquisition Only workflow, the application grays out the Group Parameters area in the Sample Setup view. You can define group information later in the Processing Only workflow to process the acquired data. When you save or submit a group for acquisition, the MetQuest application locks the group information.

You can add new groups to an existing experiment for processing using the Processing Only workflow. If you change information for processed groups, you must reprocess those groups.

Managing Groups

To add a new group

1. Click Add in the Groups area on the left.

The application displays a new group in the Groups list. Type a unique name for the group (the new name for the group in this case is Nortriptyline).

If you name a group and there is another group with the same name in the sequence, the MetQuest application displays a warning message.


2. Type a new name in the Groups area list.

3. To add information for the new group, select the new group in the Groups area list and type information in the Group Parameters area.

For more information about adding group information, see “Defining Group Information Using the Application” on page 18.

When you save or submit the group for acquisition, the MetQuest application locks the group information. You can add new groups to an existing experiment for processing using the Processing Only workflow. If you change information for processed groups, you must reprocess those groups.

To change information for a group

1. Select the group name in the Groups area.

2. Add or change information in the Group Parameters area.

3. Click Save to save the new information.

To rename a group


2. Click to the right of the group name and click Rename or double-click the group name.

3. Type a new name for the group and press RETURN.

You can change the name of a group after you have acquired raw files.

To delete a group


2. Click to the right of the group name and click Delete.

You can delete a group after you have acquired raw files.



To find a group

1. Type the group name in the search box.

Any matches automatically appear in the group list.

2. To view all groups again, remove the search information in the search box.

Defining Experiment Information for Groups

The Group Parameters information only applies to the Acquisition and Processing workflow and the Processing Only workflow. For the Acquisition Only workflow, see “Defining Sample Information Manually” on page 26.

To define information for a group of samples

1. In the Group Parameters area, select a processing method that defines how the application processes the data results.

To define a processing method, see “Creating a Processing Method” on page 42.

2. Type the parent drug name in the Parent Name box.

This field is required. The application uses the parent drug name for processing and provides information about the parent drug in the summary and detailed reports. You can use the same parent name in other groups to make it easier to use a template for group information. Use alphanumeric characters only.



3. Add the formula for the parent using one of these methods:

• If you have a .mol file for the parent, select the Mol File option and click Import. Select the file and click Open.

The application displays the structure, mass, formula, and other values from the file. To view the .mol file structure, click View Mol File to open the display box.

• If you do not have a .mol file, select the Formula option and type the formula in the Formula box.

The application calculates and displays the neutral molecular weight and other values from the formula, such as the protonated mass-to-charge value.

When you add the formula, the MetQuest application adds all elements in the formula to the Elements in Use dialog box. See “Identifying Elements in Use” on page 23 for more information.

4. (Optional) Fill in the following values as needed. These values appear in the report header for both the detailed and summary report:

a. In the Species box, specify the type of species used for each sample (for example, rat, human, or dog).

b. In the Matrix box, specify the matrix type for each sample (for example, blood or urine).

c. In the Animal # box, specify the animal ID number.

d. In the Route box, type in vitro or in vivo.

e. Type the dosage amount in the Dose Amount box and include the type of measurement after the amount (for example, μL).



Table 2. Group Parameters

Parameter Description

Processing Method Select a processing method to specify how the MetQuest application processes data after acquisition. Processing methods contain parameters used to find expected metabolites.

Parent Name Type the name of the parent drug.

Source Select a source for the molecular weight calculations. Choose from typing the parent compound formula into the Formula box or selecting a .mol file.

MW View the monoisotopic molecular weight of the parent compound. This value is displayed to four decimal points. The application updates this value as you type in the formula.

M+H+(m/z) View the positive protonated value of the parent drug mass-to-charge ratio that the application uses to process data if the experiment is run in a positive ionization mode. This value is displayed to four decimal points. The application determines the polarity of the experiment and processes data accordingly.

M-H-(m/z) View the negative deprotonated value of the parent drug mass-to-charge ratio that the application uses to process data if the experiment is run in a negative ionization mode. This value is displayed to four decimal points. The application determines the polarity of the experiment and processes data accordingly.

Species Specify the type of species used for each sample (for example, rat, human, or dog).

Matrix Specify the matrix type for each sample (for example, blood or urine).

Animal # Specify the animal ID number.

Route Type in vitro or in vivo.

Dose Amount Type the dosage amount and include the type of measurement after the amount (for example, μL).



Identifying Elements in Use

The application calculates the elemental formulas from spectral data for any metabolites found to confirm the identity of each metabolite. The application chooses probable elements from the parent formula using one of two main sources.

• Calculating Elements Automatically

• Specifying Elements Using the Elements in Use Table

Calculating Elements Automatically

The MetQuest application calculates elements automatically using the parent drug and the modification list in the processing method, adding elements that are not in the parent, but are important elements for processing. Comparing the information from these sources, the application confirms isotopic distribution effectively. If none of these sources is defined, the application cannot provide any results. You can add elements to processing by adding them in the Elements in Use table.

To automatically calculate elements

1. Click View Elements in the Group Parameters area to open the Elements in Use dialog box.

2. Make sure the Use Intelligent Elemental Calculations check box is selected.

After you select this check box, the application grays out the Elements in Use table. During each processing, the application updates this table from your latest selection and uses the current results instead of data from a previously existing customized Elements in Use table.

✔



Specifying Elements Using the Elements in Use Table

You can also specify additional elements and the minimum and maximum values for each element to be used in calculating the formula. The application provides default settings.

To identify elements to use for processing

1. To view the Elements in Use dialog box and change the list of elements used for the analysis, click View Elements in the Group Parameters area.

2. Clear the Use Intelligent Elemental Calculations check box to make changes to the Elements in Use table.

You can add or remove elements from the Elements in Use table. When you define a parent drug formula, the MetQuest application adds all elements in the formula to the Elements in Use table.

The Elements in Use table uses the following guidelines for valid values:

• If you select C, the Minimum default = 0 and the Maximum default = 100.

• If you select H, the Minimum default = 0 and the Maximum default = 200.

• If you select O, the Minimum default = 0 and the Maximum default = 15.

• If you select N, the Minimum default = 0 and the Maximum default = 10.

• If you add any other element, the Minimum default = 0 and the Maximum default = 3.

You can also add other elements in addition to those contained in the parent formula. Make sure the maximum is greater than the minimum value for any added elements. For example, if you expect a sulphation modification, click S to add sulfur.

If you change the parent drug formula, the MetQuest application changes the Elements in Use table to match the new formula.



3. To add elements from the parent formula to use in determining search results, click Add to display the periodic table. Select an element and click Apply to add the element to the Elements in Use table and close the periodic table.

4. If the element you select displays several isotopes, select the largest one for better results.

5. To remove elements from use in the elemental formula, select the leftmost column of the element line in the Elements in Use table and press DELETE.

6. Click Apply to save the elements in use options and return to the Sample Setup view or click Cancel to return to the previous search options without saving your changes.

Table 3. Elements in Use dialog box parameters


Element Chemical abbreviation for the element.

Valid values can be any symbol found in the periodic table. Defaults:

• C: the Minimum default = 0 and the Maximum default = 100

• H: the Minimum default = 0 and the Maximum default = 200

• O: the Minimum default = 0 and the Maximum default = 15

• N: the Minimum default = 0 and the Maximum default = 10

• Any other element: the Minimum default = 0 and the Maximum default = 3

Nominal Mass Define the atomic mass of the represented element isotope.

Minimum Set the minimum number of elements that can be used.

Valid values: 0 to 10 000 Default: 0

Maximum Set the maximum number of elements that can be used.

Valid values: 0 to 10 000



Defining Sample Information Manually

Use the following procedure to define sample information. To import a file with group and sample information, see “Importing a File Containing Group and Sample Information” on page 30.

Because a single measurement almost never provides accurate information for decisions, the MetQuest application supports multiple injections (replicates) and averages the results. You can either create multiple injections of the same sample or create replicate sample preparations, each injected once or multiple times. Using replicate samples provides more confidence in the average value. If you use multiple injections and get results for each injection, you can exclude the occasional result that comes from sample preparation errors, autosampler errors, or instrument analysis issues without losing the group results entirely. All workflows in the MetQuest application support replicate injections.

You can inject blank samples during the acquisition to wash and condition the system between samples of high versus low concentration and also to divide groups of one parent from groups of another parent. You do not need to supply a time point or replicate number to a blank. If you convert an existing record to a blank sample, the application deletes the time and replicate values.

To copy down information (Injection Volume, Sample Type), see “To duplicate the contents of a cell in successive rows using Copy Down” on page 39. To increment copied information (Sample ID, File Name, Vial Position), see “To increment the contents of a cell in successive rows using Fill Down” on page 39.

To enter sample information

1. To enter sample information in the table, type at least the following information before starting sample acquisition.

a. Type a sample ID. Use only alphanumeric characters. This value is used in acquisition, but is not shown on the final reports.

b. In the File Name column, type a unique file name to identify the raw file for the sample. File names take the format RawfileName.raw.

c. In the Position column, right-click and select Fill Down to automatically supply the tray position beginning with the first position.

You can also type the sample’s vial or plate position number for the autosampler. If the value is not in the correct format for your autosampler, the MetQuest application displays an error message when you click Submit.


For more information about the autosampler vial or plate position format and about using multiple trays, refer to your autosampler documentation.




d. In the Inj. Volume column, specify the injection volume of the sample to be injected (μL). This value is based on your autosampler limits.

e. In the Time Point column, type the time point in minutes. Enter a maximum of 15 time points for any experiment or group.

The application checks to see that the groups have the same time points to create a comprehensive .csv file. All groups in an experiment must use the same time point values in minutes. You cannot assign a time point to a blank sample in the MetQuest application. If you convert an existing record to a blank sample, delete the time point value.

When you click Submit, the application checks for the number of groups and the values in the group. If your group has a time point that is not listed in the defining group, the application prompts you to fix the time point. You can remove the group or change the time point before continuing with sample submission.

If you open an experiment that has incorrect time points, you must fix those time points before reprocessing or submitting your sample.

f. Enter an injection volume for each sample.

When you are using an autosampler, you can set the default injection volume in the Autosampler dialog box from the Instrument Setup view. The minimum and maximum injection volumes that you can use depend on the autosampler you select. The usable range depends on the injection mode and might be smaller than the range displayed in the status bar. For more details, consult your autosampler manual.

2. When you have sample replicates, specify different replicate numbers for samples at the same time point.

You can enter replicate time points in any order.

Each replicate set (each row) can have its own control at time T0. The first replicate set must start with replicate number 1. Even if you delete a replicate set, replicate numbers must be consecutive. Do not supply a replicate number for a blank sample. If you convert an existing record to a blank sample, delete the replicate values.


3. (Optional) Specify other parameter values as needed to process your acquired raw files. For specific information about other parameters, see Table 4.

If you plan to process your acquired data immediately, you must define the time point, sample ID, and sample type in the sample grid on the Sample Setup view before submitting the information to acquisition.

a. Specify the sample type. This value is required for automatic processing. Select Control, Blank, or Sample depending on the sample type. You can enter samples in any order in the table. Controls must be at T0 if there is a control for the experiment and must contain the parent drug, but controls are not required.

b. Specify the sample time point in the time series in minutes. This value is required for automatic processing.

Table 4. Samples parameters (Sheet 1 of 2)


Sample ID Specify an ID for each sample. (This value is not shown in the reports.)

Maximum length: 255 alphanumeric characters

File Name Specify a unique file name for the original raw file. The MetQuest application saves the raw file as RawfileName.raw. This value is required for acquisition. File names must be unique for each sample.

Maximum length: 150 characters

Replicate Specify a unique number for the replicates at a specific time point in a group. For single samples, set the value to 1. If you have two identical time points, the application notifies you so that you can specify a unique value. Do not supply a replicate number for a blank sample.

When you import an SLD file, there is no replicate information. The MetQuest application fills in the Replicate column with the default (1). Define replicate information after importing the SLD file.

Valid values: 1 to 7 Default: 1



Position View the sample’s vial or plate position number for the autosampler configured for your system. Right-click in this field and select Fill Down to have the software fill in the position number, or type the number in. If the value is not in the correct format for your autosampler, the MetQuest application displays an error message when you click Submit. For more information about the autosampler vial or plate position format and using multiple trays, refer to your autosampler documentation. This value is required for acquisition.


Inj. Volume (μL) Specify the injection volume of sample to be injected (μL). This value is required for acquisition.

When you use an autosampler, you can set the default injection volume in the Autosampler dialog box in the Instrument Setup window. The minimum and maximum injection volumes that you can use depend on the autosampler you select. The usable range depends on the injection mode and might be smaller than the range displayed in the status bar. For more details, consult your autosampler manual.

Sample Type Select Control, Blank, or Sample as applicable. Select Control or Sample for automatic processing. The application uses the control file for background comparison to remove peaks present in the control.

Time Point (min) Specify the sample time point in the time series in minutes. This value is required for automatic processing. Do not supply a time point value for a blank sample.

Valid values: 0 to 2880 minutes (48 hours)

All groups in an experiment must use the same time point values in minutes. The MetQuest application also limits the number of time points to 15.

Comment Type an optional comment for these samples. This information is not used in processing and does not appear in the summary or detailed report. It does appear in the CSV report.


Table 4. Samples parameters (Sheet 2 of 2)




Importing a File Containing Group and Sample Information

If you import a file containing sample information, the MetQuest application removes all currently displayed group and sample table information. You can import a file containing replicate samples by identifying the replicate number in the standard template.

To import a file for acquisition

1. Create a .csv file for the experiment and save the file.

For information about creating a file that provides group and sample information, see “Creating Files with Group and Sample Information” on page 132.

2. Select the type of experiment and workflow.

3. In the Sample Setup view, click Import, select the .csv file in the browse box, and click Open.

The application opens to the default location unless you changed the location. In that case, the MetQuest application opens to your last defined location. The file provides group and sample information. If the group information is not complete, see “Defining Group Information Using the Application” on page 18 for specific details.

4. Review group and sample information to make sure it is accurate. For the Acquisition and Processing workflow, add a processing method for each group. When you include blank samples, delete the time point and replicate number values.

5. Click the Configuration Setup tab and define instrument settings. See “Configuring Experiments” on page 31 for more information.

6. Click Submit to acquire the samples.

When you submit or save an experiment, the MetQuest application validates all file names, positions, and injection volumes to make sure the acquisition runs properly. If vial or plate positions or injection volumes are incorrect, the MetQuest application displays a warning message in the communicator bar displaying the valid range of injection volumes or a valid position value for needed corrections. You cannot save or submit an experiment for acquisition until all validation errors are corrected.

After you submit the experiment, to view acquisition status, click the Real Time Display tab. See “Viewing Acquisition Status” on page 69 for more information.


3 Defining Sample Information for AcquisitionConfiguring Experiments

Configuring ExperimentsUse the Experiment Setup view to define options for the entire experiment. You can set reporting options and configure acquisition values.

• Setting Report Options

• Specifying Configuration Options

To specify configuration options for experiments

To add or change instrument and processing information, click the Configuration Setup tab to view the Report Options and Configuration Options areas.




Setting Report Options

Use the Report Options area to select report templates to display processing results for your experiment. The application always provides a PDF summary report and a PDF detailed report.

You can choose whether the application generates reports automatically after processing or you can generate reports after you have checked processing results in Data Review. If you select not to generate reports, including the .csv report, the application opens Data Review after processing the experiment so that you can see the results, adjust them, and then generate reports. Blank samples are not included in Data Review or in reports.

To specify report types

1. From the Experiment Setup view, click the Configuration Setup tab.

2. To review experiment results before creating reports, select the Perform Data Review check box. The default option is checked.

In this case, no report is generated after processing is completed, and you will be prompted to switch to Data Review. To generate reports from Data Review, click the Generate Reports button in Data Review to select reporting options and generate reports.

To have the application automatically generates reports, clear the Perform Data Review check box.

3. Select group reports to be generated: Summary and/or Detailed reports. These reports are generated for each group of the experiment, in the group’s Reports folder (for example: C:\Thermo\MetQuest\Experiments\Example Experiment\Groups\Nortriptyline\Reports\).

4. Select formats for your group reports in the Report Options area.

• To create a Microsoft Excel™ spreadsheet (.xls) version of the report, select the .xls check box. Use this file type to create a record that you can import into a LIMS. Default: Unchecked

• The application always creates a portable document format (.pdf ) version of the report when the application generates reports.

• To export a comma-separated value (.csv) file, select the Experiment .csv Export check box. This report contains processing results for the entire experiment. For information about exporting a .csv file and the contents of that file, see “Working with an Experiment CSV File” on page 119.

To print reports, see “Printing Reports” on page 122 for instructions. Files can be quite large when the application identifies a large number of metabolites.



Specifying Configuration Options

Use this view to define general acquisition processing values for processing your experiment. For acquisition parameter descriptions, see Table 5 on page 35.

• Specifying Instrument Methods

• Defining Acquisition Parameters

• Specifying the Mass Tolerance Value

Specifying Instrument Methods

Use the Methods area to specify the instrument method to be used in data acquisition. You can only select a method that has been defined and saved before selecting it for use. Use the Methods area to specify startup and shutdown instrument methods for acquisition.

To specify instrument methods

1. To select an instrument method that defines instrument parameters for acquisition in the Method view, click Import.

To define an instrument method, see “Creating Instrument Methods” on page 127.

2. To use a startup method, select the Startup check box and click Import.

Select a method file in the browse box and click Open.

3. To use a shutdown method, select the Shutdown check box and click Import.

Select a method file in the browse box and click Open.


Defining Acquisition Parameters

To define acquisition parameters

1. To define instruments to use for acquisition, click Instruments in Use to open a dialog box displaying instruments currently configured to work with the MetQuest application.

2. Select the check box for each instrument to be used during acquisition.

3. Select the Start Instrument option to select a starting instrument. Click Save to return to the Acquisition Parameters area.

4. In the Acquisition Parameters area, view the autosampler tray that corresponds to the autosampler configured in the Instrument Configuration view.

You must have an instrument configured within the MetQuest metabolic stability screening software MetQuest application for the application to display tray names. If you are using more than one tray, indicate that by changing the vial or plate position as indicated in your autosampler documentation.




5. To define instrument status after processing is complete, choose one of these options for the system power scheme:

• To keep the instrument in active mode to load more samples immediately, select the On option.

• To place the pump in standby mode to stop the flow of solvents, select the Standby option.

• To turn the instrument off at the end of a run to save electricity, select the Off option. This options is rarely used because the instrument vents and the process of restoring the vacuum takes a long time.

Table 5. Acquisition parameters

Parameter Definition

Tray Name View the autosampler tray type in the Tray Name list for the samples.

You must have an instrument configured for the MetQuest application for tray names to be displayed. If you are using more than one tray, indicate that by changing the vial or plate position as indicated in your autosampler documentation.


System Power Scheme Define instrument status after processing is complete. Select one of these options:

• To keep the instrument in active mode to load more samples immediately, select the On option.

• To place the pump in standby mode to stop the flow of solvents, select the Standby option.

• To turn the instrument off at the end of a run to save electricity, select the Off option. This options is rarely used because the instrument vents and restoring the vacuum takes a long time.


3 Defining Sample Information for AcquisitionSubmitting and Reviewing an Experiment

Specifying the Mass Tolerance Value

Use the Mass Option area to specify the mass tolerance. To maximize the performance of these algorithms, the application returns results of the elemental composition search only if the computed formula matches the observed mass within the specified tolerance. Specify the mass tolerance (extraction width) used during component detection.

To specify a mass tolerance

Enter a value in the Mass Tolerance (ppm) box to detect components. The value is used to create the chromatogram.

Valid values: 1 to 500 ppm Default: 5 ppm

Submitting and Reviewing an ExperimentIf you have made changes to the Processing Setup view, you must save the processing method changes before acquiring data.

After submitting an experiment for acquisition and processing, you cannot change parameters in the instrument configuration until the MetQuest application has finished processing. After acquisition and processing, you can change the instrument configuration.

To save an experiment without submitting, click Save or Save As. If you click Save As, the application copies all necessary files (including method files) to a folder with the new experiment name.

IMPORTANT When defining information in the Sample Setup view after changing a processing method, you must also import the saved method even if it replaces a method of the same name.


3 Defining Sample Information for AcquisitionSubmitting and Reviewing an Experiment

To submit an experiment

1. Click Submit to process the samples.

When you click Submit, the MetQuest application saves the raw files from the acquisition process to the \Thermo\MetQuest\Experiments\experiment_name\group_name\Acquired folder on the drive where you installed the MetQuest software. If you choose an experiment that includes data processing, the MetQuest application begins processing when a file from a sample is acquired. The application names the raw file RawfileName.raw as specified in the sample setup grid.

When you submit or save an experiment, the application validates all file names, positions, and injection volumes so that the sequence runs properly. If vial or plate positions or injection volumes are incorrect, the communicator bar displays a range of valid values.

When validating a sequence, the application also checks for these requirements:

• Maximum time points are 15.

• All groups within an experiment must use the same time points. The application defines the specific time points in an experiment using the group with the largest number of time points. If two groups both have the largest number of time points, the application selects the first group in the group list, using time points from this group. If a group does not have a time point, the application treats that time point as missing and marks all component values as no value (NV).

• Controls are at T0.

The MetQuest application provides import and submission errors through the Import Validation dialog box.

If the application is currently acquiring data, it waits to acquire data for the new experiment until the first one is finished. Processing for an experiment begins immediately after acquisition has started. Depending on the number of samples in the first experiment, your acquisition might be delayed.

After submitting an acquisition and processing experiment, if you get a “Processing Failed” message, the application has not created a list of valid peaks. This could be because there were no valid components in the file or it could not find the parent drug within the mass tolerance specified in the Metabolite Filtering Options area.

During processing, you can open a different processing method and make changes to it without impacting the currently processing sequence.

2. To view real-time processing, click the Real Time Display tab.

The Real Time Display view displays current acquisition and processing details. If you selected the Data Review option, the application moves to Data Review after finishing acquisition and processing. For information about using Data Review to modify processing data, see “Using Data Review to Review and Modify Test Results” on page 80. To view reports, see “Viewing Experiment Reports” on page 110.


3 Defining Sample Information for AcquisitionWorking with the Sample Table Columns

Working with the Sample Table Columns Use the following instructions to make changes to the columns, rows, and data in the sample view. You can make the following changes within the sample table:

• Add or delete rows• Copy down• Fill down• Copy and paste rows

To move among cells

1. To move from cell to cell, press TAB.

2. To move backward through the cells, press SHIFT+TAB.

To add a new row

1. To add a row to the bottom of a table containing the Add Row icon ( ), enter the sample information in the empty bottom row and press TAB.

The application adds a new row to the bottom.

2. To insert a copied row in the center of a table, right-click the row above the paste location and choose Paste Below from the shortcut menu to paste the row information in the selected location. You can also choose Paste Above to insert a row above the selected row.

To copy the contents of a row

1. Type the contents and select a row by selecting the row pointer in the left margin.

For multiple rows, click in the left margin and drag the cursor down to select more rows.



2. Right-click the left column of one of the selected rows and choose Copy from the shortcut menu.

The application copies the row contents to the Clipboard.

To duplicate the contents of a cell in successive rows using Copy Down

1. Type the contents in the cell.

You can copy values in the Injection Volume and Sample Type fields.

2. To select rows to hold the copied information, select the row pointer ( ) on the left of the first column and drag it down the left column to select the rows to paste to.

3. Right-click the cell of the starting row that you want to copy from and choose Copy Down from the shortcut menu.

The application copies the row contents to successive rows.

To increment the contents of a cell in successive rows using Fill Down

1. Type the contents in the cell.

You can increment values in the Sample ID, File Name, and Position fields.

2. To select the rows for the incremented contents, select the row pointer ( ) and drag the cursor down the left column to select the rows to receive the incremented value.

3. Right-click the cell of the row that you want to copy from and choose Fill Down from the shortcut menu.

The application increments the row contents in the successive rows.




To remove rows completely

1. Select a row or rows by selecting the row pointer ( ) on the left of the first column to remove.

2. Press the DELETE key or right-click the row pointer of one of the selected rows ( ) and choose Delete from the shortcut menu.

3. Click Yes when prompted to delete the rows.

To remove the contents of a field

1. Select the contents of the field.

2. Press CTRL+X or right-click and choose Cut from the shortcut menu to remove the contents and copy the contents to the Clipboard.

You can use CTRL+C keys or the Copy command to copy the content to the Clipboard and the CTRL+V keys or the Paste command to paste the content from the Clipboard.

4

Working with Processing Methods

The processing method determines how the MetQuest application processes the data. A method (.proc file extension) defines the data processing and reporting parameters for a sample. When you process a sample, the application provides a set of result files and a set of electronic reports. Review the report files (.pdf and .xls) using the Report view.

To process your acquired data directly after acquisition, choose a processing method and specify sample values in the Sample Setup view before submitting the samples to acquisition (see “Defining Sample Information” on page 16 for more information about setting up samples).

To process previously acquired data, see “Processing Data Acquired Using the MetQuest Application” on page 60.

The Processing Setup view displays the following options and parameters to be used for each of the data processing steps that are available as part of the overall workflow configuration.

• Parameters for comparing components in samples to components in a control

• A list of expected modifications

• Data entry for an internal standard

• Elemental Composition limits

• Options to include adduct peaks in area calculations

• Advanced spectral fit settings

• Peak acceptance criteria

You can create new methods, modify existing methods, and save method files.

Follow these major steps to create or change a method. You must create at least one method before processing samples.

Contents

• Creating a Processing Method

• Changing a Processing Method


4 Working with Processing MethodsCreating a Processing Method

Creating a Processing MethodTo create a processing method, complete these procedures:

• Setting Parameters for Metabolite Searching

• Specifying Internal Standard and Elemental Composition Settings

Setting Parameters for Metabolite Searching

Use the Metabolite Options view to set parameters for metabolite searching. The MetQuest application uses the control file within each group for background comparison.

The application provides a list of possible metabolites to search for. When found, these metabolites are included in the search results.

• Defining Control Comparison Values

• Defining the Retention Time Range

• Specifying Expected Metabolites

After you save the processing method, you can save and reopen the method containing your search parameters and specify which processing method to use for each group in the experiment in the Sample Setup view.



Defining Control Comparison Values

To set up the processing method to search for metabolites

1. Start the application by clicking or choosing Start > Programs > Thermo > MetQuest.

2. To provide more room on the screen for your data, click in the upper right corner to hide the Thermo banner.

3. Select a workflow type.

To change or create a processing method, select the Acquisition and Processing workflow.

4. Click the Processing Setup tab to open the Metabolite Options view.




5. In the Control Comparison area, specify the Drift Tolerance (sec) +/- (the size of the retention time window in seconds).

Change the value of drift tolerance to adjust component grouping as needed.

The application uses the drift tolerance window to look for peaks that might have slightly shifted in retention time from sample to sample. If you select too large or too small a drift tolerance window, the application might group together components that should not be grouped or separate components that should be grouped. The application adds the value before and after the specific retention time to widen the search area. For example, if your peak is at 3.00 minutes and you choose an offset of 30 seconds, the application searches from 2.5 to 3.5 minutes. It does not include search results outside the changed retention time. If you select a value smaller than the default, the application can separate components that should be grouped together. For example, in a sample, +O_6 and +O_7 might be separated using the default (providing no value for the peak).

Grouping works only when samples are entered in the sample setup grid in ascending or descending injection order so that samples acquired in order are compared in that same order. For example: Sample for first injection, sample for second injection and so forth, down to the sample for last injection.


If you set the Drift Tolerance to more seconds (in this case, 20 seconds), the application groups +O_6 and +O_7, providing a component with a value.

Range: 0.5 to 30.0 seconds Default: 2.4 seconds

6. Set an intensity ratio threshold to include peaks that would be excluded from the final results because they are in the control file but are larger in the sample file by a specific ratio.

If at least one time point area is the selected value (n) times larger than the area of the control, the component is kept and displayed in Data Review.

Valid values: 2x, 3x, or 5x Default: 2x



Defining the Retention Time Range

To define the retention time range

1. In the Retention Time Range area, select the RT (min) check box to exclude peaks that are outside the range for the parent drug and metabolites.

2. Specify a low and high value for the retention time area to define the area.

The low value must be lower than the high value.

Specifying Expected Metabolites

When creating a list of expected modifications, use these rules:

• Start all formulas with a positive or negative (+ or –) sign indicating whether to add or subtract from the parent formula. Example: +CH

• Type valid element symbols from the periodic chart. The application displays an error for invalid formula entries after you leave the cell. Example: +QH +N displays an error.

• Repeat the same element as needed. Example: +CHCH–CH is valid.

• To change from addition to subtraction or the reverse, specify the sign for the change just before the first compound with a new sign. Otherwise, the application defines the first encountered sign for all compounds that follow. Example: +CH2NH2–CH2 is not the same as +CH2NH2 CH2.

• For compound formulas, the application accepts spaces within the formula.



To use a list of expected metabolites

1. From the Expected Metabolites area, click Import to select a modification list from the Template folder (MetQuest_PhaseI_Common.csv is the default file) or a custom list file for use in the current processing method.

The application opens to the default location unless you changed the location. In that case, the MetQuest application opens to your last defined location.

2. Click Open.

To create a new list file or add metabolites to the file, see “Working with a Metabolite Modification List for Processing” on page 136.

3. To delete a row, select the row using the left column, right-click the row, and choose Delete.

4. To add, copy, or delete information in a single cell, select the cell, right-click the cell, and choose the appropriate command.

5. To add a row, add cell information in the first row and press ENTER.

Column Description

Modification The name of the expected modification.

Formula The formula change (with respect to the parent formula) for the type of modification.

Mass Shift The change in theoretical mass-to-charge ratio (m/z) associated with a metabolite modification when calculated with the parent drug.



Specifying Internal Standard and Elemental Composition Settings

The MetQuest application uses the parent drug at time point 0 as the reference for relative quantitation. The application reports all peaks with relative % area and absolute area.

Select an internal standard to normalize the peaks within each data file to account for variability among runs caused by changes in instrument conditions. Place an equal amount of the internal standard in each sample.

Use these procedures:

• Defining an Internal Standard

• Setting the Sum Area Option

• Defining Elemental Composition Values

• Changing Advanced Spectral Fit Values

• Defining Peak Acceptance Criteria

To open the ISTD/Elemental Comp view

From the Processing Setup view, click the ISTD/Elemental Comp tab.



Defining an Internal Standard

To define the internal standard

1. From the Processing Setup view, click the ISTD/Elemental Comp tab to open the ISTD/Elemental Comp view if it is not already open.

2. Type a name for the compound in the Name/ID box.

By default, this box is empty.

3. Add the formula for the ISTD using one of these methods:

• If you have a .mol file for the ISTD, click Import, select the file from the browse box, and click Open.

The application displays the mass, formula, and other values in the appropriate area from the file.

• If you do not have a .mol file for the ISTD, type the formula in the Formula box.

The application calculates the mass and other values from the formula.


Name/ID Type a name for the internal standard compound.

Formula Type a formula for the calculations or select a .mol file.

MW View the molecular weight of the internal standard compound. This value is displayed to four decimal points. The application displays this value as you type a formula or select a file.

M+H+ (m/z) View the positive protonated value of the internal standard m/z to be used for data processing if the experiment is run in a positive ionization mode. The application displays this value as you type a formula or select a file.

M-H- (m/z) View the negative deprotonated value of the internal standard m/z to be used for data processing if the experiment is run in a negative ionization mode. The application displays this value as you type a formula or select a file.



Setting the Sum Area Option

In the Sum Area Option area, you can increase the area of the component (metabolite or parent drug) by adding adduct areas related to the component. Increasing the area leads to a better signal-to-noise ratio and makes low level components less likely to be hidden below a sum area threshold.

If you select this option, the MetQuest application adds the adduct areas to the component area to increase the size of the component when calculating relative quantitation values. The application provides a positive adduct list for positive polarity data and a negative adduct list to be used for negative polarity data. The following table lists adducts in the adduct list. You cannot change adduct information in this table.

To specify the sum area option

Select the Include Adducts check box to add adduct values to the component area.

The default is unchecked.

Formula Polarity Mass Difference

NH4 Positive 17.0265

Na Positive 21.9819

K Positive 37.9559

H3C2O2 Negative 58.0066

HCO2 Negative 43.9909

Cl Negative 33.9610

H Negative 2.0156



Defining Elemental Composition Values

Use the Elemental Composition Limits area to specify values for spectral fit parameters that the application uses when calculating formulas (see Table 6).

The application does the following:

• Locates the spectral peak by its theoretical m/z value.

• Computes elemental composition based upon average scans acquired across a peak.

• Examines a set of ions around the spectral peak, including the isotope pattern.

• Calculates all elemental composition candidates based on an isotope cluster and the intensities of the isotopes (usually the monoisotopic mass). The calculation is bounded by the elements in use chosen in the Sample Setup view.

• Scores the comparison between the measured isotope pattern and the theoretical isotope pattern of each composition candidate with a fit factor. A good fit factor (100 percent is the maximum) is reached only if both the measured and the theoretical isotope patterns and intensities are almost identical. If the patterns do not match exactly, the fit factor is less than 100 percent.

• Sorts the elemental composition candidates in terms of decreasing fit. The most likely candidate chosen is the candidate with the best fit factor.

To enter elemental composition values

1. Select whether to use the Nitrogen Rule in the formula calculation.

For an LC/MS analysis, select Even Electrons because the sample has an even number of electrons.

Valid values: Do Not Use, Odd Electrons, or Even Electrons Default: Even Electrons

2. To restrict the number of possible elemental compositions, enter a value in the Mass Tolerance (ppm) box.


The application returns results of the elemental composition search only if the computed formula matches the observed mass within the specified tolerance. The application uses this value for mass tolerance throughout the MetQuest application for elemental composition (acceptance and monoisotopic mass determination).



3. To set the values for double bonds and ring equivalents—a measure of the number of unsaturated bonds in a compound—and limit the calculated formulas to only those that make sense chemically in this regard, type values in the RDB Equiv Low box and the RDB Equiv High box.

Valid values: –1 to 100 Default (low): –0.5 Default (high): 100

4. Specify the total number of results by typing a value or clicking the up and down arrows in the Max Results box.

Valid Values: 1 to 10 Default: 10

Table 6. Elemental Composition Limits parameters (Sheet 1 of 3)


Nitrogen Rule Select whether to use the Nitrogen Rule in the formula calculation. For an LC/MS analysis, select Even Electrons because the sample has an even number of electrons.

Options: Do Not Use, Odd Electrons, or Even Electrons

Default: Even Electrons

Mass Tolerance (ppm) Specify a mass tolerance to restrict the number of possible elemental compositions. The application returns results of the elemental composition search only if the computed formula matches the entered mass within the specified tolerance. Type an integer in the box.


The application uses this value for mass tolerance throughout the MetQuest application for elemental composition. If the instrument is not well calibrated, you might need to increase the mass tolerance.



RDB Equiv Low View or change the low value for double bonds and ring equivalents—a measure of the number of unsaturated bonds in a compound—and limit the calculated formulas to only those that make sense chemically.

Valid values: –1 to 100 Default: –0.5

The application uses the following formula:

Where

D is the value for the RDB

imax is the total number of different elements in the composition

Ni is the number of atoms of element i

Vi is the valence of atom i

The calculation results in an exact integer such as 3.0, indicating an odd-electron ion or an integer with a remainder of 0.5, indicating an even-electron ion. A value of –0.5 is the minimum value and corresponds to a protonated, saturated compound (for example, [H3O+]).

Valid values: –1 to 100 Default: –0.5

This value must be less than the RDB Equiv High value.



D 1

Ni Vi 2–

i

imax

2-----------------------------------------+=




RDB Equiv High View or change the high value for double bonds and ring equivalents—a measure of the number of unsaturated bonds in a compound—and limit the calculated formulas to only those that make sense chemically.


This value must be greater than the RDB Equiv Low value.

Max Results View or change the maximum number of results you want the MetQuest application to display. To increase or decrease the value, change the value.





Changing Advanced Spectral Fit Values

Use these parameters to find the best match.

To change values for advanced spectral fit parameters

1. To measure an observed mass error on an isotopic peak based on its abundance, type a value in the Expected Mass Error (ppm) box.


This option ensures that more abundant masses have a smaller percentage of errors than less common masses.

2. To measure an observed intensity error on an isotopic peak upon its abundance, type a value in the Expected Intensity Error (%) box.

More abundant intensity readings have a smaller percentage of error than less common ones. Valid values: 0 to 100 Default: 10


Expected Mass Error (ppm)

The Spectral Fit algorithm compares the differences between theoretical and measured isotope masses and intensities. To decide whether two peaks have a good or bad mass fit, the MetQuest application must know the expected mass error of the mass spectrometer. This parameter specifies a value for the expected mass error in the spectrum data to be used by the algorithm.

For best results, set the expected mass error to a value that causes less than 98 percent of all mass errors to be smaller than the value for the Expected Mass Error. Type an integer to specify the expected mass error in ppm.


Expected Intensity Error (%)

The application compares the differences between theoretical and measured isotope masses and intensities. In order to decide whether two peaks have a good or bad intensity fit, the MetQuest application must know the expected intensity error of the mass spectrometer. This parameter sets a value for the expected intensity error in the spectrum data to be used by the algorithm.

For best results, set the expected error to a value that causes less than 98 percent of all intensity errors to be smaller than the value for the Expected Intensity Error value. Specify the expected intensity error as a percentage. Type an integer.

Valid values: 0 to 100% Default: 10%



Defining Peak Acceptance Criteria

To define peak acceptance criteria

To specify the minimum number of points a peak must have in order to be defined as a peak (component, isotope, or adduct) in the search results and in the reports, enter a value in the Min. # of Points Across a Peak box.


If you set this value too high, you can miss some or all of your peaks.

The application reports this parameter in the header information of the detailed and summary reports.

Saving Processing Methods

To save a processing method

1. In any processing method view, click Save to open a dialog box.

2. Give the method a unique name so that it is available for future processing.

3. Click Save.


4 Working with Processing MethodsChanging a Processing Method

Changing a Processing MethodUse the Processing view to review or change a processing method.

To review or change a method

1. Click Open in the Processing view and browse to open a method file. Click OK.

2. Select an existing method.

The saved method opens, displaying the current parameters.

3. Change parameter values in any view and click Save in any processing method view to save the method under the same name.

• To change parameters for metabolite searching, see “Setting Parameters for Metabolite Searching” on page 42.

• To change parameters for elemental composition, see “Specifying Internal Standard and Elemental Composition Settings” on page 48.

• To change report settings, see “Setting Report Options” on page 32.

4. To save a modified method under a new name, select Save As, type a new name, and click Save.

IMPORTANT After changing a processing method, you must also import the saved method into the Sample Setup view even if it replaces a method of the same name.


5

Processing Previously Acquired Data

To process acquired data or reprocess data, use the Processing Only workflow. If you plan to import a file containing sample information for the first time, click New Experiment. You might process information in these situations:

• To process acquired data from time course samples when you did not define a processing method (continuing an experiment)

• To process samples acquired and processed using the MetQuest application so that you can change processing parameters (a continuing experiment)

• To process samples acquired using a different Thermo Scientific application that creates an .sld file to define acquisition and processing information (a new experiment)

• To process samples acquired using a different Thermo Scientific software product using raw files (a new experiment)

For information about creating or changing a processing method, see “Working with Processing Methods” on page 41.

Contents

• Processing Data Acquired Using the MetQuest Application

• Processing Data Acquired and Processed Using the MetQuest Application

• Processing Data Acquired Using an .sld File

• Processing Data Using Raw Files Acquired in Xcalibur


5 Processing Previously Acquired DataProcessing Data Acquired Using the MetQuest Application


Processing Data Acquired Using the MetQuest ApplicationThis section describes how to process data acquired using the Acquisition Only experiment type. You can define or change processing parameters, but you cannot change acquisition parameters.

To process data acquired using the Acquisition Only experiment type

1. Start the MetQuest application by clicking or choosing Start > Programs > Thermo > MetQuest.

2. To process previously acquired data, select the Processing Only workflow.

3. To process an experiment, select an experiment name in the Select an Experiment list and click Continue Experiment.

The application displays the Sample Setup view.

4. In the Groups area on the left, select the group name.

5. Click Import, browse to a processing method, select the method, and click Open.

For an experiment with multiple groups, select a group name in the Groups area and then click Import from the Group Parameters area to select a processing method (.proc). Do this for all groups in the experiment. To create a processing method, see “Creating a Processing Method” on page 42.

5 Processing Previously Acquired DataProcessing Data Acquired and Processed Using the MetQuest Application


The MetQuest application displays the method name in the Processing Method box and opens to the default location unless you changed the location. In that case, the application opens to your last defined location.

6. View or change configuration options in the Configuration Setup view.

7. Click Save to save the experiment or click Submit to process the data.

When you submit an experiment for reprocessing, the application removes processing files for all the submitted groups and creates files using the new parameters. The application processes files from changed or new groups, whether you are adding groups, deleting groups, or processing only part of an experiment.

If the MetQuest application is currently acquiring data for an acquisition-only experiment and if you then send a processing-only experiment to processing, the application waits to process the processing-only experiment until the acquisition-only experiment is complete. Depending on the number of samples still being acquired, processing might be delayed.

Once you have reviewed the results, you can make changes to the processing method to refine data selection and process it again using new parameter values.

Processing Data Acquired and Processed Using the MetQuest Application

Use these methods to make changes and reprocess experiments acquired and processed using the MetQuest application.

• Processing Complete Experiments

• Processing Partial Experiments

Processing Complete Experiments

This process describes how to reprocess data originally acquired and processed using the MetQuest application. You can change processing parameters, but you cannot change acquisition parameters.

To process data acquired and processed using the MetQuest application


2. To process previously acquired data, select the Processing Only workflow.

The application displays existing experiments.


3. Select an experiment name in the Select an Experiment list to define processing parameters and click Continue Experiment.

The application displays the Sample Setup view.

4. To select a different processing method (.proc) for a group, select the group name, click Import in the Group Parameters area, select a method, and then click Open.

The application displays the method name in the Processing Method box. Repeat this step if needed for each group. To change an existing processing method, see “Changing a Processing Method” on page 57.

5. View or change configuration options in the Configuration Setup view.

6. Click Save to save the experiment and click Submit to process the data.

7. To copy the experiment, click Save As.

The MetQuest application prompts you for a new name for the experiment. Type a unique experiment name and click OK.




Processing Partial Experiments

Once you have acquired an experiment, you cannot delete or make changes to the groups that have been acquired unless you reprocess the experiment. However, you can add groups and acquire and process those groups.

You might want to process only part of an experiment if you add new groups to the experiment. You can select only the new groups to process or select to reprocess any of the processed groups. You would reprocess processed groups if you made an error in acquisition or processing. To reprocess groups that have already been processed, see “Processing Complete Experiments” on page 61.

To add a group after processing an experiment

1. Select the Processing Only workflow.

The application displays existing experiments.

2. Click Continue Experiment and select an experiment to open the experiment.

3. Click the Sample Setup tab.

4. In the Group area, click Add to add a new group and name the group.

To process new groups, import raw files.

5. Import or type in group and sample details.

For information about adding group and sample details, see “Defining Sample Information for Acquisition” on page 15. Before clicking Submit, you can delete any newly added groups. The application validates group and sample information when you import the data, before saving if you try to save, and before submitting the experiment. You can continue making changes to an experiment with errors after importing the file. The application provides warning errors. You cannot submit or save an application if there are errors.

6. To process the group and generate reports, select the check box for that group and click Submit.

If you made no changes to any of the other groups, the application does not change the reports or reprocess the groups.



To change group information after processing

1. Open the experiment.

2. Click the Sample Setup tab.

3. In the Group area, select the group name to display group and sample information.

Change group and sample details. When you click Submit, the MetQuest application validates the new information for the selected group.

For information about changing group and sample information, see “Defining Sample Information for Acquisition” on page 15.

4. To process the group, select the check box for any group you want to reprocess and click Submit.

When you click Submit, the MetQuest application validates the new information for the selected group.


In the Review view, the application displays current valid results for all groups in the experiment, including original processing results for the groups that were not resubmitted and new processing results for the groups which were resubmitted or added.

When you click Generate Reports from Data Review, the application creates reports for all checked groups in the experiment. The MetQuest application also creates PDF reports for all checked groups in the experiment if you skip Data Review.


5 Processing Previously Acquired DataProcessing Data Acquired Using an .sld File

Processing Data Acquired Using an .sld FileYou can use the MetQuest application to process data acquired using other Thermo Scientific software products that produce an .sld file. You can process data acquired using the LTQ Orbitrap family of mass spectrometers or an Exactive instrument that is not running the MetQuest application.

The processing does not change or move your original files. After you save or submit the experiment, the MetQuest application copies your raw files, processes them, and places the copies in the Thermo\MetQuest\Experiments directory. Then the application lists them in the Select an Experiment list so that you can open them, view the results, and process them again.

Your .sld file might contain an instrument method or processing method, but you must identify these methods in the MetQuest application after importing the file. The application puts all samples into the currently selected group, specifying default values for group information.

To process data acquired using a different Thermo Scientific software


2. To process previously acquired data using an .sld file, select the Processing Only workflow.

3. Click New Experiment to open the Sample Setup view.

You can rename the group.

4. Select or create a group for the sample information.

All information from an .sld file goes into one group.

5. From the Sample Setup view, click Import and select .SLD Sample List, browse to a list, select the list, and click Open.

The application fills in the sample information in the sample list.

You can create a template based on the sample information by exporting the sample information to a .csv file. Click Export to create a .csv file, including the parent information, group information, and sample information associated with each group in the experiment. The exported file uses the same format as the Import template. For information about using the Import template to create a file to import, see “Creating Files with Group and Sample Information” on page 132.

6. Add parent information for each group and any group information that is needed.


5 Processing Previously Acquired DataProcessing Data Acquired Using an .sld File

7. To select a processing method (.proc) for a group, click Import in the Group Parameters area, browse to a method, select the method, and click Open.

The application fills in the method name in the Processing Method box.

8. In addition to the information provided by the .sld file, add or change sample information:

• Add a time course.

• Select the sample or control sample type for each sample.

• Provide a fully justified path and file name for every raw file.

9. Click Submit to process the data.

If the application prompts you for a name for the experiment, type a unique experiment name and click OK.

The application validates group and sample information at the time of import, before saving if you try to save, and before submitting the experiment. You cannot submit or save an experiment if there are errors.

The application stores a copy of your raw files and processed files in C:Thermo\MetQuest\Experiments\experiment_name\group_name\Acquired.

10. To view reports for the experiment, click the Reports tab. If the experiment is not listed, click the Workflows tab, select Processing Only, and select the experiment name in the Select an Experiment area.

Once you acquire, process, or save the experiment, you cannot make changes to the existing groups. You can add groups to the experiment and delete or rename these added groups until you save the experiment again.

If the MetQuest application is currently acquiring data for an acquisition-only experiment and if you then send a processing-only experiment to processing, the application waits to process the processing-only experiment until the acquisition-only experiment is complete. Depending on the number of samples still being acquired, processing might be delayed.

For more information about finished reports, see “Viewing Experiment Reports” on page 110 and “Printing Reports” on page 122.


5 Processing Previously Acquired DataProcessing Data Using Raw Files Acquired in Xcalibur

Processing Data Using Raw Files Acquired in XcaliburYou can use the MetQuest application to process data from raw files acquired using other Thermo Scientific software products, such as the Xcalibur data system. You can process full-scan AIF data acquired using the LTQ Orbitrap family of mass spectrometers or an Exactive instrument that is not running the MetQuest application.

The processing does not change or move your original files. The MetQuest application copies your raw files from the location you specified in the sample grid to the Thermo\MetQuest\Experiments\experiment_name directory and adds them to the Select an Experiment list so that you can open them, view the results, change the processing information, and process them again.

To process raw files acquired using a different Thermo Scientific software


2. To process previously acquired raw data files, select the Processing Only workflow.

3. Click New Experiment to open the Sample Setup view.

4. Select or create a group for the sample information. Enter group information before identifying sample files. For more information about defining group information, see “Defining Group Information Using the Application” on page 18.

5. From the Sample Setup view, define sample information in the sample list. Specify a raw file name and a fully justified path for each sample.

6. Add or change any needed sample information:

• Add time course information for each sample.

• Provide a fully justified path and file name for every raw file.

7. Set or display configuration options in the Configuration Setup view.

8. Click Submit to process the data.

Based on the size of your sample files, the number of groups, the number of replicates, and so forth, this process can be lengthy because the MetQuest application performs component detection.

9. If you have not already saved the experiment, the MetQuest application prompts you for a name for the experiment. Type a unique experiment name and click OK.

The application stores a copy of your raw files and processed files in the C:Thermo\MetQuest\Experiments \experiment_name\group_name folder.


5 Processing Previously Acquired DataProcessing Data Using Raw Files Acquired in Xcalibur

Once you have reviewed the results, you can make changes to the processing method options to refine peak selection and process the data again using this procedure.

For more information about finished reports, see “Viewing Experiment Reports” on page 110 and “Printing Reports” on page 122.


6

Reviewing Acquisition and Processing Status

This chapter describes how to view the acquisition and processing status for a sample, a group of samples, or an experiment. Before viewing data, submit your samples following the instructions in “Defining Sample Information for Acquisition” on page 15 and “Working with Processing Methods” on page 41.

Viewing Acquisition StatusUse the Real Time Acquisition Display view to see acquisition status for samples to evaluate acquisition status. You can stop, start, pause, and delete samples from the acquisition queue.

The application displays the following:

• TIC chromatogram

• Data from other detectors, such as UV, A/D cards, PDA

• Mass spectral data

• Device status

• Acquisition queue Start/Stop/Pause buttons

• Sample and Sequence information

Contents

• Viewing Acquisition Status

• Viewing Processing Information


6 Reviewing Acquisition and Processing StatusViewing Acquisition Status

To view acquisition status

1. Click Submit to send an experiment for acquisition.

The application moves automatically to the Real Time Display view.

The application displays the Real Time Acquisition Display view.

The Status view displays acquisition status and the Data view shows chromatographic and mass spectral data as it is acquired.

For the following options, right-click the Data view to open the shortcut menu:

• Show Spectrum Trace: Choose Show Spectrum Trace to hide or display the spectrum trace.

• Copy to Clipboard: Choose Copy to Clipboard to copy an image. Paste the image into a document or spreadsheet.

• Reset Scaling: To change the scaling and widen a chromatogram segment, drag the cursor along the time line. To return to the original scaling, right-click the chromatogram window and choose Reset Scaling.

Status view Data view



The upper graphic shows the original view. When you zoom in on a segment, the view (see the lower graphic) shows a closer view of the selected area (4.45 to 5.12).

2. Click the Acquisition Queue tab to open the list of submitted samples.

When the sample has finished, the list displays an X before the name of all acquired samples.



3. To view device status below the run status in the acquisition queue, click the device name.

The application displays acquisition status for that device in the Status area.

To view information about the sample, right-click the check box and choose Sample Info from the shortcut menu. The application displays information about the sample.

4. To remove a sample or sequence from the queue, select the check box next to the name. Click Delete to remove the sample from the list.

The application marks the sample as deleted and does not process it.


6 Reviewing Acquisition and Processing StatusViewing Processing Information

The following buttons at the top of the Real Time Display view help you control acquisition.

Viewing Processing InformationThe MetQuest application provides information about status and errors that occur during the processing of samples in two ways: you can view status information on the screen in the Processing Display area, or you can open the processing log in the experiment folder after the application finishes processing the experiment. (See “Viewing Information in the Processing Log” on page 77 for more information about locating and viewing the log.)

• Viewing Information in the Processing Display Area

• Stopping Processing Activity

• Viewing Information in the Processing Log

• Understanding Error Handling

Button Action

Restarts acquisition from a pause.

Pauses the acquisition queue. If the application is currently acquiring a sample, it continues acquiring. If you click Go, the application continues acquiring the next sample.

Stops acquisition for the sample in progress and pauses the acquisition queue. The application opens a menu with these choices:

• Stop acquisition for current sample and pause.

• Stop acquisition for current sequence and pause.

• Stop acquisition for all sequences.

Stops updating the Real Time Acquisition view but has no impact on acquisition.

Hides the spectral data view.




Viewing Information in the Processing Display Area

Use the Real Time Processing Display area to determine whether the acquisition data from a sample or group of samples has been processed successfully. The application continues displaying the information whether you are viewing the Processing Display area or not. You can also tell which experiment and sample the application is processing. This feature is helpful when trying to determine how much time the processing task takes. The application also provides a way to cancel processing at the group or experiment level. However, there is no resume function after you cancel processing.

To open the Real Time Processing Display area

1. Open the Real Time Processing Display area when you are in the experiment or after you have left the experiment.

If you have submitted the experiment, the application moves automatically to the Real Time Processing Display view.

When you have left an experiment but while the experiment is still processing, to view real-time processing information about an experiment, do the following:

a. Open the application.

b. Select Acquisition and Processing or Processing Only.

c. Select an experiment from the Select an Experiment list and click Continue Experiment.

d. Click the Real Time Display tab.

e. Click the Processing tab.

The Processing Status view opens, showing the experiments that are actively processing or are waiting for processing.

Experiment list Log information


2. Select an experiment to review processing status.

The application displays all groups in the experiment and displays processing information about the experiment to the right of the experiment list.

3. Select a group to view processing information for that group and the samples in the group. To view specific processing information for a sample, select the sample.

When the application is processing a unit, the green circle next to it continues to move. If the selected unit has a solid green circle next to it, the application has completed processing that unit. Once all samples and groups of an experiment have been processed, the application removes the experiment from the display. The related processing information for the last experiment processed remains in view on the screen.

In the Real Time Processing Display area, the MetQuest application displays information for each specific event:

• Event start time

• Event end time

• Event outcome, successful or not successful and, when not successful, the reason it did not succeed

• Messages that the activities are publishing (for example, acquisition activity reports when the sequence is queued, acquisition is started or finishes, and so forth)

The application displays information in the processing status area while it is still processing the experiment. When you process multiple experiments, the MetQuest application displays the processing information for the last experiment processed in the Processing Status area.



Stopping Processing Activity

You can cancel the processing of data within the MetQuest application. This action is useful if you are processing previously acquired data or decide to change processing parameters in the middle of processing or need to process a different experiment or group out of order. Canceling processing has no effect on acquisition because the application only displays experiments that have already been acquired. To stop acquisition, see “Viewing Acquisition Status” on page 69.

To stop processing activity

1. Select an item or items (either sample, group, or experiment) from the processing list.

2. Right-click and choose Cancel from the shortcut menu to stop processing.

The MetQuest application stops all processing activity for the selected items and does the following:

• Deletes all processing results for the selected unit.

• Removes the selected group or experiment from the processing queue.

If you cancel acquisition of one or several groups in an experiment, the application processes the groups that have been acquired.



Viewing Information in the Processing Log

In addition to viewing processing information in the processing status area, the application records a log for each experiment that shows the final processing status for samples and groups in the experiment. The application stores this log (ProcessingLog.xml) in the experiment folder. The log contains the same information that was displayed in the processing status area. After the application has finished processing or if the application cannot process the information, you can open the processing log to review status. Use this log to review errors that occurred during processing.

To open the processing log from the application

1. Open the experiment if it is not already opened.

2. Click either the Data Review or Reports tab.

3. When the display opens, click Processing Log.

4. To check processing information for a specific sample, click the sample name from the sample list.

To open a processing log in the experiment folders

From the C:\Thermo\MetQuest\Experiments\experiment_name folder, click the ExperimentProcessLog.xml file.



Understanding Error Handling

For some sample processing errors, the MetQuest application stops processing the entire group. However, the application can process groups that have some sample errors and still generate the data for a group, but the information will not be complete.

While processing an experiment, the application responds to the following types of errors:

Problem Response

The MetQuest application cannot process a sample in an experiment (see below for specific examples).

The application continues processing the remaining samples within the group and calculates % Remaining values for only the processed samples. For samples that could not be processed, the application provides no value (NV).

• The MetQuest application cannot process a time point sample in a single injection analysis with no replicates.

The application calculates the % Remaining values for all the time points that did process. The application identifies the sample as Excluded, displaying the value as NV. You cannot include this sample after that point.The time point does not appear in the Time Course plot.

• The MetQuest application cannot process a control sample replicate.

The application calculates the % Remaining values based on those replicate controls that successfully processed. The application provides NV for the control and you cannot include it in the sample results.

• The MetQuest application cannot process a time point sample (in Replicate analysis).

The application calculates the % Remaining values for all processed time points and samples. Using the values for the samples from the replicate set that processed successfully, the application calculates the average % Remaining value for the time point, marking the non-processed sample NV. You cannot include this sample after that point.


7

Reviewing Results from the Application

This chapter describes how to view the results of processing a sample or samples. Click the Data Review tab to view, filter, or modify experiment results or click the Reports tab to examine finished reports. You can also view the log file describing events and errors in acquisition and processing. Before viewing data, process your sample following the instructions in “Defining Sample Information” on page 16 and “Working with Processing Methods” on page 41.

Contents

• Using Data Review to Review and Modify Test Results

• Viewing Experiment Log Results

• Viewing Experiment Reports

• Working with an Experiment CSV File

• Printing Reports


7 Reviewing Results from the ApplicationUsing Data Review to Review and Modify Test Results

Using Data Review to Review and Modify Test ResultsClick the Data Review tab to review and modify data for all experiments before or after creating reports. You can copy graphic elements from Data Review to the Clipboard and paste them into another software product. If you do not have any experiments to review or want to review a successfully processed experiment, the application provides sample data with reports that you can open and review.

• Viewing Component Results

• Viewing Elemental Composition Information

• Working with Component Results in Data Review

• Changing Components

• Saving Changes in Data Review

• Defining Report Options for Data Review

Viewing Component Results

If you are still in the experiment and data processing is completed, the application moves automatically to Data Review.

To view Data Review

1. To see results for an experiment after you have closed the application, open the application and select a workflow: select Acquisition and Processing or Processing Only.

2. Select an experiment from the Select an Experiment list and click Continue Experiment.



3. Click the Data Review tab to open Data Review, displaying the Component Review table that shows the results of the first sample from the selected experiment.

To copy graphic elements from Data Review

1. Right-click any of the graphics from the top of the page.

2. Choose Copy to Clipboard from the shortcut menu.

3. Paste the graphic into a file.

Time course plot Extracted ion chromatogram (XIC) MS/MS or AIF spectrum

Component review table



Viewing the Component Review Table

The application stores processing results in a table that is sorted by retention time. Using this table, you can select a time point and the application displays graphics for the selected time point. The table lists components in the order of parent, ISTD, and then metabolites, sorting them from low to high retention time within the group. Blank samples are not included in Data Review or in reports.

For experiments with replicates, the results table provides data for each specific replicate under an average row that shows the averaged results based on the included replicates and time points.

You can exclude components, injections, replicates, or time points from processed data; however, you cannot copy, change, or paste component table values. For more information about excluding components, see “Changing Components” on page 99.



Selecting Components

When you open an experiment result, the application highlights the Tn% for the first time point in the first group. If the experiment has replicates, the application highlights the Tn% for the first time point in the first replicate of the first group.

To select a component

When you open the experiment in Data Review, select one of these options for a component:

• Tn% to see the ratio of the component peak area to the parent drug at T0

• TnArea to see the area of the component peak

To help you view and understand the relationship between values for Tn% and TnArea, when you select a Tn% cell, the application colors that cell blue and highlights and colors the corresponding TnArea cell orange. When you select a TnArea cell, the application colors that cell blue and highlights and colors the corresponding Tn% cell orange. The application updates all displays based on the selected cell. Using the tab key and right and left arrow keys you can move along the row within %T and TnArea blocks, respectively. In addition, you can use the up and down arrow key to move within a single column. You can only select cells from these two areas.

For more information about moving around the table, see “Navigating the Component Table Using Arrow Keys” on page 87.



When your experiment has replicates, the application averages values for replicate samples and displays those values in a row above the replicates. You cannot select an averaged value, but if you make changes to the include or exclude value for a selected component, the application recalculates all related average values.

Table 7. Component parameters

Parameter Definition

Group View the name of the group.

Replicate View the number of the specific replicate. The application shows the default value 1 for non-replicated experiments.

Component View the name of the component.

RT (Retention Time) View the apex retention time for the component peak. The application filters the data to show only the metabolites within the range of RT specified in the Metabolite Filtering Options section of the Processing setup. The value is calculated in minutes. The application reports the retention time rounded to 2 decimal places.

Theoretical m/z View the theoretical m/z value (based on component detection rather than a calculated value). The application reports this value rounded to 4 decimal places.

Mass Shift View the mass shift between the parent and expected components. The application subtracts the theoretical value of the parent m/z from the putative metabolite component.

Tn% (For each time point) View the ratio of the component peak area to the parent drug at T0 (n = time of sample).

TnArea (For each time point) View the area of component remaining at the chosen time point as an integer (n = time of sample).

%RSD View the relative standard deviation to the mean (%RSD) for the internal standard. This value measures the accuracy and reproducibility of a test, defining how precise the test will be. This is especially useful when working with replicates. If the ISTD %RSD value is low, the precision is high. The application reports this value to 1 decimal place.

Group Parameters You entered these values on the Sample Setup page before processing the data.

Species View the type of species used for each sample (for example, rat, human, or dog).

Matrix View the matrix type for each sample (for example, blood or urine).

Animal # View the animal ID number.

Route View the route: either in vitro or in vivo.

Dose Amount View the dosage amount and the type of measurement.



Reviewing Results from Replicate Samples

The application averages the results from replicate samples after component detection is finished and provides area values for all successfully acquired components and processed time points.

The application groups items in the result table first by group and then by component type and name, ordering time points from shortest to longest. The application averages the following values for replicates: RT, Tn%, and TnArea.

To view results from replicate samples

1. Open a processed replicate experiment if one is not already opened.

2. Click the Data Review tab and click Component Review.




The component table for replicate experiments identifies each replicate by number. When averaging each group of replicates, the application calculates the average value of each column for each replicate within a component and displays it in the respective column of the average row:

To calculate individual values for Tn% and TnArea for a given replicate component, the application

• Calculates the percent remaining value for each individual replicate at each time point using this formula:

When no ISTD is present:

The application calculates the percent remaining of replicate y at time T this way:

% remaining = 100 * (area at time x, replicate yaverage area of T0s)

When an ISTD is present:

The application averages the percent remaining of replicates x, y, and z at time T this way:

% remaining = 100 * ([area at time t, replicate yISTD area at time t, replicate y][area t0 replicate ISTD t0 replicate x] + [area t0 replicate yISTD t0 replicate y] +[area t0 replicate zISTD t0 replicate z]3)


During processing the application can support the situation where a parent is not present for each and every time point and replicate.

• When calculating percent remaining values, the application treats a missing control as an excluded control.

• If every control in every replicate set of a group is missing, the application can process the group and provide area values but cannot calculate a value for the % remaining.

• The application names metabolites by adding an ordinal to the end of the modification name.

For information about the impact of excluding replicate time points, see “Excluding Replicate Samples” on page 104.

Navigating the Component Table Using Arrow Keys

To move around the table

• Press the left arrow key (or SHIFT + TAB) to move to the left in a row. When the cursor reaches the leftmost value, press left to move to the rightmost value of the row above. You cannot select or navigate to an average row.

• Press the right arrow key (or TAB) to move to the right in a row. When the cursor reaches the rightmost value, press right to move to the leftmost value of the row above.

• Press the up arrow to move up in a column.

• Press the down arrow to move down in a column.



Viewing the Time Course Plot

The Data Review function limits the number of components in the time course plot, plotting only the currently selected component and the parent for that group. If you selected the parent, the application displays only the parent in the time course plot.

This plot compares the amount of the component at any time point to the parent amount at T0 (100%) and displays a percentage of the component. When replicates exist, the application averages the values of the replicates to get the value. The time course plot adjusts the y axis to the height of the highest currently plotted item. If you select a component and the plot displays a percentage of several hundred of the parent drug, you might need to review your information or processing method. You can copy the time course plot and paste it into another software product.

To exclude a time point, see “Excluding Time Points from Groups” on page 101.



Viewing the Extracted Ion Chromatogram (XIC)

Use Data Review to display a chromatogram for the base peak of each component. The extracted ion chromatogram (XIC) is sometimes called an individual ion profile. The application provides an XIC chromatograph in Data Review and in the detailed report for the selected component. The mass range is the theoretical m/z applied to the mass tolerance. The time range is the integrated peak apex RT plus or minus (1.5 * [peak width]). If you selected to include adducts in the sum area option, then the application includes the adduct peak area in the component area. The application draws the chromatogram using a line to define the chromatogram shape and area and excludes anything that is not related to the compound, providing a simple chromatogram. Check this view to see that the integrated area goes from the baseline to the baseline for a peak of interest. You can make changes to the integration and zoom in to see details more closely. You can copy the chromatogram and paste it into another software product.

You can change the chromatogram in the following ways:

• Zoom to view parts of the chromatogram to make sure all components have been included.

• Manually integrate the chromatogram to create a more desired integration (see “Manually Integrating Components” on page 107 for details).

When you click on a cell that has no peak found (NV), the application uses the most intense peak’s apex retention time together with the theoretical m/z to create the XIC, MS/MS scan, full MS scan, and elemental composition results.

When you manually integrate a peak, the application uses the manually integrated peak’s apex retention time together with the theoretical m/z to create the XIC, MS/MS scan, full MS scan, and elemental composition results.



To make visual changes to a chromatogram

1. When the experiment is open, click the Data Review tab.

2. To make changes, select the chromatogram area.

• To make part of the chromatogram larger for a clearer analysis, click the x axis or a section of the chromatogram and drag the cursor across the area you want to look at in an expanded view.

• To return to the original graphic, right-click and choose Reset Scaling.

You can also choose Copy to Clipboard to paste the chromatogram to another document format.



Viewing the MS/MS or AIF Scan

To determine the metabolite relative structure or to determine whether a component (a putative metabolite) is a true metabolite for the parent compound, use the fragmentation data found in the MS/MS scan. The application displays this scan in both Data Review and in the detailed report.

You can copy the MS/MS plot and paste it into another software product.

The application selects scans for Exactive, Q Exactive, or LTQ Orbitrap hybrids in the following order:

• If available, the number 1 option is displayed

• If not, then if available, the number 2 option is displayed, and so forth

1. Single selected FTMS MS/MS scans (high-resolution, accurate mass data selected using precursor ions)

2. Single selected ITMS MS/MS scan (low-resolution data selected using precursor ions)

3. All Ion Fragmentation (AIF) scan (no precursor selected fragmentation). If the AIF scan is data dependent, the precursor ion m/z needs to be within the scan range of the AIF scan.

4. No scan (no fragment scan of any type is available)

The application displays the following details in the MS/MS spectrum header:

• Raw file name

• Scan number

• Retention time

• Nominal level (NL)

• Scan filter



Viewing Elemental Composition Information

To view information about the accurate mass, full MS scan profile, and calculated elemental composition of a selected component, click the Elemental Composition Review tab. You can also make changes to the component grid. For information about managing the component grid, see “Viewing the Component Review Table” on page 82.

• Viewing Elemental Composition Results

• Viewing the Full MS Scan Display

For information about making changes to the component table, see “Changing Components” on page 99.

To review composition results

In Data Review, click the Elemental Composition Review tab to see composition results.

Full MS scan display Elemental composition table

Component table




Viewing Elemental Composition Results

The elemental composition table contains the calculated elemental compositions. You cannot make changes to the elemental composition table.

The application calculates accuracy for the elemental composition determined for the parent and expected modifications, measuring how far each identified elemental compound is from the theoretical m/z of the target compound. The application displays that value in the ppm accuracy column in the elemental composition table. If measuring against a real value, 100.0000, and the application displays a value of 100.0001, the measurement is off by 0.0001. The measurement is equal to 1 part per million (0.0001/100*1000000). In this case the result is positive because the measurement is higher than the expected value. If the measurement is 99.9999, the application reports the same 1 ppm difference, reporting it as negative because the measurement is lower than the real value.

To view the elemental composition table

In Data Review, click the Elemental Composition Review tab to view composition results.

The application adjusts the entries in the elemental composition table when you navigate through the component table or change peak integration in the XIC plot.



Viewing the Full MS Scan Display

The application displays the full MS scan at the peak apex retention time for the selected component. The full MS scan is displayed in Data Review under the Elemental Composition Review tab and in the detailed report. The default view displays the x axis and you can zoom in on the x axis and y axis to clearly view the theoretical component m/z value and baseline.

The application provides the following information in the header of the full MS scan display: the raw file name, scan number, retention time, nominal level (NL), and scan filter. If there is no full MS scan available, the application notifies you about this issue in the graphic location.

To scale the full MS scan display

1. To make part of the full MS scan display larger, click the x axis and drag your cursor across the area you want to increase.

2. To return to the original graphic, right-click and choose Reset Scaling.

3. Select Copy to Clipboard to paste the full MS scan display to another document format.


Working with Component Results in Data Review

The MetQuest application can filter data displayed in Data Review using custom filters. The application applies these filters at the component level and filters the entire component either in or out. To make it easier to interpret data, the application can also sort data within groups by these values:

• Retention time

• Intensity

• Mass shift from parent

• m/z value

To filter or sort viewed data in Data Review, open the Filtering and Sorting Options window by clicking Filter and Sort and make your selections.

See these sections for information about filtering or sorting in Data Review.

• Filtering Components in Data Review

• Sorting Elements in Data Review



Filtering Components in Data Review

To filter viewed data

1. In Data Review, click Filter and Sort to open the Filtering and Sorting Options window.

2. To hide peaks that are smaller than a specified percentage of the area of the parent, select the % Parent Area Threshold check box in the Filtering Options area and type a value in the box.

The application grays out the check box if there is no T0 available or if T0 is not found.

Valid values: 0.0 to 100.0 Default: 5.0

3. To view a specific number of components, select the Top N Components check box and select the number of components to display.

Valid values: 1.0 to 10.0 Default: 10.0

When multiple components have the same most intense relative area, they are all displayed in Data Review and in the reports.

4. To view peaks that fit better (fit > 0) with the suggested component formula by having the application discard the 0 fit peaks, select the check box for the Elemental Composition Spectral Fit Score > 0 check box.

The application compares the differences between theoretical and measured isotope masses and intensities, creating a spectral fit score. If you select the check box, the application displays only peaks with a positive spectral fit score.

Default: Unchecked

Changes you make in Data Review, including selected filtering options, are reflected in the reports.



If a component doesn't have at least one non-excluded cell which has numerical absolute area associated with spectral fit score greater than 0, the application filters out the entire component.

If a component has at least one area cell that is non-excluded and has a numerical absolute area value associated with at least one spectral fit score greater than 0, the application includes the entire component (all replicates) in the view.

The application only applies filters to non-excluded cells.

Sorting Elements in Data Review

For easier interpretation of the data, the MetQuest application can sort data within groups by these different values:

• Retention time

• Intensity

• Mass shift from parent

• m/z value

To sort viewed data

1. In Data Review, click Filter and Sort to open the Filtering and Sorting Options window.

2. In the Sorting Options area, select a method to use as a sort mechanism:

• Retention Time

• Most Intense (%)

• Mass Shift

• m/z

3. Select a sorting order, either Ascending or Descending.

• Descending (the largest value to the smallest value)

• Ascending (the smallest value to the largest value)

After selecting the method for sorting the data, you can sort by descending order (the largest value to the smallest value) or ascending order (the smallest value to the largest value).

When two components have the same value, the one with the lowest retention time appears first in the grid.



4. Click OK.

The application sorts the grid according to the order you defined.

• The application always sorts the rows below an average row based on the replicate number from lowest to highest.

• The application continues to enforce specified sort order preferences. If you exclude or include a cell, the application adapts the table order to match the changes.

• The application saves the sort order with the experiment.

Within a group, components are displayed as follows:

• The parent is always the first component.

• The internal standard, when present, is always the second component.

• The average rows of all other components are sorted according to your selections.

In the following example, Data Review displays the original data order:

After sorting the data by most intense peak in descending order, Data Review displays the following:



Changing Components

Use the component grid to exclude components, injections, replicates, or time points from processed data based on examination of data results. You might exclude a component or time point for one of these reasons:

• The formation (appearance/disappearance) of the component might not be consistent with a real metabolite.

• The fragments observed might be inconsistent with the parent drug.

• The mass shift of the component from parent drug might be inconsistent with any known or explainable biotransformation.

After you exclude a unit from the calculation, the application retains the unit but prevents it from appearing in any report generated after you exclude it. Excluded units appear in red.

For instructions about saving changes that you make in Data Display, see “Saving Changes in Data Review” on page 108.

To include any excluded unit (highlighted in red), right-click and choose the appropriate option from the shortcut menu.

See these topics for changing components.

• Excluding a Single Sample

• Excluding Time Points from Groups

• Excluding Time Points from All Groups in an Experiment

• Excluding a Time Course from Groups

• Excluding Replicate Samples

• Manually Integrating Components



Excluding a Single Sample

Use Data Review to exclude a single sample (single cell exclusion). You might choose to exclude a single injection because of an injection error, an unusual internal standard performance, or a sampling error.

The application excludes the excluded injection from the calculation of averages. In addition, if the excluded cell is an internal standard, the application excludes all cells calculated using values from that internal standard. If the cell represents the most intense peak for a replicate, then the next most intense peak for that replicate determines the retention time for that replicate, recomputing retention time.

To remove a single sample

1. Select the sample in the sample grid.

2. Right-click the time point and choose Exclude Component From Injection to exclude it.

To include the time point again, right-click the time point and choose Include Component From Injection.

When the time point is a single occurrence of T0, the application excludes the group. When it is a single occurrence for another time point, the point values are excluded from calculations and from the AD plot. The application automatically draws a straight line between the two points on either side of the excluded point.The excluded point is not included in reports.

When you exclude the last defined area or % from a time point, the application reports the average as 0, rather than NV.

When the time point is a replicate, the application automatically recalculates all averages and does not include any excluded components in reports.

When the values of an area or % for an excluded time point across an experiment are all NV, then the summary row reports the average as NV. If you exclude only some of these NV cells, the Average row displays NV.

When the value of an area or % has a value for that time point, and you exclude that time point, the application excludes the value from the calculation of the average in the summary row.

When a cell has NV in the cell, something happened during acquisition or processing and there is no value for that sample. This might be because a sample got dropped before acquisition. You can include or exclude columns, rows, or cells with NV in them. The application uses the following rules for cells containing NV.

• When a column (time point) within a group or experiment has all NV values, the application does not remove the time point. You can manually exclude this time point (see “Excluding Time Points from Groups” on page 101).



• You can click in an NV cell to see the XIC chromatogram based on the most intense time point (retention time and mass) and full MS scan for the cell to verify that nothing existed at that point. If a peak exists, you can manually add it (see “Manually Integrating Components” on page 107).

Excluding Time Points from Groups

Use Data Review to exclude a time point (a single column) from a group due to injection error, sampling error, internal standard error, instrument error, and so forth. This procedure excludes the time point across the entire group. The application does not show excluded time points on generated reports.

When the values of an area or % for an excluded time point across an experiment are all NV, then the summary row reports the average as NV. If you exclude only some of these NV cells, the Average row displays NV. If the value of an area or % has a value for that time point, and you exclude that time point, the application excludes the value from the calculation of the average in the summary row and does not display the point in the appearance/disappearance plot. If you exclude the last defined area or % from a time point, the application reports the average as 0, rather than NV.

To exclude a time point in a group

1. Select a single time point in the sample grid.

2. Right-click the time point and choose Exclude Time Point From Group to exclude it.

3. To include the time point again, right-click the time point and choose Include Time Point From Group.



Excluding Time Points from All Groups in an Experiment

Use Data Review to exclude a time point (a single column) from the entire experiment due to injection error, sampling error, internal standard error, instrument error, and so forth. This procedure excludes the time point across the entire experiment. The application does not show excluded time points on generated reports or on the appearance/disappearance plot.

When the values of an area or % for an excluded time point across an experiment are all NV, then the summary row reports the average as NV. If you exclude only some of these NV cells, the Average row displays NV. If the value of an area or % has a value for that time point, and you exclude that time point, the application excludes the value from the calculation of the average in the summary row. If you exclude the last defined area or % from a time point, the component grid reports the average as 0, rather than NV.

To exclude a time point in an experiment

1. Select a single time point in the sample grid.

2. Right-click the time point and choose Exclude Time Point From All Groups to exclude it.

To exclude or include all time points, use a time point from the T0 column.

3. To include the time point, right-click the time point and choose Include Time Point From All Groups.



Excluding a Time Course from Groups

Use Data Review to exclude a single replicated time course (a single row) due to injection error, sampling error, internal standard error, instrument error, and so forth. This procedure excludes the time course from the entire group. The application does not show excluded time courses on generated reports.

When the values of an area or % for an excluded row are all NV, then the summary report displays a blank. If you exclude only some of these NV cells, the Average row displays NV. If the value of an area or % has a value within that row, and you exclude that row, the application excludes the value from the calculation of the average in the summary row. If the last defined area or % is excluded from that row, the application reports the average as 0, rather than NV.

To exclude a time course in a group

1. Select a single point in the time course.

2. Right-click the time point and choose Exclude All Components From Injection to exclude the time course. The application requests confirmation.

3. Select Yes to exclude the row or No to leave it in the group or experiment.

If you delete an average time course in a replicate study, the application excludes all replicates that provided calculations used for the average values.

The application displays excluded points in red.

To include the time course again, right-click the time point and choose Include All Components From Injection.

To retain a cell in the time course, see “Manually Integrating Components” on page 107 to manually include it.




Excluding Replicate Samples

For an experiment with replicate samples, the application supports excluding replicates that cannot be used due to these common errors:

• Poor injection performance

• Sampling error

• Internal standard error

• Instrument error

Excluded replicates are not used in the calculation of average results. The application automatically calculates these results when you exclude a replicate. If you exclude a component, the application excludes any replicates that exist for that component. When excluding replicate samples, these rules apply:

• You can exclude a T0 control because the value is calculated from the average of the remaining T0 controls, even if only one T0 remains.

• When you exclude a T0 value, the application calculates a new average T0 value and recalculates all time points based on the new average.

• When you exclude any replicate at a time point, the application calculates a new average for that time point.

• When a single sample cannot be acquired or processed, the application displays it as NV. This sample is not used for any calculations, including the number of replicates.

• To exclude an average row, exclude all related replicates of the sample.

The following example shows a data point at 8152.09% of the parent drug. This point is probably a contaminant, so you can remove it from the data review plot and the reports.



To remove this point and see the appearance/disappearance curve of the parent more clearly, right-click the area value of the point and choose Exclude Component From Injection.

After excluding the point, the application colors the point red, excludes the point from the related average, and corrects the plot from the updated average row. If you select the deleted point, the application displays the spectrum so that you see why you deleted the point.


To exclude replicates

1. Select the replicate.

2. Remove the replicate using one of these options:

• To remove the single time point, right-click the time point and choose Exclude Component From Replicate to exclude it.

To include the time point, right-click the time point and choose Include Component From Replicate.

• To remove an entire time course series (a row), right-click the time point and choose Exclude All Components From Replicate to exclude it.

To include the time point, right-click the time point and choose Include All Components From Replicate.

• To exclude all entries for a time point from the group, right-click the time point and choose Exclude Time Point From Group to exclude it.

To include the time point, right-click the time point and choose Include Time Point From Replicate.

• To exclude all entries for a time point from the experiment, right-click the time point and choose Exclude Time Point From All Groups to exclude it.

To include the time point, right-click the time point and choose Include Time Point From All Groups.

After excluding the requested unit, the application moves you to the last selection after setting the check/uncheck state and recalculating averages. The application also changes all visual displays to respond to the changes.



Manually Integrating Components

Use Data Review to modify any incorrect integration. A naturally occurring component might have been divided between two or more smaller peaks. To include the area of the smaller peaks with the larger component peak, you can zoom in on the peak (both x and y axes zoom) and manually join or exclude the peak area using the chromatogram view. The application immediately updates the integration on the % area table and on the Time Course plot.

To perform manual integration in the chromatogram view

1. If an experiment is not displayed in Data Review, open an experiment.

2. Select a sample.

3. To include an adjacent peak, right-click and choose Use Manual Peak Integration.

4. Starting from the baseline, drag the cursor to include the new area.

When you release the mouse button, the application updates values for the sample to include the second peak. The application shows the newly defined area in dark blue.

When you manually integrate the parent at T0, the application recalculates all relative areas.

When you manually integrate any ISTD, the application recalculates all relative areas of components that calculate values using that ISTD.

When you manually integrate any peak that is not an ISTD or Parent at T0, the application updates the absolute area and relative area of that peak.

To check your decision, right-click the new peak and choose Use Automatic Integration to display the original peak. To save your changes and display them in the reports, click Save.

For all manually integrated peaks, the application updates the apex retention time, the area, the relative area, the full MS scan, the best MS/MS scan, and the elemental composition results.



Saving Changes in Data Review

To save your Data Review changes

After making changes to both views in Data Review, click Save.

The application saves the current state of Data Review to a file.

When you click Save, the application includes the automatic integration results, manual integration results, include/exclude state, recalculated areas, and percent remaining.

When you click Generate Reports, the application automatically saves your changes. If you switch between tabs or close the application, the application reminds you to save your changes.

Defining Report Options for Data Review

After making changes to your experiment processing results, you can generate reports that contain your changes. Report types include group-level summary and detailed reports and experiment-level CSV reports.

To create reports after reviewing data

1. To create reports from Data Review, click Generate Reports.

The application opens the Report Options dialog box displaying report options. These options are set to the choices in the processing method.

To change report options, see “Setting Report Options” on page 32. To display report processing, the application opens the Processing area in the Real Time Processing Display area.

Changes in the dialog box do not change the experiment setup settings. To change your experiment setup, click the Experiment Setup tab and click Configuration Options.


7 Reviewing Results from the ApplicationViewing Experiment Log Results

2. Click OK to generate reports or click Cancel to close the dialog box without creating reports.

When you click OK, the application processes the reports and saves them to the experiment folder. After processing the reports, the application displays the following message:

3. Click Yes to go to the Reports view.

The application takes you to the report page and displays an active list of reports where you can click a report name to view the report (see “Viewing Experiment Reports” on page 110).

Viewing Experiment Log ResultsThe application keeps a record of acquisition and processing events and errors for all experiments. The application stores the log (ProcessingLog.xml) in the experiment folder after an experiment is processed. The log contains information on the processing of each sample within a group (events and errors), the processing of a group (creation of time courses, reports, and so on), and the completion of each experiment.

To view the experiment log within the application

1. Open an experiment.

2. In Data Review or in the Reports view, click Processing Log.

The application displays the log for the open experiment.

3. To view acquisition and processing information about a specific time point, select the time point.


7 Reviewing Results from the ApplicationViewing Experiment Reports

Viewing Experiment Reports You can view reports generated for finished sequences in the Reports area. The application provides two levels of reports for each experiment if you selected those levels. It also provides a CSV file for importing into other data systems. You can also view a page containing links to all reports for that experiment.

The application lists all reports for an experiment, providing hypertext links to specific reports. Use the Reports view to access these reports. The application by default displays the Report List when you open the Reports view.

• Viewing Summary Reports

• Viewing Detailed Reports

To view a list of reports for an experiment

1. If the experiment is not open, click the Workflows tab and double-click the experiment name from the Select an Experiment list to open it.

2. Click the Reports tab to view available reports.

3. In the Reports view, select a group name to display a list in the Group Reports area of all reports from that group's directory. The column headers are Group, Report Type, Report Format, Directory, and File.

4. Select the report name to open the report in the appropriate application for that file type:

• .pdf files in Acrobat viewer

• .xls files in Microsoft Excel

• .csv files in Microsoft Excel



For more information and a summary report view, see Viewing Summary Reports.

The detailed report contains specific metabolic information broken down by individual time points, a chromatogram, more detailed information on parent elemental composition with a full MS scan spectrum, and, if available, an MS/MS or AIF scan spectrum. The application provides elemental composition information with a spectrum for each identified metabolite later in the detailed report. For more information and a detailed report view, see Viewing Detailed Reports. Blank samples are not included in Data Review or in reports.

To print reports, see “Printing Reports” on page 122.



Viewing Summary Reports

The summary report contains the following:

The application displays NV cells and excluded cells in a summary report using the following rules:

• When you exclude an NV cell, there is no change in the report.

• When you exclude a cell at a time point that has an area value but the component has an area value for any other time point, the application displays a blank for the area % at the excluded time point.

• When you exclude a cell at a time point that has an area value, the application recomputes the average and displays the average in the summary report. If you exclude the most intense time point cell, the application changes the retention time of that replicate to the next most intense time point that is not excluded.

• When you exclude all values for a component or if the component has NV for all replicates and all time points in Data Review, the application omits the component in all reports.

• For any replicate (row) whose time points contain all NVs, the application shows that replicate with an NV in the report and displays the average of the area and % area calculations for all replicates for each time point in the table, and the title of the table changes to indicate there is an average value in the table.

• However, if all replicates are NV or excluded in all time points, the application omits that component from reports.

• When in Data Review, you have excluded all time points in a summary row (making them NV), the time points in the summary report table will show NV as well.

• The application never hides columns (time points) from a report even if all values are NV.

Header section Provides a summary of the group information and processing and acquisition settings.

Time course plot Shows the appearance and disappearance of the parent drug and metabolites.

Component average by % Shows the % to parent area calculation for all time points, including the parent drug and all found metabolites.

Component average by area Displays the peak area for all time points for all found metabolites.

ISTD compound summary table

Displays RSD% and peak area for all time points and replicates if an ISTD is present.



To view summary reports

1. Select a group name from the Groups list on the left.

2. To display group information, click Summary Report in the dropdown box above the List area.

–or–

Click the link to a summary report in the Reports List view.

The application displays the summary report for the group.



The report contains these items:

• Summary information about the group, containing acquisition and processing settings, including filter and sort options.

• The time course plot of expected metabolites showing the amount of parent drug and metabolites over time.

In this section of the report, you can see the metabolic rate of the parent drug and the appearance and disappearance over time of expected metabolites. The metabolites in the time course plot are normalized to the parent drug (100 percent at T0). To indicate potential large impurities that might indicate a problem with the sample, the application plots these in a strict relationship to the parent drug at 100 percent (at T0). The plot displays the parent drug and the 9 most intense metabolites. The report does not display the ISTD compound.



• A table of the parent drug and all found metabolites with the area% (relative to the parent at T0) of each metabolite at each time point. The table is useful for viewing time course behavior of low-level metabolites. If the experiment has replicates, the report displays the average area%.

• A table of metabolites with the area of each metabolite at each time point. The table lists the peak area over the time course for the parent drug and metabolites.

• A table containing the internal standard areas for each injection if you included an internal standard. Use this table to check the sample preparation, autosampler performance, instrument analysis, and area response for all internal standards as a review of performance.



Viewing Detailed Reports

To view detailed reports

1. Select a group name in the Groups list to display group information.

2. Click Detailed Report.

The application displays a multi-paged detailed report for the group.



The detailed report contains these items:

• Header section, displaying summary information about the group with details from the acquisition and processing workflows.

• Detailed table for each time point in the time course that lists the parent drug and metabolites. This table also includes the area and the area percent for all metabolites and the internal standard (if there is one) when compared to the parent drug peak area. The actual area of a peak helps to calculate formation rates and half-lives, generate more absolute quantitation, and so forth.

The table shows the retention time and theoretical m/z for all components and the amount of the parent drug present.

The Relative to T0% column shows the relative quantity as a percentage of the metabolites based on the quantity of the parent drug (always listed as 100 percent at T0).



The table displays a breakdown by time course by metabolite. Each time course starts with the parent compound followed by the ISTD, and then by metabolites (in order of increasing RT).

When a component in a replicate row is excluded or has NV at a time point, it is omitted from detailed reports at that time point for that replicate.

• Detailed information about the compounds at the most intense time point across all replicates. It shows the parent drug first, followed by the ISTD if available, and then expected metabolites by retention time.


7 Reviewing Results from the ApplicationWorking with an Experiment CSV File

The information includes the following:

• Name or metabolite ID

• Time point number and replicate number of the sample with the most intense peak area for the compound

• Theoretical m/z

• Elemental composition results table

– Calculated formula

– Spectral fit score

– Parts per million (ppm)

• Extracted ion chromatogram (XIC)

• Full MS scan spectrum

• MS/MS scan or AIF scan if available

Working with an Experiment CSV FileTo generate a report that transfers results from a single experiment into other processing tools or a database, use the Experiment CSV Export option in the Report Options area. The application names the report ExperimentName_YYYYMMDDHHMMSS.csv and saves it to the experiment_name\Experiment Reports folder.

You can select the experiment CSV option before acquisition or after data review. The file contains all data in the Data Review component review table reported to full precision as well as sample setup information and sort or filter settings. The application displays all excluded cells and NV cells in the detailed report as blanks.

When the area for all time points for all replicates become NV or are excluded, the application does not display that metabolite in the CSV report.



When a replicate at all time points has a value of NV or has been excluded, but another replicate has an included value, the application displays all replicate rows in the report. When an imported CSV file contains a blank with a time point or replicate assigned to it, the application grays out the columns and ignores the time value or the replicate number.

Reports with excluded or NV cells that you can open in an Excel spreadsheet show an empty cell.

To open an experiment CSV file

1. If the experiment is not open, click the Workflows tab and select the experiment name from the Select an Experiment list to open it.

2. Click the Reports tab to view available reports.

3. Click the experiment CSV title for the report.

The file looks similar to this example report.

To make changes to an experiment and regenerate a CSV file

1. Open the experiment if one is not already opened.

2. To make changes and reprocess the experiment, click the Experiment Setup tab.

3. Make changes to the processing method, if needed.

4. Click Submit to reprocess the experiment. The application displays the results in Data Review.

5. Make changes to the filtering options in Data Review, if needed.

6. To generate the report from the existing Data Review display, click Generate Reports.



Your file contains the following column headings with sample information.

Column heading Definition

Group Name of group.

Replicates Number for replicate within the group.

Component Name of component.

Formula Parent drug formula.

Retention Time Apex retention time for the component peak.

Theoretical m/z Theoretical mass-to-charge ratio (m/z) for the component.

Mass Shift The mass shift from the parent for expected components.

Tn % (For each time point)

Percent of component remaining, normalized to parent at the time for each time point (n = time of sample).

Tn Area (For each time point)

Area of component remaining at the chosen time point as an integer (n = time of sample).

RSD (%) The relative standard deviation from the mean (RSD) for the internal standard. This value measures the precision of the internal standard area across all the injections for a group. If the RSD% value is low, the precision is high. This value is blank for every column except the internal standard. The application reports this value to 1 decimal place.

Group Parameters

Species Type of species used for each sample (for example, rat, human, or dog).

Matrix The matrix type for each sample (for example, blood or urine).

Animal # The animal ID information.

Route In vitro or in vivo.

Dose Amount Dosage amount (include the type of measurement after the amount, for example, μL).

Time The time point definition.

Replicate When replicates exist, the replicate number.

Sample ID The ID for each sample. (This value is not shown in the reports.)

Maximum length: 255 alphanumeric characters

File Name A unique file name for the original raw file. The MetQuest application saves the raw file as RawfileName.raw. This value is required for acquisition. File names must be unique for each sample.


Sample Type Select Control, Blank, or Sample as applicable. This value is required for automatic processing. The application uses the control file for background comparison to remove peaks present in the control.


7 Reviewing Results from the ApplicationPrinting Reports

Printing ReportsFrom the Reports view, you can scale a report within the reports view or print a report. See “Setting Report Options” on page 32 for information about setting or changing report print options.

To print reports

1. Display the report in the Reports view.

2. Click to print from your default printer.

3. Click OK.

Comment Type an optional comment for these samples. This information is not used in processing.


Metabolite Sort Options The options selected for sorting the data: Ascending or Descending.

Metabolite Filtering Options

The filtering options selected for data display.

Column heading Definition


8

Configuring Instruments for Acquisition

Use the Instrument Setup view to create an instrument method to process samples and acquire data. The MetQuest application can acquire data using the Exactive or Q Exactive instrument. For more information about processing data from the LTQ Orbitrap family of mass spectrometers, see “Processing Data Acquired Using the MetQuest Application” on page 60.

Configuring Instruments To configure instruments for use with the MetQuest application, add those instruments to the system.

After submitting a batch for acquisition and processing, you cannot change the instrument configuration until after the application has finished processing.

To add or remove instruments, follow the appropriate procedure:

• Adding Hardware Devices to Your System Configuration

• Removing Hardware Devices from Your System Configuration

Contents

• Configuring Instruments

• Working with Instrument Methods


8 Configuring Instruments for AcquisitionConfiguring Instruments

Adding Hardware Devices to Your System Configuration

To add hardware devices

1. To make changes to your hardware configuration, close any open Thermo Scientific software applications.

2. Choose Start > Programs > Thermo Foundation n.n > Instrument Configuration where n.n is the version of Thermo Foundation.

The Instrument Configuration window opens.

3. From the Device Types list, select the type of hardware device to add to your instrument configuration.

The Instrument Configuration window displays all available configurable devices as icons in the Available Devices area.

4. Select the icon of the device you want to add.

If you do not see the device you want to configure, you might need to install the device driver.

5. To add the device to the Configured Devices area, click Add.

The Instrument Configuration application copies the selected Available Devices icon to the Configured Devices area, displayed as a Configured Devices icon.

To quickly add a device from the left column, double-click the device icon.



6. To configure a device, select the device icon in the Configured Devices area and click Configure or to quickly configure a device, double-click the device icon in the right column.

The configuration dialog box for the selected device opens. This dialog box is for the Thermo Exactive instrument.

7. Enter all required configuration information for the device. Complete entries and options for all pages.

The options on each page are different for every device.

8. To save settings and close the DeviceName Configuration dialog box, click OK.

The Instrument Configuration window stays open.

9. Repeat steps 3 through 8 for all devices to be configured.

10. To save settings and close the Instrument Configuration window, click Done.

Once you have added all hardware devices to the system, see “Working with Instrument Methods” on page 127 for information about setting up an instrument method to work with the application.



Removing Hardware Devices from Your System Configuration

To remove hardware devices from the system

1. To make hardware changes effective, close any open Thermo Scientific applications.

2. Choose Start > Programs > Thermo Foundation n.n > Instrument Configuration where n.n is the version of Thermo Foundation.

The Instrument Configuration window opens.

3. To select a device to remove from the configuration, select the device icon in the Configured Devices area.

4. Click Remove.

The Instrument Configuration application removes the selected Configured Device icon from the Configured Devices area.

5. Repeat steps 2 through 4 for all devices to be removed.

6. To save settings and close the window, click Done.


8 Configuring Instruments for AcquisitionWorking with Instrument Methods

Working with Instrument MethodsUse the Instrument Setup view to define or change a set of analytical parameters for the configured instrument. These parameters include the operating settings for instruments you plan to use (collectively referred to as the instrument method).

• Creating Instrument Methods

• Changing an Instrument Method

• Importing an Existing Instrument Method

Creating Instrument Methods

Use the Instrument Setup view to define a set of analytical parameters for the configured instrument. The parameters that are displayed in the Instrument Setup view depend on the instrument that you select from the vertical tab bar at the left of the window. If the instrument you want to configure is not displayed in this vertical tab bar, see “Configuring Instruments” on page 123 for information about adding the instrument. To view supported instruments, see “System Requirements” on page ix.

You can also define instrument settings to be used before or after the application runs an experiment (the startup method and shutdown method, respectively).

• Define and use a startup method when the instrument has been idle for a length of time that might impact results. A startup method generally performs all functions in the analytical method including injecting a sample, but it does not acquire a raw file. Because the system injects fluid during the startup method, use a blank to condition or wash the autosampler. This blank does not get processed.

• Define and use a shutdown method when there are no samples immediately following so that the instrument is clean and ready for more processing.

To create an instrument method

1. Double-click to open the MetQuest application. From the Workflows view, choose a workflow type. Select Acquisition Only or Acquisition and Processing to view, define, or make changes to an instrument method.

2. Click the Instrument Setup tab to display the Instrument Setup view.

3. Click an instrument device tab from the vertical bar on the left.

The application displays the method editor for that device under the tab and displays the setup view for the instrument method. You can open a previously opened instrument method by clicking Open.



4. Make changes to the instrument parameter values.

The options displayed are related to the specific requirements and capabilities of the device. For help setting parameter values, refer to the documentation for the specific hardware device by going to Start > Thermo Instrument > Manuals > LC Devices.

If the device for the instrument method you want to define is not on the list, make sure the device has been added to the system. See “Configuring Instruments” on page 123.

5. To set parameter values, repeat steps 2 to 4 for each device on your configured system. When you have defined parameters in an existing instrument, save the method using one of these options:

• To save changes to an existing method, click Save above the Instrument Setup view.

• To save the method with a different name, click Save As, type a new name, and click Save.



Changing an Instrument Method

You can change an instrument method after creating it to optimize the data acquisition conditions. After submitting an experiment for acquisition and processing, you cannot change the instrument configuration until the application has finished processing. After acquisition and processing, you can change the instrument configuration.

To change an instrument method

1. In the Instrument Setup view, click Open to open a browse box displaying current instrument methods.

The application opens to the default location unless you changed the location. In that case, the application opens to your last defined location.

2. Select an existing method.

The Instrument Method view displays the current values for that method.

3. Click an instrument tab to display the method parameters for a specific instrument.

4. Change parameter values for any of the devices and click Save Instrument Method in the Instrument Setup view.

The application prompts you to save the method.

If you click No, choosing to not save the instrument method, the application restores instrument default values rather than your original method.

Importing an Existing Instrument Method

Instead of creating a new method, you can import an existing instrument method created from outside the MetQuest application. You can use the method with existing parameter settings or you can edit the method.

To define instruments in use and select a starting instrument, see “Defining Acquisition Parameters” on page 34.

To import an instrument method

1. In the Instrument Setup view, click Open to open a browse box.

2. Select the name of an instrument method (.meth) that was created in another application.

After the MetQuest application displays the method, make changes to it and click Save.


9

Working with Templates

The MetQuest application provides example templates to make data entry easier. You can enter sample information or expected modifications using templates in C:\Thermo\MetQuest\ Templates\. To import information from your LIM (Laboratory Information Management) system, use the defined column headings in the order presented in the template.

Use these procedures to create templates containing commonly used information.

Contents

• Creating Files with Group and Sample Information

• Working with a Metabolite Modification List for Processing


9 Working with TemplatesCreating Files with Group and Sample Information

Creating Files with Group and Sample InformationTo enter a group of samples for acquiring or processing an experiment easily, use the ImportCSVTemplate.csv sample template in C:\Thermo\MetQuest\ Templates\. To enter a group of replicate samples, use the sample template and identify replicates by number. The template includes the following:

All samples must be part of a group. Do not create an import file that has more than 100 groups or 1500 samples. You can enter up to 15 time points in one group or a combination including one control and 14 samples.

To create a file defining acquisition information for multiple samples

1. Open ImportCSVTemplate.csv to create import files for individual samples or replicate samples.

You can open any of the templates in any type of editor, including Microsoft Notepad. You can also open a .csv file in Microsoft Excel to manage information more easily.

2. Enter the group and sample information to define acquisition.

For more information about valid values, see “Defining Experiment Information for Groups” on page 20 and “Defining Sample Information Manually” on page 26. Follow these tips:

• When you reference a .mol file, you must include a fully qualified path to that file.

• When you import a .csv file, the application removes all existing groups and sample information in the current experiment, including the processing method. Add a processing method for each group. The application fills in a 5 μM default for the injection volume unless you provide a value.

• Do not put spaces in file names. Do not include file paths for an Acquisition Only or Acquisition and Processing workflow. The application checks for path names and you must remove the path information before submitting the experiment to acquisition.

group name time point

processing method sample type

parent name species

formula matrix

sample ID dose amount (μL)

file name.raw (including a full path for the Processing Only workflow)

animal #

replicate number if there are multiple samples at the same time point in the group

route

vial position MOL file path

Injection Volume



• For processing non-MetQuest files, type the full path name including the .raw extension.

• If you selected an Acquisition Only or Acquisition and Processing workflow, the application ignores file paths for samples (if provided in the .csv file) and stores processing results in C:Thermo\MetQuest\Experiments\experiment_name under the group file name. Each group folder contains a folder for acquired data and one for reports.

• If you selected a Processing Only workflow, you must include a fully justified file path for each sample, storing processing results in C:Thermo\MetQuest\Experiments\Groups\ under the group file name.

• Using definitions from the first record, the application replaces group information for the remaining records in that group in the columns for Group Name, Parent Name, Formula, Species, Matrix, Dose Amount (μL), Animal #, and Route.

• If you provide an inaccurate vial position, the application displays an error message when you submit the samples. For more information about the autosampler vial or plate position format, refer to your autosampler documentation.

• All samples in an experiment must use time points from the same group of time points.

• When your experiment uses replicates of samples within a group, provide a replicate number for identification. Each replicate set (each row) can have its own control at time T0. The first replicate set must start with replicate number 1. Even if you delete a replicate set, replicate numbers must be consecutive.

After you fill in the information, the file should look similar to the following:



For an experiment containing replicates, after you fill in the information, the file should look similar to the following:

3. Click Save As to give the file a unique name and save it as a .csv file in the project folder. Refer to Excel documentation for instructions about saving a file as a .csv file.

To use this sample list, see “Submitting and Reviewing an Experiment” on page 36. If you import the file, the application removes all currently displayed group and sample information and prompts you to save the sequence under a unique name.

To export a .csv file to use for a new experiment

1. In the Experiment Setup view, click Export.

2. Give the file a name and save it to a location.

3. Make changes for the new experiment.

4. Save the file.



After you fill in the information, the file should look similar to the following:

For an experiment containing replicates, after you fill in the information, the file should look similar to the following:

5. Open a new experiment.

6. Import the changed file into Sample Setup.


9 Working with TemplatesWorking with a Metabolite Modification List for Processing

Working with a Metabolite Modification List for ProcessingYou can use one of the modification lists provided by the MetQuest application or make changes to a list and save it under a different name. To populate the expected metabolite list, use the .csv files to contain information. Once you have created a file, you can import it into the processing method.

• Phase I modifications includes oxidative, reductive, and hydrolytic reactions.

• Phase II modifications involves conjugative reactions, for example, glucuronide conjugation.

To open a modification list

1. In the application, click the Processing Setup tab and click Metabolite Options.

2. In the Expected Metabolites area, click Import and browse for one of the metabolite modification lists. Select the list name and click OK.

Choose from five factory modification lists, stored in the C:\Thermo\MetQuest\Templates\ directory.

Default: MetQuest_PhaseI_Common.csv (The application uses this list unless you select another one.)

• MetQuest_PhaseI_Common.csv

• MetQuest_PhaseI_Complete.csv

• MetQuest_PhaseI+II_Common.csv

• MetQuest_PhaseII_Common.csv

• MetQuest_PhaseII_Complete.csv


9 Working with TemplatesWorking with a Metabolite Modification List for Processing

To make changes to the list

1. Open one of the modification list template files in either the Excel application or a text editor.

If you open the file as a .txt file in a text editor, you do not need to reformat the file. If you import the report in .xls format, you must format the formulas in the file for import.

2. To import a file to an Excel spreadsheet, do the following:

a. Open a blank work book in the Excel application.

b. Click the Data tab and click Get External Data.

c. Click From Text to display a browse window.

d. Browse to the C:Thermo\MetQuest\Templates folder.

e. Select the ImportModificationListTemplate.csv file.

f. Click Import.

g. Choose the Delimited option and click Next.

h. Select the Comma delimiter and click Next.

i. Select the Formula column.

j. Change the column data format to Text and click Finish.

3. Add metabolites, identifying the following values:

• Modification

• Formula

• Mass shift

4. Save the list under a different name before importing it into the MetQuest application.


I

Index

Symbols.mol file

internal standard 49parent drug 21

%Parent Area Threshold, setting 46

Aabundance

intensity error weighted 55mass error weighted 55

acquired data, processing 60acquiring data 61acquisition

defining a sequence 15queue, removing samples 72

Acquisition and Processing workflow 3Acquisition Only workflow 3adding

groups 18hardware devices 124instruments to MetQuest 123

animal #, group information 22arrow keys in the component table, using 87

Bbest fit score, setting 46

Ccalculating averages in the component table 86changing

chromatogram in the Data Review area 89group information 19instrument method 129processing method 57

changing modification lists 136chromatogram

changing in the Data Review area 89Reset Scaling 90

Component Review, graphic 81

component tablecalculating averages 86Data Review

parameters 84selecting components 83using the arrow keys 87

componentschanging in Data Review 99integrating in Data Review 107manually integrating 107

configuringhardware devices 124multiple instruments 128

contacting us xiiControl Comparison

Drift Tolerance 44Intensity Ratio Threshold 45

control file, using 42creating

processing method 42templates 132

DData Review

changing components 99changing the chromatogram 89Component Review area 81component table

calculating averages 86parameters 84using the arrow keys 87

defining report options 108elemental composition results 93Elemental Composition results, viewing 92elemental composition table 93excluding

replicate samples 104replicates 106single sample 100time course 103time points 101time points from all groups 102


Index: E

extracted ion chromatogram (XIC) 89filtering components 96full-scan display 94integrating components, introduction 107manually integrating components 107MS/MS scan 91recalculation when excluding replicate samples 104reviewing results from replicate samples 85saving changes 108selecting components 83sorting components 97time course plot 88viewing an experiment 80

definingelemental composition values 49experiment information 15group information 19internal standard 49parent information 19

deleting groups 19detailed reports

contents 117parent drug information 118viewing 116

documentation survey xiidosage, group information 22double bonds 52–54Drift Tolerance, setting values 44

EElemental Composition area

charge states 51double bond and ring equivalents 52mass tolerance 36, 51Max Results 52Nitrogen Rule 51RDB Equiv 52

elemental composition, Data Review results 93excluding

replicate samples in Data Review 104replicates in Data Review 106time course in Data Review 103time points from experiments 102

Expected Intensity error (%), Spectral Fit area 55expected metabolites list

editing 137using 47

experiment log, viewing in MetQuest 109experiments

defining 15export 65import 65

extracted ion chromatogram (XIC), viewing in DataReview 89

FFiltering and Sorting Options window 96filtering components in Data Review

% Parent Area Threshold 96Elemental Composition Spectral Fit Score > 0 96Top N Components 96

filtering metabolites 46finding groups 20formula

ISTD 49parent drug 8, 21

full-scan displayin Data Review 94scaling 94

Ggetting a license xgroup information

animal # 22dosage 22matrix 22molecular weight (MW) 22name 22polarity 22, 49route 22source 22species 22

groupsadding 18changing information 19defining parent drug 20deleting 19finding 20renaming 19

Hhardware devices, adding and configuring 124


Index: I

Iimporting

files from templates 132instrument method 129

ImportModificationListTemplate.csv, using 47instrument method

changing 129creating 127definition 127importing 129Instrument Setup window overview 127selecting for acquisition 33

instrument statusacquisition queue 71changing scaling 70copying 70device status 72viewing 70

instrumentsadding to MetQuest 123configuring multiple 128selecting (Instruments in Use) 34selecting starting instrument 33

Instruments in Use 34integrating components 107Intensity Ratio Threshold

including peaks 45values 45

internal standard.mol file 49defining 49for processing 49molecular weight 49MW 49name 49polarity 49source 49

ISTDformula 49using 42

Llicense, getting xlist, expected metabolites 47, 136

Mmass tolerance 36, 51matrix, group information 22Maximum Results 52

metabolitesfiltering 46finding 42list 42

methodchanging 57reviewing 57

modification listsexpected metabolites list 47opening 136

molecular weight (MW), group information 22MS processing, specifying values 48MS/MS scan in the Data Review 91

Nname, group information 22Nitrogen Rule 51

Pparent drug

.mol file 21defining processing 20formula 8, 21Mol file 8, 21, 49

PDF reports, selecting 32polarity

group information 22, 49internal standard 49

printing reports 122processing

acquired data 60configuration file, setting parameters for 57data acquired using MetQuest 60data in an .sld file 65managing errors 78previously acquired data 60stopping processing 76viewing log 77viewing saved log 77

processing methodcreating 42finding metabolites 42for previously acquired data 60, 66using 41

Processing Only workflowdefining 3processing data acquired using MetQuest 60

processing previously acquired data 61Processing Setup, accessing 43


Index: R

RReal Time Acquisition Display

acquisition queue 71controls 73device status 72introduction 69status information 72viewing instrument status 70

Real Time Processing Displayerror management 78introduction 73open area 74stop processing 76viewing log 77viewing processing status 77

relative quantitation, calculating 48renaming groups 19replicate experiments, component table 82replicate samples

defining 27excluding 106excluding in Data Review 104recalculating when excluding 104reviewing results 85

reportsintroduction 110options

Data Review 108Report Options area 32

printing 122saving 122

reprocessing processed data 61retention time window 44reviewing a method 57ring equivalents 52–53route, group information 22

SSample Setup view 6, 17samples

defining experiment information 15defining group information 18defining information in sample grid 26defining information using an imported file 30excluding in Data Review 100real-time sample acquisition information 72real-time sample processing information 77

saving changes, Data Review 108

Select an Experiment list, using 60, 62selecting components from the component table 83shutdown method, defining 127sorting components in Data Review

intensity 97m/z value 97mass shift 97retention time 97

source, group information 22species, group information 22Spectral Fit area, Expected Intensity error (%) 55startup method, defining 127Sum Area Option (adding adducts) 50summary reports

appearance/disappearance plot 114contents 114viewing 113

survey link xii

Ttemplate, types 132theoretical m/z range, setting 46Time course plot, Data Review 88time course, excluding in Data Review 103time points

excluding from all groups 102excluding in Data Review 101

Vviewing

an experiment in the Data Review area 80experiment log 109

Wworkflow

Acquisition and Processing 3Acquisition Only 3Processing Only 3

XXLS reports, selecting 32


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