pathfinder 1.0 administrator guide version...

PathfinderVersion 1.0

Administrator Guide

XCALI-97242 Revision A June 2009

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2009 Thermo Fisher Scientific Inc. All rights reserved.

Xcalibur is a registered trademark of Thermo Fisher Scientific Inc. in the United States.

Windows, Excel, and Windows Vista are registered trademarks of Microsoft Corporation in the United States and other countries. Adobe, Acrobat, and Reader are registered trademarks of Adobe Systems Incorporated in the United States and other countries.

Pathfinder is a trademark of Thermo Fisher Scientific Inc.All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

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: Pathfinder Version 1.0.

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

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vRelated Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viSystem Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viContacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chapter 1 Getting to Know Pathfinder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Understanding Pathfinder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2How Pathfinder Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Quick Access to Pathfinder Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Using the Pathfinder Administrator Application . . . . . . . . . . . . . . . . . . . . . . . . . 5

Chapter 2 Getting Started with Pathfinder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Opening Pathfinder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Analyzing a Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Analyzing Groups of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Viewing Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Chapter 3 Working with Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Working with Instrument Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Creating an Instrument Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Changing an Instrument Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Working with Pathfinder Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Creating a Pathfinder Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Changing a Pathfinder Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Working with Method Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Creating a Method Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Modifying a Method Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Changing the Active Method Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Deleting a Method Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Chapter 4 Working with Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Submitting a Single Sample for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Submitting Multiple Samples for Analysis Manually . . . . . . . . . . . . . . . . . . . . . 52Creating Files for Importing Lists of Multiple Samples . . . . . . . . . . . . . . . . . . . 54Submitting an Imported List of Samples for Analysis . . . . . . . . . . . . . . . . . . . . 55Viewing Instrument Status During Processing. . . . . . . . . . . . . . . . . . . . . . . . . . 56

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Contents

iv Pathfinder User Guide Thermo Scientific

Chapter 5 Viewing Workflow Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59Viewing Data Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Viewing Results for a Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Viewing Peak Details. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Viewing Specific Details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Viewing Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Modifying Sample Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Working with peaks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Reprocessing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Changing Parameter Values for a Single Sample . . . . . . . . . . . . . . . . . . . . . . 76Reprocessing a Group of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Chapter 6 Managing Users and the Pathfinder System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79Working with Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Adding Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Changing User Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Removing a User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Defining System Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Configuring Pathfinder on Remote Workstations . . . . . . . . . . . . . . . . . . . . . 83Setting Up a Virtual E-Mail Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85Configuring Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Initializing Pathfinder Using Xcalibur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Setting Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Appendix A Pathfinder Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99Pathfinder Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Tools Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Go Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Method Development View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Method View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Instrument Configuration Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116Method Group Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Analysis View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Data Review Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Reports Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Real Time Status View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

User Administration View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121User View Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121User Mode Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Survey Screening Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Target Matching Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Target Screening Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125

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Preface

This guide describes how to use Thermo Pathfinder to analyze a variety of samples.

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

Contacting Us

Thermo Scientific Pathfinder User Guide v

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Preface

Related DocumentationPathfinder includes Help and these manuals as PDF files:

Pathfinder Quick Start

Pathfinder Administrator Guide

To view product manuals

Go to Start > Programs > Thermo Pathfinder 1.0 > Manuals.

To open Help

From the Pathfinder window, choose Help > Contents.

In a specific window or dialog box, click Help or press F1 for information about setting parameters.

For more information, including upcoming application notes, visit www.thermo.com/appnotes.

System RequirementsIn addition, your system that supports online instruments must meet these minimum requirements.

System Requirements

PC 2 GHz processor with 2GB RAM CD-ROM drive Video card and monitor capable of 1280 1024 resolution (XGA) 75 GB or greater available on the C: drive NTFS format

Software Microsoft Windows XP Professional with Service Pack 3 Microsoft Office 2003 or 2007 Xcalibur 2.1

vi Pathfinder User Guide Thermo Scientific

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Preface

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 copy manuals from the Internet

Go to mssupport.thermo.com and 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.thermo.com/lcms-techpubs.

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

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1

Getting to Know Pathfinder

Pathfinder is a data acquisition, processing, and reporting system for users who have a range of experience levels with liquid chromatography mass spectrometry (LC-MS).

This chapter provides a basic overview of Pathfinder and demonstrates how you can use Pathfinder in the work environment. For information about configuring Pathfinder, see Defining System Information on page 82.

Contents

Understanding Pathfinder

How Pathfinder Works

Quick Access to Pathfinder Tasks

Using the Pathfinder Administrator Application

Thermo Scientific Pathfinder User Guide 1

1 Getting to Know PathfinderUnderstanding Pathfinder

Understanding PathfinderYou can access Pathfinder in one of two different environments, as Administrator or User. These names represent two different and nonoverlapping access modes. The following table describes what each account name allows.

You can use Pathfinder to develop and save multiple sets of experimental definitions (methods) and related information. Then you can run these saved instruction sets to automatically process and report on LC-MS data. Pathfinder works with high-mass accuracy and high-resolution data sets, such as elemental composition for data analysis, reduction, review, and reporting.

Table 1. End-user Modes for Pathfinder

Account name Tasks and permissions

Administrator Develops and tests methods for sample analysis with manual and automated data processing.

Develops data review and reporting formats. Analyzes sample run data. Manages the user mode accounts.

User Submits samples for analysis. Receives the results of automated sample analysis and

data processing.

2 Pathfinder User Guide Thermo Scientific

1 Getting to Know PathfinderHow Pathfinder Works


How Pathfinder WorksFigure 1 illustrates the workflow to use when setting up and analyzing your data. It includes the workflow for both administrators and users. Click workflow entries in bold to go to the section describing that process.

Figure 1. Pathfinder workflow overview for both Administrator and User accounts

Pathfinder

LC-MS technique produces raw data

Specify general method information

Save and distribute method

View spectra graph and reports

Administrator mode

Define peak processing

Define MS processing

Define report settings

Test method

Analyze results

Click workflow

User mode

Select method

Enter user and sample ID

Enter sample file name (Enter target molecular weight)

Collect printout

Click Go to run sample

Note Optional steps are italicized.

1 Getting to Know PathfinderQuick Access to Pathfinder Tasks

Quick Access to Pathfinder TasksThe following table shows you the location in Pathfinder to perform tasks in Administrative mode:

Task Task location in Pathfinder

To run a sample See Getting Started with Pathfinder on page 9.

To create or change a processing methodSee Working with Pathfinder Methods on page 21.

To specify general method information Click Method Development. Select New method or Open Method in the Method Development view. Click the General tab.

To define processing values for a Target Screening method

Select Target Screening as a workflow on the General page and click the MS Processing tab.

To define peak processing for Target Matching and Survey Screening methods

Select Target Matching or Survey Screening as a workflow on the general page and click the Peak Processing tab.

To define MS processing for Target Matching and Survey Screening methods

Click the MS Processing tab.

To define report settings for Target Matching and Survey Screening methods

Click the Reports tab.

To save method Click Save method in the Method Development view.

To test method Click User Administration in the Method Development view. Select a test type under User Mode in the User Administration view.

To view results See Viewing Workflow Results on page 59.

To view test results Click Analysis in the Method Development view. Click Open sequence. Click Data review or Reports view to verify test data.

To configure instruments See Configuring Instruments on page 89.

To add, open, or save instrument methods to configure instruments

Click Method Development. Click Instrument Methods in the Method Development view.

To add or change method groups Working with Method Groups on page 44.

To add, modify, activate, or delete method groups

Click Method Development. Click Method Groups in the Method Development view.


1 Getting to Know PathfinderQuick Access to Pathfinder Tasks

To add or change users See Working with Users on page 80.

To add, modify, delete users Click User Administration in the Method Development view. Click User View in the User Administration view.

Task Task location in Pathfinder


1 Getting to Know PathfinderUsing the Pathfinder Administrator Application

Using the Pathfinder Administrator ApplicationThis section describes the main sections of the Pathfinder Administration GUI.

After you log into Pathfinder, the main window opens.


1 Getting to Know PathfinderUsing the Pathfinder Administrator Application

This page displays three main buttons.

Icon Description Areas

Opens the Method Development view where you can do the following:

Create, modify, and save instrument methods

Create, modify, and save processing methods.

Define and activate method groups

Click the button at the left of a view to hide the options. Click the name of a view to display the options.

Opens the Analysis view where you can do the following:

View real time status and instrument processing

Open, save, or reprocess a sequence

View data information for a specific sequence

View reports for a sequence



Opens the User Administration view where you can do the following:

Process sequences using the User Mode

Create, modify, and delete users


Icon Description Areas

2


Getting Started with Pathfinder

Pathfinder guides you in the analysis of one or more LC-MS samples using predefined methods to produce automated reports. This quick start chapter describes how to submit samples in User mode.

Click the icon below to see a video explaining how to process data.

Contents

Opening Pathfinder

Analyzing a Sample

Analyzing Groups of Samples

Viewing Reports

2 Getting Started with PathfinderOpening Pathfinder

Opening PathfinderTo open Pathfinder

1. Choose Start > Programs > Thermo Pathfinder 1.0 > Pathfinder or double-click the Pathfinder icon on your computer desktop. The Pathfinder logon screen opens.

2. Click User. The Pathfinder User window opens.


2 Getting Started with PathfinderAnalyzing a Sample

Analyzing a SampleUse one of the Pathfinder workflows to submit a sample for analysis. You must define a processing method before the user can analyze a sample (see Creating a Pathfinder Method on page 21).

To run a single sample

1. From the Pathfinder User window, choose a workflow.

To view peak results, click Survey Screening. Adjust peak processing values to define an area of interest.

To search for a target compound with a specified molecular weight, click Target Matching.

To search for several compounds in a library, click Target Screening.

The page for that workflow opens.

2. Type a unique personal identifier in the User name box. Pathfinder uses this ID to notify you when reports are available.

3. Type the sample identification in the Sample ID box.

4. Type the autosampler vial position in the Vial position box.

If the vial position value is not in the correct format for your autosampler, Pathfinder displays an error message when you click Go. For more information about the autosampler vial position format, see your autosampler documentation.


2 Getting Started with PathfinderAnalyzing a Sample

5. Type the name of the sample data file in the File name box, unless Pathfinder grays out the file name.

6. If the workflow is Target Matching, type the monoisotopic molecular weight that you want to match in the Target MW box.

The molecular weight entered must be the monoisotopic molecular weight and not the average molecular weight.

7. Select a method from the Method area. When you click a method name, Pathfinder displays a method description if available to help you choose the appropriate method.

8. Click Go to start the sample analysis. If the method specifies a printed report, Pathfinder prints a report using the printer selected in the method.


2 Getting Started with PathfinderAnalyzing Groups of Samples

Analyzing Groups of SamplesTo run a group of samples using an imported sample list

1. From the Pathfinder User window, choose a workflow. The page for that workflow opens.

2. Type a user name in the User name box.

3. Select a specific method from the Method area.

4. Click Import to add a file containing sample information to the method. A browse window opens. Browse to where your sample files are stored. Sample files are initially stored in C:\Thermo\Pathfinder\Templates.

5. Select the samples file and click Open. In the Sample ID table, make sure each sample has the correct vial position.

6. To change the information on any sample, select the sample and enter data in the boxes to the left. Click Update to save the changes.

7. Click Go. If the method specifies a printed report, Pathfinder prints it using the printer selected in the method.


2 Getting Started with PathfinderAnalyzing Groups of Samples

To analyze a group of samples using a manually created sample list

1. From the Pathfinder User window, choose a workflow. The page for that workflow opens.

2. Type the user name in the User Name box.

3. Select a specific method from the Method area.

4. Enter sample information:

a. Type the first sample ID in the Sample ID box.

b. Type the first autosampler vial position in the Vial position box.

c. Type the file name of the sample data file in the File name box, unless Pathfinder supplies the name. Each sample must have a unique file name.

d. If you chose Target Matching, type a molecular weight in the Target MW box. The molecular weight entered must be the monoisotopic molecular weight. It cannot be the average molecular weight.

e. Click Add. The sample information is displayed in the table to the right.

5. Type information for the remaining samples, repeating all of step 4 for each sample.

6. To change the information on any sample, select the sample and enter data in the boxes to the left. Click Update to save the changes.

7. Click Go. If the method specifies a printed report, Pathfinder prints it using the printer selected in the method.


2 Getting Started with PathfinderViewing Reports

Viewing ReportsWhen viewing a Target Screening report, look for the following:

In the summary report, a table of all compounds from the library with the retention times when they were found, the m/z value found, the difference from the expected value, and an intensity calculation. Also, information on the adducts found and any matching HCD fragments.

In the long report, all the information from the summary report for each compound. Also, if adducts are identified, the mass chromatogram for each adduct and, if matching fragments are found, the mass chromatogram for the fragment.

There is an option to generate reports on only found compounds or on all compounds in the user library.

When viewing a Survey Screening report, look for the following:

Information on each peak found including a chromatogram that highlights the peak found, a mass spectrum of that chromatographic peak, HCD spectrum (if applicable), and area and intensity calculations for that peak.

A list of calculated formulas for the most intense peak in the mass spectrum.


2 Getting Started with PathfinderViewing Reports

When viewing a Target Matching report, look for the following:

Information on any peaks that match the target m/z.

For each match, a chromatogram showing the match, the mass spectrum, the HCD spectrum (if applicable), and area and intensity calculations for the matched peak. Also, a graphical representation of the peak area integration.

A list of calculated formulas for the target match candidate from the mass spectrum.

A table on the first page of the target matching report that shows all peaks found for the target mass. If the peak matches the boundary conditions specified in the method, then Pathfinder displays Yes in the match column.


3

Working with Methods

A Pathfinder method represents the set of instructions for the acquisition, processing, and reporting to be performed by the LC/MS system on a sample submitted for analysis. This chapter describes how to create and modify methods.

Working with Instrument MethodsAn instrument method defines the data acquisition parameters for a sample, resulting in a data file that can then be processed using the other elements of a Pathfinder method. You can use this data file in other software applications, such as Xcaliburs Qual Browser.

Use the Instrument Method view to change the options and parameters for each of the devices that are available as part of the system configuration. The devices include the autosampler (AS), liquid chromatograph (LC), and mass spectrometer (MS).

You can create new instrument methods, modify existing instrument methods, and save instrument method files. When you define an instrument method, it resides on the instrument where you define the method.

Contents

Working with Instrument Methods

Working with Pathfinder Methods

Working with Method Groups

Note Before you use the Instrument Method View, use the Instrument Configuration program to select the instrument and devices for your analyses. For more information, see Configuring Instruments on page 89.


3 Working with MethodsWorking with Instrument Methods

On the left, the Instrument Method View displays the icons of all available devices you have selected using the Instrument Configuration window. Each tab in the horizontal tab bar opens the setup parameters for a specific available device.

To enter the setup parameters for a particular instrument, click the tab for that device. The Instrument Method View then displays one or more pages of parameters that you can set.

Use the following steps to create or change an instrument method. You must create at least one instrument method before running samples.

Creating an Instrument Method

Changing an Instrument Method




Creating an Instrument Method

Use an instrument method to define the data acquisition options and parameters that are available on the individual devices that make up the analytical system, that is, the autosampler, HPLC pump, mass spectrometer and so forth.

To create an instrument method

1. In the Method Development area, click New instrument method in the Instrument Method view.

2. Click an instrument device tab on the horizontal tab bar. Pathfinder opens the method editor for that device in the workspace and displays options related to the specific requirements and capabilities of the device. For help setting parameter values, see the documentation FOR that hardware device.

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 89.

3. To set parameter values, repeat steps 2 and 3 for each device on your configured system. When finished, click Save instrument method in the Method Development area.

4. Type a name in the Save dialog box and click OK.


Changing an Instrument Method

You can change an instrument method after creating it to optimize the data acquisition conditions.

To change an instrument method

1. In the Method Development mode, click Open instrument method in the Instrument Method view. The Open Method dialog box opens.

2. Select an existing method. The Instrument Method view opens, displaying the list of configured devices in a horizontal tab and the method editor in the workspace. Select an instrument tab to open the method page for a specific instrument.

3. Change parameter values for any of the devices and click Save instrument method in the Method Development area.

4. To save this modified method under a new name, choose File > Save as.


3 Working with MethodsWorking with Pathfinder Methods

Working with Pathfinder MethodsA Pathfinder method, created in the Method View, specifies the instrument method used for data acquisition and defines the data processing and reporting parameters for a sample. When you process a sample, Pathfinder provides a set of result files and, if you specify it, a set of electronic and hardcopy reports. Review these result files using Pathfinder's Analysis mode.

The Method View displays the options and parameters to be used for each of the data processing steps that are available as part of the overall workflow configuration. These include descriptive information, data acquisition, peak processing, mass spectrometer-specific processing, and reporting. You can create new Pathfinder methods, modify existing methods, and save method files.

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

Creating a Pathfinder Method

To create a Pathfinder method, complete these procedures:

Defining General Values for a Method

Defining Processing Values for Target Matching and Survey Screening

Defining Target Screening Settings

Choosing Report Types

Contents

Creating a Pathfinder Method

Creating a Processing File for Target Screening

Changing a Pathfinder Method

Working with Method Groups



Defining General Values for a Method

Pathfinder requires some information, such as the selection of an instrument method, when using a Pathfinder method. Other information, such as the assay type, is optional. Use the General tab of the Method View to enter these values.

To enter general information about the method

1. Double-click the Pathfinder icon on your desktop or choose Start > All Programs > Thermo Pathfinder 1.0 > Pathfinder 1.0.

2. Click Administrator on the opening screen and enter the password.

For access to the Administrator mode, type your system password (thermo is the default password and might be different on your system. Contact your system administrator if the password is different.).

3. Select Method Development on the left side of the window.

4. Select New method. Method View opens to the General Page.

5. Type the name of the lab in the Lab name box.

6. Type the name of the test in the Assay type box.

7. To define the amount of the sample used for analysis, click the up and down arrows in the Injection Volume (L) box or type in a specific value. Enter a value from .1 to 100.

8. Select an instrument method from the Instrument method box. For more information about instrument methods, see Creating a Pathfinder Method on page 21.



9. To select a processing workflow, select from the Workflow list:

To perform overall peak processing and elemental composition (for the mass spectrometer data), click Survey Screening.

To perform overall peak processing and elemental composition (for the mass spectrometer data), and then compare each MS detected peak to a user-supplied target molecular weight with a goal of finding a target compound in a sample, click Target Matching.

To find compounds in a database in the sample, click Target Screening.

To specify descriptive information about this method, enter text into the Description box. Users will see this information when selecting a method for submission of a sample or a set of samples.

Defining Processing Values for Target Matching and Survey Screening

Use the following steps to define processing values for Target Matching and Survey Screening. For a Target Screening workflow, see Creating a Processing File for Target Screening on page 41.

Setting Peak Processing Values

Defining Peak Detecting and Integration Values

Setting High (ICIS) Integration Values

Setting Standard (Genesis) Integration Values

Specifying MS Processing Values



Setting Peak Processing Values

On the Processing page in the Peak Processing view, you can select one or more data sources to be used for peak detection. Use the other pages to define specific peak detection parameters for the data sources selected. If you choose the Target Matching workflow, Pathfinder hides peak processing options because they are selected using a different view. Options that are grayed out are not available for your selected workflow.

To detect peaks

1. Click the Peak Processing tab to define peak parameters. The Processing Page opens.



2. In the Data Source area, select the data traces Pathfinder will process for peak detection. For each of these detector types, you might have one or multiple channels of data that need to be processed, stored, and visualized.

Select an MS option to designate one or more data traces for Pathfinder to process for each of the available scan filters from the MS detector. When processing negative and positive data, you can get elemental composition results for the positive mode or the negative mode, but not both. To get elemental composition results for both the negative and positive mode, process the modes separately, being sure to set the correct charge state in elemental composition for each polarity. Processing an MS data source results in a peak list with associated mass spectra:

To process a specific mass range, select the Mass Range check box. Use this option with a Target Matching workflow only.

To process a total ion current [TIC] chromatogram, select the TIC check box. Use this option for a Survey Screening workflow.

To process a base peak chromatogram, select the Base Peak check box.

For PDA data, valid trace types are Total Scan or Spectrum Maximum. Processing a PDA data source results in a peak list with associated PDA spectra:

To process the data trace for the summed wavelength intensities, select the Total Scan check box.

To process the data trace for the spectrum maximum, select the Spectrum Maximum check box.

To select UV detector data, select a channel in the UV channel area. For UV detector data, Pathfinder supports four channels (labeled Channel A-D). UV results only provide a peak list.

To select data, select from the A/D Card area. For data from an A/D card, Pathfinder supports two channels (labeled A/D 1-2). A/D results only provide a peak list.

3. To set delay offsets:

To set the offset for PDA data, type a value in minutes in the PDA offset (min) box.

To set the offset for UV data, type a value in minutes in the UV offset (min) box.

To set the offset for analog data, type a value in minutes in the Analog offset (min) box.

To set the offset for A/D data, type a value in minutes in the A/D offset (min) box.



If you selected a Target Matching workflow, Pathfinder also displays options for target matching.

4. For the targeting range, select an adduct in either the Positive ion adducts or Negative ion adducts area to generate a target m/z value. You can select more than one adduct. The most common adduct is H.



Pathfinder calculates the m/z of the ionized molecular ion with the specified adduct. This step is necessary to compensate for the difference between the measured m/z value of an ion for a particular compound (resulting from the ionization process) and that compounds non-ionized molecular weight.

For example, in LC-MS using positive polarity API, the ion can be formed by the addition of a positive charge (proton) to the compound. In this instance, the observed ions m/z value for that compound is shifted by 1.0073 amu relative to its molecular weight. Since the values that you submit for target matching are given as the monoisotopic molecular weight of the compound of interest, the measured ion m/z value must have the value of any adduct present subtracted from it to determine the observed non-ionized molecular weight and make the appropriate comparison to the target molecular weight.

Define the value to add or subtract by selecting one or more adducts from the available list or by specifying a custom adduct if the choice is not available (for example, a unique LC mobile phase chemistry).

Pre-defined adducts and their adjustments include the following:

5. Define the width of the mass window. Pathfinder uses your value above and below the mass. The default is 5 ppm. Pathfinder uses this value for the mass tolerance for target matching.

6. If you are specifying details for a Target Matching workflow, set a value for the Minimum acceptable relative intensity by entering a value from 1 to 100. Compounds that match the mass specified in the workflow but that fall below the intensity threshold will not appear in the report because the peak was not defined as a match. This setting is not available for survey screening.

Positive ion Negative ion

Adduct Adjustment Adduct Adjustment

H + 1.0073 H - 1.0073

H3O + 19.0178

NH4 + 18.0338

Na + 22.9892

K + 38.9632



7. To enter specific values for finding peaks, in the Peak Processing view, click the tab for a type of detector (MS, PDA, UV, or A/D Card). The page for that detector type opens.

8. Specify a sensitivity level for the peak detector algorithm by selecting from the Sensitivity list. For information about setting peak integration values, see Defining Peak Detecting and Integration Values on page 29.

To use high sensitivity levels for detection, select High (ICIS). This setting, designed for mass spectrometers, performs a more exhaustive peak processing pass.

To use standard sensitivity levels for detection, select Standard (Genesis). This setting performs a comprehensive peak processing pass.



9. To define the retention time range for peak processing, select the Limit the retention time range check box. Specify a start (Min RT) and end time (Max RT) for the range.

10. To turn on a peak threshold for defining the lower limit for peak retention, select the Enable peak threshold check box.

Define the lowest value of the threshold as a percentage of the largest peak.

To evaluate peaks by height, select the By height option, or to evaluate them by area, select the By area option. By area is the default option.

11. To store a defined number of peaks by size, starting with the largest peak, select the Only select top peaks check box.

Define the number of largest peaks to store by clicking the up or down arrow in the Only select top peaks box. You can also type a value using the keyboard.

To evaluate peaks by height, select the By height option, or to evaluate them by area, select the By area option.

Defining Peak Detecting and Integration Values

Follow these procedures to set detection and integration values for algorithms that control the sensitivity of the peak processing results. If you choose the target matching workflow, Pathfinder hides peak detection and integration options. Options that are grayed out are not available for your selected workflow.






1. Select High (ICIS) in the Sensitivity list. The ICIS Peak Integration Area opens.

2. In the Smoothing points box, define the number of points to use for chromatogram smoothing. Type an odd number from 3 for minimum smoothing to 15 for maximum smoothing. To perform no smoothing, type 1 in the box.

3. To specify the number of scans to review, looking for a local minima, type a value in the Baseline window box. The valid range is 1 through 500. The default value is 40 scans.

4. To define the noise level multiplier used to determine the peak edge after the location of the possible peak, type a value from 1 through 500 in the Area noise factor box. Pathfinder excludes peaks that are less than the selected threshold.

5. To specify the noise level multiplier used to determine the potential peak signal threshold, type a value from 1 through 1000 in the Peak noise factor box.

6. To limit the peak width of a component during peak integration of a chromatogram, select the Constrain peak width check box. You can then set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor.

7. To change the peak height value and test the width of target peaks, enter a value in the Peak Height box. You can enter any value from 0 to 100 percent.

8. To control how Pathfinder integrates the tail of a peak, enter a value from 0.5 through 9.0 in the Tailing factor box. This factor is the maximum ratio of the trailing edge to the leading side of a constrained peak.




To set Genesis peak integration values

1. Select Standard (Genesis) in the Sensitivity list. The Genesis Peak Detection Area opens.

2. Define the number of points to use for chromatogram smoothing. Type an odd number from 3 for minimum smoothing to 15 for maximum smoothing in the Smoothing points box. To perform no smoothing, type 1 in the box.

3. Define the signal to noise (S/N) threshold for detecting peak edges. The default value is 0.5 and the valid range is 0.0 to 999.0. Pathfinder excludes peaks that are less than the selected threshold.

4. To help detect and separate unresolved peaks, select the Enable valley detection check box to use the Genesis valley detection approximation method. This method drops a vertical line from the vertex of the valley between unresolved peaks to the baseline. The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak.

5. To set the expected peak width parameter (in seconds), type a value in the Expected width (sec) box. This value defines the minimum width that a peak is expected to have and is used with the valley detection option to determine unresolved as opposed to asymmetric peaks.

6. To limit the peak width of a component during peak integration of a chromatogram, select the Constrain peak width check box. You can then set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor.

7. To change the peak height value and test the width of target peaks, type a value in the Peak Height box. You can type any value from 0 to 100 percent.

8. To control how Pathfinder integrates the tail of a peak, enter a value from 0.5 through 9.0 in the Tailing factor box. This factor is the maximum ratio of the trailing edge to the leading side of a constrained peak.



Specifying MS Processing Values

Use the MS Processing tab of the Pathfinder method to define the unique processing requirements for the mass spectrometer data of the sample. If you are defining processing requirements for a Target Matching or Sample Survey workflow, select the Elemental Composition tab to specify the options for the Spectral Fit algorithm used for determining elemental formulas based on mass spectral ions and their intensities. Use the following sections to define processing values for mass spectrometry processing.

Use the MS Processing: Elemental Composition Page to set values that calculate one or more possible empirical formulas for a particular chromatographic peak. Options that are grayed out are not available for your selected workflow.

When you process a Survey Screening sample, the base peak is the largest peak in the sample defined by height. For a Target Matching method, Pathfinder finds the peak closest to the value entered in the Target MW field. The resulting list of peaks is sorted according to the spectral fit value.

Pathfinder uses the isotope patterns and intensities surrounding the base peak to provide a more rigorous match than the standard elemental composition models. In the Elemental Composition view, you specify spectral fit parameters. The algorithm does the following:

Identifies the base peak (by height) in a particular peaks mass spectrum.

Determines the monoisotopic peak, if not the base peak.

Examines a set of ions around the monoisotopic peak, including the monoisotopic cluster.

Calculates all elemental composition candidates based on a single exact mass (usually the monoisotopic mass).

Scores the match 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 are not similar, the fit factor is 0 percent.

Sorts the elemental composition candidates in terms of decreasing fit. The candidate with the best fit factor is considered to be the most likely match.



To define elemental composition values for survey screening and target matching

1. Click the MS Processing tab. The MS Processing: Elemental Composition Page opens.

2. Define the expected charge state by entering a value from -99 to 99.

3. Select whether or not to use the Nitrogen rule in the formula calculation. Select from Do not use, Odd electrons, or Even electrons.

4. To restrict the number of possible elemental compositions, enter a value in the Mass tolerance box. Pathfinder returns results of the elemental composition search only if the computed formula matches the observed mass within the specified tolerance. Pathfinder uses this value for mass tolerance throughout the application for elemental composition (acceptance and monoisotopic mass determination).

5. To set the values for double bonds and ring equivalentsa measure of the number of unsaturated bonds in a compoundand 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.

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



7. To use the standard carbon isotope distribution for elemental composition, select the Use representative elements check box (13C abundance = 1.070%). To use the carbon isotope distribution that is common in proteins, clear this option (13C abundance = 1.086%).

8. To select elements to use in the element formula candidate, click to display the periodic table. Select an element and click Apply to add the element to the list. If the element is already in the grid, you can activate it by selecting the use check box.

To remove elements from use in the elemental formula, select the element in the grid and click .You can also turn off the element by clearing the use check box.

To define the number of elements in a calculated candidate, specify a maximum or minimum value in the grid.



9. To set advanced parameters, including those for spectral fit results, click Show Advanced. The Advanced area opens. To close the Advanced area, click Hide Advanced.

10. To specify the expected mass error in the spectrum data, enter a value in the Expected mass error box. This value is 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 expected mass error. The expected mass error must be specified in ppm (parts per million).

11. Enter a value in the Expected intensity error box so that the algorithm knows the expected intensity error of the mass spectrometer. 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 expected intensity error value.

12. Set a penalty to identify peaks that are specifically searched for but not found. If the theoretical isotope pattern of a candidate and the measured isotope pattern are compared, Pathfinder might not find a measured packet for a theoretical isotope peak. This is called a missing peak.

13. To measure an observed mass error on an isotope peak upon its abundance, select the Mass error weighted by abundance check box. This ensures that more abundant masses have a smaller percentage of errors than less common masses.



14. To measure an observed intensity error on an isotope peak upon its abundance, select the Intensity error weighted by abundance check box. More abundant intensity readings have a smaller percentage of error than less common ones.

15. To measure intensity errors by absolute value rather than by percentage, select the Absolute intensity errors check box.

16. To specify the threshold spectrum enhancement method as automatic, select the Automatic intensity threshold check box. This method limits the number of ions in the final spectrum prior to library searching by applying an intensity threshold. If the intensity of an ion is below the specified threshold, the ion is discarded from the spectrum. To set a value for the intensity threshold, use the up and down arrow or type a value.



Choosing Report Types

Use the Reports tab to set report values for Survey Screening and Target Matching workflows. Report values for the Target Screening workflow are set on the Target Screening page, so you cannot set report values for Target Screening on this page.

To specify report types

1. To select a report, click the Reports tab. The Reports Page opens.

2. To generate a particular report, select the Active Report check box next to the report name. You can also select the Print, Create PDF, and Create XML check boxes as needed to specify the output type of final report.

To create the report, select the Active Report check box. You must select this box to create any type of report.

To print the report, select the Print check box.

To create a PDF version of the report, select the Create PDF check box. This report file resides in the Reports folder in the batch folder.

To create an XML version of the report, select the Create XML check box. Use this version to create a record that you can import into a LIMS or electrical LIMS notebook. This report file resides in the Reports folder in the batch folder.

3. To select a printer for the method, select a printer from the printer list. If you do not select a printer after selecting the Print check box, Pathfinder alerts you when you try to save the method.

Do not choose a printer driver that requires user entry into a dialog box before generating electronic files. An example of this type of printer is the Microsoft XPS Document Writer.

4. In the Method Development area, click Save Method to save the method and make it available for users.



Defining Target Screening Settings

To set values for Target Screening

1. Click the MS Processing tab. The Target Screening page opens.

2. Browse to select a .csv file as the target database. To edit this file, click Edit. The file opens in Excel and you can add or change values.

3. To get a report, choose from the listed options. Select Summary report: Create PDF to get a one page report of the processing results, select Long report: Create PDF to get a detailed report of reprocessing. You can select to print summary and long reports.

4. To specify a local printer that Pathfinder uses for printing reports, select from the printer list. If you do not select a printer, Pathfinder has no specified font size. The report might have small text.



5. Click Setup to set parameters for target screening processing. The Setup dialog box opens. For parameter definitions, see Target Screening Setup Dialog Box on page 40. Once you define the screening method as Based on Exactive screening method, set other available options.

6. Click OK to save Setup values and close the Setup dialog box.

7. Click Save to save the method. The Save Method box opens. Name the method and click OK.



Target Screening Setup Dialog Box

Parameter Description

Screening Method Select Based on Exactive screening method to set values for Target Screening.

Company name Specify the name of company.

Laboratory name Specify the name of laboratory.

Company logo Specify the location of the logo file for the company. For example: C:\logo.jpg.

RT Window (min) Set the size of the RT window in minutes.

Exact Mass Window (ppm)

Specify the exact mass variability window. This is the value above and below the center m/z value.

Exactive

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

No Specified Retention Time

Select the No Specified Retention Time check box if you do not wish to have retention time specified. Selecting this option allows Pathfinder to search the entire raw file for the peak specified. Select this option when you do not know when the target compound will elute.

Report retention time based on

If you have selected the No specified retention time option, then select the First Peak or Highest Peak option to provide guidance for Pathfinder. This is necessary in case more than one m/z match is found in the raw file to guide Pathfinder in selecting a single peak to use for processing. Select this option when you know when the target compound will elute.

Report All compounds Listed in Configuration File

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



Creating a Processing File for Target Screening

A Target Screening workflow requires experiment- and compound-specific information that you define in the processing configuration file. Name and save the processing configuration file as a .csv file before setting up a Target Screening workflow.

Define target screening workflows on the computer managing the instruments. You cannot define target screening workflows on a remote computer.

To define the Pathfinder processing configuration file parameters

1. Open Excel and then open the Target Screening processing configuration file, called Target Database, for the experiment you want to perform. The template for the Target Screening workflow is in the C:\Pathfinder\Examples directory.

Use the ExactiveTest_Config.csv file in screening experiments that identify compounds based on accurate mass of parent ion with adducts, retention time, or both. You can view the processing configuration file in Figure 2, a sample summary report in Figure 5 on page 67, and a sample long report in Figure 6 on page 68.

2. Set the compound-specific parameters as shown in Table 2 on page 42. Observe the following:

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

When you save a .csv file, you lose any changes you make, such as enlarging or shrinking columns or rows.

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

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

Figure 2 provides an example for the Target Screening processing configuration file (name.csv).

Figure 2. Exactive processing configuration file



Table 2 describes the compound-specific parameters for the Target Screening workflow.

Table 2. Compound-specific parameters

Parameter Description

Compound Name (all experiments)

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

Chemical Formula (Exactive) Chemical formula of the compound.

Polarity (Exactive) Positive or negative ionization.

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

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

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

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

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

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

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

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



Changing a Pathfinder Method

Use the Method view to review or change a method.

To review or change a method

1. In the Method Development mode, click Open method in the Method view. The Open Method dialog box opens.

2. Select an existing method. The Method view opens, displaying the current parameters.

3. Change parameter values on any page and click Save method in the Method Development area.

4. To save this modified method under a new name, choose File > Save as


3 Working with MethodsWorking with Method Groups

Working with Method GroupsCreate method groups that define a set of methods to be used for a specific instrument configuration or task. Certain methods might require that API sources, HPLC systems, or HPLC solvents be in place. Once you create a method group, you can activate it for users so that they can only select a method that works with the current configuration of the instrument.

When creating groups, remember these concepts:

A method can exist in more than one group.

You can only make one group at a time available to users by activating it.

You do not need to create method groups. If this is the case, Pathfinder displays all methods to the user.

You can deactivate methods within a group so that they remain in the group but are not available to users.

The user only sees available methods, whether part of a group or not.

The method group capability can be turned off so that all methods are available.

The method group filter is only active in the User account. An administrator always sees all methods.

Creating a Method Group

To create a method group

1. From the Method Development view, click Method Groups in the Method Development area. The Method Groups page opens.

2. To activate the method group capability, select the Enable method group filtering for users check box.

3. Click New Group. The New Method Group area opens.

4. Type a name for the group in the Name box and click Create. The Method Groups page displays the new group as the active group.

The Available list box displays all Pathfinder methods that are currently available.



5. To add methods from the Available box, select a method name and click to add the method to the Selected box.

6. When the Selected box displays the list of chosen methods, click Save to save the group with the methods.



Modifying a Method Group

To add methods in a group

1. Select the method group in the Active group list.

2. Add methods by selecting the names in the Available box and clicking .

3. Click Save.

To remove a method from a group


2. Select the name of the method in the Selected box and click to remove the method.

3. Click Save.

To deactivate a method from a group


2. Clear the check box next to a method in the Selected area. The user will not see the method when the methods from the group are displayed.

To rename a group


2. Click Rename. The New Method Group dialog box opens.

3. Type a new name for the group in the Name box and click Change to rename the group.

4. Click Save.



Changing the Active Method Group

Use the following procedure to make a different method group active. The Method Development area displays the active method group in the method group view.

To change the active method group

1. Click Method Group view.

2. If the Enable method group filtering for users box is not selected, select it.

3. Select the method group name in the Active group list.

4. Close the dialog box. The new method group appears when you submit samples.

Deleting a Method Group

Deleting a method group does not delete the methods in the group. The methods remain available for other method groups or as individual tests.

To delete a method group

1. Select the method group name in the Active group list.

2. Click Delete. Pathfinder requests confirmation of the action.

3. Click Yes in the confirmation box. Pathfinder deletes the method group.


4

Working with Samples

Follow these procedures to test methods and perform analyses for both single samples and sets of multiple samples.

Contents

Submitting a Single Sample for Analysis

Submitting Multiple Samples for Analysis Manually

Creating Files for Importing Lists of Multiple Samples

Submitting an Imported List of Samples for Analysis

Viewing Instrument Status During Processing


4 Working with SamplesSubmitting a Single Sample for Analysis

Submitting a Single Sample for AnalysisUse the User Mode in the User Administration view to analyze a single sample using a Pathfinder method.

To perform an analysis on a single sample

1. Select User Administration and select the type of workflow in the User mode area.

a. To test a survey screening method, click Survey Screening in the User Administration view to open the Survey Screening page and specify sample information.

b. To test a target matching method, click Target matching in the User Administration view to open the Target Matching page and specify sample information and the molecular weight.


4 Working with SamplesSubmitting a Single Sample for Analysis

c. To test a target screening method, click Target screening in the User Administration view to open the Survey Screening page and specify sample information.

To clear all sample information and start over, click Clear. To stop the submission process completely, click Cancel.

2. Type the user name in the User name box.

3. Type the first sample ID in the Sample ID box.

4. Type the autosampler vial position in the Vial position box. If the vial position value is not in the correct format for your autosampler, Pathfinder displays an error message when you click Submit. For more information about the autosampler vial position format, refer to your autosampler documentation.

5. Type the file name with the information for the sample in the File name box. File names must be unique for each sample. If Pathfinder provides automatic file naming, the File name box is grayed out. See Defining a File Naming Convention on page 94.

6. If you selected Target Matching, type a molecular weight in the Target MW box. Specify the level of accuracy in the method.

7. Click Go to submit the sample. If the Go button is grayed out, you are missing information required for the analysis. Place your mouse over the button and Pathfinder displays a list of needed information.

8. To view results, click Analysis to open the Analysis view. Click Open sequence and select a results file from the list displayed. For more information about viewing test results, see Viewing Workflow Results on page 59.


4 Working with SamplesSubmitting Multiple Samples for Analysis Manually

Submitting Multiple Samples for Analysis ManuallyUse the User Mode in the User Administration view and the Add option to analyze a set of samples using a Pathfinder method.

To perform an analysis on a group of multiple samples by manually adding entries

1. Click User Administration to open the User Administration view. Select the type of workflow in the User mode area. The page for that method opens.

2. Type the user name in the User name box.

3. Type the first sample ID in the Sample ID box.

4. Type the autosampler vial position in the Vial position box. If the vial position value is not in the correct format for your autosampler, Pathfinder displays an error message when you click Submit. For more information about the autosampler vial position format, refer to your autosampler documentation.

5. Type the file name with the information for the sample in the File name box. File names must be unique for each sample. If Pathfinder provides automatic file naming, the File name box is grayed out. See Defining a File Naming Convention on page 94.

6. If you selected Target Matching, type a molecular weight in the Target MW box. Specify the level of accuracy in the method.

7. Click Add. The sample information is displayed in the table to the right and Pathfinder clears the user entry parameter boxes.

8. Type information for the rest of the samples, clicking Add after each sample.


4 Working with SamplesSubmitting Multiple Samples for Analysis Manually

9. To edit information for a sample that you have added to the list, click the sample to move the sample information back to the sample information area. Make changes and click Update.


10. To submit all samples, click Go. If the Go button is grayed out, you are missing information required for the analysis. Place your mouse over the button and Pathfinder displays a list of needed information.



4 Working with SamplesCreating Files for Importing Lists of Multiple Samples

Creating Files for Importing Lists of Multiple Samples To enter groups of samples easily, use the templates in C:\Thermo\Pathfinder\Templates\ImportTemplates. There are three options for entering data:

ImportTABTemplate.csv: a tab-delimited template file

ImportCSVTemplate.csv: a comma-delimited template file

ImportXMLTemplate.xml: an easily editable xml file

To create a file for the analysis of multiple samples

1. Open a template. You can open any of the templates in any type of editor, including Microsoft Notepad. You can also use Microsoft Excel to make it easy to work with tables of information.

2. Enter the sample ID, vial number, and file name for each sample in the designated column for that row. If the vial position value is not in the correct format for your autosampler, Pathfinder displays an error message when you submit the samples. For more information about the autosampler vial position format, refer to your autosampler documentation.

3. If you are doing a target matching test, enter the molecular weight in the TargetMW area. If you are not doing a target matching test, delete the portion of the template that requests a molecular weight. The following example is a .csv batch file for a target matching test.

If Pathfinder provides file naming and you put file names in the batch file, Pathfinder uses the file names from the batch file. If the batch file has no file names, Pathfinder provides file names. For more information about file naming, see Defining a File Naming Convention on page 94.

4. Click Save As to name the file and save it in a convenient location. To use this sample list, see Submitting an Imported List of Samples for Analysis on page 55.


4 Working with SamplesSubmitting an Imported List of Samples for Analysis

Submitting an Imported List of Samples for AnalysisUse the User Mode in the User Administration view and the Import option to analyze a set of samples using a Pathfinder method.

To perform analysis using an imported sample list

1. Click User Administration to open the User Administration view. Select the type of workflow in the User mode area. The page for that method opens.

2. Type a user name in the User name box.

3. Select a specific Pathfinder method from the Method area.

4. Click Import to select a file containing sample information. A browse window opens. For information about creating the file, see Creating Files for Importing Lists of Multiple Samples on page 54.

5. Select the multiple samples file and click Open.

6. To edit information for a sample that you have added to the list, click the sample to move the sample information back to the sample information area. Make changes and click Update.


7. To submit all samples, click Go. If the Go button is grayed out, you are missing information required for the analysis. Place your mouse over the button and Pathfinder displays a list of needed information.



4 Working with SamplesViewing Instrument Status During Processing

Viewing Instrument Status During ProcessingUse the Real Time Status view to monitor sample analysis.

To view instrument status

1. After clicking Go to start an analysis, click or click Real Time Status in the Analysis view. The Real Time Status view opens.

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

To view device status below the processing status, click the device name. The status page for that device opens in the lower left area (Status).


4 Working with SamplesViewing Instrument Status During Processing

2. Click the Acquisition Queue tab to open the list of submitted samples. When the sample has finished, the list displays Complete after the sample name.

3. To remove a sample or sequence from the queue, select the check box next to the name. Right-click the check box and choose Remove checked from the shortcut menu. Pathfinder removes the sequence item.

You can only delete an entire sequence before the instrument begins to acquire data in that sequence. Also, if you submit a sequence of samples, you can only delete samples before the instrument begins to acquire data from those samples.

The following buttons at the top of the Real time status view help you control acquisition.

Button Action

Go Restarts acquisition from a pause.

Stop Stops acquisition for the sample in progress and pauses the acquisition queue. If you click Go, Pathfinder continues with the next sample in the queue.

Pause Pauses the acquisition queue. If Pathfinder is currently acquiring a sample, it will continue acquiring. If you click Go, Pathfinder continues acquisition on the next sample.

Note You can get a quick look at the system status at any time by looking in the lower right corner of the Pathfinder window. This area displays a green or red LED and a description of the number of samples in the queue (if any).


5

Viewing Workflow Results

This chapter describes viewing the results of processing a sample or samples using a method. The results appear on the Data Review page and the Report page. Before viewing data, process your sample following the instructions in Working with Samples on page 49.

Contents

Viewing Data Results

Viewing Reports

Modifying Sample Results

Reprocessing Samples


5 Viewing Workflow ResultsViewing Data Results


Viewing Data ResultsThis section provides information about viewing the results of a sample after processing. Pathfinder provides sample details at a variety of levels.

Viewing Results for a Workflow

Viewing Peak Details

Viewing Specific Details

Viewing Results for a Workflow

You can view data results for Target Matching or Survey Screening workflows in the Data Review area. When viewing these results, you can zoom in on a spectrum by placing the mouse at the outside edge of the area you want to see in greater detail and sliding the mouse over the area. If you zoom a full scan spectrum, Pathfinder retains the zoom frame when you select different peaks in the navigational chromatogram. When you switch from sample to sample, zoom resets to defaults. When you switch filters within a sample, the zoom resets to defaults. When you switch to another view and return, Pathfinder maintains the zoom setting.

To view reports from the Target screening workflow, go to the Thermo\Pathfinder\Batches directory. Open the batch folder and open the Reports folder. Double click to open the .pdf file.

To view data results

1. From the Administrator view, click Analysis. The Analysis view opens.

2. Click Open sequence. The Open sequence dialog box opens. The most recently acquired sequence is at the top of the dialog box.

3. To select a data results file, select the file name and click Open. The Data Review page opens, displaying workflow data.


a. If your method was Survey Screening, the Survey Screening page opens.

Figure 3. Survey screening data review

Navigational chromatogram

Integrated peak chromatogram

Spectrum

Peak listSample list

Active data source



b. To view Target matching values, click the Target Matching tab. The Target Matching page opens.

Figure 4. Target matching data review

Active data source

Navigational chromatogram


Peak ListSample list

Elemental Composition results



If the report is for Target matching, the Peaks box identifies in blue any peaks that satisfy the matching criteria. To make sure Pathfinder has identified a peak, hover over the peak with a mouse. Pathfinder highlights the peak in the Peak list if the peak has been identified.

On the Data Review page for both Target Matching and Survey Screening, you can view the results of using a supplemental detector to define the data field by clicking the arrow in the Data source list.

For a detector type of Analog, A/D, or UV detector channel, Pathfinder stores only the peak list. For a detector type of PDA, Pathfinder stores only the peak list, but you can view a PDA spectrum. You cannot perform any other processes (for example, target matching, target screening, or elemental composition) from any of these supplemental detectors because they do not have a mass data spectrum.



Nachr


Viewing Peak Details

To view peak information

1. To view peak information, click a specific peak number. Pathfinder identifies the peak in the navigational chromatogram using a red line that marks the retention time.

vigational omatogram




2. To view a different part of the peak and review the spectrum for that area, click an area in the integrated peak chromatogram.

The retention time marker in the integrated peak chromatogram moves to the area you clicked and the red line in the navigational chromatogram moves with it.

The Spectrum box displays spectrum information for the selected retention time.

To reset the peak, select another peak in the Peak list and then select the original one.

Viewing Specific Details

If your sample contains HCD data, Pathfinder displays an HCD spectrum in the data review. The HCD spectrum displayed should be one scan number greater than the full scan of the target match. Use the HCD scan only to recognize fragments.


Click the Elemental Composition tab to view the values that meet the window of the elemental composition parameters. The Elemental Composition page opens. The peak with the best match has a checkmark in the Index box according to the elemental composition criteria. Pathfinder sorts the peaks by the spectral fit value, from high to low.

You can select a different peak from the Elemental Composition list. To change the best match choice, select the check box of the compound that is more accurate. Elemental composition results are also available in the Survey Screening and Target Matching reports.

For Target Matching results, click the Target Matching tab. The Target Matching view shows detailed information about the selected peak.


5 Viewing Workflow ResultsViewing Reports

Viewing ReportsTo view the test report for Target Screening workflows

1. To view reports from the Target screening workflow, go to the Thermo\Pathfinder\Batches directory.

2. Open the batch folder and open the Reports folder.

3. Double-click to open the .pdf file.

4. From the .pdf file, choose File > Print to print the file. Even if you did not select a type of file, Pathfinder creates a .pdf file.

Figure 5. Target Screening sample summary report




Figure 6. Target Screening sample long report



5. Target screening reports display the following information so that you can review the data.For each processed data source:

The summary report shows

Header information

A trace for each target compound

A table showing matches to the selected database

The long report shows

One page for each compound

Detailed results of all adducts and fragments identified using the database


To view the test report for Target Matching or Survey Screening workflows

1. In the Analysis view, click Reports view. The Reports page opens.

2. Select a report in the Reports box. Select a specific file in the Sample file box.

3. All reports display the following information so that you can verify the data:

A batch header at the top with the batch name, the Pathfinder method, the methods lab and assay name, and the user who submitted it

A sample header with sample specific information, such as the sample ID, the date and time of acquisition, the raw data file name, and the vial position

Figure 7. Survey screening report



Survey screening reports display the following information so that you can review the data. For each processed data source, Pathfinder displays a chromatogram followed by a report block for each peak detected in that data source, including

Textual peak information, such as peak retention, height, and area

An enlarged view of the detected peak (the same data that is in Data Review)

A mass spectrum taken from the peak apex

For HCD processing, an HCD trace if one was acquired

The HCD scan shown is one scan number greater than the mass spectrum taken from the peak apex.

Elemental composition results

This report block repeats once for each data source that was selected on the Peak Processing tab of the Method view.



Figure 8. Target matching report




Target matching reports display the following information so that you can review the data for each processed data source:

A table listing al

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