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What’s New in Empower 3

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Copyright © Waters Corporation 2010All rights reserved

Copyright notice

© 2010 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use.

Trademarks

Waters is a registered trademark of Waters Corporation, and Empower and “THE SCIENCE OF WHAT’S POSSIBLE.” are trademarks of Waters Corporation.

Other registered trademarks or trademarks are the sole property of their owners.

Customer comments

Waters’ Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it. Please help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usability.

We seriously consider every customer comment we receive. You can reach us at [email protected].

Contacting Waters

Contact Waters® with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail.

Waters contact information

Contacting medium Information

Internet The Waters Web site includes contact information for Waters locations worldwide. Visit www.waters.com.

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Empower 3 software

Safety information

See the operator’s guides of the instruments or devices associated with this software product for information on how to safely operate and maintain them.

Intended use

Use the Waters Empower™ 3 software for acquiring, processing, reporting, and managing your chromatographic information.

Audience and purpose

This guide addresses novice and experienced Empower users. It explains in detail Empower 3 features that were not included in earlier versions of Empower software. It is intended as a supplement to the Empower 3 online Help.

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Table of Contents

Copyright notice ................................................................................................... ii

Trademarks ............................................................................................................ ii

Customer comments ............................................................................................ iii

Contacting Waters ............................................................................................... iii

Empower 3 software ............................................................................................ iv Safety information .............................................................................................. iv Intended use........................................................................................................ iv Audience and purpose......................................................................................... iv

1 Process-only sample sets ...................................................................... 1-1

Overview ............................................................................................................. 1-2

Process-only sample set privileges ............................................................... 1-3

Creating a process-only sample set .............................................................. 1-4 Process-only sample set editor considerations ............................................... 1-5 Altering process-only sample set information................................................ 1-6

Acquiring a new sample set based on process-only sample sets ........... 1-6

Reporting process-only sample sets ............................................................. 1-7

Process-only sample set considerations ...................................................... 1-7 Saving process-only sample sets ..................................................................... 1-7 Copying and deleting process-only sample sets ............................................. 1-8 Custom fields.................................................................................................... 1-8 Audit trail and revision history comments..................................................... 1-9 Audit trail records............................................................................................ 1-9

Table of Contents 1

2 Calculating USP, EP, and JP signal-to-noise ratio ........................ 2-1

Calculation method .......................................................................................... 2-2

Calculating the signal-to-noise ratio using a blank value ...................... 2-5

3 Mass spectrometry ................................................................................. 3-1

Ion ratio processing (Peak ratio processing) ............................................. 3-2 Using the Peak Ratios (MS Ion Ratios) tab.................................................... 3-2 Entering peak ratio tolerances and reference injections ............................... 3-3 Peak ratio results............................................................................................. 3-4 Peak Ratio view in Review and QuickStart ................................................... 3-5 Peak Ratio (MS Ion Ratio) Report groups ...................................................... 3-5

Target Mass ........................................................................................................ 3-7 Using the sample table and a single method to process multiple base masses ...

3-7 Configuring the MS Processing method ......................................................... 3-8 Using the Include the Uncorrected Target or Base Mass option .................. 3-9 Specifying base masses according to the expected mass injection.............. 3-11 Specifying the “Ionization” mode .................................................................. 3-12 Using the Percentage of Base Peak field ...................................................... 3-12 Reporting expected mass results .................................................................. 3-14

MS library changes ......................................................................................... 3-14

4 Fraction Collection with WFC III ...................................................... 4-1

Collecting fraction data ................................................................................... 4-2 Adding and updating fraction data................................................................. 4-2

Viewing fraction data ...................................................................................... 4-2 Viewing fraction data in Review ..................................................................... 4-2 Viewing fraction data in the Project window ................................................. 4-3

2 Table of Contents

Formatting fraction data in Review ............................................................. 4-4

Reporting fraction data ................................................................................... 4-6

5 Method validation .................................................................................. 5-1

Accuracy profile capability ............................................................................ 5-2 Reporting Accuracy Profile Plot information ................................................. 5-6

Linearity of the Method ................................................................................... 5-7 Linearity processing steps............................................................................... 5-7 Linearity and Linearity Comparison report groups ...................................... 5-9

Limits of Quantitation ................................................................................... 5-10

6 ACQUITY UPLC Enhancements ......................................................... 6-1

eCord data in the Empower 3 database ....................................................... 6-2 Adding eCord data to the database................................................................. 6-2 Updating eCord data ....................................................................................... 6-3 Viewing and managing eCord data................................................................. 6-4 Reporting eCord data....................................................................................... 6-5 Deleting eCord data from the Empower database......................................... 6-7

ACQUITY UPLC sample loop support .......................................................... 6-7

ACQUITY UPLC sample queue functionality ............................................. 6-8 Auto-addition ................................................................................................... 6-8 Fixed vials ........................................................................................................ 6-8 Column selection.............................................................................................. 6-8

7 Post Run Report group ......................................................................... 7-1

Post Run Report group .................................................................................... 7-2

Table of Contents 3

4 Table of Contents

1 Process-only sample sets

This chapter describes the process-only sample set feature, which you can use to create sample sets from previously acquired data.

Contents

Topic Page

Overview 1-2

Process-only sample set privileges 1-3

Creating a process-only sample set 1-4

Acquiring a new sample set based on process-only sample sets 1-6

Reporting process-only sample sets 1-7

Process-only sample set considerations 1-7

1-1

Overview

The process-only sample set feature helps you create a sample set from previously acquired injections, channels, or sample sets. The data in a process-only sample set is a copy of the original data. Changes you make to process-only sample sets do not affect the original sample sets.

You create a process-only sample set by selecting existing raw data from the Project window or the QuickStart application and bringing it into the Process-Only Sample Set Editor, where you can rearrange the order of the data, add function lines, split replicate injections from one row into multiple rows, and combine replicate injections from multiple rows into a single row.

Reviewing and processing process-only sample set data

You review and process process-only sample sets in the same way that you process regular sample sets. Locked channels are not processed when a process-only sample set containing those channels is processed.

You cannot export process-only sample set data. While you can preview results created from a process-only sample set, you cannot preview the process-only sample set itself (raw, unprocessed data).

Viewing process-only sample set data

The Sample Set Type column in the Sample table on the Project Window’s Sample Set tab indicates the sample set type. The software sets the field to Acquired, for regular sample sets, and Process Only, for process-only sample sets. When you copy a process-only sample set to another project, the software sets the sample set type to Copied Process Only.

When viewing information relating to process-only sample sets in the Project Window or QuickStart interface, bear in mind these restrictions:

• You cannot use the View As functionality to view a process-only sample set as injections or channels. You see process-only sample set information only in the Sample Set, Result Set, and Results views.

• When you view sample sets as injections or channels, you cannot view the injections or channels related to the process-only sample set. If process-only sample sets are among the rows selected, only conventional sample sets appear as injections or channels.

• You cannot view the generated results of process-only sample sets as injections or channels. If multiple results are selected, only results generated from regular sample sets appear as injections or channels.

1-2 Process-only sample sets

The Number of Process Only Sample Sets field tracks the number of times each injection or channel is associated with a process-only sample set. You can access this field from the following areas in the Empower 3 interface:

• Injection view

• Channel view

• Review

• Reports

Process-only sample set privileges

The Manage Process Only Sample Set privilege controls user access to process-only sample set functionality.

You can view and copy process-only sample sets in the Project Window and QuickStart Sample Set view even if you do not have the Manage Process Only Sample set privilege. Nevertheless, you must have the privilege to perform actions relating to creating a process-only sample set.

To delete process-only sample sets, you must have both the Manage Process Only Sample Set privilege and the Delete Data privilege.

To enable or disable the Manage Process Only Sample Set privilege:

1. In the Configuration Manager tree, click User Types.

2. Select the user type for which you want to grant process-only privileges, right click, and then select Properties.

3. In the User Type Properties dialog box, click the Methods tab.

4. In the list of properties, select or clear the check box Manage Process Only Sample Set.

5. Click OK.

6. Supply a comment and a password, if required, and then click OK.

Process-only sample set privileges 1-3

Creating a process-only sample set

A process-only sample set is comprised of existing injection, channel, or sample set data you select from the Project window or the QuickStart application. You can mix any sample types in a process-only sample set.

When you create a process-only sample set, the Empower 3 software makes a copy of the original data. The injection ID of the data in the process-only sample set is the same as the injection ID of the native data. However, the vial IDs of the data in the process-only sample set are different from the vial IDs of the native data.

Built-in sample information fields such as Node and System Name are populated when the information is relevant to all data within the process-only sample set. Otherwise, these fields are blank.

To create a process-only sample set:

1. In the Project window or the QuickStart application, select the injections, channels, or sample sets you want to include in the process-only sample set.

2. Click Tools > Create Process Only Sample Set.

3. In the Name Sample Set dialog box, enter a name for the sample set.

Tip: The name you select also becomes the name of the process-only sample set’s sample set method.

Restriction: The name “Untitled” is not a valid process-only sample set name.

4. Click OK.

Result: The process-only sample set editor appears and searches for duplicate entries and for sample lines that can be split into individual sample lines.

5. If you selected replicate injections that share the same vial ID, the software prompts you to merge the injections into one sample line.

• To merge the injections, click Yes.

• To bring the injections into the editor individually, click No.


6. Add or rearrange data in the Process-Only Sample Set Editor window as follows:

• To add data to the process-only sample set, in the Project Window or the QuickStart interface, select the injections, channels, or sample sets you want to add to the process-only sample set and drag them into the process-only sample set editor. If you add injections generated from the same vial, the editor prompts you to merge them into one sample line.

• To add a function to the process-only sample set, select a row in the process-only sample set editor, and then select Edit > Insert Row. The row is added to the process-only sample set above the row you selected.

• To delete a row from the process-only sample set, select the row you want to delete, and then select Edit > Delete Row. Deleting an item from a process-only sample set does not delete the item from the original sample set or from the project.

• To merge sample lines, select the lines you want to merge, right-click, and then select Merge Sample Lines.

• To rearrange rows, select a row and then drag it to the desired position. You can select and move one or more rows at the same time.

7. Select File > Save to save the process-only sample set.

Important: Be sure the process-only sample set is configured as desired before you save it. After you save the sample set, you cannot reopen the process-only sample set editor. You must make all subsequent changes to the process-only sample set using the Alter Sample function.

Process-only sample set editor considerations

You cannot create new sample lines in the editor. You must bring existing injections, channels, or sample sets into the editor.

The Auto Fill, Add Row, Insert Rows, and Delete Rows functions exactly as they do in the Alter Sample editor.

The Copy, Paste, and Cut menu items and their corresponding key combinations are not supported in the process-only sample set editor. Use the drag-and-drop function to rearrange items in the editor.

You cannot change injection volumes in the process-only sample set editor.

Creating a process-only sample set 1-5

Altering process-only sample set information

After you save a process-only sample set, you cannot edit it in the process-only sample set editor. You must make all changes using the Alter Sample function.

Use Alter Sample to edit the process-only sample set in the same way that you modify native sample sets. Because the data within a process-only sample set is a copy of the original data, altering the process-only sample set does not alter the original data and vice versa.

When you modify sample or sample set information in a full audit trail project that contains a process-only sample set, the software generates sample/sample set history information, starting from the point of process-only sample set creation.

Acquiring a new sample set based on process-only sample sets

Empower 3 software allows you to reacquire a sample set based on process-only sample sets, in the same manner as regular sample sets. There are two ways to acquire process-only sample sets.

Tip: Ensure that the system configuration used for all samples in the sample set are compatible.

To acquire a process-only sample set from the Run Samples window:

1. In the Empower 3 Pro window, click Run Samples.

2. In the Run Samples dialog box, select the chromatographic system on which you want to run samples, and then click OK.

3. In the Run Samples window, select File > Load Samples.

4. In the Load Sample dialog box, select Reinject samples from a previously run sample set, and then click OK.

5. In the Project window, click the Sample Sets tab, and then select the process-only sample set.

6. Click the Load into Run Samples button.

7. Run the samples as you would run a regular sample set.


To run a process-only sample set from the Project window:

1. In the Project window, click the Sample Sets tab, right-click the process-only sample set, and then select View as > Sample set methods.

2. Right-click the sample set method, and then select Method Properties.

3. In the Method Properties dialog box, select the method, and then click Save as current.

4. Click OK. The software saves the process-only sample set as a sample set method, which now appears in the Methods tab in the Project window.

5. Run the process-only sample set as a normal sample set method.

Reporting process-only sample sets

You can create reports only by using results or result sets that were generated from process-only sample set. You cannot print the process-only sample set (the raw, unprocessed data) or bring it into Preview or Report Publisher.

Results and result sets generated from process-only sample sets are reported in the same manner as native Empower results.

Process-only sample set considerations

This section provides details about how Empower 3 software behaves with process-only sample sets.

Saving process-only sample sets

When you save a process-only sample set, the software behaves as follows:

• Generates a new sample set method associated with the sample set.

• For Full Audit Trail projects, removes existing sample history of the data within the process-only sample set. The sample history remains with the original sample set.

• Assigns the same date and time to the sample set’s start and end dates.

Reporting process-only sample sets 1-7

• Stores the name of the user who created the sample set in the Acquired By column.

• Sets the System and Node names of the process-only sample set to empty, unless the System and Node information is the same for all data included in the process-only sample set.

Copying and deleting process-only sample sets

When you copy a process-only sample set to another project, the software generates a regular sample set by cloning associated injections, vials, and methods. The copied sample set, indicated by the Copied Process Only sample set type, behaves like a conventional sample set in the destination project.

Before deleting individual injections or channels that are associated with a process-only sample set, you must delete any associated process-only sample sets.

Custom fields

Empower 3 software populates process-only sample sets with sample set type custom field data as follows:

Sample type custom field information is retained when you create a process-only sample set.

To supply or edit custom field values in process-only sample sets, use the Alter Sample function.

When you create a process-only sample set from...

Empower 3 software

Injections or channels Does not copy sample set custom field information.

Multiple sample sets Copies only the sample-set-level custom fields from the first selected sample set to the new, process-only sample set. Custom fields from other sample sets are ignored.


Audit trail and revision history comments

When you create, alter, or delete a process-only sample set, the Comment dialog box allows you to enter comments for the process-only sample set and underlying sample set method. These comments are separate from and in addition to audit trail comments.

When you save a process-only sample set, the software prompts you for the revision history reason and the audit trail reason based on the sample object comment setting or the method object comments setting, as follows:

• Both settings Silent – The software requests neither sample history reason nor audit trail reasons. The software does not enter default comments into the audit trail.

• Both settings not Silent – The software requests reasons for both sample revision history and audit trail.

• Either setting Restricted – The software considers audit trail commenting as restricted and the Save dialog box displays the audit trail strings associated with the Sample and/or Method objects in the Default Comments area of the dialog box. If no default string is available, then the software prompts you to enter comments.

• Either setting Unrestricted, with neither setting set to Restricted – The software considers the audit trail commenting as unrestricted, and the Save dialog box allows you to use the Default Comments drop down list or the comments area of the Save dialog box to create comments.

The audit trail record and the sample/method revision history record comments share these comments description.

Audit trail records

Process-only sample sets have the following audit trail records types:

Record type Action

Created Process Only Sample Set

Records the creation of a process-only sample set. The details section of this audit trail record shows all injection IDs associated with the process-only sample set, the sample set ID, the sample set method ID, and an audit trail reason.

Process-only sample set considerations 1-9

Altered Process Only Sample Set

Records the modification of a process-only sample set in the Alter Sample editor. The details section of the audit trail record shows the sample set name and ID, sample set method name and ID, and an audit trail reason.

Deleted Process Only Sample Set

Records the deletion of a process-only sample set. The details section of this audit trail record shows the sample set name and ID and an audit trail reason. Injection IDs are not listed in this entry because actual injections are not deleted.

Record type Action


2 Calculating USP, EP, and JP signal-to-noise ratio

This chapter describes USP, EP, and JP signal-to-noise calculations that are new or changed in Empower 3.

Contents

Topic Page

Calculation method 2-2

Calculating the signal-to-noise ratio using a blank value 2-5

2-1

Calculation method

The software calculates signal-to-noise ratio according to the latest US, European, and Japanese pharmacopeias, as shown by this equation

s/n = 2h/hn

where

h = the height of the peak corresponding to the component

hn = the difference between the largest and smallest noise values observed over a distance equal to at least five times (USP) or 20 times (EP and JP) the width at the half-height of the peak and, situated equally around the region of the peak of interest in a blank injection.

You can specify whether to calculate USP, EP, and JP s/n using the Calculate USP, EP, and JP s/n (formerly Calculate EP s/n) check box on the processing method’s Suitability tab.

You also can specify whether to calculate USP s/n, EP s/n, and JP s/n using noise values calculated from the peak regions centered on a blank injection.

Noise regions are unique to each peak. You determine noise regions by means of a corresponding blank injection that centers the noise region at the retention time of each peak. You define the noise region by specifying the half-height multiplier parameter.

USP s/n

The new suitability peak field USP s/n is implemented using the signal-to-noise (s/n) formula in the US Pharmacopeia. USP s/n is calculated as

2 height/(noise/scaling)

where:

height = the absolute value of the height of the peak

noise = the peak's noise value (peak-to-peak noise)

scaling = the scale to µV value

By default, the software reports the USP s/n value to a precision of 6, not in scientific notation, and with no units.

2-2 Calculating USP, EP, and JP signal-to-noise ratio

The noise value used to calculate USP s/n is determined according to the state of the “Use noise centered on peak region in blank injection” option:

• When you select this option, the software calculates a noise value for each peak using the peak-to-peak noise determined in the blank injection. The value is calculated for a region in the same channel in a separate blank injection. This region is centered around the peak's retention time and has a width equal to the peak’s width at half height the Half Height Multiplier for USP Noise Region value. The software reports this noise value in the result as USP Noise. By default, the software reports the value to a precision of 6, not in scientific notation, and with the units “Plot Units”.

• When you clear the option, the software uses the result's peak-to-peak noise value; noise is not calculated using the blank injection. You specify the start and end times of this region on the processing method’s Noise and Drift tab.

The Half Height Multiplier for USP s/n Noise Region field, located on the processing method’s Suitability tab, has a range of 1 to 99 and defaults to 5. The field is inactive when you clear the option “Use noise centered on peak region in blank injection” and when the pharmacopoeia choice is JP or EP.

EP s/n

The EP s/n suitability peak fields are implemented using the signal-to-noise (s/n) formula in the European Pharmacopoeia. The EP s/n is calculated as

2 (height - (0.5 noise/scaling))/(noise/scaling)

where:




By default, the software reports the EP s/n value to a precision of 6, not in scientific notation, and with no units.

Calculation method 2-3

The noise value used to calculate EP s/n is determined according to the state of the “Use noise centered on peak region in blank injection” option:

• When you select this option, the software calculates a noise value for each peak using the peak-to-peak noise determined in the blank injection. The value is calculated for a region in the same channel in a separate blank injection. This region is centered around the peak's retention time and has a width equal to the peak’s width at half height the Half Height Multiplier for EP Noise Region value. The software reports this noise value in the result as EP Noise. By default, the software reports the value to a precision of 6, not in scientific notation, and with the units “Plot Units”.

• When you clear the option, the software uses the result's peak-to-peak noise value; noise is not calculated using the blank injection. You specify the start and end times of this noise region on the processing method’s Noise and Drift tab.

The Half Height Multiplier for EP s/n Noise Region field, located on the processing method’s Suitability tab, has a range of 1 to 99 and defaults to 20. The field is inactive when you clear the option “Use noise centered on peak region in blank injection” and when the pharmacopoeia choice is JP or USP.

JP s/n

The new JP s/n suitability peak field is implemented using the signal-to-noise (s/n) formula in the 16th JP Pharmacopoeia. The JP s/n is calculated as

2 (height - (0.5 noise/scaling))/(noise/scaling)

where:




By default, the software reports the JP s/n value to a precision of 6, not in scientific notation, and with no units.


The noise value used to calculate JP s/n is determined according to the state of the “Use noise centered on peak region in blank injection” option:

• When you select this option, the software calculates a noise value for each peak using the peak-to-peak noise determined in the blank injection. The value is calculated for a region in the same channel in a separate blank injection. This region is centered around the peak's retention time and has a width equal to the peak’s width at half height the Half Height Multiplier for JP Noise Region value. The software reports this noise value in the result as JP Noise. By default, the software reports the value to a precision of 6, not in scientific notation, and with the units “Plot Units”.

• When you clear the option, the software uses the result's peak-to-peak noise value; noise is not calculated using the blank injection. You specify the start and end times of this region on the processing method’s Noise and Drift tab.

The Half Height Multiplier for JP s/n Noise Region field, located on the processing method’s Suitability tab, has a range of 1 to 99 and defaults to 20. The field is inactive when you clear the option “Use noise centered on peak region in blank injection” and when the pharmacopoeia choice is USP or EP.

Calculating the signal-to-noise ratio using a blank value

You can use noise values calculated for a blank injection(s) to calculate USP, EP, and JP signal-to-noise ratios.

When the software calculates the noise value from a blank injection(s), it behaves as follows:

• If the sample set includes a single blank injection, the software uses the injection to determine the peak-to-peak noise values for each peak in the sample set.

• If the software finds multiple blank injections in the sample set, it calculates multiple noise values for each blank injection (of the same channel), averages them, and then uses the average noise value.

• The s/n Blank Channel IDs field displays the IDs of the blank channels used to determine USP, JP, and EP noise, and subsequently USP, JP, and EP s/n.


Blank injections are defined by selecting the Blank check box in Run Samples, the sample set method editor, or Alter Sample. The blank injection(s) must occur in the same sample set and must be located before the injections for which USP, JP, and EP s/n is being determined. By default, this check box is cleared.

Use the Calculate USP, EP, and JP s/n check box on the processing method’s Suitability tab to specify whether to calculate USP s/n, EP s/n, and JP s/n.

Use the “Use noise centered on peak region in blank injection” check box on the processing method’s Suitability tab to specify whether to calculate USP s/n, JP s/n, and EP s/n using noise values calculated from a blank injection.

Use the USP Half Height Multiplier for Noise Region, EP Half Height Multiplier for Noise Region, and JP Half Height Multiplier for Noise Region to specify the noise region in the corresponding blank chromatogram. Empower determines the half height of each integrated peak in the chromatogram and uses a noise interval in the blank equal to a distance of the half height the multiplier specified in the processing method. The noise region is determined in the blank(s) and is centered around the retention time of each peak of interest.

Tip: The Blank check box is also available for use in custom field formulas.

When signal to noise is calculated with the “Use noise centered on peak region in blank injection” option selected, the noise value is either a noise value calculated from a single blank channel or an averaged noise value calculated from multiple blank channels. The new suitability result field s/n Blank Channel IDs holds the IDs (separated by spaces) of the blank channels used. The s/n Blank Channel IDs field is blank if USP s/n, JP s/n, and EP s/n are not calculated using noise values from blank channels.

Rule: The blank channel must be placed earlier in the sample sequence than the channels that use it to calculate USP s/n, EP s/n and JP s/n.

If you do not apply the noise from a blank injection(s), the software uses the peak-to-peak noise, as determined using the parameters specified on the processing method’s Noise and Drift tab. The Peak to Peak Noise field is used as the noise value for USP s/n, EP s/n, and JP s/n only if you select “Calculate USP, EP, and JP s/n” but do not select “Use noise centered on peak region in blank injection” in the Processing method’s Suitability tab.

The noise region in the blank requires a minimum of five data points to determine noise. Ten or more are recommended.


To calculate signal to noise using a blank value:

1. Include one or more blanks in your sample set to inject before injection of the samples for which you want to calculate signal to noise. Select the Blank check box for the blank samples.

2. On the processing method’s Suitability tab, select both Calculate USP, EP, and JP s/n, and Use Noise Centered on Peak Region in Blank.

3. Process the sample set with the processing method from step 2 or a method set that contains the processing method from step 2.


3 Mass spectrometry

This chapter describes mass spectrometry functionality that is new or changed in Empower 3.

Contents

Topic Page

Ion ratio processing (Peak ratio processing) 3-2

Target Mass 3-7

MS library changes 3-14

3-1

Ion ratio processing (Peak ratio processing)

Use the new ion ratio processing parameters to calculate ion ratio peak values for LC, PDA, GC, IC, CE, CIA and MS results. Ion ratio processing produces multi-channel results similar to cross channel internal standard processing. Ion ratios are available for native 2D channels, derived 2D channels, and 2D channels extracted from 3D data.

Tip: Although the main function of this functionality is to determine MS ion ratios, you also can use it to determine peak ratios for non-MS data.

Using the Peak Ratios (MS Ion Ratios) tab

The LC Processing Method window includes the new Peak Ratio (MS Ion Ratios) tab. Use this tab to select a processing method component and enter the information necessary to complete cross-channel peak ratio calculations with acceptance tolerance checking on data related to an injection. The Peak Ratios (MS Ion Ratios) tab includes the following components:

Components of the Peak Ratios tab:

Component Description

Primary Peak Table (Primary MS Ion Table)

Enables you to specify one or more existing components and the primary channel.You can specify a channel by name or by description.You also can specify whether the software determines the peak ratio by area or height.

Secondary Peak Table (Secondary MS Ion Table)

Contains the secondary peaks for the component that you select in the Primary Peak table.This table can contain up to four secondary peaks.

Peak Response field Enables you to specify determination of the peak (ion) ratio by peak area or peak height.

3-2 Mass spectrometry

Tips:

• In Review and QuickStart, you can specify the channel description by selecting it from the drop down list. The software does not store channel descriptions in the processing method. The purpose of channel descriptions is only to help you specify the appropriate channel name.

• When you create derived channels, ensure that each channel description is unique. The software requires unique channel names and descriptions when it loads data into the Peak Ratio Tab.

Entering peak ratio tolerances and reference injections

You can enter peak ratio acceptance tolerances for each peak ratio channel.

The acceptance tolerances are percentages of the reference injection’s peak value. For example, if peak ratio tolerance is 20, the peak ratio for the unknowns must be within 20% of the peak ratio for the same component in the reference injection.

The system flags results outside of the specified peak ratio tolerance as faulted. A fault is a condition (detected by Empower software) that results during processing or acquisition when either a peak field value is outside the limits set in System Suitability, or the Internal Standard, RT Reference, Default, or Must peak is missing. Because the peak ratio fields exist only in the main results, the system flags only those results when peak ratio faults exist.

See also: “System Suitability Limit fields” in the Empower 3 online information system.

Although the Peak Ratio Tolerance field instructs the software to assess the peak ratios relative to a reference injection, you also can use the System Suitability Limits tab to define absolute limits on the four peak ratio value peak fields.

To define the reference injection:

1. Open the sample set in the Run Samples sample table or the Alter Sample editor.

2. For each sample you want to designate as a reference injection, select the Peak Ratio Reference check box.

When processing a sample set that contains more than one reference injection, the software processes the samples in the order specified in the sample set,


averaging the peak ratio(s) of the reference injections with those of previous reference injections, and applying this running peak ratio average for subsequent samples.

Peak ratio results

Peak ratio processing produces multi-channel results similar to those produced by cross-channel, internal standard processing. The multichannel result includes a main result that is quantitated or calibrated as in normal single channel processing, and a list of one or more sub results. The sub results include a result for each of the primary and secondary peak ratio channels for all the components listed in the Peak Ratio tab, unless one of the peak ratio channels is the same as the main result’s channel.

Main results are displayed in the Project window. To display sub results, bring the main result into Review, click the 2D Channels tab, and then select the Sub Results check box in the table.

Tip: It is often helpful to display main and sub results in a stack plot.

Results include:

• Peak Ratios (ion ratios) 1 - 4.

• Peak Ratio % Difference 1 - 4 (the difference between the peak ratio of the current result, as compared with the reference, expressed as a percentage).

Peak Ratio Tolerance (the tolerance specified in the processing method). The software displays faulted results (results that are outside of the tolerance limit) in bold, red font.

The software determines each Peak Ratio value by dividing the Peak Response of the appropriate primary channel by the Peak Response of the secondary channel. For example, to calculate a ratio when the Peak response is set to Height, the software uses the height of the peak in the primary channel as the numerator and the height of the peak in the secondary channel as the denominator. Both channels are always from the same injection.

Peak Ratio Reference injections are used only under the following conditions:

• Peak Ratio Reference injections must be in the same Sample Set as the sample injections that refer to them.

• Peak Ratio Reference injection results must have been processed with the same processing method as the sample injections.


• Peak Ratio Reference injection results must have been processed before the sample injections.

• If a Peak Ratio Reference injection was processed multiple times, only the last saved (non-superseded) result for the injection is used.

• When Cross Channel Peak Ratio processing is performed in Review, the software ignores Peak Ratio Reference injection results unless they have been saved to the database.

Tip: You must ensure that the Peak Ratio Reference injections are processed and the results saved to the data base before the non Peak Ratio Reference injections.

When the software processes peak ratios for a Peak Ratio Reference injection, the Peak Ratio Reference values in the Peak Ratio Reference result’s peaks are blank. The values are blank because a Peak Ratio Reference injection cannot reference another Peak Ratio Reference injection.

Peak Ratio view in Review and QuickStart

In “Peak Ratio View” mode, the Chromatogram plot displays the channels in the current result that are associated with the selected peak ratio peak. These channels include the main result channel and the sub results associated with the primary and secondary peak channels named in the component’s peak ratio parameters.

To turn on and turn off “Peak Ratio” mode:

Perform one of the following steps:

• To turn on “Peak Ratio” mode, in the Chromatogram plot, right-click within a peak, and then select Show Peak Ratio (MS Ion Ratio) View.

• To turn off “Peak Ratio” mode, in the Chromatogram plot, right-click a peak, and then select Return from Peak Ratio (MS Ion Ratio) View.

Peak Ratio (MS Ion Ratio) Report groups

The Processing Method report group tree includes the new Peak Ratio Plot report group and Peak Ratio Table group. Use these groups to report peak ratio (MS ion ratio) parameters for all processing method types except GPC.


Peak Ratio Plot group

Use the Peak Ratio Plot group to report, in plot format, a component and its peak ratio results. Each plot reports only one component overlaid in a single or stack plot.

This group includes the following sub-groups:

• Overlay Chrom All Peaks

• Overlay peak Ratios

You can use the Peak Ratio Plot properties dialog box to specify whether to plot one peak and its ratio peaks per plot, or to plot the entire chromatogram and any associated chromatograms that contain ratio peaks related to the peaks in the main result.

For example, consider a result that contains two peaks, “Component A” and “Component B,” each with one associated peak ratio. If you overlay one peak per plot, the result produces two plots, one for Component A and its associated ratio peaks, and another plot for Component B and its associated peaks. The plots show only the peaks. They do not show full chromatograms.

If you overlay chromatograms, the result produces two plots, one for Component A, and another plot for Component B. Each plot shows full chromatograms for the main peak and related peak ratios.

To report and customize Peak Ratio Plot information:

1. In Report Publisher, perform one of the following procedures:

• Create a Peak Ratio Plot report group method.

• Open a report method that includes a Peak Ratio Plot report group.

2. Right-click the Peak Ratio Plot group, and then select Peak Ratio Report Properties.

3. In the Peak Ratio Plot Properties dialog box, use the Peak Ratio Plot tab to select one of the following:

• Overlay Peak Ratio (1 peak/plot)

• Overlay chromatogram with All Peak Ratios

4. Use the tabs on Peak Ratio Plot Properties dialog box to customize the Peak Ratio Plot report.


Peak Ratio Table group

Use the Peak Ratio Table group to report, in tabular format, a peak and its peak ratio results. Each table reports information for only one peak and its associated peak ratio results.

To report and customize Peak Ratio Table information:


• Create a Peak Ratio Table report group method.

• Open a report method that includes a Peak Ratio Table report group.

2. In the report, right-click the Peak Ratio Table group, and then select Peak Ratio Table Properties.

3. In the Peak Ratio Table Properties dialog box, use the tabs to select customization options.

Target Mass

Using the sample table and a single method to process multiple base masses

In previous versions of the Empower software, you can enter only one target mass per processing method. To target a different base mass, you must use a different processing method and reprocess the same data.

In Empower 3 software, you can enter as many as five target masses in the sample table together with a method set containing the corresponding processing method. As a result, you can use the same processing method to process multiple target masses in one sample or different target masses in different samples.

Enter target mass information when you use Run Samples, QuickStart, or Open Access to acquire data. You also can use the Alter Sample function to add or modify masses to data that has already been acquired.

When the sample table contains target masses, and the target mass functionality is enabled in the processing method, the software calculates target masses. You also can specify a target mass in the processing method, in the same way that you did in previous versions of Empower. Target masses

Target Mass 3-7

specified in the sample table supersede the single base mass specified in the processing method.

To enable the Target Mass functionality:

1. Ensure that the Mass Spectrometry option is enabled in the project.

2. Add the target masses to the samples.

3. As in Empower 2, configure the MS processing method for injection-based expected mass processing.

4. As in Empower 2, add the processing method to the Expected Mass dialog in the Method Set Editor. (To open the Expected Mass dialog box, click the Expected Mass button.)

Configuring the MS Processing method

To accommodate multiple base masses, the MS processing method includes the following additions and changes.

New MS processing method additions and changes:

Addition/Change Description

Include the Uncorrected Target or Base Mass in the list of expected masses option (new functionality)

If selected, uses only the adduct-corrected target or base masses in the list of expected masses. If you do not select this option, the software includes the target or base mass and also the adduct-corrected target or base mass in the list of expected masses.

Expected Mass Injection-based base processing (changed functionality)

Enables you to specify expected mass injection-based base masses in either the MS processing method or the sample table.

Injection-based Base Mass and Intensity fields (changed functionality)

Accept blank input.

Ionization Mode (new functionality)

Specifies the ionization mode (positive or negative) of the channel that contains the adduct. You can leave this field blank.


See also: The “Expected Mass Tab” topic in the Empower 3 online information system.

Using the Include the Uncorrected Target or Base Mass option

The Include the Uncorrected Target or Base Mass option includes the uncorrected target or base mass in the list of expected masses. By default, in existing MS processing methods, this option is selected. In new MS processing methods, by default, this option is not selected.

Enabling the Include the Uncorrected Target or Base Mass option

If you select the Include the Uncorrected Target or Base Mass option, you can enter a maximum of four adducts of the same ionization mode provided that you observe the restrictions in the following table.

Percentage of Base Peak column in the Injection-based Adduct table (new functionality)

When the Include the Uncorrected Target or Base Mass option is selected, enables you to specify, in the Injection-based Adduct table, the percentage of base peak for the adduct. The software faults the adduct's expected intensity value in the peak when the value, as a percentage of the peaks's Expected Intensity 1 value, is less than the value that you specify here.

Allowable entries when Include Uncorrected Target or Base Mass is selected:

Condition Allowable entries

Only positive adducts Maximum of four positive adducts

Only negative adducts Maximum of four negative adducts

Positive and negative adducts

Maximum of four positive adducts plus a maximum of four negative adducts

No positive or negative adducts

Maximum of four unspecified (blank) adducts

New MS processing method additions and changes: (Continued)

Addition/Change Description

Target Mass 3-9

Additionally, the software allows the adduct table to remain empty.

To enable the Include the Uncorrected Target or Base Mass option:

1. Create an MS processing method, or open an existing MS processing method in the processing method editor.

2. On the MS Expected Mass tab, select the “Include the Uncorrected Target or Base Mass in the list of expected masses” check box.

3. Make other desired changes to the processing method, and then save it.

Clearing the Include the Uncorrected Target or Base Mass option

If you clear the Include the Uncorrected Target or Base Mass option, you can enter a maximum of five adducts provided that you observe the restrictions in the following table.

Rule: You must add at least one adduct to the table.

Positive and blank adducts

Maximum of three positive and one blank adducts

Negative and blank adducts

Maximum of three negative and one blank adducts

Allowable entries when Uncorrected Target or Base Mass is cleared:


Only positive adducts Maximum of five positive adducts

Only negative adducts Maximum of five negative adducts

Positive and negative adducts

Maximum of five positive adducts plus a maximum of five negative adducts

No positive or negative adducts

Maximum of five unspecified (blank) adducts

Positive and blank adducts Maximum of four positive and one blank adducts

Negative and blank adducts Maximum of four negative and one blank adducts

Allowable entries when Include Uncorrected Target or Base Mass is selected: (Continued)



To clear the Include the Uncorrected Target or Base Mass option:


2. On the MS Expected Mass tab, clear the “Include the Uncorrected Target or Base Mass in the list of expected masses” check box.


Specifying base masses according to the expected mass injection

You can specify base masses according to the expected mass injection in both the MS processing method and sample table. You can add an expected mass processing method to a Method Set via the Expected Mass Processing button in the Method Set editor. This function is available for all MS detectors.

When processing an MS scan channel with a method set containing an expected mass processing method, if the sample table contains target masses, the software creates an MS Result for each target mass. If the sample table contains no target masses, the software creates an MS result for the base mass in the processing method.

Tip: The target masses that you specify in the sample table supersede the single-base mass listed in the processing method.

If you select Include the Uncorrected Target or Base Mass in the List of Expected Masses, the target or base mass is the first mass in the list of expected masses.

If you clear Include the Uncorrected Target or Base Mass in the List of Expected Masses, the target or base mass is not included in the list of expected masses.

To specify expected base masses in the processing method:


2. On the MS Expected Mass tab, select Injection-Based Processing.

3. Select or clear the “Include the Uncorrected Target or Base Mass in the list of expected masses” check box.

Target Mass 3-11

4. Enter a base mass in the Base Mass box.

Alternative: Enter the base mass in the Target Mass 1-5 fields in the Sample Table.

5. In the Adduct table, enter an adduct delta mass value, in Daltons, and then enter an adduct intensity in percent.


Specifying the “Ionization” mode

Use the “Ionization” mode option to match the mode of the adducts of the MS data.

To specify the “Ionization” mode:

In the Expected Mass tab of the MS Processing Method for injection-based processing, use the Ionization mode field in the Adduct table to set the Ionization mode for the adduct.

• If the adduct's Ionization mode is Positive, the adduct is applied only to positive Ionization Mode 3D MS channels.

• If the adduct's Ionization mode is Negative, the adduct is applied only to negative Ionization Mode 3D MS channels.

• If the adduct's Ionization mode is blank, it is applied to all 3D MS channels.

Using the Percentage of Base Peak field

When the Include the Uncorrected Target or Base Mass in the List of Expected Masses option is selected, use the Percentage of Base Peak field to specify a percent value used to fault an adduct's expected intensity value in the peak. The the adduct's percentage of base peak is defined as the adduct's expected intensity value as a percentage of the peaks's Expected Intensity 1 value. If the percentage of base peak is less than this entered value, then the adduct's expected intensity value is faulted.


The following table explains the results of the Percentage of Base Peak search. These results appear in the Expected Mass Found (Peak) field.

Percentage of Base Peak search results:

Result Description

Found Indicates that one or more of the following conditions are true:• If Include the Uncorrected Target or Base Mass in

the List of Expected Masses is selected,

– the system found a target or base mass that does not have a specified percentage intensity value.

– the system found a target or base mass that has a percentage intensity that exceeds the specified percentage intensity value.

• The system found an adduct mass that does not have a specified percentage intensity value.

• The system found an adduct mass that has a percentage intensity value equal to or greater than the specified percentage intensity value.

• The system found an adduct mass that has a percentage of base peak equal to or greater than the specified percentage intensity value.

Target Mass 3-13

Reporting expected mass results

You can use the Expected Mass dialog box in the method set editor to specify an individual or summary report method for each expected mass processing method.

MS library changes

The Spectra to Average field on the MS processing method’s MS Library Search tab now defaults to 5.

Not Found The following conditions are true:• If Include the Uncorrected Target or Base Mass in

the List of Expected Masses is selected:

– The system did not find the target base mass.

– The system found the target base mass, but the mass has a percentage intensity that is less than the specified percentage intensity value.

• The system failed to find an adduct mass that does not have a specified percentage intensity value.

• The system failed to find an adduct mass that has a percentage intensity value equal to or greater than the specified percentage intensity value.

• The system failed to find an adduct mass that has a percentage of base peak equal to or greater than the specified percentage intensity value.

Blank The system did not perform expected mass processing.

Percentage of Base Peak search results: (Continued)

Result Description


4 Fraction Collection with WFC III

This chapter describes new fraction collection functionality in Empower 3.

Contents

Topic Page

Collecting fraction data 4-2

Viewing fraction data 4-2

Formatting fraction data in Review 4-4

Reporting fraction data 4-6

4-1

Collecting fraction data

When you use the Empower 3 software with the Waters Fraction Collector (WFC III) to collect eluent from an injection, the software stores the fraction data, in a fractions table, in the Empower 3 database. The software associates the fractions data with the injection ID.

Because fraction data is stored in the Empower 3 database, you can use the Empower 3 software to view data associated with injections.

Adding and updating fraction data

When you copy an injection to a project, the Empower 3 software copies the fractions associated with the injection into the project database.

When you delete an injection from a project, the software deletes the fractions associated with the injection.

You cannot delete or copy individual fractions between projects.

Viewing fraction data

Viewing fraction data in Review

You can view data from the Fractions tab in Review.

To view fraction data in Review:

1. Bring the desired sample sets, injections, or channels into Review.

2. In the bottom pane of the Review window, click the Fractions tab.

3. Right-click the Chromatogram plot, and then select Properties.

4. Click the Chromatogram tab, and then select the Display Fraction Regions check box.

Result: The pane displays the fraction data for the selected injection.

4-2 Fraction Collection with WFC III

Viewing fraction data in the Project window

The Empower 3 Project window includes the Fractions view, which displays the information described in the following table.

Fraction data in the Project window:


Fraction name A name that the WFC III assigns to the fraction (for example, Fraction 1).

Fraction location The location of the fraction (for example, 1)

Inj Id (Fraction) The Empower 3 software injection ID to which the fraction data is associated.

Fraction Index Numeric index of the fraction within an injection.The software assigns an index to each fraction collected from an injection. The index, which starts at 1, reveals the order in which the software detected the fraction.

Location Index Numeric index of a location within a fraction.Because fractions can span multiple physical tubes or wells, the software assigns an index number to the tube or well used for each location within a fraction. The index, which starts at 1, reveals the order of fraction locations for each fraction. For example, if a fraction spans three tubes, the fraction index has corresponding location indices of 1, 2, and 3.

Fraction Start Time The starting time of the fraction in relation to the injection start time (in minutes).

Fraction End Time The ending time of the fraction in relation to the injection start time (in minutes).

Fraction Volume (mL) The volume of eluent collected for the fraction.

Fraction ID An identification code that the Empower 3 software assigns to the fraction.

Viewing fraction data 4-3

To view fraction data from the Project window:

Open a project, and then click the Fractions tab.

Result: The Fractions Data table displays.

To view sample sets associated with a fraction:

From the Fractions Data table, right-click the row for the fraction whose data you want to view, and then click View As > Sample Sets.

Result: The Sample Sets table displays the sample sets associated with the selection fraction data.

To view injections associated with a fraction:

From the Fractions Data table, right-click the row for the fraction whose data you want to view, and then click View As > Injections.

Result: The Injections table displays the injections associated with the selection fraction data.

To view fractions data from the Injections tab:

From the Injections tab, right-click a row in the injections table, and then click View As > Fractions.

Result: The Fractions table displays the fraction data for the selected injection.

Formatting fraction data in Review

From Review, you can enable the display of fractions regions in chromatograms and format the way fraction data is displayed.

Chromatograms display fractions as regions on the plot. In overlayed chromatograms, fraction lines and labels appear in the color assigned to the chromatogram from which the lines and labels originate.

In single chromatograms, region shading is displayed in gray, if the Enable Fraction Shading check box is selected on the Chromatogram tab in the Plot Properties dialog box. In overlayed chromatograms, region shading is not displayed.

If a plot displays overlayed chromatograms, and you select “Only One Chrom in Overlay” on the Overlay tab in the Plot Properties dialog box, the plot


displays only the fractions associated with the chromatogram you selected. Otherwise, the plot displays fractions for all of the chromatograms.

When you make a selection in the chromatogram, the corresponding fraction information appears in the fractions table.

To display and format fraction regions in a chromatogram:

1. From Review, right-click the chromatogram.

The Plot Properties dialog box opens.

2. Click the Chromatogram tab.

3. In the Chromatogram Fraction Regions section, select Display Fraction Regions.

Rule: By default, this option is not enabled.

4. Select any or none of the options in the following table:

Fraction regions display options:

Options Description

Enable Fraction Shading Adds a gray-shaded background to the fraction regions for overlay stacked and non-overlay chromatograms. If you overlay chromatograms in a single plot, this option is unavailable.

Draw Fraction Lines Uses the Tick Marks/Fractions pen to draw a vertical line indicating the start and the end of each fraction region.If a fraction spans multiple vessels, it can include several “fraction locations.” In fact, the locations are actually part of the same fraction. If this is the case, the start line of the first location and the end line of the last location use the line style, defined by the Tick Marks/Fractions pen. All lines annotating the locations appear as dotted lines.

Formatting fraction data in Review 4-5

Reporting fraction data

You can report fraction regions in the following report groups:

• Chromatogram

• Zeroed Baseline Chromatogram

• Fraction Table

• Internal Standard Peak Plot

• Purity Plot

• Spectrum Index Plot

• System Suitability Peak Plot

• Validation Chromatogram Peak Ratio Plot

• Validation Purity Plot

• Validation Spectrum Index Plot

Tip: Fraction fields are not available in the Fields report group or in other table report groups.

You can use the new Fraction Table report group to report fraction information.

Label Fractions Uses the Tick Marks/Fractions font to put a label in the fraction region.The label includes a concatenated fraction name and the fraction location. For example, A,1. These values are optionally provided by the fraction collector. If the values are not provided, Empower 3 uses the fraction name and fraction location to generate a default label.The labels appear vertically along the upper left edge of the fraction region.

Fraction regions display options: (Continued)

Options Description


During data acquisition for an injection, the Empower 3 software stores the following fraction data, along with injection data, in the Empower 3 database.

The components listed above are available to add to the Fraction Table report group. By default, the Fraction Table report group contains no columns.

Default fraction data:


Fraction location The location of the fraction (for example, 1).

Fraction ID An identification code that the Empower 3 software assigns to the fraction.

Fraction Start Time The starting time of the fraction in relation to the injection start time (in minutes).

Fraction End Time The ending time of the fraction in relation to the injection start time (in minutes).

Fraction name A name that the WFC III assigns to the fraction (for example, Fraction 1).

Fraction Index Numeric index of the fraction within an injection.The software assigns an index to each fraction collected from an injection. The index, which starts at 1, reveals the order in which the software detected the fraction.

Fraction Volume (mL) The volume of eluent collected for the fraction.

Instrument Type The type of instrument that generated the fraction.

Inj Id (Fraction) The Empower 3 software injection ID to which the fraction data is associated.

Location Index Numeric index of a location within a fraction.Because fractions can span multiple physical tubes or wells, the software assigns an index number to the tube or well used for each location within a fraction. The index, which starts at 1, reveals the order of fraction locations for each fraction. For example, if a fraction spans three tubes, the fraction index has corresponding location indices of 1, 2, and 3.

Reporting fraction data 4-7

To report and customize fraction data:


• Create a Fraction Table report group method.

• Open a report method that includes a Fraction Table report group.

2. Right-click the Fraction Table report group, and then select Fraction Table Information Properties.

3. Use the tabs in the Fraction Table Properties dialog box to customize Fraction Table information.

Tip: You can report fraction data from any of the report groups listed on page 4-6.


5 Method validation

This chapter describes method validation functionality that is new or changed in Empower 3.

The new Method Validation Manager capabilities include the determination of Accuracy Profile and Linearity of the Method. These capabilities and calculations are recommended by the SFSTP Commission (France Society of Science and Techniques of Pharmaceuticals) and the UCB Pharmaceutical Society in Belgium.

Contents

Topic Page

Accuracy profile capability 5-2

Linearity of the Method 5-7

Limits of Quantitation 5-10

5-1

Accuracy profile capability

The primary calculation for the Accuracy Profile is Accuracy Total Error. Whereas the current method validation manager approach for accuracy is a risk-based approach, where Alpha represents the risk of rejecting a good result, the Accuracy Total Error approach is a beta approach (the inverse of the alpha approach), where the Beta Tolerance represents the risk of accepting bad results. This approach provides a tolerance interval rather than a confidence interval.

The accuracy profile capability allows the determination of % Total Error, % Relative Error, % Relative Error Mean, and absolute ß-expectation tolerance interval and limits, % relative ß-expectation tolerance interval and limits, and Lower and Upper Limits of Quantitation (LQL and UQL) of the amounts or concentrations at each level. Accuracy profile processing also produces an accuracy profile plot, which displays % Relative Error vs. X-Value.

See also: The “Accuracy test configuration” topic in the Empower 3 online information system.

LQL and UQL are obtained by calculating the smallest and the highest X values where the relative tolerance limits are not outside the acceptance limits.

The LQL is the X value at the low end of the region where the relative tolerance intervals are not outside the acceptance limits. The UQL is the X value at the high end of the region where the relative tolerance intervals are not outside the acceptance limits. A tolerance limit line is outside the acceptance limits at all points where its Y values are either greater than the upper acceptance limit line's Y values or less than the lower acceptance limit line's Y values.

Enabling accuracy profile capability

You enable the accuracy profile capability by selecting the Calculate Accuracy Profile check box when you define the accuracy configuration for a validation protocol method. This check box requires the use of the # Experiments and Experiment Group By fields in the Acquisition Requirements.

Enabling the Calculate Accuracy Profile check box in the Accuracy Acquisition Parameters instructs the software to perform the calculations necessary to calculate an accuracy profile for the accuracy test. The Calculate Accuracy Profile check box is unchecked by default.

5-2 Method validation

Accuracy result fields

The following results are determined during accuracy profile processing.

Accuracy result fields:

Table Field name Description

Accuracy Test Data

% Relative Error

% Relative Error is the relative % bias of the data point, which is % deviation of Calculated X Value with respect to the X Value Mean of the level:% Relative Error = 100 (Calculated X Value - X Value Mean) / X Value Mean

Experiment Group

The value in the Experiment Group By field for this data point.

Calibration Curve ID

The Id from the calibration curve used to calculate the % Recovery value for this data point.

Accuracy Test Level Summary

% Relative Error Mean

% Relative Error Mean is the % relative bias at this level, which is the % deviation of Calculated X Value Mean from X Value Mean with respect to X Value Mean:% Relative Error Mean = 100 (Calculated X Value Mean - X Value Mean) / X Value MeanNote: Calculated X Value Mean is not reported in the Level Summary table.

% Total Error

% Total Error is the sum of the errors due to bias and precision of Calculated X Value with respect to X Value Mean at this level. This precision includes both the within experiment variance and the between experiment variance.Also the absolute value of the bias is used in the calculation.% Total Error = |% Relative Error Mean| + 100 Intermediate Precision SD / X Value MeanNote: Intermediate Precision SD of Calculated X Value is not reported in Level Summary Table.



Absolute TI Absolute ß-expectation tolerance interval of Calculated X Value at this level, which is calculated based on intermediate precision one-way ANOVA for Calculated X Value (as assessed value) vs. “Experiment” (as variable) from all data points at this level. The calculation can be simplified, as follows, for a balanced design (in which the number of data points in each experiment are the same at this level):N = total number of data points at this levelp = number of experimentsn = number of replicates per experimentR = Ratio of inter- to intra-variance componentVARip = total intermediate precision variance component = inter-variance component + intra-variance componentB^2 = (R+1) / (nR+1)VAR = (1+1/(pnB^2)) VARipv = effective degree-of-freedom of above variance using Satterthwaite formula:

v = (R+1)^2 / ((R+1/n)^2/(p-1)+(1-)1/n)/(pn))Note: Above v is rounded to an integer.

Qt = quantile of one-tailed t-distribution, which is the inverse one-tailed t-distribution at given significance level alpha and degree-of-freedom v calculated above.Absolute TI = Qt SQRT(VAR)

Absolute LTL

Lower absolute ß-expectation tolerance limit of Calculated X Value at this levelAbsolute LTL = Calculated X Value Mean - Absolute TINote: Calculated X Value Mean is not reported in the Level Summary Table.

Accuracy result fields: (Continued)




Absolute UTL

Upper absolute ß-expectation tolerance limit of Calculated X Value at this levelAbsolute UTL = Calculated X Value Mean + Absolute TINote: Calculated X Value Mean is not reported in the Level Summary Table.

Relative TI % Relative ß-expectation Tolerance interval of the % Relative Error at this levelRelative TI = 100 Absolute TI / X Value Mean

Relative LTL

Lower relative ß-expectation tolerance limit of % Relative Error at this levelRelative LTL = % Relative Error Mean - Relative TI

Relative UTL

Upper relative ß-expectation tolerance limit of % Relative Error at this levelRelative UTL = % Relative Error Mean + Relative TI

LQL X value at the low end of the region where the relative tolerance intervals are not outside the acceptance limits.

UQL X value at the high end of the region where the relative tolerance intervals are not outside the acceptance limits.

Experiment Number of experiment groups in the test.

Points/ Experiment

Number of replicate data points in each experiment group. This field is empty when the number of replicate data points is not the same in all the experiment groups in a unbalanced design.

Experiment Group By

The name of the experiment field copied from method.

Acceptance Limit

The Acceptance Limit copied from the test parameters.

Accuracy result fields: (Continued)



Reporting Accuracy Profile Plot information

The new Accuracy Profile Plot report group enables you to report the information in accuracy profile plots, which are graphical depictions of the points in Data and/or Level Summary tables. This report group appears under the Accuracy Groups node of the Report Group tree.

The Accuracy Profile Plot report has the following characteristics:

• The % Relative Error is plotted on the Y axis against the X value on the X axis.

• The summary points for the % Relative Error are plotted and connected by a dashed line.

• The Lower Relative Tolerance Limit values and the Upper Relative Tolerance Limit values versus the summary points for the x axis data are shown on the plot as straight lines.

• When you enter an acceptance limit value in the validation method, the Upper and Lower Accuracy Acceptance Criteria appear on the plot.

– The Upper Accuracy Acceptance Criteria are displayed as a horizontal line on the plot at the Acceptance Criteria percentage value.

– The Lower Accuracy Acceptance Criteria is displayed as a horizontal line on the plot at the Acceptance Criteria percentage value multiplied by (-1).

• If you enable plotting of the % Relative Error data points, these values are displayed as points on the plot.

• If you enable plotting of the LQL and UQL values, these values are shown as vertical lines at the LQL and the UQL.

To report and customize Accuracy Profile Plot information:


• Create an Accuracy Profile Plot report group method.

• Open a report method that includes an Accuracy Profile Plot group.

2. Right-click the Accuracy Profile Plot report group, and then select Accuracy Profile Plot Properties.


3. In the Accuracy Profile Plot Properties dialog box, in the Items to Include in Report section, select or clear the following options:

• % Relative Error

• LQL and UQL values

4. Use the tabs in the Accuracy Profile Plot Properties dialog box to customize the Accuracy Profile Plot report according to the following properties:

Linearity of the Method

The linearity processing parameters, which assess the Linearity of the Response, include the new option Linearity of the Method.

Linearity of the Method fits a linear curve to a set of (X,Y) points where the X value is the user-entered amount or concentration of the Linearity of the Response curve, and the Y value is the calculated value of that point from the Linearity of the Response curve.

During Validation processing for linearity, if Linearity of the Method is not selected, the test produces only Linearity of the Response results (as in Empower 2 software). If the Linearity of the Method processing is enabled, the test produces both Response Function Linearity Results and Method Linearity Results.

Validation results determined using linearity of the result and linearity of the method are distinguished in the Linearity Type field, which is set to either “Result” or “Method”.

Tip: In the Empower 3 software and user documentation, the term “Linearity” generally refers to Linearity of Response.

Linearity processing steps

Use the Linearity of the Method check box on the Processing and Acceptance Criteria tab of the validation protocol methods Linearity test to indicate whether the linearity test assesses the Linearity of the Method.

When Linearity of the Method is not selected, the software processes linearity results by creating a Response Function Linearity result. Empower 3 software calculates Linearity of Response results using the same methodology as previous versions of Empower software.


When Linearity of the Method is selected, the software processes linearity results by performing the following steps:

1. Creates a Response Function Linearity result, including a linearity curve and its statistics.

2. Creates a Method Linearity result by fitting a linear curve to a set of (x, y) points where the X value is the X value of a linearity point from step 1 and the Y value is the Calculated Value of the same point, and then calculating the statistics for that curve.

Rule: If the fit type used to calculate the Linearity of the Method curve is not Linear or Linear-Through-Zero, the software calculates a linearity curve but does not assess linearity of response. Validation results determined using linearity of the result and linearity of the method are distinguished in the Linearity Type field, which is set to either “Result” or “Method”.

New processing method fields

When the Linearity of the Method function is enabled, three new fields become available in the Component Type table in the Processing and Acceptance Criteria tab:

• Fit Type

• Weighting

• Average By

These fields are equivalent to the Fit, Weighting, and Average By fields in the Component table in the LC processing method, and have the same choices and restrictions as the fields in the component table, except that they can be left blank (the default setting).

If the Fit Type, Weighting, and Average By fields in the validation protocol method are blank, the processing method's component fields are used to calculate the linearity of response. If the fields are not blank, the selections override the processing method's component fields when calculating the calibration curve (response function).

Use the Linearity Type fields in the Acceptance Criteria and Level Acceptance Criteria tables to set the type of linearity result to which the acceptance criteria applies. The Linearity Type of existing Linearity Acceptance Criteria and Linearity Level Acceptance Criteria is set to Response Function.


Linearity comparison test

When the Linearity of the Method processing is enabled, the Linearity Comparison test assesses the linearity of “Method” Linearity Results (linearity results with their linearity types set to Method).

When the Linearity of the Method processing is disabled, the Linearity Comparison test assesses the linearity of the Response Function type Linearity results.

The Linearity Comparison test compares only linearity test results with the same linearity type. The Linearity Type field in the Linearity Comparison Result indicates the type of linearity results compared.

Linearity and Linearity Comparison report groups

The following table summarizes changes, in Empower 3, to the Linearity and Linearity Comparison report groups.

Changes to the Linearity and Linearity Comparison report groups:

Report group Description of changes

Linearity and Linearity Comparison

• Support for Linearity Type.• Separation of Linearity of the Response

Function and Linearity of the Method test results.

Linearity Level Sum Table Inclusion of Linearity Type properties Response Function (default), and Method.

Linearity Plot and Linearity Comparison Plot

Inclusion of Linearity Type properties Response Function (default), and Method.

Regression ANOVA Table Inclusion of Linearity Type properties Response Function (default), and Method.

Residual ANOVA Table Inclusion of Linearity Type properties Response Function (default), and Method.

Residuals Plot and Residuals Comparison Plot

Inclusion of Linearity Type properties Response Function (default), and Method.

Response Factor Plot and Response Factor Comparison Plot

Report only response function linearity and linearity comparison results.


Tip: Other ANOVA and Test Effect Report tables not included in the preceding table do not include the new Linearity Type property page.

The Linearity Type tab on the plot properties dialog box enables you to specify the type of Linearity or Linearity Comparison test results the software reports. This tab includes the properties described in the following table.

To specify Linearity Type properties:

1. In Report Publisher, add the desired report group to the report layout.

2. Right-click the report group and select Report group name Plot Properties.

3. In the plot properties dialog box, click the Linearity Type tab and then select the desired Linearity type.

4. Click OK.

Limits of Quantitation

When the Method to Determine LOD/LOQ is set to Signal to Noise, selecting USP s/n or JP s/n in the S/N Field dropdown list instructs the software to calculate LOD and LOQ using the USP s/n or JP s/n values calculated by system suitability processing.

When the Method to Determine LOD/LOQ is set to Linearity Curve Intercept SD or Linearity Curve Residual SD, the software uses SD values from Linearity Results where the Linearity Type is Response Function.

Linearity Type properties:

Property Description

Response Function (default)

Directs the software to report test results for only response function.

Method Directs the software to report test results for only Linearity of the method.


6 ACQUITY UPLC Enhancements

This chapter describes how the Empower 3 software enhances ACQUITY UPLC.

Important: The Empower 3 software supports the features described in this chapter. However, an updated ACQUITY UPLC Driver must be installed to enable these features. The updated driver pack is expected to become available in March, 2011.

Contents

Topic Page

eCord data in the Empower 3 database 6-2

ACQUITY UPLC sample loop support 6-7

ACQUITY UPLC sample queue functionality 6-8

6-1

eCord data in the Empower 3 database

ACQUITY UPLC columns include an eCord™ column chip that tracks the usage history of the column. The eCord column chip interacts with the Empower 3 software and records column information for sample sets run on the column.

There are three types of eCord data:

• Cumulative column usage information (per column lifetime)

• Per sample set column usage information (per sample set)

• Data associated with the injections

In addition to variable column usage data, the eCord column chip also stores fixed column manufacturing data such as

• column name

• serial number

• part number

When you attach the eCord column chip to a receptacle on the ACQUITY UPLC column heater or column manager and use Empower 3 to acquire data, the system records column usage information automatically, stores it in the Empower 3 database, and displays it in Configuration Manager. The user need not perform any action to record column information.

Note: Time data stored on the eCord does not have an associated time zone. Acquisition data adopts the time zone of the computer on which the data was acquired.

Ecord information does not appear in Configuration Manager until at least one injection has been acquired using the eCord.

eCord data is not associated with projects.

For information about reporting eCord data, see “Reporting eCord data” on page 6-5.

Adding eCord data to the database

When you use an ACQUITY system that contains a column with an eCord to perform an injection, the system either updates the column usage information for an existing eCord, or adds a new eCord entry and its column usage data to the Empower 3 database.

6-2 ACQUITY UPLC Enhancements

The system displays eCord information in the eCord nodes of the Configuration Manager tree. Click the upper eCord node to display all of the ACQUITY columns that have been connected to the Empower 3 database, and to see cumulative column usage information. When you select a specific column in the tree, the Configuration Manager table displays a list of the sample sets that have been acquired on the selected column, and the column’s usage information.

Tip: The Configuration Manager presents eCord column pressure in psi regardless of the type of units selected and displayed in the ACQUITY UPLC console. Although the Configuration Manager uses a different unit of measurement than the ACQUITY UPLC console, the data displayed in Configuration Manager is correct and equivalent to the measurements presented in the ACQUITY UPLC console.

Updating eCord data

After each injection, Empower 3 software updates eCord data stored in the Empower 3 database with column usage information. The Empower 3 software also updates the database upon completion of each sample set so that the eCord sample sets table remains up to date. You can use standard database backup and restoration to back up eCord information. If you use the Empower 3 Workgroup or Enterprise configuration, you also can use a system object export procedure to back up eCord information.

Tip: System object export is not available on the Personal configuration.

If you disconnect an eCord from a database, use it on a different database, and then reconnect the eCord to the initial database, the Empower 3 software updates the database and the sample sets table with information about the new sample set entries added to the eCord flash memory. The update occurs upon submission of an injection or a sample set. The memory of the ecord chip can retain column usage data for up to 50 sample sets. Once the memory of the chip is full, new data overwrites the older data on the chip. However, in the Empower 3 database, eCord data is never overwritten. It continually is added.


Viewing and managing eCord data

Configuration Manager displays all eCord columns that are, or have been, connected to the Empower 3 database. The following table describes data that is associated with eCords.

To view eCord data:

1. In the left-hand pane of Configuration Manager, select the eCord node.

2. Select an eCord sub-node.

eCord summary data (column lifetime information):

Field Description

ID The unique ID associated with the eCord/column

Name The name associated with the eCord/column

Part Number The part number of the eCord/column

Serial Number The serial number of the eCord/column

Injection Count The number of injections for which the eCord recorded data

First Injection Date The date of the first injection for which the eCord recorded data

Last Injection Date The date of the last injection for which the eCord recorded data

Sample Count The number of samples for which the eCord recorded data

Sample Set Count The number of sample sets for which the eCord recorded data

Maximum Pressure The maximum pressure recorded on the eCord/column

Maximum Pressure Date The date on which the maximum pressure is recorded on the eCord

Maximum Temperature The maximum temperature recorded on the eCord/column

Maximum Temperature Date

The date on which the maximum temperature is recorded on the eCord


Reporting eCord data

The new eCord information report group enables you to create reports of the eCord information that is displayed in Configuration Manager. This report group appears in the main level of the report group tree.

You can create eCord reports in the same way that you create other reports. Follow the instructions in the report wizard to create a report layout, and then drag and drop rows in the eCord column database table into the report.

The eCord sample set data is updated at the end of each sample set acquisition. This information is reported as a set of all historical information for sample sets acquired with the eCord being reported, based on the serial number.

To report and customize eCord information:

1. Drag and drop rows from the eCord Column database table in Configuration Manager into the open report method in Report Publisher.

Tip: You cannot use the eCord information group to report eCord information associated with injections.

eCord sample set data:

Field Description

Sample Set Date The date on which the sample set was run.

Sample Set Name The name of the sample set.

User Name The name of the user who ran the sample set.

System Name The name of the system on which the sample set was run.

Injection Count The number of injections for which the eCord recorded data

Sample Count The number of samples for which the eCord recorded data

Maximum Pressure The maximum pressure recorded on the eCord

Maximum Temperature The maximum temperature recorded on the eCord



• Create an eCord Information report group method.

• Open a report method that includes an eCord Information report group.

3. Right-click the eCord Information report group, and then select eCord Information Properties.

4. In the eCord Information Properties dialog box, in the Information to Print section, select one or both of the following options:

• Column Summary Data

• Sample Set Data

5. Use the tabs on the eCord Information Properties dialog box to customize the eCord Information report.

During data acquisition for an injection, the Empower 3 software stores eCord data, along with injection data, in the Empower 3 database. The following fields are available in the Project window’s Injection tab and in Report Publisher.

You also can add ACQUITY column information to your chromatographic reports by adding the eCord fields to tables, plot legends, and so on. These fields are available in the Injection node of the reporting group's properties.

eCord injection data:

Field Description

eCord Name The name associated with the eCord/column

eCord Serial Number The serial number of the eCord/column

eCord Injection Count (Lifetime to date)

The number of injections for which the eCord recorded data

eCord Sample Count (Lifetime to date)

The number of samples for which the eCord recorded data

eCord Maximum Pressure (Lifetime to date)

The maximum pressure recorded on the eCord

eCord Maximum Temperature (Lifetime to date)

The maximum temperature recorded on the eCord


Deleting eCord data from the Empower database

The Delete eCords privilege allows you to delete eCord entries from the Empower database.

You must delete entire eCord entries; you cannot delete individual sample set line items within an eCord entry.

To delete eCord entries:

1. In the eCord database table, right-click the eCord entry that you want to delete, and then click Delete.

2. At the prompt, click Yes to verify that you want to delete the entry.

Depending on the policies enabled in your environment, when you delete an eCord entry from the database, the system generates audit trail entries and records them in the database. The System Audit Trail tab includes an eCord entry.

ACQUITY UPLC sample loop support

Empower 3 software supports two injection modes for the ACQUITY UPLC Sample Manager:

• Partial loop with needle overfill

• Full loop

In Partial loop with needle overfill mode, you must enter an injection volume within the valid injectable volume range defined by the instrument.

In Full loop mode, the injection volume is limited to the volume of the installed loop.

Restriction: The Sample Manager Flow Through Needle (SM- FTN) module does not support Full loop mode. The maximum injection volume is the extension loop volume, when the configured loop volume is greater than 0. Otherwise, the maximum injection volume is the configured needle volume minus 5 µL.

Empower 3 software supports these modes in both the Run Samples and QuickStart user interfaces.

ACQUITY UPLC sample loop support 6-7

ACQUITY UPLC sample queue functionality

This section describes the ACQUITY UPLC sample queue functionality supported by the Empower 3 software.

Auto-addition

The Empower 3 software supports the Auto-addition function for the ACQUITY UPLC Sample Manager Flow-Through Needle (SM-FTN) module.

Fixed vials

Fixed vials are vial locations that are built into the SM-FTN module. They are not part of a sample plate. You can include fixed vial locations in sample sets or use them for single injections. The Sample Manager uses four fixed vials positions: V:1, V:2, V:3, and V:4.

You can enter fixed vial positions in the Empower 3 software.

To enter the fixed vial position:

1. To open the single injection tab, perform one of the following tasks:

• In Run Samples, click the Single tab.

• In QuickStart, click Single Injection.

2. In the vial column, specify the fixed vial position as V:position, where position is the sequential location of the vial in the sample tray (1 through 4).

Example: V:1

Column selection

When your chromatographic system contains an ACQUITY UPLC Column Manager (CM) or Column Manager Auxiliary (CM-Aux), use the Column Position field in the Run Samples table to specify the column configuration.

The following configurations are available:

• Column Manager (CM-A) – 4 columns

• Column Manager Auxiliary (CM-Aux) – 2, 4, or 6 columns


7 Post Run Report group

This chapter describes the Empower 3 Post Run Report group.

Contents

Topic Page

Post Run Report group 7-2

7-1

Post Run Report group

The Empower 3 software stores Post Run Report information from instruments in a way that enables instruments with no detector component to report information. To facilitate this new functionality, the software includes the new Post Run Report group.

The new Post Run Report group and the Comprehensive Status Report group differ in the following ways:

• The Post Run Report group reports information acquired from instruments by the Empower 3 software.

The Comprehensive Status Report group reports information acquired from instruments by versions of Empower software prior to Empower 3.

• The Post Run Report group stores information with the injection, resulting in one set of information per injection.

The Comprehensive Status Report group stores information with the channel, resulting in one set of information per channel.

Tip: The Post Run Report group provides information only for instruments controlled through Instrument Control Software (ICS), and does not provide information for instruments for which the driver is built into the Empower 3 software.

Both the Post Run Report and Comprehensive Status Report groups provide instrument status information for injections that is collected from the instrument during acquisition. The Comprehensive Status Report does not provide information if the data is acquired using a chromatographic system with no data channels. The Post Run Report group provides information for data acquired on chromatographic systems with or without a detector component.

To report and customize a Post Run Report information:


• Add a Post Run report group to a method.

• Open a report method that includes a Post Run report group.

2. Right-click the Post Run Report group, and then select Post run Report Properties.

3. Use the tabs on Post Run Report Properties dialog box to customize the Post Run Report.

7-2 Post Run Report group

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