User Manual

Chromeleon 7 Extension Pack

ICH Templates - Specificity (PP)

Thermo Scientific Dionex Chromeleon

Version 7.1 SR1

Document Revision: 1
October 2011
© Thermo Fisher Scientific
Table of Contents
1 Introduction 1
1.1 About this Document 1
1.2 Extension Pack 1
1.3 ICH 1
1.4 Parameters in Chromeleon 2

2 Requirements 3

3 Known Issues and Limitations 4

3.1 Limitations 4
3.2 Known Issues 4

4 Getting Started 5
4.1 Importing the ICH Templates 5
4.2 Description of the ICH Specificity (PP) eWorkflow template 6
4.2.1 eWorkflow General 6
4.2.2 Sequence General 6
4.2.3 Sequence Layout 8
4.2.4 Resulting Sequence 9
4.3 Customizing the ICH Specificity (PP) eWorkflow template 9
4.3.1 eWorkflow General 11
4.3.2 Sequence General 11
4.3.3 Sequence Layout 11

5 Step By Step Guide 13

5.1 Creating a Sequence 13
5.1.1 Launching the ICH Specificity (PP) eWorkflow 13
5.1.2 Modifying the created sequence 14
5.2 Running the Sequence 15
5.3 Processing the Data 15
5.3.1 Processing Method 15
5.3.2 Updating the Component Table 16
5.4 Reviewing Results 17
5.5 Creating the ICH Specificity (PP) report 18
5.5.1 Printing the report 19
5.5.2 Exporting the report 19

6 Description of the Report Sheets 21

6.1 Report Sheet “Specificity (PP)” 21
6.1.1 Limitations 21
6.2 Other Report Sheet 21

7 Combined Validation Report 22

7.1 Importing Query 22
7.2 Modifying Query 22
7.3 Printing / Exporting the Query 22
1 Introduction

1.1 About this Document

The layout of this User Manual is designed to provide quick reference to sections of interest. However, we recommend that
you review the User Manual thoroughly before working with the Chromeleon 7 Extension Pack in order to obtain a full
understanding of the software.
This User Manual is provided "as is". Every effort has been made to supply complete and accurate information and all
technical specifications and programs have been developed with the utmost care. However, Thermo Fisher Scientific Inc.
assumes no responsibility and cannot be held liable for any errors, omissions, damage, or loss that might result from any
use of this manual or the information contained therein. We appreciate your help in eliminating any errors that may appear in
this document.
At various points throughout this document messages of particular importance are indicated by the following symbols:

Note: Indicates general information that points out specific items of interest.

Caution: Indicates areas where operations should be performed carefully.

The information contained in this document is subject to change without notice.

Chromeleon® and UltiMate® are registered trademarks of Thermo Fisher Scientific Inc.. Any other mentioned trade or company names are subject to the copyright and the
property and trademark rights of the respective companies.
All rights reserved including those for photomechanical reproduction and storage on electronic media. Without the written permission of Thermo Fisher Scientific Inc., no part
of this publication may be reproduced in any form (by means of photocopy, microfilm, or any other process) for any purpose or processed, copied, transmitted, or distributed
in any other form, independent from the means, electronic or mechanical, that is used.

1.2 Extension Pack

The Chromeleon Extension Pack contains templates that allow you to quickly and easily calculate results that require
advanced calculations, for example ICH Specificity (PP) determinations. All required items to perform the calculation are
combined in an eWorkflow:
• Sequence Templates

• Processing Methods

• Report Template

• Custom Variables

Furthermore the eWorkflow contains the following files:

• User Manual

• Example Sequence

1.3 ICH
The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH) provides recommendations on the validation of analytical procedures (Q2(R1)). Recommended tests include -

• Accuracy

• Detection Limit

• Linearity

• Precision

• Quantitation Limit

• Range

• Robustness
• Specificity

The calculation and reporting of all data requested by ICH can be very tedious and time consuming. The ICH method
validation templates provided in the Chromeleon 7 Extension Pack automate these steps and allow the quick and easy
creation of method validation reports.

1.4 Parameters in Chromeleon

To perform the ICH Specificity (PP) calculations in the Chromeleon report template, additional information that cannot be
readily entered in Chromeleon is required. Table 2 gives an overview of the required information.

Parameter Description

TEST Identifies the ICH test. This is an injection custom variable that can be entered in the injection list.

Minimum Peak Purity allowed for component. This is a component custom variable that can be entered in the
Min_PP
component table in the processing method.

Test Specifies if the test will be run for the corresponding component or not.
Table 1: ICH Parameters in Chromeleon
2 Requirements
The ICH templates can only be used with Chromeleon 7.1 SR1 or higher.
It is possible to export the ICH results in the Adobe® Acrobat® PDF format. Acrobat Reader version 5.0 or higher is needed
to read these reports.
3 Known Issues and Limitations

3.1 Limitations
• There are no limitations.

3.2 Known Issues

• The “Print Preview” option does not work for graphical objects.
4 Getting Started

4.1 Importing the ICH Templates

All files required for a particular ICH test are contained in the relevant ICH Template eWorkflow. Each eWorkflow is named
according to the test e.g. ICH Specificity (PP), and distributed as a Chromeleon backup file with .ewfx, extension. The files
are located on the Chromeleon 7 disc, in the folder \Extension Pack\ICH Templates.
1. In the Chromeleon Console go to the eWorkflows category.

2. In the Menu click File, Import From.

3. Select the relevant eWorkflow backup file and click Open.

4. Select in which Data Vault the eWorkflow must be imported and click Start (Figure 1).

Figure 1: eWorkflow import - Select Data Vault

The eWorkflow is now imported and visible in the list of eWorkflows (Figure 2A). A Chromeleon backup file of the demo data
(Figure 2B) and User Manual (Figure 2C) are directly accessible.

Figure 2: Imported eWorkflow

4.2 Description of the ICH Specificity (PP) eWorkflow template
4.2.1 eWorkflow General
The eWorkflow General page is used to specify some general settings for the eWorkflow and to associate instruments,
methods, and documents. The eWorkflow General settings for the ICH Specificity (PP) Template are displayed in Figure 3.

Figure 3: eWorkflow General page

Template settings are as follows:

Figure 3 Property Setting

eWorkflow State Approved for Use

eWorkflow Type HPLC

This eWorkflow contains all files for performing Specificity (PP) calculations. Please refer to the
eWorkflow Description
attached user manual for information.

No instruments are associated with the ICH Specificity (PP) Template eWorkflow. This
Instruments
eWorkflow can therefore be used for all instruments.

Processing Method ICH Specificity (PP), containing the required custom variables for
Specificity (PP) calculations.
Methods Report Template Method_Validation, containing all required calculations for Specificity (PP).
View Settings ICH Specificity, to display chromatogram information in the Data Processing
Category.

Chromeleon backup file ICH Specificity (PP) - Demo Data.cmbx, containing an ICH Specificity
Attachments (PP) example
PDF document ICH Specificity (PP) - User Manual.pdf
Table 2: Default eWorkflow General settings
Section 4.3 describes how these settings can be customized.

4.2.2 Sequence General

The Sequence General page is used to specify general settings for the sequence and select a default report and view
settings.
 Figure 4: Sequence General page
Template settings are as follows:

Figure 4 Property Setting

1
Default Report Template Method_Validation

2
Default View Setting ICH Specificity

3 Defines the channel to be displayed as the default channel when opening an injection.
Default Channel
As this depends on the instrument and corresponding instrument method this is empty.

‘ICH Specificity (PP)’ followed by current data/time (Example: ICH Specificity (PP)
Sequence Name
20110511 161743)

Data Vault ChromeleonLocal

ICH/Specificity is the directory that the resulting sequence is stored in. If the folder in this
Path directory path does not exist, it will be automatically created when the first sequence is
created.

All signature steps to submit electronic signatures are checked, to enable a qualified
Electronic Signature
user to electronically sign the sequence and submit, review or approve it.

Comment This sequence is created using the ICH Specificity (PP) eWorkflow.

Table 3: Default Sequence General settings

1
Report template to be used as the default template for presenting the data in a report.
2
View settings to be used as the default settings for presenting the data in the Chromatography Studio.
3
Channel to be displayed as the default channel when you open an injection.
Section 4.3 describes how these settings can be customized.

4.2.3 Sequence Layout

The sequence layout tab defines rules for the sequence layout, for example, the number of repetitions of Specificity (PP)
injections. The sequence layout helps to create a sequence with a well-defined structure. A sequence layout comprises four
block types, each marked by a different color.

Figure 5: Sequence Layout page

Template settings are as follows:

Figure 5 Property Setting

1
Sequence Header <Empty>

By default the sequence is setup to make 2 Specificity (PP) sample injections of 2 samples
prepared using two different methods (total of 2 injections). This is typically performed by creating
2 a solution of the final dosage form, and then degrading it (e.g. by using hydrogen peroxide or by
Sample Block
placing in a light box). However, it is not mandatory to follow this approach. You can add or
remove injections to meet your requirements. Injections can also be set as multiple injections from
the same vial in the same autosampler vial position.

3
Bracket <Empty>

4
Sequence Footer <Empty>

Table 4: Default Sequence Layout settings

1
Sequence Header includes injections that always appear at the beginning of a sequence, such as blank injections.
2
Sample Block includes a line for each sample that needs to be analyzed. The sample block is repeated between brackets.
3
Bracket includes injections that will be performed between sample blocks (for example, calibration standards). A bracket
separates two sample blocks from each other.
4
Sequence Footer includes injections that always appear at the end of a sequence.
The settings in the sequence columns are as follows:

Sequence Column Setting

No. of inj. 1

Name Dosage Form

 Type Unknown

Position <uneditable>

Volume 1µl

Instrument Method <empty>

Processing Method ICH Specificity (PP)

Status <uneditable>

Inject Time <uneditable>

Weight 1.0000

Dilution 1.0000

IntStd 1.0000

ID <empty>

TEST Separate injections must be assigned the test “Specificity (PP)”

Comment <empty> user can define test level conditions e.g. Degraded dosage form with H2O2
Table 5: Default sequence column settings

4.2.4 Resulting Sequence

When launching the ICH Specificity (PP) eWorkflow template without making any changes, the resulting sequence will
look like Figure 6.

Figure 6: Resulting sequence

4.3 Customizing the ICH Specificity (PP) eWorkflow template

The eWorkflow can be customized to fit any application. When doing so the number of steps to do before being able to run a
ICH Specificity (PP) analysis is reduced to a minimum.
To start customization:
1. In the Chromeleon Console go to the eWorkflows category.

2. In the navigation pane select the ICH Specificity (PP) eWorkflow template (Figure 7A).
 Figure 7: Start editing the ICH Specificity (PP) eWorkflow templates
3. In the work area click Edit (Figure 7B). The eWorkflow editor now opens.

4. To save the eWorkflow with a new name click File, Save As.

5. In the Save eWorkflow dialog select the Data Vault (Figure 8A) to store the eWorkflow and enter a name (Figure 8B)
for the new eWorkflow.

Figure 8: Saving eWorkflow with new name

The eWorkflow can now be customized. The settings to modify are described in sections 4.3.1, 4.3.2, and 4.3.3.

Tip: For an extensive explanation of modifying eWorkflows and defining the sequence layout, refer to the Chromeleon 7
online help.
4.3.1 eWorkflow General

Figure 3 Property Customization

While customizing the eWorkflow the state can be set to In Development. This way it is clear to
eWorkflow State users the eWorkflow is still being worked on. When the customization is finalized the state can
be set back to Approved for Use.

eWorkflow Description Provide a descriptive text for the eWorkflow.

Add the instrument(s) that can run this ICH analysis. The eWorkflow can only be launched on
listed instrument(s).
Instruments
When no instruments are added, the eWorkflow can be used with all instruments accessible to
the user.

Add the instrument method(s) for the instrument(s).

Modify the processing method to:
Methods - have a suitable chromatogram integration
- include the required standard components and their molecular weight
- define the ICH calibration type

The current manual can be removed or other documents can be attached, for example a
Attachments
relevant SOP about running the ICH Specificity (PP) application.

Table 6: Customization options for the eWorkflow General page

4.3.2 Sequence General

Figure 4 Property Customization

When instrument method(s) are added to eWorkflow General tab a list of corresponding
Default Channel
channels is available. Select a default channel.

If the default sequence name is not suitable, define a sequence name. Note that variables can
Sequence Name
be used for dynamic sequence names.

If the default ChromeleonLocal is not suitable, select the Data Vault where the sequences
Data Vault
should be stored.

If the default ICH/Specificity path is not suitable, select the folder location where the sequences
Path
should be stored. Note that variables can be used for dynamic folder names.

If electronic signatures are not used, uncheck the Submit electronic signatures option.
Electronic Signature
If electronic signatures are used, define the appropriate signature steps.

Comment Provide a comment for the created sequence.

Table 7: Customization options for the Sequence General page

4.3.3 Sequence Layout

Figure 5 Property Customization

Sequence Header Customize sequence header per your requirements.

Sample Block Customize sample block per your requirements.

Bracket Customize bracket per your requirements.

Sequence Footer Customize sequence footer per your requirements.

Table 8: Customization options for the Sequence Layout page

Sequence Column Setting

No. of inj. Enter the number of injections that should be injected from the same vial.

Name Enter relevant name

Type Must remain as Unknowns

Position This is a non editable field, which will be filled when creating the sequence.

Volume Enter desired injection volume

Select the correct instrument method. If the eWorkflow can be used on multiple instruments
Instrument Method
with different instrument methods, it’s better to leave this field empty.

When the processing method is updated and has a new name, select the correct processing
Processing Method
method.

Status <uneditable>

Inject Time <uneditable>

Weight 1.0000

Dilution 1.0000

IntStd 1.0000

TEST Separate injections must be assigned the test “Specificity (PP)”

Comment User can define test level conditions e.g. Degraded dosage form with light
Table 9: Customization options for the sequence columns
5 Step By Step Guide
Using the ICH Specificity (PP) eWorkflow template involves a simple process:
1. Creating a sequence, using the ICH Specificity (PP) eWorkflow template. This creates a Sequence that checks the
expected result for the analyte peak against the actual result and expresses it as a percentage (e.g. % recovery).

2. Processing the data

3. Reviewing the results

4. Creating the ICH report

This section provides a step-by-step guide for using the ICH Specificity (PP) template.

5.1 Creating a Sequence

5.1.1 Launching the ICH Specificity (PP) eWorkflow
To create a sequence using the ICH Specificity (PP) eWorkflow follow these steps:
1. In the Chromeleon Console go to the eWorkflows category.

2. Select the relevant eWorkflow (Figure 9A) and the instrument (Figure 9B) to use.

Figure 9: Launching customized eWorkflow

3. Click Launch (Figure 9C). The eWorkflow wizard is launched (Figure 10).
 Figure 10: eWorkflow wizard
4. Depending on the settings in the eWorkflow various settings have to be made. The minimum required entries are the
Number of samples and the starting position of the vials in the sampler. Optionally the setting Run sequence after
creation can be activated.

5. Click Finish. The sequence is now created (Figure 11).

Figure 11: Created sequence

5.1.2 Modifying the created sequence

Depending on the setup of the eWorkflow, some modifications to the sequence are required, e.g.:
• Modify the positions of the injections in the automated sample injector.

• Select the correct Instrument Method for all injections.

• There is a column called “TEST” available. This is used to tell Chromeleon what test an injection is assigned to.
Separate injections must be assigned the test “Specificity (PP)”.

• Define the test level conditions in the comment field for each sample e.g. Degraged dosage form with H2O2.
 Figure 12: Setting Custom Variables

Note: All information in the sequence table can be updated after the sequence has run.

Note: If you do not want to use the data from a specific test, ensure that the sample type is set to “Unknown” and that the
injection is not assigned a “TEST” value.

5.2 Running the Sequence

Now that the sequence is created and modified, it can be started. Follow these steps:

Note: When the option Run Sequence after creation is used in the eWorkflow wizard (see section 5.1.1, Launching the
ICH Specificity (PP) eWorkflow, page 14), the sequence is already running.

1. Select the sequence in the navigation pane.

2. Click Start (Figure 13A).

Figure 13: Starting the sequence

The sequence is now running (Figure 14).

Figure 14: Running sequence

5.3 Processing the Data

After the ICH Specificity (PP) data has been acquired, it can be processed. All data processing steps are performed in the
Chromatography Studio. To open the Chromatography Studio from the Console, double click any object in a sequence.

5.3.1 Processing Method

The ICH Specificity (PP) eWorkflow template contains a dedicated processing method. This processing method is designed
to show the processing parameters required by the test, and also to allow you to define your specifications for the test
(Figure 15).
 Figure 15: ICH Specificity (PP) processing method layout
The default Processing Method file for the Specificity (PP) test shows 4 tabs
Detection: Allows you to define your peak detection parameters
Component Table: Allows you to name your peaks and define their specifications (e.g. minimum correlation coefficient)
UV Spectra: Allows user to adjust “Peak Purity Threshold”.
General: Allows you to define general settings
The major steps in using the Specificity (PP) Processing Method file are to update the Component Table (Figure 15A).

Note: By default, the peak purity measurement starts at 10 % height on the left side of the peak and ends at 10 % height
on the right side of the peak. These values can be adjusted by using the detection parameter “Peak Purity Threshold”.

Note: The processing method can have different layouts, consisting of different tabs and names. Therefore the
Processing Method tables and columns you see may look different from the ones in these pictures.

Tip: Choose the processing method layout appropriate for your application, or define your own layout by selecting
individual tabs via the Processing Method layout dialog ( ).

5.3.2 Updating the Component Table

If the processing method is not already updated, the major step is to update the component table (Figure 16).

Figure 16: Component table containing standard peaks only

In this table the calibration compound names (Figure 16A) are entered, including their corresponding retention times
(Figure 16B) and retention time window (Figure 16C) for peak identification. ICH Specificity (PP) specific parameters
(Figure 16D) are entered into eight custom variables columns (Table 11).

Component Table Column Description

Name Name of the peak.

 Retention Time Expected retention time of the peak.

Window Allowed window for the expected retention time.

Test Specifies if the test will be run for this component or not.

Min_PP Minimum peak purity allowed for component.

Table 10: ICH Specificity (PP) specific custom variables in the component table

The steps involved in updating the component tables are as follows:

1. Open your sequence in the Studio, for example by double-clicking an injection in the Console.

2. Click the Data Processing category bar.

3. Show the Chromatogram Pane and the Processing Method Pane using the corresponding tools in the Data
Processing Home Ribbon tab (Figure 17).

Figure 17: Panes Group of the Data Processing Home Ribbon tab
In Component Table:
4. Ensure that your component is correctly identified by updating the retention time and retention time window.

5. Decide if you want to test the Specificity (PP) of each component or not by updating the “Test” field.

6. Define the minimum peak purity for each component.

5.4 Reviewing Results

The ICH Specificity (PP) eWorkflow template contains a report template. This report template is designed to report all ICH
test results in a convenient way including Specificity (Peak Purity). Once the sequence table and the processing method are
updated, Chromeleon immediately calculates the results.
To review the results switch to the Report Designer category (Figure 18).

Figure 18: Review results in the report designer category

In this view you will see that each ICH test has its own associated tab (e.g. Accuracy, Specificity (RS), Robustness, …).
Click on the “Specificity (PP)” tab to review your results. The ICH Specificity (PP) report will now be visible (Figure 19). For a
detailed description of the report sheet, please refer to section 6.
 Figure 19: Reviewing ICH Specificity (PP) results in the report template

5.5 Creating the ICH Specificity (PP) report

The ICH report template is designed for a quick calculation of ICH results and creation of the report. The process to
generate the report is as follows:
1. Select the sequence containing the ICH results you want to report (Figure 20) and right click. Continue at section 5.5.1
to print the report or section 5.5.2 to export the report.

Note: When only a few of several samples need reporting, select those samples, right click, and select Print or Export.

Figure 20: Starting reporting the ICH results

5.5.1 Printing the report
To print the report, follow the steps below:
2. Right click and select Print Report (Figure 20A). The Print dialog now opens (Figure 21).

Figure 21: Print dialog when printing the ICH report

3. In the Report Template & Channel section, select the correct channel to print the data (Figure 21A).

4. In the Printer section, select the correct printer (Figure 21B).

Note: The automatic reporting options are configured to print the ICH Specificity (PP) test sheet. Please refer to section 7
for instructions how to print the results of all ICH tests together.

5. Click OK to start printing.

5.5.2 Exporting the report

The report can be exported as an electronic copy of the results in Adobe Acrobat PDF format. The electronic copy of the
report is automatically stored on the hard drive in the folder C:\Chromeleon_Export. The file name is
ICH_Specificity_(PP)_yyyymmddhhmmss.pdf, where:
• yyyy = calender year, for example 2011

• mm = month, for example 06

• dd = day, for example 19

• hh = hour, for example 11

• mm = minutes, for example 59

• ss = seconds, for example 23

This ensures that every file created has a unique name. To export the report follow the steps below:
2. Right click and select Export (Figure 20B). The Export dialog now opens (Figure 22).
 Figure 22: Export dialog when exporting the ICH report
3. In the Report Template & Channel section, select the correct channel to export the data (Figure 22A).

Note: The automatic reporting options are configured to export the Specificity (PP) sheet for injections with TEST custom
variable.

4. Click OK to start exporting.

6 Description of the Report Sheets

6.1 Report Sheet “Specificity (PP)”

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be
present. Typically these might include impurities, degradants, matrix, etc. This template checks that the analyte peak does
not contain any underlying peak with a sufficiently different UV/Vis spectrum.

Figure 23: Report Sheet "Specificity (PP)"

6.1.1 Limitations
None.

6.2 Other Report Sheet

The other report sheets are described in the respective manual of each individual test.
7 Combined Validation Report

7.1 Importing Query

The templates have been designed to allow you to quickly collate all your validation data and create a combined method
validation report. The process is as follows.
1. In the Chromeleon Console go to the Data category.

2. In the Menu click File, Import From.

3. Locate ‘Collate_Data.cmbx’ file on the Chromeleon 7 disc, in the folder \Extension Pack\ICH Templates. Click Open.

4. Select the correct Data Vault and directory that contains the method validation data.

7.2 Modifying Query

1. Open query.

2. In the Sequence search replace [ENTER_PATH] with the folder directory that contains the method validation (Figure
24A).

Figure 24: Query dialog for collating results

3. Save the query.

7.3 Printing / Exporting the Query

1. Click on Query Now. The query will automatically run and retrieve the relevant injections and show all of the
data in a virtual sequence (Figure 25).
 Figure 25: Virtual sequence containing all method validation data results.

2. To print the report, select Print, or expand and select Report on the Query tool bar (Figure 26A). This will open
the Print dialog.

Figure 26: Starting the reporting process

3. Select all relevant sheets of tests that you want to report.

 Figure 27: Printing collated results

4. To export the report, select the Studio button.

5. Then select the Report Designer category.
6. Click the Application button (Figure 28A) and select Export (Figure 28B).

Figure 28: Starting the exporting process

7. This will open the Export dialog (Figure 29). Ensure the option Apply to ‘Results of current injection query’ is
selected (Figure 29A).
Figure 29: Query Export dialog