G3793-90007 Optimizer QuickStart

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Agilent MassHunter Optimizer

Automated MS Method Development Software


Quick Start Guide
What is MassHunter Optimizer? 2
Startup 3
User Interface 4
Menus and Toolbar 5
Compound Setup Tab 8
Precursor Ion Selection Tab 8f
Product Ion Selection Tab 8
Optimizer Setup Tab 7
Database Browser Window 8
Ion Breakdown Profile Dialog Box 9
Getting Started 10
Using Projects 12
Setting Optimization Parameters 14
Setting Up Compounds and Working with Databases 18
Setting Up Sequences 30
Optimization and Results 37

Where to find more information


Use the online help for in-depth information not included in this Quick Start
Guide.
Display online help by pressing the F1 key to get more information about a
window, tab, or dialog box.
What is MassHunter Optimizer?
Where to find more information

What is MassHunter Optimizer?


Automated MS MassHunter Optimizer software lets you automatically optimize the data
Method acquisition parameters for MRM mode (multiple-reaction monitoring) on a
Development triple quad mass spectrometer instrument for each individual compound
Software analyzed. Specifically, it automates the selection of the best precursor ions,
the optimization of the fragmentor voltage for each precursor ion, selection of
the best product ions, and optimization of collision energy values for each
transition for a list of compounds you specify.
Automated Automation of the following optimization tasks results in a significant time
Optimization saving over manual optimization procedures:
• Selection of optimum precursor ion via rules, such as M+H, M+Na, M+K, and
M+NH4.
• Optimization of fragmentor voltage to maximize precursor ion intensity for
up to 4 precursor ions (adducts and/or charge states) for each polarity in
MS SIM
• Selection of multiple best product ions (MS/MS full scan)
• Optimization of collision energy to maximize product ion intensity (MS/MS
full scan)
• Optionally perform fine fragmentor optimization for maximum sensitivity.
Additional • Stores optimum precursor and product ions and parameters in a compound
Features database.
• Imports optimization results into Agilent MassHunter Workstation triple
quad Data Acquisition software.
• Lets you import compounds for optimization and update compound
retention times from Agilent MassHunter Workstation triple quad Data
Acquisition methods
• Supports sample introduction via manual infusion (syringe pump),
automatic infusion (via loop injection at lower flows), and injection analysis
with column (to maximize optimization yield).
• Data that is acquired during the optimization run can be displayed with
Agilent MassHunter Qualitative Analysis software.

You can optimize other source and ion funnel parameters using the "ion funnel and source
NOTE optimization" program. For more information, see online Help and the Familiarization
Guide for MassHunter Workstation Data Acquisition software.

2 Agilent Optimizer Software Quick Start Guide


Startup
To optimize peptides

Startup
MassHunter Optimizer software is automatically installed with Agilent
MassHunter Acquisition software. Two versions of MassHunter Optimizer
software are installed on your system: one for small molecules and one for
peptides.
Open MassHunter Optimizer software in either of the following ways:
• Double-click the Optimizer icon on the desktop, or
• Select Programs> Agilent> MassHunter Workstation> Optimizer from the
Windows Start menu.

To optimize peptides
A version of MassHunter Optimizer software is available for optimizing
peptides. Open Optimizer for Peptides software in either of the following
ways:
• Double-click the Optimizer for Peptides icon on the desktop, or
• Select Programs> Agilent> MassHunter Workstation> Optimizer for
Peptides from the Windows Start menu.

Projects are specific to the version of Optimizer software that you use to create them.
NOTE In other words, projects created with Optimizer for Peptides software cannot be opened in
MassHunter Optimizer software and vice versa.

To remove the software


• Click Programs and Features in Windows 7 Control Panel.
• Select Agilent MassHunter Workstation Acquisition Software in the list
of programs, then click Uninstall.
Note that Optimizer can only be removed as part of Agilent MassHunter
Workstation Acquisition software, not separately.

Agilent Optimizer Software Quick Start Guide 3


User Interface

User Interface
The MassHunter Optimizer software has the following UI elements that let you
enter parameters, set options, view status, and perform functions:
• “Menus and Toolbar” on page 5
• “Status Bar” on page 7

Tabs
• “Optimizer Setup Tab” on page 7
• “Precursor Ion Selection Tab” on page 8
• “Product Ion Selection Tab” on page 8
• “Compound Setup Tab” on page 8

Other Windows and Dialog Boxes


• “Peptide Fragment Ion Predictor Dialog Box” on page 8
• “Database Browser Window” on page 8
• “Ion Breakdown Profile Dialog Box” on page 9

4 Agilent Optimizer Software Quick Start Guide


User Interface
Menus and Toolbar

Menus and Toolbar


The following menu items and toolbar buttons are available in MassHunter
Optimizer software main window.

File menu
New Project Clears the current settings and lets you set up a new project for
use in MassHunter Optimizer software. See “To create a project” on page 12.
Open Project Lets you open a project. See “To open a project” on page 12.

Save Project Lets you save changes to the current project.

Save As Project Lets you save the current project under a new name.

Save Compounds Lets you save compound information to a database, for use
with other projects. See “To save compounds to a database” on page 25.

Delete Projects Lets you select projects and remove them.

Exit Closes the MassHunter Optimizer software window.

Edit menu
Copy Copies the selected rows, which can then be pasted into another
position in the table.

Cut Cuts the selected rows from the table, which can then be pasted into
another position in the table.

Paste Pastes the most recently copied or cut rows into the selected position
in the table.

Delete Removes the selected rows from the table

Select All Selects all rows in the table.

Fill Copies the value in the top cell down to the rest of the cells of a
highlighted block or to the entire column.

Agilent Optimizer Software Quick Start Guide 5


User Interface
Menus and Toolbar

Clear Results Lets you clear all fragmentor values, all collision energy values,
or all results, including methods and all precursor and product ions for all
compounds in the selected row and below in the table.

View menu
Show/Hide Columns Lets you select which columns of information to display
in the table. At least one column in every row must be displayed.

Expand/Collapse All Rows Lets you display all rows in the table or collapse
the table to show only Compound rows. This affects the currently selected row
and all rows below it in the table.

Import/Export menu
Import from Database Opens the Database Browser window, which lets you
select compounds for optimization from the database.
See “Database Browser Window” on page 8.
Import from Excel Lets you import compounds from an Excel file.
See “To import compounds from an Excel file” on page 27.
Import from Acquisition Methods Lets you import all compounds from
Acquisition methods into the current project.
Export to Excel Lets you export the current compound list for use in Excel.
See “To export compound information to an Excel file” on page 27.

Optimization menu
Start Optimization Starts the optimization process with the current
parameters. The results are saved to the database and displayed in the
Compound Setup tab. A report is automatically printed on completion.
See “To start optimization” on page 38.
Ion Breakdown Profile Runs optimization with the current parameters and
displays the results in the Ion Breakdown Profile dialog box when
optimization is complete. Collision energy values can be selected from the
graphs, instead of using those determined by optimization.
See “To start optimization and display ion breakdown profiles” on page 39.
Stop Optimization Lets you terminate optimization before completion.

6 Agilent Optimizer Software Quick Start Guide


User Interface
Status Bar

Tools menu

Update RT Lets you update compound retention times from Data Acquisition
methods. See “To update retention times from a MassHunter Acquisition
method” on page 29.
The following items only appear in Optimizer for Peptides software.
Extract Peptide Opens the Peptide Fragment Ion Predictor dialog box, which
lets you extract peptide sequences for optimization from a selected
MassHunter Qualitative Analysis method. See “Peptide Fragment Ion
Predictor Dialog Box” on page 8.
Convert .csv to .psq Lets you convert multiple peptide sequences with or
without modification into multiple .psq files that can be imported into
MassHunter Qualitative Analysis software. See “To convert multiple peptide
sequences for import into MassHunter Qualitative Analysis” on page 36.

Help menu

Help Topics Opens the online help window with the Contents tab displayed.

About Optimizer Displays version information for the software.

Status Bar
The following items appear in the status bar at the bottom of the MassHunter
Optimizer software main window: Project Name, Optimizer Status, Current
Record progress bar, and All Records progress bar.

Optimizer Setup Tab


Lets you set the acquisition parameters used for optimization, including
sample introduction method, Acquisition methods, and ranges for
optimization of fragmentor and collision energy voltages. See “To set
Optimizer acquisition parameters” on page 14.

Agilent Optimizer Software Quick Start Guide 7


User Interface
Precursor Ion Selection Tab

Precursor Ion Selection Tab


Lets you select adduct species to include in the search for precursor ions. The
default is +H for positive and -H for negative. See “To select precursor ions” on
page 15.

Product Ion Selection Tab


Lets you set criteria to use in the search for product ions. See “To select
product ions” on page 16.

Compound Setup Tab


Displays the list of compounds to optimize, and lets you enter precursor and
product ion values, if known. It also displays the results of optimization, when
you mark the Show results summary check box. Sequences rather than
compounds are used for optimization in Optimizer for Peptides software.

Peptide Fragment Ion Predictor Dialog Box


Available in Optimizer for Peptides software only.
This lets you select peptide sequences for optimization from a MassHunter
Qualitative Analysis method. The selected sequences are added to the
Compound Setup tab. After you click the Extract Peptide toolbar button
shown at the left, you are prompted to select the method of interest. See “To
import peptide sequences from a MassHunter Qualitative Analysis method” on
page 32.

Database Browser Window


This window appears when you click the Import from Database toolbar
button. It lets you select compounds for optimization from the database. The
selected compounds are added to the Compound Setup tab. See “To import
compounds from a database” on page 22.

8 Agilent Optimizer Software Quick Start Guide


User Interface
Ion Breakdown Profile Dialog Box

The Database Browser window also lets you open a different database, save
the current database with a new name, and make the current database the
default database.

Ion Breakdown Profile Dialog Box


This dialog box automatically appears after optimization is complete when you
click the Ion Breakdown Profile toolbar button. The product ion profiles
determined by optimization are displayed and you can manually select
collision energy values for each ion to replace the results of optimization. See
“To start optimization and display ion breakdown profiles” and “To manually
set collision energy voltages” on page 39.

Agilent Optimizer Software Quick Start Guide 9


Getting Started
Ion Breakdown Profile Dialog Box

Getting Started
The following topics will help you get started using MassHunter Optimizer
software.
Using • “To create a project” on page 12
Projects • “To open a project” on page 12
• “To edit a project” on page 12
• “To save parameters to the current project” on page 13
• “To save parameters to a new project” on page 13
Setting • “To set Optimizer acquisition parameters” on page 14
Optimization • “To select data path and methods to use for optimization” on page 15
Parameters
• “To select precursor ions” on page 15
• “To select product ions” on page 16
• “To set Optimizer acquisition parameters for peptides” on page 16
Setting Up • “To set up a compound list” on page 19
Compounds and • “To use known ions for individual compounds” on page 21
Working with
Databases • “To open a database and make it the default database” on page 22
• “To import compounds from a database” on page 22
• “To select transitions and triggers for Dynamic and Triggered MRM” on
page 24
• “To save compounds to a database” on page 25
• “To filter compounds in Database Browser” on page 26
• “To search compounds in Database Browser” on page 26
• “To import compounds from an Excel file” on page 27
• “To export compound information to an Excel file” on page 27
• “To import compounds from a MassHunter Data Acquisition method” on
page 27
• “To update retention times from a MassHunter Acquisition method” on
page 29
Setting Up • “To set up a sequence list for peptides” on page 30
Sequences

10 Agilent Optimizer Software Quick Start Guide


Getting Started
Ion Breakdown Profile Dialog Box

• “To import peptide sequences from a MassHunter Qualitative Analysis


method” on page 32
• “To convert multiple peptide sequences for import into MassHunter
Qualitative Analysis” on page 36
Optimization and • “To start optimization” on page 38
Results • “To review optimization results” on page 38
• “To start optimization and display ion breakdown profiles” on page 39
• “To manually set collision energy voltages” on page 39
• “To import optimization results to Acquisition for dMRM/tMRM” on
page 41
• “To import optimization results to Acquisition for MRM time
segments” on page 44

Agilent Optimizer Software Quick Start Guide 11


Using Projects
To create a project

Using Projects

To create a project
Use this procedure to store all the values and settings used for optimization in
a project.
1 Click the New Project button on the toolbar.
If current parameters have not already been saved, you are prompted to
save the project. The current parameters are cleared.
2 Make the desired changes to parameters on the following tabs:
• Optimizer Setup tab
• Precursor and Product Ion Selection tabs
• Compound Setup tab
3 Click the Save Project or Save As Project button and enter a name for the
project.

To open a project
Use this procedure to use optimization values and settings from an existing
project.
1 Click the Open Project button on the toolbar.
2 Click to select the project on the Select Project Name dialog box.
3 Click the Open button.

To edit a project
1 Open the project to edit as described above.
2 Make the desired changes to parameters on the following tabs:
• Optimizer Setup tab
• Precursor and Product Ion Selection tabs
• Compound Setup tab
3 Save the parameters as described below.

12 Agilent Optimizer Software Quick Start Guide


Using Projects
To save parameters to the current project

To save parameters to the current project


1 Click the Save Project toolbar button.

To save parameters to a new project


1 Click the Save As Project toolbar button.
2 Enter a name for the project and click the Save button.

Agilent Optimizer Software Quick Start Guide 13


Setting Optimization Parameters
To set Optimizer acquisition parameters

Setting Optimization Parameters


Use the following procedures to set up the optimization parameters for
MassHunter Optimizer software.
“To set Optimizer acquisition parameters” below
“To select data path and methods to use for optimization” below
“To select precursor ions” on page 15
“To select product ions” on page 16

Workflow for Peptides


“To set Optimizer acquisition parameters for peptides” on page 16
“To select data path and methods to use for optimization” below

To set Optimizer acquisition parameters


1 Click the Optimizer Setup tab.
2 Set the desired Sample introduction method (Injection, Automatic
infusion, or Manual infusion).
3 Set the Fragmentor ramp parameters as follows:
(Skip this step if your instrument is a model 6490 LC/MS. The fragmentor
parameters are not displayed, since fragmentor voltage is not optimized
for that instrument.)
a Set the range for ramping the Fragmentor values.
b (optional) Mark the Fragmentor Fine check box and enter a Step size
for Fine ramping. Fine optimization refines the coarse ramping values
and provides better optimization but takes longer to run.
4 Set the range for ramping the Collision Energy values.
5 Continue to select data path and methods to use for optimization as
described below.

14 Agilent Optimizer Software Quick Start Guide


Setting Optimization Parameters
To select data path and methods to use for optimization

To select data path and methods to use for optimization


1 If not already displayed, click the Optimizer Setup tab.
2 Select a Path for data files to store the optimization run data. The data can
be displayed later with Agilent MassHunter Qualitative Analysis software.
3 In the table on the right side of the screen, select the acquisition methods to
use for optimization as follows. These methods will be used to optimize all
compounds, except for those that have specific methods selected on the
Compound Setup tab.
a Right-click in the table and select Add Method from the shortcut menu.
This adds a row to the end of the table for a new method.
b Click the button on the right side of the Acq Method cell to open the
Open Method dialog box.
c Select the method of interest and click OK. The Polarity and Ion Source
will be filled in from the values set in the selected method.
d Check to make sure that the Ion Source from the method matches the
physical configuration of your instrument.
e (optional) Repeat steps 3a - d to select additional methods for
optimization.
Tip Additional options are available by right-clicking in the method table, such
as Delete Methods, Copy, and Fill.

To select precursor ions


Use this procedure to specify precursor ions to use to optimize the fragmentor
voltage. The information entered here will be used for all compounds, except
for those that have precursor ions specified on the Compound Setup tab.
1 Click the Precursor Ion Selection tab.
2 Select the Positive and Negative adduct ions to include.
If necessary, add Positive or Negative adduct ions to the list by typing the
value in the box and clicking the button.
3 Set the search priority of the precursor ions in one of the following ways:
• Mark the Use most abundant precursor ion option to use the most
abundant precursor ion.

Agilent Optimizer Software Quick Start Guide 15


Setting Optimization Parameters
To select product ions

• Clear the Use most abundant precursor ion option and use the Up and
Down arrow buttons to set the search order (ions at the top of the list are
given more priority).
4 To exclude certain masses from consideration, click Exclude masses at the
bottom of the screen. Enter the m/z values to exclude separated by commas
and/or enter a Minimum abundance value in counts.

To select product ions


Use this procedure to specify product ions to use to optimize the collision
energy voltage. The information entered here will be used for all compounds,
except for those that have product ions specified on the Compound Setup tab.
1 Click the Product Ion Selection tab.
2 Enter a Low mass cut-off value. Select whether to specify the value as Mass
(m/z) or as a % Precursor mass (m/z) and enter the cut-off value.
3 To exclude certain masses from consideration, click the Exclude masses
option at the bottom of the screen. Enter the m/z values to exclude
separated by commas and/or enter a Minimum abundance value in counts.
4 If desired, you can also enter Neutral Losses to exclude, e.g. water (H20).
Enter a formula in the box and click the button to add it to the list.

To set Optimizer acquisition parameters for peptides


1 Click the Optimizer Setup tab in Optimizer for Peptides software.
2 Set the desired Sample introduction method (Injection, Automatic
infusion, or Manual infusion).
3 Set the Fragmentor Ramp Settings parameters as follows. Skip this step if
your instrument is a model 6490 LC/MS, since fragmentor voltage is not
optimized for that instrument.
a In the Fragmentor area, mark the Fixed Fragmentor check box and
enter the fragmentor voltage to use for optimization. This value will be

16 Agilent Optimizer Software Quick Start Guide


Setting Optimization Parameters
To set Optimizer acquisition parameters for peptides

used for all compounds, unless you enter specific values for individual
compounds on the Compound Setup tab.
Tip Using a fixed fragmentor voltage saves time by skipping the SIM run to
determine the optimal fragmentor voltage. However, if multiple adducts are
specified, then a SIM run is made to determine the precursor ion to use.
b Mark the Overwrite existing check box to use this value for all
compounds, even if values are set for individual compounds on the
Compound Setup tab.
4 In the Collision Energy area, mark the Start from predicted value check
box, which lets the Optimizer software set a collision energy value, using
the formula shown. In the formula, m/z is the mass of the precursor ion.
5 (optional) Mark the Put instrument into standby mode at end check box to
put the instrument into standby mode at the end of the optimization run.
See “To select data path and methods to use for optimization” on page 15.

Agilent Optimizer Software Quick Start Guide 17


Setting Up Compounds and Working with Databases
To set Optimizer acquisition parameters for peptides

Setting Up Compounds and Working with Databases


Use the following procedures to set up compounds for use in MassHunter
Optimizer software.

Setting up compounds
• “To set up a compound list” on page 19
• “To use known ions for individual compounds” on page 21

Using compound databases


• “To open a database and make it the default database” on page 22
• “To import compounds from a database” on page 22
• “To select transitions and triggers for Dynamic and Triggered MRM” on
page 24
• “To save compounds to a database” on page 25
• “To filter compounds in Database Browser” on page 26
• “To search compounds in Database Browser” on page 26

Importing and exporting compound information from Excel


• “To import compounds from an Excel file” on page 27
• “To export compound information to an Excel file” on page 27

Using compound information from MassHunter Acquisition methods


• “To import compounds from a MassHunter Data Acquisition method” on
page 27
• “To update retention times from a MassHunter Acquisition method” on
page 29

18 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To set up a compound list

To set up a compound list


Use this procedure to set up the list of compounds to optimize and enter
precursor and product ion values, if known.
1 Click the Compound Setup tab.
2 (optional) Import compounds as described in “To import compounds from
an Excel file” on page 27 or “To import compounds from a database” on
page 22.
3 Clear the Show results summary check box above the table while you set up
the compound list.
4 To add a compound:
a Right-click in the table and select Add Compound from the shortcut
menu to add a row to the end of the table.
b Enter the Compound Name for the new compound.
c (optional) If you want to be able to find groups of compounds in the
database such as sulfa drugs or pesticides, assign a group name in the
Groups column. You can then specify the desired group names when you
filter compounds to import from the database as described on page 22.
d Enter the Formula of the compound. The Mass will be automatically
calculated from the formula. If you don’t know the formula, then enter
the monoisotopic mass in the Mass column.
e Enter the Sample Position for the new compound.
f (optional) Adjust the Optimization dwell time value to set a shorter or
longer cycle time if desired; otherwise a default value is used.
g (optional) To speed up optimization, specify precursor and product ions
for the compound as described in “To use known ions for individual
compounds” below. If no methods or ions are specified here, then
optimization for the compound uses the methods from the Optimizer
Setup tab and information from the Precursor Ion Selection and Product
Ion Selection tabs to generate the ions.
h Repeat the steps above to add more compounds to the table.
Compounds are global to all projects. Compound information such as name, group,
NOTE formula, and mass in one project will be reflected in the entire database.

Agilent Optimizer Software Quick Start Guide 19


Setting Up Compounds and Working with Databases
To set up a compound list

Tip To cut or copy and paste compounds (rows) in the table:


a Select the rows of interest in the table.
b Right-click on the selected rows and select Cut or Copy from the shortcut
menu.
c Right-click on the new location for the cut or copied rows and select
Paste from the shortcut menu.
5 Mark the Select columns for the compounds (rows) to use for optimization.
6 Save the compound list as described in “To save compounds to a
database” on page 25 or “To save parameters to the current project” on
page 13.
7 (optional) Export the compound list as described in “To export compound
information to an Excel file” on page 27.

20 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To use known ions for individual compounds

To use known ions for individual compounds


Use this procedure to optionally specify methods, precursor ions, and product
ions for individual compounds on the Compound Setup tab. Using known ions
speeds up the optimization process.
1 Add compounds to the Compound List as described in “To set up a
compound list” on page 19.
2 To add methods for the current compound:
a Right-click in the Compound row of interest and select Add Method
from the shortcut menu. This adds a Method row below the selected
compound.
b Click the down arrow button on the right end of the Acq Method cell and
select an acquisition method to use for the selected compound.
If you don’t see the method of interest, add it to the Method Table on the
Optimizer Setup tab as described in “To set Optimizer acquisition
parameters” on page 14.
c Repeat the above steps to add more methods for the current compound.
3 To specify a known precursor ion for the current compound:
a Right-click in the Method row for the compound of interest and select
Add Precursor Ion from the shortcut menu. This adds a Precursor Ion
row for the current compound.
b Enter a value for the Precursor Ion.
4 To specify product ions for the current compound:
a Right-click in the Precursor Ion row for the compound of interest and
select Add Product Ion from the shortcut menu. This adds a Product Ion
row for the current compound.
b Enter a value for the Product Ion.
c Repeat the above steps to add the product ions for the current compound
and precursor ion.
5 Repeat steps 2 - 4 to specify methods and ions for other compounds in the
Compound Table.

Agilent Optimizer Software Quick Start Guide 21


Setting Up Compounds and Working with Databases
To open a database and make it the default database

To open a database and make it the default database


Use this procedure when you want to use a database other than the default
optimizer database.
1 If the Database Browser window is not already open, click the Import from
Database button on the toolbar to open it.
2 Click the Open Database button on the Database Browser toolbar.
3 Select the database of interest and click OK.
4 You are asked if you want to make this database the default database.

If you are importing additional compounds into an existing project, click No.
NOTE Otherwise, unsaved changes made to the current project will be lost.
All saved compound information in the previous project will still exist in the previous
default database, but it won't be available for use with the new default database.

Tip
If you decide later to make this database the default database, click the Set
Default Database button on the Database Browser toolbar.

To import compounds from a database


Use this procedure to import compounds that have previously been saved as
described in “To save compounds to a database” on page 25.
1 Click the Import from Database toolbar button to open the Database
Browser.
2 If not already open, open the database of interest as described on page 22.
3 Select compounds for optimization from the database.
• If desired, filter compounds as described on page 26.
• If desired, search compounds as described on page 26.
• To display all compounds in the database, mark the Show All Records
check box.

22 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To import compounds from a database

4 When the compounds from step 3 appear in the Compound table in the
lower half of the Database Browser window, select the transition ions to
import in one of the following ways:
• Individually mark the check boxes in the first column of the table for the
transition ions you want to import.
• Click the Select top ranked transitions option, then enter how many top
transitions to import for each compound, and click the Select
Transitions button. You can rank the top transitions by either
Abundance or Response Factor.
• To select the primary transitions, click the Primary Transitions option,
then click the Select Transitions button.
• To select the secondary transitions, click the Secondary Transitions
option, then click the Select Transitions button.

To set up the primary and trigger transition ions to import, see “To select transitions and
NOTE triggers for Dynamic and Triggered MRM” on page 24.

5 Do one of the following:


• If the compounds in the table are the only ones you want to import at
this time, click Import. This closes the Database Browser window and
adds the compounds to the Compound Setup tab.
• If you want to do additional search and filter operations on the current
database or a different database, click Add to Import List.
6 Do one of the following:
• To find more compounds in the same database, repeat steps 3 - 5.
• To select compounds from a different database, repeat steps 2 - 5.
7 Click the Import List tab to review the list of compounds.
• Right-click and select Remove from the shortcut menu to remove any
compounds you don’t want to import.
8 Click the Import button.
The imported compounds will appear on the Compound Setup tab.

Agilent Optimizer Software Quick Start Guide 23


Setting Up Compounds and Working with Databases
To select transitions and triggers for Dynamic and Triggered MRM

To select transitions and triggers for Dynamic and Triggered


MRM
1 If not already open, open the database of interest as described on page 22.
2 In the Database Browser window, display compounds for optimization from
the database.
• If desired, filter compounds as described on page 26.
• If desired, search compounds as described on page 26.
• To display all compounds in the database, mark the Show All Records
check box.
3 Deselect any unwanted compounds by clearing the check box next to the
Compound Name in the first column of the Compound table.
Triggered MRM 4 Select transitions for a triggered MRM method as follows:
method a In the Select top ranked transitions field, enter the maximum number
of transitions per compound that you want to show in the import list.
You can have two primary transitions, plus additional secondary
transitions (see step c below).
b Click the Select Transitions button to display the transitions in the
Compound table.
c In the Set primary and trigger flags area, specify the number of
Primary transitions to use. The rest are assigned as Secondaries.
Typically, two primaries are used.
d Click Set Primaries and Trigger. The most abundant transition is
automatically assigned as the Trigger.
e Review the compound list and edit as needed.
• A very abundant low m/z ion may be unsuitable as a Primary transition.
• You can manually select a different transition for a compound as the
Primary transition, and define a low m/z ion as a Secondary.
• You may want to select two Primaries as triggers for a compound.
Dynamic MRM 5 Select transitions for a dynamic MRM method as follows:
method a Click Primary transitions.
b Click the Select Transitions button to display the transitions in the
Compound table.
• At this point, all transitions are displayed as primaries.

24 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To save compounds to a database

• Typically, the transitions are ranked by abundance.


c Review the compound list and edit as needed.
• Deselect any unwanted transitions.
6 When the list of transitions is set, click the Add to Import List button below
the Compound table.
7 Do one of the following:
• To find more compounds in the same database, repeat steps 2- 6.
• To select compounds from a different database, repeat steps 1- 6.
8 Click the Import List tab to review the list of compounds.
• Right-click and select Remove from the shortcut menu to remove any
compounds you don’t want to import.
9 Click the Import button.
The imported compounds will appear on the Compound Setup tab.

To save compounds to a database


Do one of the following to save compounds to a database:
• To save compounds to the current database:
• Click the Save Compounds button on the main toolbar, or
• Click the Save Database button on the Database Browser toolbar.
• To save compounds to a new database, click Save As Database button on
the Database Browser toolbar, enter a new name, and click the Save button.
You can import the compounds into other projects at a later time as
described in “To import compounds from a database” on page 22.

Agilent Optimizer Software Quick Start Guide 25


Setting Up Compounds and Working with Databases
To filter compounds in Database Browser

To filter compounds in Database Browser


1 If not already open, open the database of interest as described on page 22.
2 Enter the Filter criteria in the upper left area of the Database Browser
Search/Filter tab. Note that filter criteria are unavailable if Show All
Records is selected.
• Mark the Enable Filters check box.
• Mark the check boxes for the filter criteria to use, such as Optimized
Compounds, Date, Group Name, Project Name, Polarity, Model, and
Method. Enter values for each selected criteria.
3 Click the Search/Filter button.
The compounds that pass the filter are displayed in the Compound table in
the lower part of the screen.

To search compounds in Database Browser


1 If not already open, open the database of interest as described on page 22.
2 Enter the Search Text in the upper right area of the Database Browser
Search/Filter tab, such as Compound Name and Formula. This can be
either a partial or complete entry as described in step 4.

The values you enter here for Product or Precursor ions are treated as numeric values
NOTE rather than text. Matches are returned whose values fall between 0.7 amu below the
entered mass and 0.3 amu above the specified mass.

3 Mark the columns to search or click the top entry in the list (Select
Columns) to search all columns.
4 Do one of the following:
• To find compounds that match only a partial name or formula, clear the
Match entire word for each string check box.
• To find only compounds that exactly match the text you entered in step
2, mark the Match entire word for each string check box.
5 To ignore all filters for this search, clear the Enable filters check box.

26 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To import compounds from an Excel file

If you want to display all items in the database, mark the Show All Records check box.
NOTE This automatically disables all the search and filter options.

6 Click the Search/Filter button.


• The compounds that meet the search criteria are displayed in the
Compound Table in the lower part of the screen.
• Search text that was not found in the search is shown in red.

To import compounds from an Excel file


1 Click the Import from Excel button on the main toolbar.
2 Select the .xls, .xlsx, or .csv file to import, then click Open. All compounds
from the file will be imported and listed on the Compound Setup tab.
The Select Method for Linking Compounds dialog box may be displayed to
assign methods to the compounds.

To export compound information to an Excel file


1 Click the Export to Excel button on the main toolbar.
2 Enter a name to export the compound list in .xls format for use in Excel,
click Save.

To import compounds from a MassHunter Data Acquisition


method
1 Create MassHunter Acquisition methods for the compounds you want to
import.
2 Click the Import from Acquisition Methods toolbar button in MassHunter
optimizer software.
3 Select the MassHunter Acquisition methods from step 1 that contain the
compounds of interest, then click the Open button.

Agilent Optimizer Software Quick Start Guide 27


Setting Up Compounds and Working with Databases
To import compounds from a MassHunter Data Acquisition method

All compounds in the data acquisition methods will be added to the current
project and can be seen on the Compound Setup tab.

28 Agilent Optimizer Software Quick Start Guide


Setting Up Compounds and Working with Databases
To update retention times from a MassHunter Acquisition method

To update retention times from a MassHunter Acquisition


method
1 Create MassHunter Acquisition methods for the compounds of interest.
2 Be sure that compounds whose retention times you want to update are part
of a project. Only retention times for compounds that are part of a project
will be updated. Retention times for compounds that are not in a project
will not be updated, even if those compounds are in the database.
3 Click the Update RTs button on the MassHunter optimizer software main
toolbar.
4 Select the MassHunter Acquisition methods from step 1 that contain the
compounds of interest, then click the Open button.
5 When the Update Retention Time Options dialog box appears:
a Select whether to Update in current project or to Search database for
eligible projects.
b To update retention time windows, mark the Update RT Windows check
box. If marked, the retention time windows in the acquisition method
will be used to update the retention time windows in the database.
c If transition ions are found that do not match existing transition ions,
you can add them by marking the Add new transitions found check box.
Otherwise these transitions will be ignored.
d Click OK.
6 Several scenarios may result:

Projects are found that contain the same methods and transition ions In this
case, select the project to update, and click OK.
or

No project matches for update In this case, you are prompted to select
whether to add the transition ions to the Current Project or a New Project.
Click the desired option, then click OK.
All transition ions of the same compound will be updated with the first
retention time in the acquisition method. The updated compound
information can be seen on the Compound Setup tab.
7 Be sure to save the project to save the updated retention times.

Agilent Optimizer Software Quick Start Guide 29


Setting Up Sequences
To set up a sequence list for peptides

Setting Up Sequences
Use the following procedures to set up peptide sequences for use in Optimizer
for Peptides software.
• “To set up a sequence list for peptides” below
• “To import peptide sequences from a MassHunter Qualitative Analysis
method” on page 32
• “To convert multiple peptide sequences for import into MassHunter
Qualitative Analysis” on page 36

To set up a sequence list for peptides


Use this procedure to set up the list of peptides to optimize in Optimizer for
Peptides software.
1 Click the Compound Setup tab.
2 Clear the Show results summary check box above the table while you set up
the sequence list.
3 Import sequences as described in “To import peptide sequences from a
MassHunter Qualitative Analysis method” on page 32.
4 (optional) If you want to be able to find groups of sequences in the
database, assign a group name in the Groups column. You can then specify
the desired group name when you filter sequences to import from the
database.
5 Leave the Formula column blank. The Mass is imported from the method
and displayed in the table automatically.
6 Enter the Sample Position for the sequence.
7 Mark the Select columns for the sequences (rows) that you want to use for
optimization.
8 Mark the Perform multi-compound run check box. This lets you perform
collision energy optimization when all the fragment ions are known, a
typical case in peptides using ion prediction.
9 (optional) Adjust the Optimization dwell time value to set a shorter or
longer cycle time if desired.

30 Agilent Optimizer Software Quick Start Guide


Setting Up Sequences
To set up a sequence list for peptides

10 Click the Fill Values button to calculate the Cycle Time parameters and the
peak width necessary to obtain ten data points required for optimization
when using Fixed Fragmentor or CE value formula on the Optimizer Setup
tab. See “To set Optimizer acquisition parameters for peptides” on page 16.
11 Save the sequence list as described in “To save compounds to a
database” on page 25 or “To save parameters to the current project” on
page 13.
12 (optional) Export the sequence list as described in “To export compound
information to an Excel file” on page 27.

Agilent Optimizer Software Quick Start Guide 31


Setting Up Sequences
To import peptide sequences from a MassHunter Qualitative Analysis method

To import peptide sequences from a MassHunter Qualitative


Analysis method
Applies to Optimizer for Peptides software only.
1 Create a MassHunter Qualitative Analysis method that contains the peptide
sequences you want to import.
• If desired, create the .psq files for MassHunter Qualitative Analysis as
described in “To convert multiple peptide sequences for import into
MassHunter Qualitative Analysis” on page 36.
2 If you need to add custom modifications:
a Select Open Chemical Data Dictionary Editor from the Tools menu.
b Click the + button on the Modifications tab to add a custom
modification.
c Type in a name for a new modification, then enter its Chemistry values.
Example Assume you wish to add a custom modification “heavy Proline”, where the
carbons and nitrogen in Proline are replaced with 13-C and 15-N. A Proline
residue has five Carbons and one Nitrogen. Assuming complete labeling,
specify Losses of C5 N1 and Gains of Cx5 Nx1 for a Delta mass of 6.01381.
Mark the check box for Proline and clear all others to specify this custom
modification for Proline only.

32 Agilent Optimizer Software Quick Start Guide


Setting Up Sequences
To import peptide sequences from a MassHunter Qualitative Analysis method

d Click OK to close the Chemical Data Dictionary Editor.


3 Apply the custom modification to the sequences of interest as follows:
a Open the Sequence Editor window if it is not already open, and select
the sequence of interest.
b Using the example above, select the Proline amino acid in the sequence,
then apply the custom modification created in Step 2.
c Save the method, which contains the modified sequences.
4 Be sure to observe the following caveats if you modified the Chemical Data
Dictionary as described in Step 2 above:
• Close the MassHunter Qualitative Analysis software window. Otherwise,
the custom dictionary will not be modified.
• If Optimizer was open while the custom modifications were added,
restart Optimizer to make sure that it reads the new custom dictionary.
• If you plan to use this method on a different computer, you need to add
the same custom modifications to the Chemical Data Dictionary on that
system. Otherwise Optimizer for Peptides won’t recognize the custom
modifications and an error will result.

Agilent Optimizer Software Quick Start Guide 33


Setting Up Sequences
To import peptide sequences from a MassHunter Qualitative Analysis method

5 Import the sequences into MassHunter Optimizer for Peptides software as


follows:
a Review the charge states in the Precursor Ion Selection tab. Make sure
that the desired charge states are listed, and add them if they are not.
Tip For peptide analysis, charge state precursors 2+ and 3+ are usually
preferred, and singly-charged precursors are ignored.
b Click Extract Peptide on the toolbar in Optimizer for Peptides software.
c Select the MassHunter Qualitative Analysis method from step 1 that
contains the peptide sequences of interest, then click the Open button.
All sequences from the selected method are displayed in the “Peptide
Fragment Ion Predictor Dialog Box” on page 8.
d Right-click in the table and select Expand/Collapse All from the
shortcut menu to see the Precursor Ions and Charge states for each
sequence.
e Mark the sequences that you want to use for optimization.
Tip To make it easier to edit long transitions lists, mark the Show selected ions
option above the table to display only the selected ions.
• Within each peptide sequence, mark the precursor charge state to use for
that peptide and deselect all charge states that you don't want to use. For
example, you may want to examine only the 2+ charge state precursor
because it produces abundant product ions, but the 3+ charge state
precursor does not.
• If you decide to examine multiple charge states for a given peptide, then
select all of the charge states of interest.
• Select the product ions of interest that result from each peptide
precursor. Use the following check boxes in combination with selecting
individual product ions in the table to make your selections.
• Show selected ions • Show ions > than mz value
• Show only y-ions • Show ions > than % of precursor
• Show only b-ions

f Click the Add button. The selected peptide sequences from the method
are imported and listed on the “Compound Setup Tab” on page 8.

34 Agilent Optimizer Software Quick Start Guide


Setting Up Sequences
To import peptide sequences from a MassHunter Qualitative Analysis method

6 Review the list of precursors and product ions.


a Right-click in the table and select Expand/Collapse All Rows from the
shortcut menu to show all precursors and product ions.
b To edit the list, right-click on a product ion row, and select Delete
Product Ions or Add Product Ion from the shortcut menu.
7 If you want to use only a single charge state for each peptide to optimize,
then edit the charge states on the Precursor Ion Selection tab to make sure
that only one charge state is selected for each peptide. That is, remove the
charge state selection from all but one charge state. For example, clear
charge state selections except for Charge State 2.

If more than one charge state is active, then during optimization, the
NOTE software tries to determine which charge state has the highest abundance.

8 (optional) Save the compounds.

Agilent Optimizer Software Quick Start Guide 35


Setting Up Sequences
To convert multiple peptide sequences for import into MassHunter Qualitative Analysis

To convert multiple peptide sequences for import into


MassHunter Qualitative Analysis
Applies to Optimizer for Peptides software only.
Use this procedure to convert multiple peptide sequences with or without
modification into multiple .psq files for import into MassHunter Qualitative
Analysis software.
1 Create a comma separated (.csv) file that has one sequence per line, with
the following format:
NameString, SequenceString, Modification1String, Location1String, Modification2String,
Location2String
Example

2 Click the Convert .csv to .psq toolbar button in Optimizer for Peptides
software.
3 Select the .csv file created in step 1, and click the Open button.
The file is converted into multiple .psq files, one for each sequence, which
are saved in a folder named OutputPSQFiles in the same folder that contains
the original .csv file.
4 Import the .psq files into MassHunter Qualitative Analysis software and
save them in a Data Analysis method.
5 Import the sequences for use in Optimizer for Peptides software as
described in “To import peptide sequences from a MassHunter Qualitative
Analysis method” on page 32.

36 Agilent Optimizer Software Quick Start Guide


Optimization and Results
To convert multiple peptide sequences for import into MassHunter Qualitative Analysis

Optimization and Results


Use the following procedures to run the optimization procedure, evaluate the
results, and import them into MassHunter Acquisition software.
“To start optimization” on page 38
“To review optimization results” on page 38
“To start optimization and display ion breakdown profiles” on page 39
“To manually set collision energy voltages” on page 39
“To import optimization results to Acquisition for dMRM/tMRM” on page 41
“To import optimization results to Acquisition for MRM time segments” on
page 44

Agilent Optimizer Software Quick Start Guide 37


Optimization and Results
To start optimization

To start optimization
1 Set the optimization parameters as described on page 14.
2 Set up the compound list as described in “To set up a compound list” on
page 19 or “To set up a sequence list for peptides” on page 30.
3 Start the optimization process in either of the following ways:
• Click the Start Optimization button on the toolbar.
• Click the Ion Breakdown Profile button on the toolbar if you want to
display the product ion profiles determined by optimization before they
are saved to the database. For more information, see “To start
optimization and display ion breakdown profiles” on page 39.
4 If necessary, you can stop the optimization process by clicking the Stop
Optimization button on the toolbar.
5 Review results as described below.
6 (optional) Use Agilent MassHunter Qualitative Analysis software to look at
the data acquired during the optimization run.

To review optimization results


1 Run the optimization procedure as described above.
2 Click the Compound Setup tab.
3 Mark the Show results summary check box above the table.
4 Review the following values for each transition in the Compound table:
Fragmentor—The optimum voltage to apply to the exit end of the capillary
for the compound, determined by MassHunter Optimizer software. (Does
not apply to instrument model 6490 LC/MS.)
Collision Energy—The optimum voltage to apply to the ions in the collision
cell for the compound, determined by MassHunter Optimizer software.
5 Review the printed optimization report.
6 See “To import optimization results to Acquisition for MRM time
segments” on page 44, to use optimization values for data acquisition.

38 Agilent Optimizer Software Quick Start Guide


Optimization and Results
To start optimization and display ion breakdown profiles

To start optimization and display ion breakdown profiles


Use this procedure to display the product ion profiles determined by
optimization before they are saved to the database and to manually select
collision energy values for each ion if desired.
1 Set the optimization parameters as described on page 14.
2 Set up the compound list as described on page 19.
3 Click the Ion Breakdown Profile toolbar button to start optimization.
4 When optimization is complete, the Ion Breakdown Profile dialog box is
displayed. Review the graphs, which plot collision energy steps on the x-
axis and abundance values on the y-axis for each product ion. The values
shown in the table are the collision energy values determined by
optimization. See an example of Ion Breakdown Profile results in Figure 1
on the next page.
5 If desired, you can override the collision energy values as described below.
Otherwise, click Save & Exit to close the Ion Breakdown Profile dialog box.

To manually set collision energy voltages


1 Set optimization parameters and start optimization as described above.
2 Click on the Ion Breakdown Profile graph to select the desired collision
energy value. See an example Ion Breakdown Profile graph in Figure 1 on
the next page.
3 Apply the selected value to either All ions or only the Selected ion by
clicking the appropriate option for Apply manual selection to.
4 If desired, you can reload the collision energy values set by optimization at
any time by clicking the Load Optimizer Defaults button. Doing this
overwrites the manual selections you made above.
5 Click the Save & Exit button to close the Ion Breakdown Profile dialog box.

Agilent Optimizer Software Quick Start Guide 39


Optimization and Results
To manually set collision energy voltages

Figure 1 Ion Breakdown Profile Results

40 Agilent Optimizer Software Quick Start Guide


Optimization and Results
To import optimization results to Acquisition for dMRM/tMRM

To import optimization results to Acquisition for


dMRM/tMRM
1 Display the Method pane in MassHunter Workstation Data Acquisition
software.
2 Create or open the method of interest.
3 Click the MS QQQ tab in the Method Editor pane.
4 Click the Acquisition tab on the right side of the screen.

Select the time segment of interest or create a new one.

5 Set Scan Type to Dynamic MRM as shown above.


6 For triggered MRM:
a Mark the Triggered check box in the lower right area of the Acquisition
tab.

b Notice that additional columns are now available in the Scan segments
table, including Primary, Trigger, Trigger Window, Trigger MRM
Threshold, and Trigger Entrance Delay.
7 Use the expected retention time and RT window parameters to set the
timetable for monitoring transitions. Usually only a single Time segment is
used for Dynamic MRM and Triggered MRM. Note that the Cycle time
remains constant.
• As transitions are added to the list in real time, the Dwell time is reduced
for each transition that is active to keep the Cycle time constant.
• The reverse happens for transitions whose time has elapsed, and dwell
times are increased for the remaining active transitions.
• For a single compound on the list, one transition serves as the
quantitation response. Other transitions for the same compound are
used as qualifiers.

Agilent Optimizer Software Quick Start Guide 41


Optimization and Results
To import optimization results to Acquisition for dMRM/tMRM

8 Right-click in the Acquisition tab and select Import from Optimizer from
the shortcut menu.
9 When the Database Browser window appears:
a If desired, filter the compounds as described on page 26.
b If desired, search compounds as described on page 26.
c Mark the transition ions of interest from the Compound table in the
lower half of the Database Browser window in one of the following ways:
• Manually select the transition ions you want to import for each
compound, or
• Enter the top number of transitions to import for each compound, then
click the Select button. You can select the top transitions based on either
Abundance or Response Factor. Deselect any transitions that you don’t
want to use by clearing their check box.
10 If the transition ions selected in the table are the only ones you want to
import at this time, click Import. This closes the Data Browser window and
adds the transition ions into the current time segment.
• Otherwise, to do additional search and filter operations on the current
database or a different database, click Add to Import List.
11 To select additional compounds:
a To select compounds from a different database, open the database of
interest as described on page 22.

When asked if you want to make this database the default database, click No.
NOTE Otherwise, unsaved changes made to the current project will be lost.
All saved compound information in the previous project will still exist in the previous
default database, but won't be available for use with the new default database.

b Repeat steps 10a - c to select the transition ions of interest.


c Click Add to Import List.
12 Click the Import List tab to review the list of transition ions. To remove any
transition ions you don't want to import, right-click and select Remove
from the shortcut menu.
13 Click the Import button. The selected transitions are imported into the
current time segment.

42 Agilent Optimizer Software Quick Start Guide


Optimization and Results
To import optimization results to Acquisition for dMRM/tMRM

14 Review the timings to make sure that not too many transitions are being
monitored concurrently:
a Right-click in the Scan segments table and select Edit dMRM method
from the shortcut menu.
b When the Dynamic MRM Viewer appears, review how many transitions
are active at any point in time during the run.

When too many transitions are active at the same time, the dwell time per transition may
NOTE become too short (1-2 msec per transition or less). Under these conditions, the
signal/noise ratio is low, and the quantitation accuracy and precision may suffer.

c If necessary, change the retention time windows or delete transitions to


increase the average dwell time.
d You can also use the Split Method feature in the Options and
Parameters pane of the Dynamic MRM Viewer to divide the transitions
from the current method across two or more methods.
• Select the criteria for splitting the method from one of the following:
Minimum dwell time, Maximum concurrent MRM, or Number of new
methods.
• Note that all transitions for the same compound are assigned to the same
method.
15 (optional) Save the method.

Agilent Optimizer Software Quick Start Guide 43


Optimization and Results
To import optimization results to Acquisition for MRM time segments

To import optimization results to Acquisition for MRM time


segments
1 Display the Method pane in MassHunter Workstation Data Acquisition
software.
2 Create or open the method of interest.
3 Click the MS QQQ tab in the Method Editor pane.
4 Click the Acquisition tab on the right side of the screen.
5 Select the time segment of interest or create a new one.

6 Set Scan Type to MRM.


7 Right-click in the Acquisition tab and select Import from Optimizer from
the shortcut menu.
8 When the Database Browser window appears:
a If desired, filter the compounds as described on page 26.
b If desired, search compounds as described on page 26.
c Mark the transition ions of interest from the Compound table in the
lower half of the Database Browser window in one of the following ways:
• Manually select the transition ions you want to import for each
compound, or
• Enter the top number of transitions to import for each compound and
click the Select button. You can select the top transitions based on either
Abundance or Response Factor. Deselect any transitions that you don’t
want to use by clearing their check box.
9 If the transition ions selected in the table are the only ones you want to
import at this time, click Import. This closes the Data Browser window and
adds the transition ions into the current time segment.
• Otherwise, to do additional search and filter operations on the current
database or a different database, click Add to Import List.

44 Agilent Optimizer Software Quick Start Guide


Optimization and Results
To import optimization results to Acquisition for MRM time segments

10 To select additional compounds:


a To select compounds from a different database, open the database of
interest as described on page 22.

When asked if you want to make this database the default database, click No. Otherwise,
NOTE unsaved changes made to the current project will be lost. All saved compound
information in the previous project will still exist in the previous default database, but
won't be available for use with the new default database.

• Repeat steps 8a - c to select the transition ions of interest.


• Click Add to Import List.
b Click the Import List tab to review the list of transition ions. To remove
any transition ions you don't want to import, right-click and select
Remove from the shortcut menu.
c Click the Import button. The selected transitions are imported into the
current time segment.
11 Repeat step 5 through step 10 for each time segment of interest.
12 (optional) Save the method.

Agilent Optimizer Software Quick Start Guide 45


www.agilent.com

In this guide
This Quick Start Guide
describes how to use
MassHunter Optimizer
Automated MS Method
Development software.
This guide is valid for the
B.06.00 revision or higher of
the Agilent MassHunter
Optimizer Automated MS
Method Development
Software, until superseded.
Windows® is a registered
trademark of Microsoft
Corporation in the US and/or
other countries.

© Agilent Technologies, Inc. 2012


Printed in USA
Revision A, November 2012

*G3793-90007*
G3793-90007

Agilent Technologies

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